Postpartum haemorrhage remains an important cause of maternal death despite treatment with conventional therapy. Uncontrolled studies and one randomised comparison with conventional oxytocics have reported dramatic effects with high-dose misoprostol, usually given rectally, for treatment of postpartum haemorrhage, but this has not been evaluated in a placebo-controlled trial.The study was conducted at East London Hospital Complex, Tembisa and Chris Hani Baragwanath Hospitals, South Africa. Routine active management of the third stage of labour was practised. Women with more than usual postpartum bleeding thought to be related to inadequate uterine contraction were invited to participate, and to sign informed consent. All routine treatment was given from a special 'Postpartum Haemorrhage Trolley'. In addition, participants who consented were enrolled by drawing the next in a series of randomised treatment packs containing either misoprostol 5 × 200 μg or similar placebo, which were given 1 orally, 2 sublingually and 2 rectally.With misoprostol there was a trend to reduced blood loss ≥500 ml in 1 hour after enrolment measured in a flat plastic 'fracture bedpan', the primary outcome . There was no difference in mean blood loss or haemoglobin level on day 1 after birth < 6 g/dl or blood transfusion. Side-effects were increased, namely shivering  and pyrexia > 38.5°C . In the misoprostol group 3 women underwent hysterectomy of whom 1 died, and there were 2 further maternal deaths.Because of a lower than expected incidence of the primary outcome in the placebo group, the study was underpowered. We could not confirm the dramatic effect of misoprostol reported in several unblinded studies, but the results do not exclude a clinically important effect. Larger studies are needed to assess substantive outcomes and risks before misoprostol enters routine use. Excessive bleeding from the genital tract after birth, or postpartum haemorrhage (PPH) is the major cause of maternal deaths in many low-income countries. The global estimate is 125,000 deaths per year . In the There is good evidence that a policy of active management of the third stage of labour , and oneWhen postpartum haemorrhage occurs, a number of medical and surgical interventions are used to control the bleeding . A cruciMisoprostol is a methyl ester  of natural prostaglandin E1. It is marketed and registered for use in the prevention and treatment of peptic ulcer disease caused by prostanglandin synthetase inhibitors. Administered orally or sublingually, peak plasma concentrations of misoprostolic acid are achieved in less than 30 minutes . It is aAdministered orally or vaginally, it is an effective agent for the induction of abortion  and of lOral and rectal misoprostol have been used for routine management of the third stage of labour (after the birth). Several small trials have given conflicting results. The main side-effects have been shivering and pyrexia, which are dose-dependent . In the Misoprostol has been widely recommended for the prevention of postpartum haemorrhage when other methods are not available .treatment of postpartum haemorrhage has been promoted on the basis of unblinded studies. In a systematic review [Apart from the prophylactic use of misoprostol in the third stage of labour, the therapeutic use of misoprostol for the c review  we locatc review -24 and oc review . This mec review . If effec review ,28.The most common regimens reported for the treatment of postpartum haemorrhage are 800  or 1,000The objective of this study was to determine the side-effects and effectiveness of high-dose misoprostol for the treatment of postpartum haemorrhage by means of a double-blind, placebo-controlled, randomised clinical trial. The hypothesis was that measured blood loss of 500 ml or more in the hour after enrolment would be significantly less frequent in the misoprostol than in the placebo group.The study was conducted at the East London Hospital Complex  and Tembisa and Chris Hani Baragwanath Hospitals, Gauteng, South Africa, between Jan 2002 and Dec 2003. All are busy referral hospitals.Treatment packs were prepared independently, ordered in computer-generated random sequence and numbered consecutively. The packs contained either misoprostol 5 × 200 μg or inactive placebo.The treatment sequence was kept sealed, and the code broken only after complete entry and checking of all trial data.A confidential interim analysis, blind to which group was which, was performed by AMG after the first 100 enrolments. Evidence of clear benefit or harm for either group would have prompted stopping the trial, which was not the case.rd stage was routine active management with oxytocin 10 units or syntometrine one ampoule soon after the birth. Women aged 18 or more with bleeding judged by the clinician to be more than expected at least 10 minutes after delivery, and thought to be due to uterine atony and requiring additional uterotonic therapy, were given all the routine treatment for PPH (intravenous infusion, uterotonics, etc) from a special 'PPH Trolley' kept in the labour ward. The uterotonics used were oxytocin by intravenous infusion, and/or oxytocin/ergometrine, at the discretion of the attending clinician. Prostaglandin preparations such as sulprostone were not routinely available in the labour wards, and were not part of routine management of postpartum haemorrhage in the participating units.Women in labour were given basic information about the trial in the form of notices in the labour rooms (Table 2 see ). ManagOnce all emergency treatment was instituted, and if the women were in a position to give fully informed consent, they were given detailed information about the trial and asked whether they wished to participate. Information about the trial was given verbally and in writing in English or Xhosa (Table 3 see ). ThoseA low-profile plastic 'fracture bedpan' was placed under the woman's buttocks. In previous studies we have shown this to be an efficient method of collecting ongoing blood loss with very little spillage . Any smaAll other management was by hospital staff according to the hospital routine for the management of postpartum haemorrhage.After 24 hours, blood was collected for haemoglobin estimation and the hospital records checked for use of additional uterotonics and any other complications such as blood transfusion.The primary outcome measures were specified prior to commencing the study: (1) measured blood loss ≥500 ml in 1 hour after enrolment; (2) mean measured blood loss in 1 hour after enrolment; (3) haemoglobin level day 1 after birth <6 g/dl or blood transfusion; (4) Side-effects (pyrexia 38.5°C or more, moderate or severe shivering 1 hour after enrolment).Secondary outcome measures were: (1) blood loss ≥1,000 ml in 1 hour after enrolment; (2) blood transfusion; (3) haemoglobin level 1 day after birth <8 g/dl or blood transfusion; (4) additional uterotonic given after enrolment; (5) manual removal of the placenta; (6) evacuation of retained products of conception; (7) hysterectomy; (8) maternal death.In the WHO misoprostol trial control group (routine syntocinon), significant postpartum haemorrhage (blood loss >1000 ml) occurred in 2.8% of women. Of these 25% lost >1,500 ml. To reduce blood loss >500 ml after enrolment from 25% to 10%, would require 112 per group (α = 95%, β = 80%).Data were collected on a data form, entered onto an excel spreadsheet, checked for accuracy, then analysed using Epi-info 2002  and Review Manager (RevMan) [Computer program] version 4.2 for Windows. Oxford, England: The Cochrane Collaboration, 2003. Results were expressed as relative risks or mean difference with 95% confidence intervals. Analysis was by intention to treat.All women enrolled in the trial received all the conventional management available for postpartum haemorrhage, in addition to the trial medication (misoprostol or placebo). Rapid conventional therapy was facilitated by the ready availability of the 'PPH trolley' with all the necessary materials. This trolley was also available for women not enrolled in the trial. Participation was limited to women who were in a position to give fully informed consent. Ethical approval was given by the Committee for Research on Human Subjects, University of the Witwatersrand, and the Ethics Committee, East London Hospital Complex. The trial complied with the Helsinki Declaration for research on human subjects.Altogether 244 women were enrolled in the trial. The pack numbers for 6 women were incompletely filled in on the data sheets. The group allocation of these women was therefore unknown, and they could not be included in the analysis. Of these 6 women, the highest measured blood loss after enrolment was 220 ml; none had pyrexia >37.5°C, one had moderate shivering, one had a blood transfusion and none had day 1 haemoglobin level below 8 g/dl or other complications. There was no reason to expect that these exclusions occurred in any selective way which would introduce bias into the final samples, nor could their results have materially altered the conclusions of the study. All the remaining 238 women were included in the analyses.The baseline characteristics are shown in table The outcomes are shown in table Other proxy estimations of blood loss showed no significant differences between the groups. The well-known side-effects of misoprostol, pyrexia and shivering, were significantly more frequent in the misoprostol than the placebo group.Serious morbidity or mortality occurred in 5 women, all from the misoprostol group. Two women who continued to bleed despite conservative therapy underwent abdominal hysterectomy and recovered well. There were 3 maternal deaths:A 22-year old woman with one previous pregnancy developed postpartum haemorrhage after vaginal delivery, managed with a 40-unit oxytocin infusion. She was enrolled and received the trial medication 85 minutes after delivery. Thereafter she received a second 40-unit oxytocin drip, oxytocin/ergometrine (5 units/0.5 mg) and intravenous cyclokapron. In the hour after randomisation, measured blood loss was 125 ml. Subsequently bleeding continued and a sub-total hysterectomy was performed. Coagulopathy developed, bleeding continued through an abdominal drain, and she died 2 days after delivery despite re-laparotomy and multiple blood product transfusions.A 32-year-old woman, para 2, gravida 4, delivered normally after labour was induced with misoprostol, 25 μg vaginally and 4 doses of 50 μg orally. Before enrolment she received oxytocin10 units, ergometrine 0.5 mg and a 20-unit oxytocin infusion. She was enrolled and received the trial medication 140 minutes after delivery. After enrolment she received a further 40-unit oxytocin infusion. Measured blood loss in the hour after enrolment was 380 ml. Bleeding continued and a blood transfusion was commenced. Six and a half hours after delivery she suffered a cardiac arrest and was resuscitated. Examination in theatre found the uterus to be empty and intact. The main source of bleeding appeared to be a torn cervix, which was sutured. At 7.5 hours after delivery she suffered a second cardiac arrest and could not be resuscitated. The estimated total blood loss was 3,000 ml.A woman with one previous delivery by caesarean section, developed postpartum haemorrhage of about 500 ml after a vaginal delivery. She was enrolled 85 minutes after delivery. Measured blood loss over the next hour was 425 ml, after which she developed hypotension and cardiorespiratory arrest. As no post-mortem examination was performed, the possibility of internal bleeding from a dehisced caesarean section scar could neither be confirmed nor excluded.Unresponsive uterine atony was therefore not confirmed as the main cause of any of the deaths.Pyrexia and shivering were common side-effects of misoprostol, as found in previous studies. Three women, all in the misoprostol group, had severe pyrexia >40°C. Previous studies of high-dose misoprostol for the treatment of postpartum haemorrhage have mostly used the rectal route, and none has had sufficient numbers to be likely to detect rare adverse outcomes such as severe pyrexia. These side-effects may be related to the rapid absorption of misoprostol given orally, and the rapid absorption and high bioavailability when given sublingually. Because of these serious side-effects, we would recommend that for future trials the dose be reduced.Several previous unblinded studies have reported dramatic effects of misoprostol when used to treat postpartum haemorrhage. In 6 observational studies -24, 41 wIn our double blind trial we have been unable to confirm the dramatic effects of misoprostol reported previously in unblinded studies. Our results, however, are consistent with anything from a large beneficial effect to a smaller adverse effect . It is important that this possible benefivial effect be assessed by sufficiently powered randomised trials before the treatment enters routine clinical use.PPH: postpartum haemorrhage; SD: standard deviationNone known.GJH prepared the first draft of the protocol and the final manuscript, and oversaw the clinical workCN assisted with the design of the protocol and data collection sheet, initiated enrolment at two sites, supervised some of the data collection and contributed to the manuscriptSF, ZM, LM, MS and ZJ commented on the protocol, conducted the clinical procedures and collected data.SF entered and collated the dataBM undertook the literature search and commented on the protocol.GW contributed to the protocol developmentAMG contributed to the protocol development and undertook the blinded interim analysisAll authors read and approved the final manuscriptAdditional file 1 - Table 2. Notice informing women about the trial, MS Word file: HofmeyrFile1.docAdditional file 2- Table 3: Information and consent form. MS Word file: HofmeyrFile2.docThe pre-publication history for this paper can be accessed here:Table 2. Notice informing women about the trialClick here for fileTable 3: Information and consent formClick here for file


Every sensation begins with the conversion of a sensory stimulus into the response of a receptor neuron. Typically, this involves a sequence of multiple biophysical processes that cannot all be monitored directly. In this work, we present an approach that is based on analyzing different stimuli that cause the same final output, here defined as the probability of the receptor neuron to fire a single action potential. Comparing such iso-response stimuli within the framework of nonlinear cascade models allows us to extract the characteristics of individual signal-processing steps with a temporal resolution much finer than the trial-to-trial variability of the measured output spike times. Applied to insect auditory receptor cells, the technique reveals the sub-millisecond dynamics of the eardrum vibration and of the electrical potential and yields a quantitative four-step cascade model. The model accounts for the tuning properties of this class of neurons and explains their high temporal resolution under natural stimulation. Owing to its simplicity and generality, the presented method is readily applicable to other nonlinear cascades and a large variety of signal-processing systems. Comparing auditory stimuli that give the same neural response within the framework of a computational model, the authors extract intermediary signal-processing steps with sub- millisecond temporal resolution Animals and human beings rely on accurate information about their external environment and internal state for proper behavioral reactions. This vital requirement has led to a large variety of highly sophisticated sensory systems . A commoWe here show that it is possible to extract fine temporal details of individual processes within such signal-processing chains from observing the output activity alone. This progress results from a new method that extends an experimental strategy well known from measuring threshold curves in neurobiology  or applyUnlike many classical approaches of systems identification, the method is not based on temporal correlations between the input and output; hence, the time resolution of the method is not limited by the output precision of the system under study. In a spike-based analysis of neural response properties, this allows us to assess the dynamical features of the involved processes with considerably higher resolution than suggested by the spike jitter.A particularly fine temporal resolution is needed to analyze signal processing in auditory systems that solve complex tasks such as sound localization, echolocation, and acoustic communication ,13,14,15Locusta migratoria). Its anatomy is well characterized [As a suitable model system to study signal processing in the ear, we chose the auditory periphery of the locust , the sets generally lie on straight lines, at least for low-frequency receptor cells. High-frequency receptor cells do not display straight lines even at the smallest Δt used in the experiment (40 μs) for reasons that will become apparent later. For long intervals (between approximately 400 and 800 μs, depending on the cell), the iso-response sets fall onto nearly circular curves. Note that in t = 500 μs deviates from the symmetry between A1 and A2. In t = 120 μs fell in neither of the two regimes discussed above, and the corresponding iso-response set shows a particularly bulged shape. Recordings from a total of eight cells agree with the observations from the three examples displayed in A1+A2, of both click amplitudes determines the spike probability and demonstrates that the sound pressure is most likely the relevant stimulus parameter. Such linear summation of the pressure on short time scales is not surprising, considering the mechanical properties of the eardrum; owing to its mechanical inertia, rapidly following stimuli can be expected to superimpose. This interpretation is in agreement with laser-interferometric and stroboscopic observations of the eardrum, which have demonstrated that it reacts approximately linearly to increases in sound pressure [The two prominent shapes of the iso-response sets—straight lines and circles—reflect two different processing steps in the auditory transduction chain. A straight line implies that the linear sum, pressure ,19.A12+A22, now determines the spike probability. It follows that the sound energy, which is proportional to the squared pressure, is the relevant stimulus parameter on this time scale. This quadratic summation represents a fundamentally different way of stimulus integration from that of the linear summation on short time scales and indicates the involvement of a different biophysical process. A process that can mediate stimulus integration over longer intervals is the accumulation of electrical charge at the neural membrane. According to this explanation, the electrical potential induced by a click is proportional to the click's energy; contributions from consecutive clicks are then summed approximately linearly because of the passive membrane properties. This is in accordance with earlier investigations for stationary sound signals that revealed an energy dependence of the neurons' firing rate [For the longer intervals, on the other hand, the iso-response sets are circles to good approximation, indicating that the quadratic sum, or ing rate . We concA generates a vibration of the tympanum with strength X = c1·A, i.e., linear in the amplitude with a proportionality constant c1. This mechanical vibration leads to a membrane potential, whose effect on the generation of the spike some time T after the click is given by J = c2·X2= c2·(c1·A)2, i.e., quadratic in the amplitude with an additional proportionality constant c2. The square follows from the circular shape of the iso-response sets for longer time scales, which indicated that a quadratic operation must take place before the accumulation of charge at the neural membrane. Finally, the spike probability p is given by a yet unknown function p = g(J). As J is the relevant quantity determining spike probability, we also refer to it as “effective stimulus intensity.” The model contains a freedom of scaling; any proportionality constants in J can be absorbed into the function g(J). To simplify the notation, we thus set c1 = c2 = 1 and obtain X = A for the strength of the mechanical vibration and J = X2= A2 for the effective stimulus intensity in response to a single click.The above findings motivate the following mathematical model, which describes how a stimulus consisting of two sound clicks is transformed into a spike probability. Within the model, a single click of amplitude X as describing the velocity of the mechanical vibration immediately after the click and J as capturing the membrane potential at the time of spike generation.Note that in this picture, the mechanical vibration and the membrane potential are each captured by a single quantity that does not describe the time course of the corresponding processes, but rather their integrated strength in response to a click. In general, the conversion of the mechanical vibration into a membrane potential as well as the spike generation are dynamical processes that do not happen at a single moment in time. For simplicity, however, one may think of A1 and A2, respectively, we choose the first click to be small enough so that it does not lead to a spike by itself. The measured action potential is thus elicited at some time T after the second click. To derive the model equation for this experimental situation, we divide the time from the first click to spike generation into the period between the two clicks and the period following the second click.For the two-click stimulus with amplitudes X1 = A1. However, how much electrical charge accumulates during the inter-click interval to influence spike generation at time T after the second click depends on the length Δt of the inter-click interval. This effect is incorporated by a Δt-dependent scaling factor Q(Δt) into the model and results in a first contribution from the first click to spike generation given by J1= A12·Q(Δt). Since Q(Δt) denotes the effect of the first click within the inter-click interval only, it should vanish in the limit of very small Δt.Let us start by focusing on the inter-click interval. After the first click, the mechanical vibration has the strength t. This is modeled by a scaling factor L(Δt), i.e., the vibration after the second click has a strength X2 = A1· L(Δt) + A2. Accordingly, the effect of the two-click vibration on the membrane potential at time T after the second click is J2= (X2)2= (A1· L(Δt) + A2)2. For very small Δt, L(Δt) should approach unity to account for the equal contribution of both clicks for vanishing inter-click intervals. The total effective stimulus intensity is then given byLet us now consider the remaining time before spike generation. After the second click, the mechanical vibration is due to a superposition of both clicks. For short inter-click intervals, the straight iso-response lines suggest a simple addition of the two click amplitudes; in general, however, the contribution of the first click to the membrane vibration after the second click will again depend on the inter-click interval Δp via the relation p = g(J).This quantity determines the spike probability L(Δt) = 1 and Q(Δt) = 0 (straight line) and (2) L(Δt) = 0 and Q(Δt) = 1 (circle).How does this model explain the particular shapes of the iso-response sets in t and the second case in some range of larger Δt. In our biophysical interpretation, the first case means that the two clicks are added at the tympanic membrane (L(Δt) ≈ 1), but the short interval between the two clicks prevents a substantial accumulation of charge from the first click alone (Q(Δt) ≈ 0), as already discussed above. The second case may be found for Δt long enough that the mechanical vibration has already decayed (L(Δt) ≈ 0). The two clicks are then individually squared, i.e., they independently lead to two transduction currents. The currents add up if the time constant of the neural membrane is significantly longer than the inter-click interval (Q(Δt) ≈ 1).We can therefore regard A1 and A2, reflecting the symmetry of, e.g., the data in t where neither of the two cases is strictly fulfilled, this symmetry of the iso-response sets will be distorted, as is noticable for the longer Δt in L(Δt) and Q(Δt) may lead to very different iso-response shapes, as in In the two limiting cases, L(Δt) and Q(Δt) are thus filter functions associated with the linear and quadratic summation, respectively.L(Δt) and Q(Δt) can be expected to retain the salient features of the underlying biophysical processes such as frequency content and integration time. In L(Δt) as the velocity of the mechanical vibration and of Q(Δt), at least for large enough Δt, as the time course of the membrane potential following a click. In this generalized model, the input signal is an arbitrary sound pressure wave A(t), and the effective stimulus intensity is a continuous function of time, J(t), which is given byDespite the simple structure of the model, the filters A(t) is first convolved with a temporal filter, l(τ), the result is squared and subsequently convolved with a second filter, q(τ), as depicted in l(τ) and q(τ) have characteristics similar to the click-version filters L(Δt) and Q(Δt), but are not identical to them. Their relations follow from the calculation in L(Δt) and Q(Δt).Here, the input A1, these additional spikes lead to measured values of A2 that are slightly smaller than expected for a circular iso-response set. The data in Note that we interpret t, however, their dynamics may largely overlap. Is it nevertheless possible to design an experiment that directly reveals the whole time course of the mechanical vibration L(Δt) and the electrical integration Q(Δt)? This would provide a parameter-free description of both processes and advance the quantitative understanding of the auditory transduction dynamics. To reach this goal, we again measure iso-response sets. As before, we exploit that for fixed Δt, any pair of click amplitudes (B1, B2) should result in the same spike probability p as the pair (A1, A2) as soon as J(A1, A2) = J(B1, B2). It is this straightforward relation that allows us to determine both L(Δt) and Q(Δt) independently of each other. In fact, some appropriate set of measurements that fulfill the iso-response relation is all that is needed to calculate L(Δt) and Q(Δt). Illustrating this concept, we now proceed with a particularly suited choice of stimulus patterns, which keeps the mathematical requirements for the calculation at a minimum. For each Δt, we measure two different iso-response stimuli, and as a key feature, one of these has a “negative” second click, i.e., a sound-pressure pulse pointing in the opposite direction as the first click, as depicted in L(Δt) and Q(Δt), which can be solved explicitly, as explained in t, the whole time course of L(Δt) and Q(Δt) is obtained.The previous experiment showed that the separate effects of the two summation processes can be discerned for short and long time intervals. For intermediate ΔL(Δt) and Q(Δt) for three different cells. L(Δt) displays strong oscillatory components, as was observed for all cells. This property presumably reflects the eardrum's oscillation at the attachment site of the receptor cell. The detailed temporal structure of L(Δt) now allows us to investigate the salient features of this oscillation. To quantify our findings, we fit a damped harmonic oscillation to the measured data for L(Δt) and extract the fundamental frequency as well as the decay time constant. We can use these values to predict the neuron's characteristic frequency (the frequency of highest sensitivity) and the width of its frequency-tuning curve. L(Δt). The remarkable agreement confirms that the new analysis faithfully extracts the relevant, cell-specific properties of the transduction sequence. The correspondence between the tuning characteristics and the filter L(Δt) also explains why high-frequency receptor cells do not feature straight lines for their iso-response sets even at the shortest inter-click interval (40 μs) used in the experiment. For those cells, L(Δt) decays rapidly, thus not allowing access to the region where L(Δt) ≈ 1.Q(Δt) in The short initial rise phase of the measured L(Δt) was considerably smaller than that of Q(Δt). This usually leads to a region around a Δt of 400–800 μs, depending on the specific cell, where L(Δt) ≈ 0 and Q(Δt) is still near unity. These findings correspond to the circular iso-response sets of the initial experiment.In most of our recordings, the temporal extent of the filter t, on the other hand, the data show that Q(Δt) usually decreases strongly. As explained earlier, this is expected from the linear iso-response sets, and it is observed exemplarily in the data shown in Q(Δt) for some recordings, as in Q(Δt). (1) The electrical potential might be shaped by further dynamics in addition to the low-pass properties of the neural membrane, such as inactivation of the transduction channels or electrical resonances as found in some hair cells [Q(Δt)—though too small to be picked up reliably by the present experiments—can be observed in simulations of the processing cascade, see Towards very small Δir cells . (2) TheL(Δt) and Q(Δt), completes the model. In order to test its validity and suitability to make quantitative predictions, we investigated the model's performance on a different class of stimuli, namely combinations of three short clicks. Having measured the required values for L(Δt) and Q(Δt) with two-click stimuli as in the previous experiment (see L(Δt) and Q(Δt) to calculate these predictions and experimentally test them by performing a series of three-click iso-response measurements. This experiment was performed on three different cells; one cell featured an unusually high response variability, and results from the other two cells are shown in Measuring the mechanical and electrical response dynamics, ment see , we now We have presented a novel technique to disambiguate single processing steps within a larger sensory transduction sequence and to analyze their detailed temporal structures. Our approach is based on measuring particular iso-response sets, i.e., sets of stimuli that yield the same final output, and on specific quantitative comparisons of such stimuli to dissociate the individual processes. For the investigated auditory transduction chain in the locust ear, this strategy led to a precise characterization of two consecutive temporal integration processes, which we interpret as the mechanical resonance of the eardrum and the electrical integration of the attached receptor neuron. The method revealed new details of these processes with a resolution far below 1 ms. The results for the time course of the mechanical resonance agree with traditional measurements of tuning curves and show the decay of the oscillation with a temporal precision much higher than expected from the jitter of the measured output signal, the spikes. The time constants of the electrical integration that were extracted from the data had not been accessible by other means.t and is in accordance with the energy-integration model that was found to capture the sound-intensity encoding of stationary sound signals in these cells [The analysis resulted in a four-step model of auditory transduction in locusts. The model comprises a series of two linear filters and two nonlinear transformations. The quadratic nonlinearity that separates the two linear filters suggests that the mechanosensory transduction can be described by an energy-integration mechanism, as the squared amplitude corresponds to the oscillation energy of the tympanum. This quadratic form was derived from the circular shape of the iso-response sets for longer time scales Δse cells . Furtherse cells , and in se cells ,24,25,26se cells . This efAlthough the quadratic nonlinearity is fully consistent with our data, there is a second possibility within the general cascade model, L(Δt) and Q(Δt) were interpreted as the mechanical oscillation of the tympanum and the electrical integration at the neural membrane. Their oscillatory and exponential decay characteristics, respectively, support this view. In principle, however, other processes may well contribute to these characteristics, e.g., electrical resonances as seen in hair cells of the turtle and bullfrog [Q(Δt), but our data generally provide little evidence for such effects. Deviations from the exponential decay characteristics in Q(Δt) may in part be attributable to the oscillatory influx of charge resulting from the tympanic vibration. This may lead to a small oscillatory component in the early phase of the filter , which includes thresholding and saturation. By always comparing stimuli that yield the same output activity, our analysis is independent of the actual shape of g(J). Preceding integration steps may thus be analyzed without any need to model g(J). This feature is independent of the specific output measure and applies to spike probabilities, firing rates, or any other continuous output variable.Finally, in many input–output systems, the last stage of processing can be described by a monotonic nonlinearity. Here, this is the relation between the effective stimulus intensity t. Instead, performing more than the two measurements at each intermediate Δt in the second experiment  cascade, composed of two linear temporal integrations and two nonlinear static transformations . SimilarComplementary to the correlation techniques underlying the parameter estimations in those models, the method presented in this work provides a new way of quantitatively evaluating and testing cascade models. The increased complexity of the LNLN cascade identified in the present case was made accessible by invoking particular iso-response measurements, and a higher temporal resolution was achieved by focusing on how spike probabilities depend on the temporal stimulus structure instead of relying on temporal correlations between stimulus and response.Our experimental technique will be most easily applicable to systems whose signal processing resembles the cascade structure investigated here. The general concept of combining different measurements from within one iso-response set covers, however, a much larger range of systems. With increasingly available high-speed computer power for online analysis and stimulus generation, this framework therefore seems well suited to solve challenging process-identification tasks in many signal-processing systems.Locusta migratoria. Details of the preparation, stimulus presentation, and data acquisition are described elsewhere [We performed intracellular recordings from axons of receptor neurons in the auditory nerve of adult lsewhere . In shorSpikes were detected online from the recorded voltage trace with the custom-made Online Electrophysiology Laboratory software and used for online calculation of spike probabilities and automatic tuning of the sound intensities. The measurement resolution of the timing of spikes was 0.1 ms. During the experiments, the animals were kept at a constant temperature of 30 °C by heating the Peltier element. The experimental protocol complied with German law governing animal care.p of the studied receptor neurons increases monotonically with stimulus intensity, parameters of iso-response stimuli corresponding to the same value of p can be obtained by a simple online algorithm that tunes the absolute stimulus intensity. For fast and reliable data acquisition, we chose p = 70%. The response latency of the neurons varied by 1–2 ms, so that spike probabilities could be assessed by counting spikes over repeated stimulus presentations in a temporal window from 3 to 10 ms after the first click.Since the spike probability A1 and A2, and the tuning was achieved by adjusting the two amplitudes simultaneously. The ratios were chosen so that the angles α in the A1–A2 plane given by tanα = A2/A1 were equally spaced. In the second set of experiments, A1 was kept fixed, and only A2 was adjusted; similarly, in the three-click experiments, only A3 was adjusted. In the following, the intensity I always refers to the peak amplitude Amax of the stimulus pattern, measured in decibel sound pressure level (dB SPL),In the first set of experiments, stimulus patterns were defined by fixed ratios of I70 corresponding to a spike probability of 70% was determined online in the following way. Beginning with a value of 50 dB SPL, the intensity was raised or lowered in steps of 10 dB, depending on whether the previous intensity gave a spike probability lower or higher than 70% from five stimulus repetitions. This was continued until rough upper and lower bounds for I70 were found. From these, a first estimate of I70 was obtained by linear interpolation. Seven intensity values in steps of 1 dB from 3 dB below to 3 dB above this first estimate were then repeated 15 times. From the measured spike probabilities, a refined estimate of I70 was obtained by linear regression. Nine intensities from 4 dB above to 4 dB below this value were repeated 30 times (in some experiments 40 times). The final estimate of I70 was determined offline from fitting a sigmoidal function of the formFor each stimulus, the absolute intensity α and β to these nine intensity-probability pairs. This relation between p and I was then inverted to find the intensity and thus the absolute values of the amplitudes that correspond to p = 0.7.with parameters L(Δt) and Q(Δt) can be obtained independently of each other by combining the results from different measurements within one iso-response set. Here, we derive explicit expressions based on a specific choice of stimuli that are particularly suited for our system. Two measurements are needed to obtain both L(Δt) and Q(Δt) for given time interval Δt. Each stimulus consists of two clicks. The first click has a fixed amplitude A1; the amplitude A2 of the second click at time Δt later is adjusted so that a predefined spike probability p is reached. For the second measurement, the experiment is then repeated with a “negative” second click, i.e., a click with an air-pressure peak in the opposite direction from the first click. The absolute value of this click amplitude is denoted by Ã2. We thus find the two pairs (A1,A2) and (A1, Ã2) as elements of an iso-response set. Since the spike probability increases with the effective stimulus intensity J, equal spike probability p implies equal J. The two pairs (A1,A2) and (A1, Ã2) therefore correspond to the same value of J. According to the model, The response functions Setting the two right sides equal to each other, we obtainorÃ2 = − A2, does not correspond to a physical situation as both A2 and Ã2 denote absolute values and are therefore positive. The remaining, second solution readsThe first solution of this mathematical equation, L(Δt), we obtainSolving for L(Δt) from Substituting This yieldsc = J/A12. As we keep A1 and J constant throughout the experiment, this determines Q(Δt) up to the constant c. It can be inferred from an independent measurement with a single click: by setting A1 = 0 in J corresponds to the square of the single-click amplitude that yields the desired spike probability. Alternatively, c can be estimated from the saturation level of Q(Δt) for large Δt, as was done in the present study.with L(Δt) and Q(Δt); see The specific form of the effective stimulus intensity, L(Δt) were fitted with velocity response functions of a damped harmonic oscillatorThe datasets for ω and δ were optimized for minimizing the total squared error. From these, the fundamental frequency f and the decay time constant τdec were determined as f = ω/(2π) and τdec = 1/δ. A simpler fit function of the formwhere f and τdec.led to essentially indistinguishable results for fCF, of the tuning curve, and the tuning width, Δf3dB, can be predicted from the fitted values of ω and δ according to the theory of harmonic oscillators:The resonance frequency, which corresponds to the characteristic frequency, Q(Δt) were fitted with an exponential decayThe datasets for a, τint, and c were adjusted. Here, only data points for Δt > 150 μs were taken into account, as Q(Δt) initially shows a rising phase. The obtained value for c was used to determine the constant J/A12 in where the parameters For comparing these predicted values with measurements, the minimum and width of the tuning curves see A were deA1, A2, and A3 that are separated by time intervals Δt1 and Δt2, respectively  and is augmented by A2. This yields a membrane potential proportional to (A1·L(Δt1) + A2)2. After the third click, the tympanic deflection has evolved to A1·L(Δt1 + Δt2) + A2·L(Δt2) so that the membrane potential is increased by (A1·L(Δt1 + Δt2) + A2·L(Δt2) + A3)2. Summing up the different contributions and approximating the influence of the inter-click intervals on the membrane potential by appropriate factors of Q, we find for the effective stimulus intensityFor stimuli consisting of three clicks with amplitudes vely see A, an appJ for a predefined spike probability can be measured from a single-click experiment by setting A1= A2 = 0 and tuning A3 until the desired spike probability is reached. After having measured L(Δt) and Q(Δt) from two-click experiments, the above equation can be used to predict the amplitude A3 needed to reach this predefined spike probability for any combination of A1, A2, Δt1, and Δt2.The value of Protocol S1(50 KB PDF).Click here for additional data file.Figure S1t = 50 μs, (C) a double click with Δt = 500 μs, and (D) another double click with Δt = 500 μs whose second click points in the oppositve (“negative”) direction. The measurements of air-pressure fluctuations indicate a slight broadening of the click width and some residual vibrations, but they nevertheless present a good approximation of the sharp original pulses.The four panels show different examples of stimuli used in our study. Each panel illustrates the computer-generated pulse signal that drives the loud speaker (upper trace) and the resulting air-pressure fluctuations as measured with a high-precision microphone at the site of the animal's ear (lower trace). The computer-generated clicks are triangular with a total width of 20 μs. The stimuli shown are (A) a single click, (B) a double click with a peak-to-peak interval Δ(10 KB PDF).Click here for additional data file.Figure S2l(t) = sin(2πft)exp(−t/τdec) and q(t) = exp(−t/τint). The parameters were taken from the first two cells presented in detail in the main text: f = 14.5 kHz, τdec = 100 μs, and τint = 300 μs for Cell 1 (left column) and f = 5.1 kHz, τdec = 154 μs, and τint = 590 μs for Cell 2 (right column).The general cascade model, x(t) denotes the signal after application of the linear filter l(t), arbitrary units, for positive second click (solid line) and negative second click (dashed line). Inter-click intervals in these two shown examples were Δt = 80 μs for Cell 1 and Δt = 130 μs for Cell 2.(A and B) Responses of tympanic vibration. J(Δt). The second click was tuned so that the maximum of J(Δt) was equal for positive and negative second clicks. This required click amplitudes of size 1.92 and −2.49 relative to the first click for Cell 1 and 2.09 and −1.27 for Cell 2.(C and D) Corresponding responses of L(Δt) and Q(Δt) extracted according to J(Δt) for many different values of Δt (gray dots). The parameters f, τdec, and τint indicated in the plots were obtained by fitting a damped harmonic oscillator and an exponential function to L(Δt) and Q(Δt), respectively (black lines). The initial part of Q(Δt) shows small fluctuations that result from the oscillatory influx of charge following the tympanic vibrations. In (G), a magnified view of the initial section is shown in the inset.(E–H) Filters (138 KB PDF).Click here for additional data file.


However, a new study suggests that Tax preferentially transactivates the 21-bp repeats through CREB1 and not other bZIP proteins. The additional transactivation of Tax-responsive promoters subsequent to initiation is also presented. This result highlights a potentially novel role of Tax following TBP recruitment ( Viruses have long been a source of key scientific discoveries. Historically, they have contributed to our knowledge of transcription, cell cycle, and apoptosis. To date activated transcription in higher eukaryotic cells with or without chromatin is a great area of active research and many researchers use viral activators, including herpes virus VP16, adenovirus E1A, HIV-1 Tat and HTLV-I Tax to not only understand viral, but also basic mechanisms related to host control of vital cellular machineries, including transcription. Eukaryotic transcription has five distinct phases, pre-initiation, initiation, promoter clearance, elongation and termination, and is a tightly regulated and coupled process [et al. [in vivo on the 21-bp repeats  in HTLV-I infected cells through chromatin immunoprecipitation (ChIP) assays [et al. demonstrated that among the various bZIP proteins, CREB1 was clearly favored for Tax transactivation of the 21-bp repeats. Additionally, CREB1 has also been found to primarily bind at the 5' LTR (rather than the 3' LTR) in vivo within HTLV-I infected cells, lending support to the idea that CREB1 is important for HTLV-I activated transcription [In the report by Ching et al.  the authet al. -5. It wa) assays . By usincription .et al. [in vitro and in vivo).If CREB1 is the dominant bZIP protein that is needed for Tax transactivation of the LTR, then what is the purpose of the additional bZIP proteins? Besides contributing to Tax transactivation, could these bZIP proteins help to exclude negative regulators from the LTR? A report by Basbous et al.  suggesteet al. presented the intriguing possibility of Tax enhancing transcription following transcription initiation. To determine whether Tax functioned solely to target TBP to the TATAA-element or if additional events subsequent to TBP (TFIID) recruitment were promoted by Tax, the authors constructed four independent reporters. Each promoter contained the minimal TATAA-element from HTLV-I, HIV-1, SV-40, or E1b promoters, two 21-bp repeats, and five copies of the Gal4-binding site. TBP was artificially targeted to the TATAA-element thru Gal4-TBP. The authors reasoned that if Tax functioned strictly to recruit TBP to the TATAA-element, then additional enhancement of transcription would not be observed when Tax and Gal4-TBP were present. Interestingly, only the Tax-responsive promoters, i.e. HTLV-I and HIV-1, were both synergistically stimulated by the addition of Tax and Gal4-TBP. These results suggest that Tax may control downstream transcription subsequent to the initiation phase.Lastly, Ching 2/cdk4 complexes [Other viral transactivators have been shown to have a role at initiation and downstream events, such as elongation. The most notable of these has been Tat, the viral transactivator of HIV-1. Without cellular stimulation and Tat expression, RNAP II transcriptional elongation was shown to be inefficient, producing only short transcripts . One majomplexes ,14. Thiset al. [hsp70 mRNA. Tax has been shown to associate with BRG1 components of the ATP-dependent chromatin remodeling complex, SWI/SNF, and increase Tax transactivation [et al. are preliminary at this time, Tax transactivation post-initiation is indeed a novel concept. Further detailed analysis of Tax at both the LTR of HTLV-I and downstream of this region will help to resolve many of these questions and provide important insight into the transcription field.Processive elongation is highly dependent on remodeling of chromatin structure ,10. A stet al.  demonstrtivation . DisruptHTLV-I, human T cell leukemia virus, type ICRE, cAMP response elementCREB, cAMP response element binding proteinChIP, chromatin immunoprecipitationRNAP II, RNA polymerase IICTD, C-terminal domainHIV-1, human immunodeficiency virus, type 1LTR, long terminal repeatTBP, TATA binding proteinTxREs, Tax-responsive elementsGTFs, general transcription factorsTAR, transactivation regionNone declared.Both authors contributed equally to the structure and content of the manuscript.


The pulmonary residence time of inhaled glucocorticoids as well as their rate and extend of absorption into systemic circulation are important facets of their efficacy-safety profile. We evaluated a novel approach to elucidate the pulmonary absorption of an inhaled glucocorticoid. Our objective was to monitor and compare the combined process of drug particle dissolution, pro-drug activation and time course of initial distribution from human lung tissue into plasma for two different glucocorticoid formulations.®/Becloforte™ and Ventolair®/Qvar™). Initially we developed a simple dialysis model to assess the transfer of BDP and its active metabolite from human lung homogenate into human plasma. In a novel experimental setting we then administered the aerosols into the bronchus of an extracorporally ventilated and reperfused human lung lobe and monitored the concentrations of BDP and its metabolites in the reperfusion fluid.We chose beclomethasone dipropionate (BDP) delivered by two different commercially available HFA-propelled metered dose inhalers (Sanasthmax®/Becloforte™ and Ventolair®/Qvar™ in both the dialysis as well as in the human reperfusion model. The HFA-BDP formulated as Ventolair®/Qvar™ displayed a more rapid release from lung tissue compared to Sanasthmax®/Becloforte™. We succeeded to explain and illustrate the observed differences between the two aerosols with their unique particle topology and divergent dissolution behaviour in human bronchial fluid.Unexpectedly, we observed differences between the two aerosol formulations Sanasthmax®/Qvar™ are beneficial for high lung deposition, they also yield a less desired more rapid systemic drug delivery. While the differences between Sanasthmax®/Becloforte™ and Ventolair®/Qvar™ were obvious in both the dialysis and lung perfusion experiments, the latter allowed to record time courses of pro-drug activation and distribution that were more consistent with results of comparable clinical trials. Thus, the extracorporally reperfused and ventilated human lung is a highly valuable physiological model to explore the lung pharmacokinetics of inhaled drugs.We conclude that though the ultrafine particles of Ventolair Current asthma management guidelines recommend inhaled glucocorticoids as preferred therapy for control of mild persistent, moderate and severe asthma ,2. GlucoThe time course of inhaled drug absorption has been frequently studied in life animals . For thiin vivo distribution of inhaled glucocorticoids between human lung tissue and plasma has been determined in patients undergoing thoracotomy. During surgery tissue and plasma samples were obtained and analyzed for drug concentrations which were found to be significantly higher in lung tissue compared to plasma [The o plasma -11. The Plasma concentrations of glucocorticoids after inhalation of therapeutic doses are low and thus highly sensitive analytical methods are required . Blood sThe purpose of this study was to monitor and compare the combined process of drug particle dissolution, pro-drug activation and time course of distribution from human lung tissue into plasma in the absence of hepatic metabolism. We chose beclomethasone dipropionate (BDP) as a model compound because different formulations are commercially available and pharmacokinetic data from clinical studies is accessible for comparison. BDP is activated in human lung tissue to yield its active metabolite beclomethasone-17-monopropionate (17-BMP) . We init®/Becloforte™ 250 μg/dose [Asche Chiesi GmbH, Hamburg, Germany] and Ventolair®/Qvar™ 100 μg/dose [3 M Medica, Neuss, Germany]) or chlorofluorocarbon (CFC) propellant (Sanasthmax®/Becloforte™) were obtained from a local pharmacy. BDP, beclomethasone-17-propionate (17-BMP), beclomethasone-21-propionate (21-BMP), beclomethasone (B) and fluticasone propionate (FP) were a generous gift from GlaxoSmithKline (Greenford, England). Diethylether (HPLC grade) was purchased from Fluka (Buchs, Switzerland) and acetonitrile (ACN, HPLC gradient grade) from Fisher Scientific (Schwerte, Germany). Water was obtained from a Millipore™ water purification unit. Bovine serum albumin (BSA), dextrane 70000, and N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) were purchased from GERBU (Heidelberg, Germany), glucose monohydrate from Gruessing GmbH (Filsum, Germany), and stock solution containing 10000 IU/mL penicilline and 10000 μg/mL streptomycine in 0.9% NaCl from Biochrom AG (Berlin, Germany). All other chemicals were obtained from E. Merck (Darmstadt, Germany).Beclomethasone dipropionate (BDP) pressurized metered dose inhalers (MDI) with hydrofluorocarbon (HFA) propellant . Homogenization was performed under continuous cooling using an Ultraturrax (Janke & Kunkel, Staufen, Germany). Before starting the series of dialysis experiments the required amount of human lung homogenate was estimated and subsequently a sufficient amount was prepared and divided into aliquots. Since all aliquots descended from this preparation protein content and enzymatic activity of the homogenate was identical for each experiment.Human lung tissue specimen were obtained from patients with bronchial carcinomas who gave informed consent. Only cancer-free tissue was used for the experiments. None of the patients was treated with glucocorticoids for the last 4 weeks prior to surgery. Tissue samples were shock frozen in liquid nitrogen after resection and stored at -70°C until usage. To collect sufficient material for the experiments, tissue samples of three or more patients were pooled. Tissue was thawed and cut into small pieces. One part of the tissue pieces was homogenized in two parts of Krebs-Ringer-HEPES buffer , wedged into a subsegment bronchus. Small bronchial fluid aliquots of four patients were collected and pooled. The specimen was frozen and stored at -70°C until usage.Six patients with a bronchial tumour undergoing standard thoracotomy, were included in the study. None of the patients was treated with glucocorticoids for the last 4 weeks prior to surgery. Only patients with tumours that were located peripherally within the lung lobe were included. Each patient signed a written informed consent before surgery, and a local Ethics Committee approved the use of resected human lungs for perfusion. Immediately after perfusion, the lung preparations were examined as usually by a pathologist.Dialysis was performed with a dialysis unit (designed by our working group) consisting of two individual tightly fitting Teflon chambers separated by a dialysis membrane Figure . One chaIn initial experiments we evaluated the functionality and reliability of the experimental setting with respect to the convection, appropriate sample recovery and absence of trapped air. Therefore, the upper chamber was replaced by a chamber with a top made of acrylic glass instead of Teflon to monitor the processes within the unit. After filling the lower chamber with buffer the dialysis membrane was placed on this chamber and the dialysis unit was closed by attaching the second chamber. The complete and air bubble free filling of the chamber by smooth negative pressure was visually controlled. To check whether there was sufficient convection for a homogenous distribution of the analyzed compounds in the plasma and whether replacement of the sample volume was readily achieved, buffer in the reservoir was stained with a green dye. Then sampling was performed as described above. The appropriate moment for closing the valve was defined as the point of time where no visible flow of stained buffer into the chamber could be observed. It was also determined if any air bubbles gained access through the sampling aperture during sampling or after removing the pipette. Control experiments with the dye verified that the sample was not adulterated during the process of sampling and synchronous replacement of the sample volume with fresh solution. The degree of convection was controlled by following the distribution of the green dye into the clear buffer after sampling. A visually homogenous solution was obtained within a few minutes under experimental conditions. Accurate and reproducible sample volume was assured by weighing ten replicates of samples drawn under experimental conditions. Parameters for accuracy and reproducibility were within the specifications of the manufacture of the used pipettor.ex valve was applied to the homogenate employing a dosing device  was filled into one chamber. 500 μg BDP 18 column (150 × 4.6 mm I.D., 5 μm particle size, Waters, USA). Typically, 20 μL of sample were injected, a flow rate of 1 mL/min was used, and detection wavelength was set to 254 nm. Mobile phase consisted of water containing 0.2 % (v/v) acetic acid (A) and acetonitrile (B). The gradient elution started at 62 % eluent A, decreasing nonlinearly to 53 % A by 22 min and finally decreasing linearly to 28 % eluent A by 18 min. The lower limit of quantification of the assay was 20 ng/mL for all glucocorticoids.Samples of the dialysis experiments were mixed with 50 μL internal standard solution (3 μg/mL FP in methanol) and extracted twice with 2 ml diethylether for 20 min using a roller mixer, followed by centrifugation at 3000 rpm (Labofuge II, Heraeus-Christ GmbH, Osterode am Harz, Germany) for 5 min. The combined organic phases were evaporated to dryness under a gentle stream of nitrogen at 25°C. The resulting residue was reconstituted in 0.2 mL methanol and analyzed by liquid chromatography using a Waters HPLC (Milford, USA) consisting of a 1525 binary pump, a 717plus autosampler and 2487 dual wavelength absorbance detector. Data collection and integration were accomplished using Breeze™ software version 3.30. Analysis was performed on a Symmetry Ci of drug related to the parent compound found in whole plasma at i-th sample was calculated by the following equations:The amount of the parent compound BDP and its metabolites 17-BMP (active metabolite), 21-BMP and beclomethasone that were distributed from the lung tissue into plasma were determined and calculated as percentage of the applied dose. Amount AP V= r2• π • h(1) i Concentration of the compound at the i-th sample [m/V]ch Inner height of the plasma containing chamberMW Molecular weight of the compoundr Inner radius of the plasma containing chamberP Calculated volume of plasma in the dialysis unitVi the release of the drug into plasma Di at the i-th sample can be expressed as percentage of applied dose using equation (3) or (4):On basis of the calculated amount Aj-1 Amount of drug related to the parent compound found in whole plasma at the (j-1)-th sampleAapplied Actually applied dose to the human lung homogenateD2 × 2 H2O, 1.18 mM MgCl2 × 6 H2O, 2.5 mM KH2PO4, 20 mM NaHCO3, 5.5 mM glucose, 5 % bovine serum albumin and 2 % dextran; 100 μl of a stock solution containing 10,000 IU penicilline and 10,000 μg/mL streptomycine in 0.9% NaCl were added to 1 L perfusion buffer and pH was adjusted to 7.4 by addition of 10% NaHCO3), it was placed within the perfusion apparatus in a tempered water bath (37°C) and ventilated using a respirator  with air  and registered via a computer system. By addition of CO2 using a conventional oxigenator, perfusate pH was maintained within the physiological range of about 7.4. Lung preparations were weighed before and after perfusion to check for oedema formation during perfusion.During lung perfusion, pH, pOexvalve was 500 μg BDP for each MDI. Samples of 5 mL were drawn from the main venous output and the sample volume was replaced with fresh perfusion buffer. Samples were immediately frozen and transported on dry ice, and stored at -20°C until further analysis. In order to calculate the fraction of dose reaching the human lung lobe the spacer and all fittings were rinsed quantitatively and analyzed for BDP. The fraction of BDP that was determined in the spacer and connecting fittings was subtracted from the dose of 500 μg applied ex valve. The amount of the parent compound BDP and its metabolites 17-BMP (active metabolite), 21-BMP and beclomethasone that were distributed from the lung tissue into perfusion fluid were determined and calculated as percentage of the applied dose analogous to the dialysis experiments.After ventilation and perfusion were established the system was equilibrated for 5 min, and a 5 mL sample (blank sample) was drawn before administration of the glucocorticoid. Dosing was performed using a glass spacer (constructed by our group) that was placed between the respirator and the ventilated lung tissue. During application the tidal volume was increased 1.5-fold in comparison to the volume during perfusion. The dose applied 18 column (150 × 4.6 mm I.D., 5 μm particle size, Waters, USA). The mobile phase was a mixture of water containing 0.1 % (v/v) formic acid (A) and acetonitrile (B), the flow rate was 0.6 mL/min. A gradient elution started at 50 % eluent B, increasing linearly to 65 % B by 8 min and then increasing linearly to 80 % B by 8 min. A total sample volume of 25 μL was injected. The mass spectrometer was operated in selective reaction monitoring observing the transitions from 465 m/z to 355 m/z, 501 m/z to 313 m/z, and 521 m/z to 411 m/z for 17-BMP, FP  and BDP, respectively. The lower limit of quantification of the assay was 400 pg/mL for BDP and 17-BMP.Perfusion samples of 1 mL were mixed with 25 μl internal standard solution (300 ng/mL FP in methanol). Glucocorticoids were extracted twice with 3 mL diethylether for 20 min using a roller mixer, followed by centrifugation at 3000 rpm (Labofuge II, Heraeus-Christ GmbH, Osterode am Harz, Germany) for 5 min. The organic layers were evaporated to dryness under a gentle stream of nitrogen at 25°C. The residue was reconstituted in 50 μl methanol and analyzed by HPLC – MS/MS using an Agilent 1100 HPLC system  consisting of a binary pump, a vacuum degasser, a temperature-controlled autosampler and a variable wavelength absorbance detector coupled with an Agilent LC/MSD Trap SL mass sensitive detector. An ESI interface was used in the positive ionization mode. Data collection and integration were accomplished using ChemStation-for-LC-3D™ and LC/MSD-Trap™ version 4.2 software. Analysis was performed on a Symmetry C3. The aerosol device was actuated and the glucocorticoid particles were allowed to alight on the glass slide. Number and distribution of BDP particles was visually controlled by light microscopy (ECLIPSE TS100, Nikon, Düsseldorf, Germany). After BDP application onto human bronchial fluid the glass microscopy slide was sealed with silicone paste and a cover slide and incubated at 37°C for one hour. Thereafter the cover slide was removed and the fluid was evaporated under a gentle stream of nitrogen. Subsequently the slide was carefully washed twice with 50 μL purified water to remove bronchial fluid proteins. The water was evaporated under a stream of nitrogen.Beclomethasone dipropionate (BDP) from different devices was either directly applied on regular glass microscopy slides or on microscopy slides with incubation slots of 15 × 2 mm (Karl Roth GmbH, Karlsruhe, Germany) containing human bronchial fluid. For application of BDP the glass microscopy slide was placed in a plastic spacer of about 900 cmA Zeiss DSM 962 scanning electron microscope (Carl Zeiss, Oberkochen, Germany) was used to obtain the SEM photographs. The samples mounted on the glass microscopy slides were coated with palladium/coal for 3 min using a Baltec SCD 005 sputter-coater in an argon atmosphere (45 Pa and 50 mA).®/Becloforte™ and 5 × 100 μg Ventolair®/Qvar™) ex valve were applied to each 4.8 mL human lung tissue homogenate  were analyzed by SEM. To visualize the particle topology the devices were actuated and the particles were allowed to alight directly on regular glass microscopy slides. Representative HFA-BDP particles delivered by Sanasthmax®/Becloforte™ were about 2 μm  particles delivered by pressurized metered dose inhalers (MDI) with hydrofluorocarbon (HFA) propellant  into a reperfused and ventilated lung lobe and measured the rate and extend of pulmonary absorption. We succeeded to explain kinetic differences we observed between the two aerosols with unique particle topology and divergent dissolution behaviour in human bronchial fluid.In the present study we successfully demonstrated that the intial distribution of beclomethasone dipropionate (BDP) from lung tissue into extrapulmonary circulation can be assessed employing a human lung perfusion model. Therefore, we applied BDP delivered by two different commercially available HFA-propelled aerosols  and the distribution of BDP and 17-BMP from lung tissue into plasma or perfusion fluid, respectively.We evaluated novel approaches to elucidate pulmonary absorption of an inhaled glucocorticoid max) of BDP and its ester metabolites after inhalation of HFA-BDP in various clinical studies [We used the newly developed dialysis model to monitor the kinetics of drug diffusion over six hours. During this time we observed constant drug transfer into plasma and continuous generation of the active BDP metabolite. The latter suggests that the non-specific esterases responsible for pro-drug activation were functional within the chosen time interval. We ventilated and reperfused the resected human lung lobe over about one hour to be as close as possible to physiological conditions and avoid problems such as oedema formation that might be associated with prolonged perfusion times. The time interval of one hour is consistent with the time to reach maximal plasma levels  of 17-BMP or beclomethasone esters, respectively, after inhalation of HFA-BDP compared to CFC-BDP [max of 17-BMP after inhalation of CFC-BDP was not significantly altered when activated charcoal was co-administered and thus gut absorption was prevented [max of 17-BMP after inhalation of CFC-BDP is the particle size as it should be expected that bigger drug crystals dissolve more slowly. HFA-BDP with its smaller particles exhibited a faster and more efficient systemic drug delivery [In contrast to the unanticipated results of our absorption experiments with two HFA formulations, differences in pulmonary absorption after administration of CFC-BDP compared to HFA-BDP would have been expected based on the results of clinical studies. After inhalation of HFA-propelled BDP higher lung deposition rates were achieved and higher plasma concentrations of 17-BMP were observed in adults ,26 and c CFC-BDP ,26. The revented . A plaus®/Becloforte™ or Ventolair®/Qvar™, respectively. Scanning electron microscopic experiments illustratively confirmed this postulate. Particles of BDP delivered by Ventolair®/Qvar™ were significantly smaller and displayed faster dissolution in human bronchial fluid compared to particles delivered by Sanasthmax®/Becloforte™. Additionally, for the first time we provide evidence that BDP particles delivered by HFA-propelled aerosols are subjected to a re-crystallization process once they come in contact with the bronchial fluid. Obviously, the particles as delivered from the device dissolve rapidly due to their large surface area. Subsequently, locally oversaturated solutions tend to crystallize and crystals with a thermodynamically preferred smaller surface area are formed. It seems very likely that this might also occur in the life human lung since the volume of bronchial fluid is very limited. This re-crystallization of particles with high surface area to solid cubic particles with smaller surface area would be a highly desired effect as it slows down the further dissolution and therefore prolongs the pulmonary residence time.Based on the comprehension of the interrelation of aerosol particle size and rate of pulmonary absorption we suspected that the different time courses of distribution into extrapulmonary compartments we observed were due to different particle sizes of HFA-BDP formulated as Sanasthmax®/Qvar™ with its smaller BDP particles. The fact that we also detected the active metabolite 17-BMP in the perfusion fluid of the extracorporally perfused and ventilated lung lobe after only 2–3 min allows the conclusion that the hydrolysis of BDP is an extremely rapid process. This is in excellent agreement with the results of earlier in vitro assays with human lung tissue [The notion that BDP delivered by HFA-propelled inhalers initially rapidly dissolves and then locally re-crystallizes would also explain the detection of BDP in reperfusion fluid of the human lung preparation after only 2–3 min. It seems plausible that a certain amount of dissolved drug is instantly distributed into extrapulmonary compartments since the human lung has a high inner surface area and is the organ with the highest blood flow of about 15 L/min/kg . The detg tissue . Hence, ®/Qvar™ displayed a rapid release from lung tissue while Sanasthmax®/Becloforte™ exhibited a prolonged residence time in tissue. We succeeded to explain the observed differences in lung tissue residence time on the basis of the particle size and dissolution behaviour in human bronchial fluid. We conclude that though the ultrafine particles of Ventolair®/Qvar™ are beneficial for high lung deposition [®/Becloforte™ and Ventolair®/Qvar™ were obvious in both the dialysis and lung perfusion experiments, the latter allowed to record time courses of pro-drug activation and distribution that were more consistent with results of comparable clinical trials. Though it is not clear whether the kinetic differences observed between Sanasthmax®/Becloforte™ and Ventolair®/Qvar™ might be of clinical relevance the detection of divergent absorption kinetics confirm the functionality and sensitivity of our experimental setting. Thus, the extracorporally perfused and ventilated human lung is a highly valuable physiological model to explore the comparative lung pharmacokinetics of inhaled drugs.To summarize, we employed two different models to monitor the distribution kinetics of two HFA-propelled BDP aerosols from human lung tissue into extra-pulmonary compartments. With both the dialysis setting as well with the human lung perfusion model we detected significant differences between the two BDP formulations. The HFA-BDP formulated as Ventolairposition ,35, they


Checkpoint mechanisms prevent cell cycle transitions until previous events have been completed or damaged DNA has been repaired. In fission yeast, checkpoint mechanisms are known to regulate entry into mitosis, but so far no checkpoint inhibiting S phase entry has been identified.Schizosaccharomyces pombe spores to UV irradiation in G1. When germinating spores are irradiated in early G1 phase, entry into S phase is delayed. We argue that the observed delay is caused by two separate mechanisms. The first takes place before entry into S phase, does not depend on the checkpoint proteins Rad3, Cds1 and Chk1 and is independent of Cdc2 phosphorylation. Furthermore, it is not dependent upon inhibiting the Cdc10-dependent transcription required for S phase entry, unlike a G1/S checkpoint described in budding yeast. We show that expression of Cdt1, a protein essential for initiation of DNA replication, is delayed upon UV irradiation. The second part of the delay occurs after entry into S phase and depends on Rad3 and Cds1 and is probably due to the intra-S checkpoint. If the germinating spores are irradiated in late G1, they enter S phase without delay and arrest in S phase, suggesting that the delay we observe upon UV irradiation in early G1 is not caused by nonspecific effects of UV irradiation.We have studied the response of germinating S. pombe spores to UV irradiation in G1 and shown that S phase entry is delayed by a mechanism that is different from classical checkpoint responses. Our results point to a mechanism delaying expression of proteins required for S phase entry.We have studied the response of germinating  They prevent cell cycle transitions until previous events have been completed or damaged DNA has been repaired . Checkpoand cds1 -13 The cewed in . Rad3 i protein ,16. Rad3 protein  The Rad1 protein -21. Rad1 protein , a compl2 in a Rad3 dependent manner [There are two known effector kinases downstream of the checkpoint Rad proteins, Chk1 and Cds1. Chk1 is phosphorylated in response to DNA damage induced in late S or Gt manner ,23,24. Pt manner  and is ot manner ,26 Activt manner -29.Crb2 and Mrc1 act upstream of Chk1 and Cds1, respectively. Crb2 shares homology with the budding yeast RAD9 protein , which icdc10 arrest point was shown to depend on Chk1 [orp1 mutant cells depends on the checkpoint Rad proteins and on Chk1 [In addition to the G2/M, S/M and intra-S checkpoints, three papers have reported the existence of G1 checkpoints that inhibit mitosis when the cells are arrested in G1 using cell cycle mutants. Arrest at the  on Chk1  and Rum1 on Chk1 . Arrest  on Chk1 . It shouS. pombe. The drop of CDK activity at the M/G1 transition allows the assembly of the pre-Replication Complex, preRC, which is the first step leading to initiation of S phase. The preRC consists of the ORC (Origin Recognition Complex), Cdc18, Cdt1 and the MCM proteins. Expression of Cdc18 and Cdt1 is cell cycle regulated, thus providing one of the means to regulate initiation of S phase [Xenopus that the intra-S phase checkpoint cannot be engaged until polα-primase is loaded and replication begins [A G1/S checkpoint has so far not been detected in  S phase ,38 Once n begins ,40 This S. pombe due to poor nutrient availability. We decided to use several approaches to synchronise the cells and/or to extend G1. Recently we reported the existence of a mechanism that delays entry into S phase when cycling cells are UV-irradiated in G1, using cdc10 and cdc25 mutants to synchronise the cells or growing the cells in medium where G1 is extended [G1 in fission yeast is very short under standard laboratory growth conditions, rendering the investigation of a G1/S checkpoint(s) difficult. However, G1 might be much extended in the natural habitat of extended .S. pombe spores delay entry into S phase upon UV irradiation in early but not late G1. We demonstrate that there is a G1/S delay that is not dependent on any of the known checkpoint proteins and does not target Cdc2 phosphorylation. We argue that the delay is due to a novel mechanism that leads to delayed expression of Cdt1 and possibly other replication proteins.Here we show that germinating 2, which gave a cell survival of about 30% in wild type cells (data not shown). At the time of irradiation, the majority of germinating spores had a 1C DNA content. The timing of S phase was measured, by flow cytometry, as an increase in cellular DNA content from 1C to 2C. The decrease of the 1C population was plotted against time, and the graphs for unirradiated control and UV irradiated cells were compared at the point where 50% of the cells had 1C DNA content.Spores made from diploid cells were allowed to germinate for 3.5 h at 30°C before UV irradiation. At this time point, 1 – 2 hours before S phase entry, the spores showed visible signs of germination by phase contrast microscopy. The dose of UV light was 1200 J/mAn inherent problem in the present experiments is that the time of germination varies both within each population of spores (low degree of synchrony) and between the different preparations (experiment-to-experiment variation). Thus, the time from resuspension in medium until the cells enter S phase is variable, and it is difficult to ensure that irradiation occurs at exactly the same time point relative to S phase entry. Therefore the experiments were repeated at least twice and the averages were determined Table . The expde novo synthesis of Cdc18 and Cdt1, which in turn are required for MCM loading. We investigated Cdt1 levels in germinated spores treated as above to establish the timing of the 1C delay relative to Cdt1 expression. Wild type spores carrying myc-tagged Cdt1 were UV irradiated as described above and samples of irradiated and control cells were removed for analysis by flow cytometry and by immunoblotting. Cdt1 expression was induced already at 30 minutes in the control cells, but not until 55 minutes later in the UV irradiated cells . We conclude that Rad26, Rad1, Rad9, Hus1 and Rad17 are required for at least a part of the 1C delay.We have investigated whether the other checkpoint Rad proteins are involved in the G1/S delay. cds1 and chk1 are both known downstream targets of the Rad3 protein kinase and they are required for Cdc2 phosphorylation in the DNA damage and replication checkpoints. We irradiated germinating spores carrying mutations of cds1, chk1 or both. In cds1 spores the delay was reduced to 45 minutes . We conclude that Mrc1 and Cds1 are required for part of the delay, while Crb2 and Chk1 are not required.Crb2 and Mrc1 are required for activation of Chk1 and Cds1, respectively. We irradiated germinating 1 spores . ConsistCdc2 kinase activity is required for the initiation of S phase and is inhibited by phosphorylation on Tyr15 as DNA replication commences ,47. We ires2 mutant should override the UV-induced G1 delay. We have irradiated germinating res2Δ spores as described above and found that S phase entry was not delayed compared to unirradiated control cells . We conclude that Cdc10 dependent transcription is not the mechanism of the delay.The above result indicates that either constitutive expression of Cdc10 dependent genes required for S phase entry can override the delay or inhibiting Cdc10 dependent transcription might be the mechanism of the delay. A prediction of the latter alternative is that cells arrested with 1C DNA content upon UV irradiation should not have performed the Cdc10-dependent transcriptional events, including induction of the We have provided evidence for the existence of a mechanism in germinating fission yeast spores that delays entry into S phase upon UV irradiaton in early G1. Germinating wild type spores displayed a pronounced delay in entering S phase after UV irradiation. The delay was observed only when irradiation was carried out in early but not in late G1. We have investigated the dependence of the delay on classical checkpoint proteins and showed that they are required for some but not all of the delay with 1C DNA content. We argue that the observed delay is caused by two separate mechanisms, the first taking place before entry into S phase, and the second in early S phase (see below).rum1 mutant, which presumably loses the G1 part of the delay. Third, mutants lacking Mrc1 or Cds1, which are essential for S-phase checkpoints reported so far in fission yeast [res2 mutant, which can not turn off the Cdc10 dependent transcription signal. The finding that a mutation affecting expression of proteins crucial for preparation for S phase abolishes the delay argues that the wild type cells first stop in G1 and only later stop in S phase.The delay in exit from the 1C population was demonstrated by means of flow cytometry, which does not allow us to distinguish between a G1/S and an early S delay. The following data represent strong evidence that part of the delay takes place before entry into S phase. First, the irradiated cells delay expression of Cdt1. In the absence of Cdt1 the cells cannot form preRCs and thus cannot initiate S phase. Second, the irradiated cells express Rum1 longer than unirradiated control cells. Since Rum1 expression is cell cycle regulated such that it is only expressed in G1, , this obon yeast , still don yeast . Fourth,on yeast ,47, argurad, cds1 and mrc1 mutants delay 30–50 minutes and rum1Δ cells lose ca 40 minutes of the delay, compared to wild type cells. The remaining ca 40 minutes of the total delay requires the checkpoint Rads, Mrc1, Cds1 and Cdc2 is phosphorylated. We argue that this part of the delay is brought about by the intra-S checkpoint. However, the resolution of our experiments is not high enough to exclude the possibility that some of the checkpoint Rad- and Cds1-dependent part of the delay occurs in late G1. We consider this possibility unlikely for two reasons; first, previous work has shown that the role of Cds1 is specific for S phase [On the basis of these results we conclude that there is a UV-induced G1 delay, which is not checkpoint Rad dependent and is brought about by an as yet undescribed mechanism. This part of the total delay with 1C DNA content is ca 40 minutes, since  S phase  and secoSince the level of synchrony is low in germinating spores, we have not emphasised minor differences in the timing of S phase entry. In spite of poor synchrony, we deem germinating spores a good model system, since spore germination is a natural phenomenon, it involves an extended G1 period and we observed clear-cut effects. Furthermore, this model system allowed us to investigate the effects of a number of mutations that would have not been possible using synchronisation by other methods.rad3 mutation does not abolish all of the G1/S delay. Since we have not identified a checkpoint mutation which abolishes the delay, we attribute the effect of caffeine to another effect than the inhibition of checkpoint proteins. Interestingly, recent data suggest that caffeine inhibits checkpoint responses without inhibiting the ATR and ATM kinases in human cells [We have explored whether the G1/S delay is caused by a checkpoint mechanism. We have shown that caffeine abolishes the delay, but this is not entirely due to inhibition of Rad3 activity, since a an cells ,52.Previously, Rhind and Russell  showed tWe have recently discovered a mechanism that delays entry into S phase in cells irradiated in early G1 in synchronised or in cycling cells  This inhS. pombe, Cds1, is not involved in the present pathway. The budding yeast G1/S checkpoint delays entry into S phase by phosphorylating and thereby downregulating Swi6, the homologue of Cdc10 [In budding yeast there is a G1 DNA damage checkpoint response that depends upon Mec1 ,32,54, aof Cdc10 . In contres2Δ overrides the delay, (3) transcription of Cdc10 dependent genes is not downregulated during the delay and (4) expression of Cdt1 is delayed, suggest that the G1/S delay is caused by delayed expression of Cdt1 and probably also of Cdc18 and Cig2. Since we have shown that transcription of Cdc10 regulated genes is not downregulated, the most likely mechanism of the delay is reduced translation rate of Cdt1 and possibly other proteins required for initiation of DNA replication.Other possible mechanisms for the G1/S delay include inhibition of Cdc2 by Rum1 or an as yet unidentified mechanism such as preventing the formation of Cdc2-cyclin complexes or by restricting the availability of cyclins. We have shown that Rum1 is expressed during the delay and is required for the G1 delay. This observation does not imply that Rum1 is a direct target of the G1/S delay, but this remains an attractive possibility. Another possible mechanism for the delay is delaying expression of proteins required for the initiation of DNA replication. In particular, the findings that (1) irradiation in late G1 does not cause a delayed entry into S phase, (2) increased transcription of Cdc10 dependent genes in Schizosaccharomyces pombe cells to UV irradiation in G1. We used germinating spores to exploit a natural phenomenon where the cells have a long G1. In this paper we provide evidence for the existence of a mechanism in fission yeast that delays entry into S phase upon UV irradiaton in early G1. The G1 delay is independent of classical checkpoint proteins and Cdc2 phosphorylation. Our results point to a mechanism that delays translation of proteins required for S phase entry.We studied the response of Schizosaccharomyces pombe L972h- strain. All basic growth and media conditions were as described [All our strains are derivatives of the escribed .h+ and h- strains carrying the met3 or ade1 complementing markers. The rum1:::ura4+/rum1+ diploid was made by protoplast fusion since rum1Δ is sterile [res2Δ  at 30°C overnight. The spores were washed twice in water and resuspended in EMM2 supplemented with adenine and methionine for germination and UV irradiation.Diploids were made by interrupted mating of  sterile . All dip meiosis ) and rum2. Cell survival was monitored by conventional plating on YE plates. The incident dose does not reflect the dose absorbed by the cells because UV light of this wavelength penetrates poorly into water. However, since irradiation conditions were constant, the incident dose was proportional to the absorbed dose.Cells were irradiated with 254 nm UV light while rapidly stirred in a thin layer (3 mm) of liquid medium. The dose administered was measured with a radiometer (UVP instruments) and an exposure time of 4 minutes gave an incident dose of about 1100 J/mProtein extracts for western blotting were made by TCA extraction, as described previously . For wes32P-labelled RNA probes, generated with T7 RNA polymerase (Riboprobe System T7 Kit, Promega). For cig2 and cdc18, the ORFs were inserted into pGEM-3 MCS to serve as template for producing the RNA probes. For cdt1, a PCR fragment of the ORF with T7 promoter sequence attached to the lower primer was used as template. Hybridisation was carried out using standard procedures and visualised by a STORM 860 Phosphoimager (Molecular Dynamics).Total RNA was isolated as described , resolve7 cells were spun down for each sample and fixed in 70% ethanol before storing at 4°C. Samples were processed for flow cytometry as described [About 10escribed  and staiescribed , and anaEAN showed the existence of the delay and investigated the roles of Rad3, Chk1 and Cds1, Cdc2 phosphorylation and Cdc10-dependent transcription as a potential mechanism. MS, TT and HV investigated the roles of further checkpoint proteins and that of Res2. EB participated in the design and coordination of the study and in writing the manuscript. BG devised the study, analysed Rum1 and Cdt1 expression and drafted the manuscript. All authors read and approved the final manuscript.


Adenoviral vectors have been frequently used for gene delivery to vein ex vivo and pre-clinical studies have shown effective blockade in neointima development by overexpression of candidate therapeutic genes. However, high titers of adenovirus are required to achieve sufficient gene delivery to provide therapeutic benefit. Improvement in the uptake of adenovirus into the vessel wall would therefore be of benefit. Here we determined the ability of an adenovirus serotype 5 vector genetically-engineered with the RGD-4C integrin targeting peptide inserted into the HI loop (Ad-RGD) to improve the transduction of human saphenous vein smooth muscle cells (HSVSMC), endothelial cells (HSVEC) and intact saphenous vein compared to a non-modified virus (Ad-CTL). We exposed each cell type to virus for 10, 30 or 60 mins and measured transgene at 24 h post infection. For both HSVSMC and HSVEC Ad-RGD mediated increased transduction, with the largest increases observed in HSVSMC. When the experiments were repeated with intact human saphenous vein , again Ad-RGD mediated higher levels of transduction, at all clinically relevant exposures times (10, 30 and 60 mins tissue:virus exposure). Our study demonstrates the ability of peptide-modified Ad vectors to improve transduction to human vein graft cells and tissue and has important implications for gene therapy for CABG.The establishment of efficient gene delivery to target human tissue is a major obstacle for transition of gene therapy from the pre-clinical phases to the clinic. The poor long-term patency rates for coronary artery bypass grafting (CABG) is a major clinical problem that lacks an effective and proven pharmacological intervention. Late vein graft failure occurs due to neointima formation and accelerated atherosclerosis. Since CABG allows a clinical window of opportunity to genetically modify vein  This unique "clinical window" has clear safety advantages over in vivo gene delivery since excess vector can be removed from the graft prior to coronary grafting. However, the clinical window is short (likely 10–60 minutes) and therefore necessitates the use of an efficient vector system for gene delivery. Adenoviral vectors have proven efficient for gene delivery in this context [ex vivo [v integrins [ex vivo, the ultimate clinical target.Long term patency rates for CABG using autologous saphenous vein are poor, showing 1, 5 and 10 years post-CABG rates of 93%, 74% and 41%, respectively -4, a pat context . The lat context . Any imp context , dramati[ex vivo . Likewis[ex vivo , the pre[ex vivo . In the ntegrins , this hantegrins  althoughntegrins . Since Sntegrins , it is p2 at 37°C. Cells were plated to reach 80% confluence 24 hours later. HSVEC or HSVSMC were infected in 96 well plates with increasing doses [plaque forming units (pfu) / cell] of Ad vectors for 10, 30, 60 mins at 37°C. The cells were washed twice in PBS and the media changed. 24 hours post-infection, the cells were again washed in PBS, lysed in PBS/0.2% Triton-X-100 and transduction quantified using the Wallac 1420 (Victor2) Multilabel Counter with recombinant eGFP (Clontech, Basingstoke, UK) as a standard. Reporter gene expression was normalised for total protein using the bicinchoninic acid (BCA) protein assay (Perbio, UK) with bovine serum albumin as standard, measured using a VICTOR2 plate reader. Exposure of HSVEC to Ad-CTL or Ad-RGD [50 plaque forming units (pfu) / cell] resulted in a time-dependent increase in the level of transduction  supplemented with 20% (v/v) foetal calf serum . HSVSMC were grown from medial explants from the same material and maintained in Dulbecco's modified Eagle's medium (DMEM) with 4500 mg/l glucose supplemented with 20% (v/v) FCS and 100 IU/ml penicillin, 100 μg/ml streptomycin and 2 mmol/l L-Glutamine. All cells were grown in a humidified atmosphere with 5% COn Figure . At eachn Figure . Fluoresn Figure  at all tn Figure . HSVSMC n Figure , consistn Figure . Again, 9 pfu / ring) before being washed twice in PBS and maintained in organ culture for 5 days. Rings were maintained in wash medium supplemented with 30% (v/v) FCS. Vein rings were snap frozen in liquid nitrogen and homogenised using a mortar and pestle for determination of reporter gene expression 5 days post-infection. Ex vivo homogenates were suspended in 100 μL reporter lysis buffer (RLB) and kept on ice for 1 hour before supernatants were analysed for luciferase expression using the Luciferase Assay System (Promega). 96 well plates were prepared using 10 μL/well of homogenate suspension diluted to a total volume of 100 μL with RLB. 100 μL of Luciferase Assay Reagent was added to each well and the plate immediately read for 10 seconds per well. Detection was achieved using a Wallac 1420 (VICTOR2) Multilabel Counter with recombinant luciferase (Promega) as a standard and normalised for total protein. Ad-RGDLuc mediated a time-dependent increase in the level of transgene expression that was evident at all exposure times studied – 10, 30 and 60 minutes . Individual vein rings were incubated with Ad vectors for 10, 30 or 60 minutes  and the British Heart Foundation (PG03/031 to A.H.B.)