The bell prover is widely used for gas flowrate measurements by timing a known stroke of the bell as it rises, presumably with a constant speed, from a bath of sealing liquid. A differential equation for the bell motion (from the previous paper of the same title) is used together with the previous equations of motion for the gas and sealing liquid modified here to provide the basis for a computer simulation of the prover performance. Examples of the results of the computations show substantial fluctuations in all of the motions and modified measurement procedures for improved accuracy are discussed. Proposed modifications to the prover itself are shown by the computations to dampen the fluctuations and to improve the measurement accuracy. Only a limited number of changes of the prover design and of the initial conditions were researched for their effect. Vb), and thermal insulation are installed in the inlet pipe. These are suggested to help attenuate the fluctuations of pressure that are generated in the bell and also to assure equality of temperature of the bell and of the gas input. A second essential addition consists of two annular orifice plates mounted in the two sealing liquid spaces between the bell and its tank. It’s expected that the constricted liquid flow area there will help to attenuate the liquid motion which is shown in the subsequent computations herein to be a major source of flow measurement inaccuracy.The bell prover of iV each time a different meter is installed for calibration. A third benefit arises in that the computations that are to follow here will be more likely to represent actual events for this restricted system as compared to the usual large complicated piping system.Three other additions of lesser importance are shown in More than one nozzle (size) may be needed to be able to cover the full range of flow of any one prover. Other basic requirements are that, at the flow rate of choice, the gas velocity in the nozzle throat be equal to the velocity of sound there, and that the gas at the entry point to bell be at the temperature of the bell. Another concept may prove useful here. Simultaneous measurements of temperature and pressure can be made at the sonic nozzles to calibrate them along with the meter. These calibrated nozzles then should prove useful as secondary flow standards for calibration of meters without the necessity of always exercising the bell prover. A second addition to the ordinary prover should include a thermocouple placed at the exit of the inlet pipe so that it can provide a confirmation that the gas and bell are at equal temperatures all during the measurement stroke of the bell. An average of measurements taken at different times and even places in the bell may not be sufficiently accurate if bell and gas temperatures are not equal.The third difference between the modified and ordinary prover is in the location and design of the diverter valve. It is placed on top of the bell instead of on that portion of the inlet pipe that is external to the prover’s tank. It is proposed that it be an electro-mechanical device designed to be able to remotely select and replicate the stroke time and flow rate history of the gas through the valve. A lateral discharge of the gas from four ports spaced 90° around the new valve will be necessary to avoid a variable and disturbing force on the bell during its upward motion. A rotatable collar or other device with equally spaced and appropriately sized ports together with, possibly, a spring and/or an electric and/or a pneumatic motor to rotate the collar, will be needed to complete the essential parts of the valve. It’s not necessary that the new valve be located as shown in The differential equation of motion for the bell from reference , togetheSpecial integrals of the Navier-Stokes (NS) equation will be used  to derive the liquid drag forces for use in the bell’s equation of motion. Forces connected with entrained liquid and effects not included in the special integrals will be estimated for the same application. The NS equation also provides a justification to treat the motion of the sealing liquid as if it is a “solid” and thus a one-dimensional, second-order differential equation is used for the liquid motion to calculate its effect on the gas volume under the bell.Each set of differential equations will be subjected to numerical integration using the Runge-Kutta fourth-order method outlined in .Av then can be calculated as Av=Avi (1–γ). Here γ is used as t/tv, the time t from start of the stroke divided by the total stroke time tv, and Avi is the initial open area. For a constant acceleration stroke, Av=Avi (1−γ2). The latter assumption is used in the subsequent calculations of the flow as its accomplishment seemed to present a simpler valve design problem, and possibly because it would provide a “smoother” acceleration of the bell upward from its float position. A valve might be designed for a still smoother operation to close slowly at both the beginning and end of its stroke, with a port open area represented, for example, by a function such as Av=Avi (1/2−(1/4) cos3πγ+(3/4) cos πγ). The rate of area change with a valve so designed would be ≃0 at the beginning and end. Effects of valve design on prover performance probably will be researched better by calculations than by experiment.The flow measurement process is initiated by a gradual closing of the diverter valve to start the upward motion of the bell from its balanced position. A calculation of the net mass flow into the bell during the stroke of the diverter valve is based on the difference between the initial steady state mass flow out of the valve and the smaller flow out during its stroke. If the ports are rectangular and if the stroke is assumed to be one of constant velocity the orifice port area D, asP is (P−Pa) and ΔPi is (Pi−Pa) and the first term on the right side of FRI. This rate is to be compared to that established by the calculations from the derived motion of the bell and sealing liquid and for the change of state of the gas in the bell. The mass of gas in the bell and inlet pipe is represented by m and ρ is its density.The net mass flow rate into the bell can be written, using the well known equation for the orifice with coefficient of discharge CP to the bell and oil motions so that it can be inserted into Pi as the initial balance pressure, asmt and Vt are the initial mass and gas volume under the bell and inlet pipe, and Ax is the volume change under the bell due to its motion x from x=0. ΔV is the change of gas volume from oil entrainment on the bell as it moves upward plus the volume change caused by motion of the liquid seal. The exponent k may be taken as the ratio of gas specific heats when an apparatus is used that would give adiabatic conditions. For a very small apparatus in which the process might be one of almost constant temperature, k should approach unity. Note here that A is the effective area of a bell prover and is given in ρ in terms of its pressure in the bell and inlet pipe and then the now CD will be specified as equal to zero at the instant that γ= 1 to simulate a closed diverter valve. V into P/Pi which will be used in The gas law is used to express the gas density Vb and is expressed asm under the bell isCD should be made equal to zero at the instant γ is specified to equal unity. Note that (Pbi‒Pi) is the initial pressure drop that is to be specified across the porous plug and that the flow rate through it is proportional to (Pb−P). This requires a plug designed for viscous flow which probably would be more effective than one designed for turbulent flow.A computer simulation of the modified prover’s performance requires that P as expressed by the equationP, m, mi are for conditions under the bell only. For the specified constant gas temperature in Vb, Pb can be written asAn It is intended here to derive the forces on the bell that are associated with the motion of the bell in the sealing liquid. For this purpose some aspects of Couette flow in both steady and unsteady state motion between parallel plates will be discussed. It is also necessary to look at the development of the laminar boundary layer on a flat plate in the event that this could be dominant as to the force it applies to the bell. These considerations might be useful in examining further the forces from oscillatory liquid motion, but these will be ignored as they tend to cancel when both sides of the bell are considered. However, the liquid oscillation would affect the volume of gas under the bell and an attempt will be made to account for this effect. Other force aspects of the liquid motion, such as from entrained oil on the sides of the bell and motion of the oil at its bottom edge are estimated to include them in the bell’s motion equation.r > >2b and (L−x)> >2b. These length conditions would limit the influence of the complex flows that exist at the free surface and at the bottom edge of the bell. Thus the applicable equation is written asy is the distance from the bell’s surface, z is along its circumference and u and υ are the liquid velocities in the x- and y-directions, respectively, as illustrated in u2/∂x2, ∂2u/∂z2, ∂u/∂x, υ and ∂u/∂t in u = U at y =0 (or that the liquid on the bell’s surface is carried up with the bell whose velocity is U) and second that u =0 at y=2b are now used in the integration of P/∂x was assumed to be constant across y in the derivation of The well-known momentum equation of NS may be used to derive the velocity profile for a fully-developed, steady flow between parallel plates. This condition is practically satisfied if, in P/∂x such that the liquid up-flow adjacent to the bell is balanced by a down-flow adjacent to the tank wall. Thus, an integration of the flowrate across y derives the volume rate total which, when set to zero, gives ∂P/∂x ash is derived from µU(L−x)/2b2ρ0g. The up-flow must spill over at the free surface to build a liquid head at the tank wall comparable to Δh to provide for the flow down along the tank wall and for its recirculation at the bottom of the bell. When the value of ∂P/∂x is inserted into u =0 point (y=2b/3), is calculated to be equal toℓ is an average circumference of the bell. Third, this volume rate can be compared to the entrainment rate df/dt of liquid that adheres to the bell as its surface leaves the liquid. Reference  and α [=y/2(v0t)1/2]. Partial differentiation (with respect to y) gives the velocity gradient at the moving wall (y=0) ast→0, and as t→∞, the gradient decays to that for the steady moving wall.With that modified NS equation and using appropriate boundary conditions the derived velocity profile is given as a series of complementary error functions (erfc) of the quantities αt =0 to t=Δt, and to use the “averaged” gradient to calculate force F2 on the bell. The time-averaged value calculates to bet to be used here. This result is taken as most applicable during the acceleration phase of the motion of the bell. Thus It is proposed here to calculate a time-averaged value of the velocity gradient from h3 as used in h3 and h3 by considering the oil as a solid in motion.(b) The “sudden” concept is also regarded as applicable to estimate the motion of the oil seal (as caused by pressure changes in the bell) and the resultant change of b. The integration limits for η are calculated from α1 and α as given previously. A numerical integration of t assumed equal to integration time intervals Δt to be used here does show an almost constant velocity across practically all of the distance 2b. Thus, a value of h3 derived from the “solid” equation of motion should be reduced by a factor derived from an average value of b. It should be noted here that the oil motion effect on the gas volume is expected to have most significance while the diverter valve is closing. If the subsequent motion of the bell is at constant velocity the motion of the oil seal should have insignificant influence. Note the big “if” in the previous sentence.A velocity profile in this “sudden” case is adequately represented  asA. H. Shapiro  presentsF3 calculated below:Sealing liquid that is entrained on the walls of the bell previously was stated to modify the indicated volume of gas collected. Here the liquid acts to modify the mass of the bell by way of the force of gravity on the liquid. This will be the third non-conservative force (b) Liquid at the bottom edge of the bell is sucked upward by it which creates a drag force there which can be stated asThe equation of motion 13) of [3 of [1] xi=0, to calculate h4i at the initial balance condition using an assumed (or specified) value of (Pi−Pa). Second, eqs (15) and (16) of  A3=A4. For the two “solids” case the equation is quite complicated and contains a product of the oil acceleration and elevation and also its velocity squared. This seems an unwarranted complication at the present time and the single “solid” will be used.Two different approaches might be selected to set up the oil motion equation, one regarding the oils on the two sides of the bell as two separate “solids”, or by treating the oil as a single “solid” using the assumption that h4−h4i)≃−(h3−h3i), to giveρ0(A3+A4)(L−x), and that the term containing h4 is the liquid’s back pressure. The factor 2  arises from the pressures acting on the assumed area=(1/2)(A3−A4). A “Force Loss” is included in the equation to include the effect of the turn that the oil must make at the bottom of the bell of the ordinary prover. This loss is assumed to be equal to one-half of the velocity pressure, or (1/2)(1/2) A3, which then gives the last term on the right side of A5 on each side of the bell to modify this part of the prover, a pressure drop will be introduced into the flow there. This drop will arise from the frictional loss of mechanical energy mostly on the downstream side of each constriction. For an area constriction down to about 10 percent, a possibly conservative estimate of the pressure drop would correspond to one velocity head pressure based on the liquid’s average velocity in the constricted area. On this bases the last term of The equation of motion for the single “solid” is derived using the familiar concept that acceleration is given by force per unit mass and that  assumes the use of a constant acceleration diverter valve. P/Pi and V/V1 as follows:Z1 = [(A3−A4)/A3+A4] (Z3) (4πA1)1/2 ν00.644;Z2 = a correction factor for lack of oil “solidity”;Z3 = a constant whose value depends on the system of units that will be used.Note above that h4 has replaced (h3i−h3) because A3 is assumed equal, in A4 and that the motion equations when integrated will give h4=(h4−h4i).The gas flow P/Pi and Pb/Pi, respectively, and A5 is constructed to be equal to 0.1 (A3+A4). With this substitute, last term of The gas flow CD. A3 was assumed equal to A4 with corresponding assumptions f3=f4 as in h3=h4 as in Vb.A recapitulation of the main assumptions used in the derivations of the above equations seems advisable. It does not seem advisable at the present time to develop a change to ρiA (x2−x1) with corrections applied to it for the fluctuations of P and ΔV. Subscripts 1 and 2 are used on x to signify values at the beginning and end of the chosen timing interval, respectively. It seemed preferable to separate the P and ΔV corrections, and to do this the pressure ratio in P−Pi)kP). With some use of algebra, Vb = 0 in the far right-hand term and Vi should be replaced by Vt which is given by (Vi+Vb).The numerical integrations of the equations of motion are used to derive the performance of the provers which will be illustrated by various curves showing the motions of the bell and sealing oil and the derived gas pressure and gas volume correction all expressed as a function of the time. See  to 5. ThV1 of 5 ft3 (0.1415851 m3) and these were used in three computer runs for the first look at the utility of the proposed analytical method. Results of these runs indicate the effectiveness of the integration method as well as demonstrate the size and sources of errors likely to be encountered with the usual laboratory procedures. The prover specifications are listed in Almost complete physical data were available to the authors on a bell prover of nominal capacity The two runs at the high rate of flow  were done with different time intervals in the integration process, namely 0.002 and 0.01 s. Rates of flow derived from use of these two integration processes differed by about 0.01 percent. Comparison of other physical variables also showed differences near 0.01 percent. On the bases of this good agreement it was decided to use the 0.01-s time interval for the low (50 s collection) flow rate calculation. It seemed reasonable to expect much larger differences than 0.01 percent between prover performance at the two rates of flow.Pi from Pi with a relatively long period, initially about 1.8 s and decreasing to about 1.1s. The pressure fluctuation decreases to about ±30 percent of ΔPi from the mean pressure, but the curve near 7 s shows a hint of a possible increase at that point. Higher frequency fluctuations of the pressure, with periods of about 0.3 s, are also evident. The amplitude of the oil motion as converted from values of h4 to pressure is about 120 percent of the pressure fluctuation. It is evident that the pressure fluctuations are strongly modulated by the oil fluctuations. It is also strongly evident from V under the bell fluctuates practically in unison with h4. This fact suggests that failure to measure h4 possibly can be the major source of error.Pi from Pi, and again a relatively long period initially of about 1.8 s which decreases to about 1.4 s. The pressure fluctuation and the pressure level both decrease, the former to ±1.3 percent of ΔPi about the mean pressure which is about 9 percent of ΔPi below Pi. These fluctuation amplitudes are about six percent of those at the high rate of flow, as compared to the flowrate ratio of 10 percent. The amplitude of the sealing liquid motion (not shown) begins at about 115 percent of the pressure, but winds up near the end of the measurement stroke at about twice that of the pressure fluctuation. As at the higher flow rate, the gas volume correction (not ordinarily measured) fluctuates practically in unison with h4 and with the low frequency pressure fluctuations. The high-frequency fluctuations have periods of about 0.19 s which increases to about 0.31 s.At the low rate of flow x as equal to V is the volume of air and x is the motion of the bell about a stationary position. The vibration period for this well known case is The high frequency motion calculated for the bell can be shown to be real by considering the bell to be vibrating as if suspended from a “spring” which can “replace” the air enclosed in the bell. For the case of the very low values of the pressure fluctuations prevailing here, this concept is sufficiently accurate to derive the “spring constant” on An inspection of P and either Δh4 (or ΔV) are accurate at any instant and accurately correlated with the bell positions x1 and x2, If the measurements of ρiA(x2−x1) are necessary if the initial and final timing points  can be identified by the instrumentation when the variables of (P−Pi) and Δh4 both equal to zero at both bell positions (a zero-zero criterion). In the more general case  this criterion does not prevail. However, pressure transducers probably are available with capability to follow the low frequency fluctuations of the pressure (as caused by the motion of the oil) with good accuracy especially at the fluctuation midpoints. If the timing points x1 and x2 are made to coincide in time with those mid-point pressures, the corrections derived with V Fand the ΔP motions are shown here to coincide. These methods should be used but apparently are not because of the practical difficulties. A third procedural compromise, Method 3, is usually used to derive the corrections.AFP) at two preselected, convenient positions x1 and x2. These measured AFP values are used in V (based on AFP) to derive “corrections” to ρiA(x2−x1). The “worst” timing point choices that can be derived from Measurements are made with a water manometer to give (what is believed to be) mid-point values of the high-frequency fluctuating pressures . This should be and is confirmed if some bell positions, particularly near x2, can be found where AFP values or transducer measured values are equal to Pi. These positions need not necessarily be those that are finally used for the rate measurements.It might be possible to implement the zero-zero criterion as described in method 2 to use what is essentially a “correctionless” procedure. Two requirements are necessary for this purpose. First, the prover must be modified to maintain the average of the maximum/minimum pressures, P and Δh4 is the optical encoder used by G. Kulin, P. Huang, and G. Mattingly [V and values of (P−Pi) both were zero. Therefore, presumably, the encoder’s indication of the maximum bell velocity also occurs at the zero-zero condition. This leads to the suggestion that the timing points should be made to coincide with the maximum velocity indications of the optical encoder to try to eliminate the otherwise needed, but in accurate, AFP corrections to derive the true mass of gas collected. As an alternative to the above, more attention should be given to fully implement Method 2.An instrument which might be used for phase indications of Δattingly  to betteAFP procedure of Method 3 which can be modified to measure the true collected mass of gas. Two modifications appear of possible utility, both needing a constant value of Pm=Pi during the stroke of the bell. The first would use computed motions of Δh4 (as described herein) to identify and use the bell positions x1 and x2 where Δh4 values are expected to be zero. AFP values would then also equal Pi and corrections to ρiA(x2−x1) would also equal zero. Results from use of this method could then be compared to results obtained below with the second modified AFP procedure.It appears to be possible to implement the zero-zero criterion as described in Method 2 using the AFP procedure would utilize a multiplicity of strokes of the bell each with positions x1 and x2 systematically moved until both give AFP values equal to Pi. Pm must also be maintained equal to Pi with this method (see Suggestion 2). This method is based on the assumption that AFP values of pressure fluctuate about Pi with a smaller amplitude than those of the high frequency pressure fluctuations, and with a period of the low frequency fluctuations of Δh4. It’s expected that on average five different strokes of the bell would be needed with x1 and x2 each moved each time a distance corresponding to the distance traveled by the bell during 0.05 period of the Δh4 fluctuation. It’s assumed in the experimental situation that the pressure measuring instrument actually does indicate the AFP values. If all of these criteria prevail the modified, multiple AFP method is also correction less. An advantage of these two procedure modifications is that neither requires apparatus changes.The second modified Pi, and tv was reduced to 0.2 s (from 2 s as used in HIFLO and LOFLO). These three changes were made simultaneously and “almost” arbitrarily, and hence the name HOLHOG was chosen. Note that ΔV increased by only about 50 percent over that in HIFLO in spite of the 14-fold increase of ΔP at the end of 0.2 s. These larger upsurges, however are subsequently efficiently dampened. Although the frequencies of the motions of ΔV and ΔP are the same as in the ordinary prover, the practically constant phase difference between the two motions is not evident here.AFP Method 3 is gratifyingly reduced to 0.053 percent from the 0.586 percent needed for the ordinary prover. This possible “worst” result for HOLHOG is improved to a needed remaining correction of only 0.006 percent if a “best” timing choice happens to be made. Phase differences between the ΔV and ΔP motions that change during the stroke of the bell limit suggestions for improvement in methodology to Suggestion 3 above. However, the need for improvement is much, much less than for HIFLO at its worst.The calculated “worst” corrections for HOLHOG listed in the table above demonstrate that the correction remaining and needed after application of the x1 to make a significant improvement. A third preliminary calculation was done to evaluate the effect of a single change in an initial condition, namely a reduction of tv from 2 to 0.2 s in program HIFLO. This change produced a two-fold increase in the initial upsurge of ΔV over the already large increase evident in HOLHOG. This large increase illustrates the importance of the initial condition, and second that the orifice effectively dampened the motion of the oil in HOLHOG and especially the initial upsurge motion.It’s appropriate at this point to discuss the “almost” arbitrary group of apparatus changes (specified for HOLHOG) and the problem of setting the initial conditions. As a preliminary to HOLHOG, computations of the effect of the orifice installation alone, and next of the effect of the porous plug alone, indicated that dampening was not effective enough before the bell reached Vi to Vb to V1, also could have significant effects on the performance of both provers studied here. This discussion should now revert back to the need to use a motorized and stroke-designed diverter valve as previously suggested herein. This discussion also leads to the conclusion that prover performance cannot be adequately specified until the initial conditions are too.Other changes of initial conditions, such as in the flow rate characteristics of the diverter valve, and changes in the relationship of It appears that the equations of motion for the moving elements of the prover system and the procedure used in their numerical integration can produce calculated results which offer a reasonable chance that they can be used to design improvements in the apparatus and measurement procedures. “Dynamic errors” in the measurement of flow rates can arise primarily from the fluctuating motions of the sealing liquid, second from fluctuating velocity of the bell, and third and usually least in magnitude from entrainment of liquid on the bell. Proposed additions to the prover can be used to (1) assure needed equality between gas and bell temperatures and (2) to reduce the amplitude of the fluctuating motions as one way to reduce the magnitude of measurement errors. Three other suggestions for improved measurement procedures include an ideal situation in which pressures and oil motions are accurately measured at any two bell positions of choice. A second more practical suggestion would require that the timing points for the bell motion be properly correlated with indications of the mid-point pressures from a pressure transducer or correlated with indications of the maximums in the fluctuating bell velocity. A third suggestion for improved accuracy entails the utilization of two different approaches both being modifications of that currently in use which relies on manometer reading of the average indications of the high-frequency fluctuating pressures at the two bell positions of choice. Some changes in the initial conditions produced performance changes which emphasizes that blanket performance and accuracy statements cannot be specified unless all initial conditions are also specified.


With the advent of new and more efficient anti-androgen drugs targeting androgen receptor (AR) in breast cancer (BC) is becoming an increasingly important area of investigation. This would potentially be most useful in triple negative BC (TNBC), where better therapies are still needed. The assessment of AR status is generally performed on the primary tumor even if the tumor has already metastasized. Very little is known regarding discrepancies of AR status during tumor progression. To determine the prevalence of AR positivity, with emphasis on TNBCs, and to investigate AR status during tumor progression, we evaluated a large series of primary BCs and matching metastases and recurrences.AR status was performed on 356 primary BCs, 135 matching metastases, and 12 recurrences using a next-generation Tissue Microarray (ngTMA). A commercially available AR antibody was used to determine AR-status by immunohistochemistry. AR positivity was defined as any nuclear staining in tumor cells ≥1 %. AR expression was correlated with pathological tumor features of the primary tumor. Additionally, the concordance rate of AR expression between the different tumor sites was determined.AR status was positive in: 87 % (307/353) of primary tumors, 86.1 % (105/122) of metastases, and in 66.7 % (8/12) of recurrences. TNBC tested positive in 11.4 %, (4/35) of BCs. A discrepant result was seen in 4.3 % (5/117) of primary BC and matching lymph node (LN) metastases. Three AR negative primary BCs were positive in the matching LN metastasis, representing 17.6 % of all negative BCs with lymph node metastases (3/17). Two AR positive primary BCs were negative in the matching LN metastasis, representing 2.0 % of all AR positive BCs with LN metastases (2/100). No discrepancies were seen between primary BC and distant metastases or recurrence (n = 17).Most primary (87 %) and metastasized (86.1 %) BCs are AR positive including a significant fraction of TNBCs (11.4 %). Further, AR status is highly conserved during tumor progression and a change only occurs in a small fraction (4.1 %). Our study supports the notion that targeting AR could be effective for many BC patients and that re-testing of AR status in formerly negative or mixed type BC’s is recommended. The androgen receptor (AR) is located on the long arm of the X chromosome Xq12) and acts, upon ligand binding, as a transcription factor 2 and act–5, respeThe patient cohort described in this study has been previously reported . BrieflyPrior to ngTMA construction all primary BCs, positive lymph nodes, distant metastasis, and recurrences underwent pathological review  for diagnostic confirmation. The primary tumors were classified according to the WHO classification 2013 . ngTMA cFor the assessment of AR status a monoclonal anti-human androgen receptor antibody  was used (1:100 dilution). A positive AR status was defined as average of ≥1 % positive tumor nuclei regardless of staining intensity as previously described . The AR Results on ER, PgR, Her2, and MIB-1 were available from our previous study , and thep-value <0.05 was considered statistically significant. Analyses were carried out using SPSS 21 .The Chi-Square test was used to calculate significant differences between categorical variables. A AR status was informative in 99.2 % (353/356) of primary BCs, 90.4 % (122/135) of metastases  and in 100 % (12/12) of the recurrences . The majority of BCs were AR positive (≥75 %) with the exception of BCs with medullary-like features (36.4 %).Primary BCs were AR positive in 87 % (307/353) and showed a significant (p < 0.001) correlation with a positive ER  and PgR  status, and a low proliferation index of <20 % .p < 0.001) with high tumor grade (G3) (37/120).ER negative primary BCs showed a positive AR status in 25 % (12/48), PgR negative BCs in 61.2 % (60/98), while ER and PgR negative BCs had a positive AR status in 26.7 % (12/45). A negative AR status correlated significantly , luminal B (Her2 negative) in 91.4 % (74/81), and all luminal B (Her2 positive) were AR positive (24/24). The Her2 and TNBC subtype were AR positive in 80 % (8/10) and in 11.4 % (4/35), respectively.Lymph node metastases were AR positive in 85.5 % (100/117), distant metastases in 100 % (5/5) and recurrences in 66.7 % 8/12). The mean percentage of AR positive cells among recurrent BCs was: 43.1 % in primary tumors and 39.9 % in recurrences. AR negative BCs stayed negative in primary tumors and recurrences (mean: 0 % AR staining each). The results are summarized in Table . The meaA discordant AR status between primary BC and matched metastatic samples was observed in 4.1 % (5/122) of cases tested. However, a discrepant AR status was only seen between primary BC and matched lymph node metastases , but not between primary BC and distant metastasis. Two AR positive primary BCs had a negative corresponding lymph node metastasis . Three negative primary BCs had an AR positive lymph node metastasis . No discordant AR status was observed between the primary BC and distant metastases or recurrences.To confirm the discordant results, we re-evaluated the 5 discordant primary BC and their matching lymph node metastases. In two discordant cases ID 237, 248), the primary BC was negative and their matched metastases were positive, but the AR status was close to the cut-off of ≥1 % for each. In another case (ID 204), the primary was negative but the lymph node metastasis was positive. In this case, the final score was not close to the cut-off of ≥1 %. In the fourth BC (ID 47), the primary BC was positive but the lymph node tissue was negative. The lymph node metastasis in this case was frozen prior to formalin-fixation (sentinel) and showed some crush artifacts, which could explain the negative score. The fifth case (ID 356) was of the ductulo-lobular histological subtype. The primary BC showed punches with both, a negative and a positive AR status. The primary tumor was signed off as AR positive but the metastasis was negative. The results are summarized in Table 7, 248, tFor decades, the steroid hormone receptors ER and PgR have been well known therapeutic targets in BC  but consn = 16) and lymph node metastases (n = 46), it was shown that AR discrepancies between primary tumors and metastasis did not occur [n = 5) with discordant AR status from the primary tumor to the lymph node metastases. Due to the small number of distant metastases (n = 5) and recurrences (n = 12) included in our study and in the study by McNamara et al. [From a biological and therapeutic point of view, the dynamics of AR status during tumor progression is an important consideration. In a recent study, which included a small series of TNBC with matched recurrences  in BC , 21.Other issues leading to potential discordant scores between matched samples is pre-analytical handling of the specimens and whether the specimens were optimally fixed, which can impair staining . Tumor hLastly, since we observed that 3/17 (17.6 %) AR negative primary BCs showed an AR positive lymph node metastases, we recommend re-testing of previous AR negative tumors. Additionally, re-testing of BCs with morphologically mixed components would be desirable. It has to be discussed with the oncologists if AR positive primary tumors need to be re-assessed on metastases or recurrences.In summary, AR positivity is very frequently found in BC regardless of the disease site and immunophenotype. Therefore, AR targeted therapy might be a warranted treatment option for many BC patients. Furthermore, it appears that AR status is highly preserved during tumor progression but discordance between primary and metastatic sites may occur in a small fraction of tumors. Hence, we recommend re-testing of AR status in previously AR negative primary tumors and BCs with morphologically mixed components.


Although the new generation of androgen receptor (AR) antagonists like enzalutamide (ENZ) prolong survival of metastatic castration-resistant prostate cancer (CRPC), AR-driven tumors eventually recur indicating that additional therapies are required to fully block AR function. Since DNA topoisomerase II (Topo II) was demonstrated to be essential for AR to initiate gene transcription, this study tested whether catalytic inhibitors of Topo II can block AR signaling and suppress ENZ-resistant CRPC growth. Using multiple prostate cancer cell lines, we showed that catalytic Topo II inhibitors, ICRF187 and ICRF193 inhibited transcription activities of the wild-type AR, mutant ARs (F876L and W741C) and the AR-V7 splice variant. ICRF187 and ICRF193 decreased AR recruitment to target promoters and reduced AR nuclear localization. Both ICRF187 and ICRF193 also inhibited cell proliferation and delayed cell cycling at the G2/M phase. ICRF187 inhibited tumor growth of castration-resistant LNCaP and 22RV1 xenografts as well as ENZ-resistant MR49F xenografts. We conclude that catalytic Topo II inhibitors can block AR signaling and inhibit tumor growth of CRPC xenografts, identifying a potential co-targeting approach using these inhibitors in combination with AR pathway inhibitors in CRPC. Androgen deprivation therapy (ADT) is the first line of treatment for locally advanced, recurrent or metastatic prostate cancers. It aims at suppressing transcriptional activity of the androgen receptor (AR). Despite frequent and often durable responses, progression to castration-resistant prostate cancer (CRPC) invariably occurs, most often driven by reactivation on the AR pathway via mechanisms involving AR amplification, overexpression, mutations as well as intratumoral steroidogenesis –3. TheseThe transcriptional activity of AR requires DNA Topoisomerase II (Topo II) to be recruited to target promoters –10. TopoThere are two types of Topo II inhibitors, Topo II poisons and catalytic inhibitors. Topo II poisons are exemplified by the cytotoxic chemotherapeutic, etoposide. When DNA breaks are created by Topo II, etoposide inhibits Topo II to re-ligate DNA breaks resulting in DNA damage. Such unfixable DNA damages cause cell cycle arrest and subsequently apoptosis –14. CataIn this study, we demonstrate that catalytic Topo II inhibitors can block AR signaling and inhibit prostate cancer cell proliferation and CRPC growth, provide proof-of-principle that co-targeting Topo II in combination with AR pathway inhibitors may cooperatively de-activate the AR and delay CRPC progression.Two Topo II isoforms, alpha and beta, are widely expressed in prostate cancer cells . We confICRF187 and ICRF193 are catalytic inhibitors to both Topo II isoforms [v567ed. Co-treatment of ICRF187 or ICRF193 with 5uM of ENZ induced a further 60% decrease in luciferase activity driven by the AR splice variants in LNCaP95 cells  of the AR. However, these drugs induce resistance via adaptive changes in the AR genome including AR amplification, mutations and constitutively active AR splice variants –21. For In contrast to AR pathway inhibitors, catalytic Topo II inhibitors target not only AR-mediated transcription initiation but also suppress cancer cell mitosis. Since a downstream effector of re-activated AR signalling in CRPC tumors is acceleration of tumor cell mitosis , 7, targThe knowledge that Topo II is required for AR transcriptional activity may lead to two possible therapeutic strategies for prostate cancer patients. One possibility is to utilize super physiological doses of androgen in combination with Topo II poisons, as proposed previously , to capiIn summary, we demonstrate that catalytic Topo II inhibitors can block AR signaling and inhibit tumor growth of castration-resistant xenografts, identifying catalytic Topo II inhibitors as potentially novel drugs to treat in patients with CRPC.Human LNCaP, 22RV1, VCaP, PC3 and 293T cell lines were from American Type Culture Collection. LNCaP C4-2, LNCaP95 and LNCaP(AI) were from Drs. Leland Chung (Cedars-Sinai), Alan Meeker (Johns Hopkins University) and Ralph Buttyan (Vancouver Prostate Centre). LNCaP95 and LNCaP(AI) cell were maintained in RPMI1640 medium with 5% charcoal stripped serum (CSS) (Hyclone) as we reported , 24. MR4Real-time PCR assays were performed as described , 27. TotPlasmid DNA were transfected into cells using Lipofectmine 2000 (Invitrogen), while siRNA was transfected by siLentFect reagent (Bio-Rad). Luciferase activities were determined using the luciferin reagent (Promega) according to the manufacturer's protocol. Transfection efficiency was normalized to Renilla luciferase activities.ChIP assays were performed with AR antibody (Santa Cruz) as we described . To deteCell proliferation rates were measured by using the 3--5-(3- carbo xymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium (Promega) assay according to the manufacture's protocol.http://www.meduniwien.ac.at/user/johannes.schmid/PIstaining3.htm). Relative DNA contents from 10,000 cells were analyzed by FACSCanto II flow cytometer and BD FACSDiva software v5.0.3 (Becton Dickinson) as we reported [Cell cycling was analyzed by FACS with 40ug/mL propidium iodide staining following the protocol (reported .6 LNCaP cells in 0.1mL Matrigel (Becton Dickinson Labware) were inoculated subcutaneously in bilateral flanks of 6-8 week old male athymic nude mice (Harlan Sprague Dawley Inc.). Tumor volume (V= L*W*D*0.5236) and body weight were measured weekly. Serum PSA levels were determined by ELISA. When serum PSA concentrations reached 50 ng/mL, mice were castrated. CRPC LNCaP tumors were defined when PSA levels relapsed to pre-castrated levels. Animals were randomly separated into four groups treated daily with control, 10mg/kg ENZ, 50mg/kg ICRF187 or 5mg/kg ENZ plus 25mg/kg ICRF187. To establish ENZ-resistant MR49F xenografts, castrated mice were inoculated subcutaneously with MR49F cells and treated with 10mg/kg ENZ daily till endpoint. When tumor volumes reached 100 mm3, mice were also treated daily with vehicle, 25mg/kg or 50mg/kg of ICRF187. Tumor volumes and PSA levels were measured. CRPC 22RV1 xenografts were constructed by inoculating 22RV1 cells subcutaneously in bilateral flanks of nude mice. When mice were castrated and tumors reached to 100 mm3, mice were treated daily with vehicle or 50mg/kg ICRF187. PC3 xenografts were constructed by inoculating PC3 cells subcutaneously in male athymic nude mice. When tumors reached 200 mm3, mice were treated daily with vehicle or 50mg/kg ICRF187. In all experiments, animals will be sacrificed if tumors mass is ~10% of the body weight or any endpoint situation according to the animal protocol. At the endpoint, tumors were harvested for evaluations of mRNA levels of AR targeted genes by real-time PCR. Tumors samples were also used to construct tissue microarray to measure Ki67 expressions by immunohistochemistry (IHC). All animal procedures were under the guidelines of the Canadian Council on Animal Care.To construct CRPC LNCaP xenografts, a total of 1×10P<0.05 as *, P<0.01 as ** and P<0.001 as ***. One-way ANOVA plus student's t-test were used to compare among groups when the means follows the normal distribution. Detail statistical analyses were addressed in each figure legend.Statistical analysis was carried out using GraphPad Prism (version 6) with the level of significance set at


Although chronic adrenocorticotropic hormone (ACTH) and androgen hyperstimulation are assumed to be involved in the pathogenesis of adrenal myelolipomas associated with poor-compliance patients with congenital adrenal hyperplasia (CAH), the expression of their receptors has not yet been demonstrated in these tumors so far.Melanocortin 2 receptor (MC2R), Androgen Receptor (AR), Leptin (LEP), and Steroidogenic factor 1 (SF1) expression using real-time qRT-PCR in two giant bilateral adrenal myelolipomas from two untreated simple virilizing CAH cases and in two sporadic adrenal myelolipomas. In addition, the X-chromosome inactivation pattern and CAG repeat numbers in AR exon 1 gene were evaluated in the 4 cases.We analyzed MC2R gene was overexpressed in myelolipomas from 3 out of 4 patients. AR overexpression was detected in 2 tumors: a giant bilateral myelolipoma in a CAH patient and a sporadic case. Simultaneous overexpression of AR and MC2R genes was found in two of the cases. Interestingly, the bilateral giant myelolipoma associated with CAH that had high androgen and ACTH levels but lacked MC2R and AR overexpression presented a significantly shorter AR allele compared with other tumors. In addition, X-chromosome inactivation pattern analysis showed a polyclonal origin in all tumors, suggesting a stimulatory effect as the trigger for tumor development.The MC2R or AR overexpression in giant bilateral myelolipomas from poor-compliance CAH patients.These findings are the first evidence for  CT scan revealed giant bilateral adrenal myelolipomas  with fat component density. She did not present to clinical follow-up and had not received any medications in the last 15 yr. Hormonal evaluation revealed extremely increased levels: basal 17OH-progesterone (17-OHP) 192 ng/mL, ACTH 1,172 pg/mL, total testosterone 949 ng/dL and androstenedione 17 ng/mL . CT scan revealed giant bilateral adrenal myelolipoma  with fat component density  and patient 4  presented with incidental findings of sporadic unilateral adrenal myelolipoma ranging from 8 to 10 cm in the major diameter. MRI demonstrated fat tissue signal in both masses. The patients did not present any clinical presentation of hyperandrogenism and non-classical CAH was rule-out by clinical evaluation.Briefly, the histopathological analysis of all tumors showed lobes of mature adipose tissue mixed with abundant hematopoietic tissue consisting of all three hematopoietic elements with mature and precursor cells. There were islets of cells from the zones of the adrenal cortex in the periphery of tumors as well as between hematopoietic and lipoid cells.MC2R gene was overexpressed in the myelolipomas of 3 out of 4 patients . AR expression was correlated with LEP expression in all tumors.The X-chromosome inactivation pattern analysis revealed a polyclonal origin in all tumors. In addition, all tumors were informative, and nCAG repeat numbers varied from 20 to 30, except in the tumor from patient 1, which had a short allele (15 repeats) (Table MC2R gene was overexpressed in 3 out of 4 myelolipomas. Among these 3 cases, 1 giant bilateral myelolipoma was associated with CAH, and two patients presented sporadic myelolipomas. Chronic ACTH hyperstimulation has been proposed as the main hypothesis to explain the higher frequency of giant and bilateral myelolipomas in poor-compliance CAH patients, but it has never been previously demonstrated. Lack of MC2R gene expression was demonstrated in a giant adrenal myelolipoma associated with CAH employing a semi-quantitative approach to analyze mRNA expression [MC2R overexpression in myelolipomas. MC2R overexpression was found in all but one case. Despite of chronic ACTH hyperstimulation, myelolipoma from patient 1 did not present MC2R overexpression.The pathogenesis of adrenal myelolipomas is unclear, but several mechanisms have been proposed to underlie the etiology. One of the hypothesis is that hematopoietic and fat elements could derive from common progenitor cells secondary to stimulatory factors . In thisAR overexpression was detected in 2 tumors: a giant bilateral myelolipoma in a CAH patient and a sporadic case. AR expression was previously assessed in a single case of bilateral myelolipoma associated with CAH using a semi-quantitative technique, but the results were negative [AR and MC2R expression. The overexpression of AR and MC2R genes was concomitantly found in two of the cases.Androgen receptors have also been implicated in the pathogenesis of myelolipomas associated with poorly controlled CAH patients . In the negative . Here, wAR gene contains highly polymorphic trinucleotide repeats, and the length of the nCAG repeat segment is inversely correlated with the transactivation function of the AR gene [MC2R and AR overexpression had a shorter AR allele compared with the other tumors, suggesting that this AR genotype in the context of very high androgen levels may play a causative role in the development of myelolipomas. The stimulatory effect of MC2R and AR overexpression or increased AR transactivation activity in the development of adrenal myelolipomas could be reinforced by the finding of a polyclonal origin in all tumors described here.It has been postulated that androgens, through their interaction with androgen receptors, may play an important role in the development of specific tumors, such as ovarian and prostate cancer ,17. Exon AR gene . InteresMC2R or AR overexpression in giant bilateral myelolipomas from poor-compliance CAH patients. Therefore, we can speculate that chronic ACTH and androgen stimulation may play a causative role in myelolipomas of poorly controlled CAH patients. In addition, X-chromosome inactivation pattern analysis revealed a polyclonal origin in all tumors, suggesting a stimulatory effect as a trigger for tumor development.In conclusion, we first demonstrated here CAH: Congenital adrenal hyperplasia; ACTH: Adrenocorticotropic hormone; MC2R: Melanocortin 2 receptor; SF1: Steroidogenic factor 1; LEP: Leptin; AR: Androgen Receptor; ACTB: Beta-actin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.The authors declared that they have no competing interest.MQA: data analysis, interpretation, drafting the manuscript. LCK: data acquisition, data analysis. LPB: data acquisition, data analysis, statistical analysis. AML: data acquisition, statistical analysis. BMP. Mariani: data acquisition, data analysis. MR: data acquisition, critical revision. OM: data acquisition, critical revision. FTD: performed the laparoscopic procedure of patients included in this study. BBM: critical revision, supervision. TASSB: conception and design, drafting the manuscript, critical revision, supervision. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6823/14/42/prepub


Objective: The purpose of the study was to determine what patients, professionals and significant others regarded as the most important positive- and challenging aspects of Pulmonary Rehabilitation Programmes for patients with Chronic Obstructive Pulmonary Disease (COPD) and to gain insight into how such programmes could be developed and improved.Method: A modified Nominal Group Technique method was used in three consultation workshops .Results: Each of three workshops resulted in the production of approximately ten positive- and ten challenging aspects related to Pulmonary Rehabilitation Programmes.  These were further developed by a process of thematisation into seven broad themes.  The most important was ‘the patient’, followed by ‘physical health’; jointly ranked as third were: ‘mental health’ and ‘knowledge and education’.  ‘The programme’ and ‘professional characteristics’ were jointly ranked as fifth, with ‘the future’ being ranked as the least important theme.Conclusions: The modified Nominal Group Technique method allowed the development of a ranked thematic list that illustrated the important positive- and challenging aspects of Pulmonary Rehabilitation Programmes for patients with COPD. These themes should be core to planning future Pulmonary Rehabilitation Programmes, particularly if patients and carer views are to be considered. COPD results in a reduced capacity for functional activities and performance of daily activities with a corresponding impairment in Health Related Quality of Life2. Current figures show 900,000 people have been diagnosed with and are receiving treatment for COPD within the United Kingdom3. However, due to under reporting or under diagnosis, the actual number of those suffering with COPD is estimated to be as high as 3 million4. Stopping smoking is crucial and is the only intervention that influences the natural history of lung deterioration, with current pharmacological treatment being aimed at reducing symptoms and exacerbations5.Chronic Obstructive Pulmonary Disease (COPD) is a progressively disabling condition characterised by impaired respiratory function associated with physical limitations and psychological co-morbidity8. Pulmonary Rehabilitation Programmes have been shown to be cost-effective and are now recommended for all patients who remain breathless despite optimal bronchodilators, irrespective of severity and age9. Pulmonary Rehabilitation Programmes are also being effectively applied to non-COPD causes of pulmonary impairment10.Pulmonary Rehabilitation Programmes are multi-disciplinary interventions individually tailored to optimise each patient’s physical and social performance. Rigorous evidence from randomised controlled trials demonstrates that Pulmonary Rehabilitation Programmes for COPD can improve dyspnoea, exercise tolerance, Health Related Quality of Life, and reduce the number of days spent in hospital and the utilisation of healthcare resources8. Research exploring the benefits following Pulmonary Rehabilitation Programmes has predominantly been quantitative in nature. There have been some qualitative studies with COPD patients, but these have focused largely on specific aspects of patient experience12 and barriers to participation in Pulmonary Rehabilitation Programmes or other self-management programmes14. There has been some exploration of the effectiveness of self-management programmes from the patient perspective17. However, none of these studies have combined patient, carer, and professional perspectives, particularly in an in-depth analysis regarding the long-term impact of Pulmonary Rehabilitation Programmes in relation to personal needs and issues such as perceived patient benefits, and expectations and challenges of Pulmonary Rehabilitation Programmes. It has been recognised that a better understanding of how Pulmonary Rehabilitation Programmes improve Health Related Quality of Life could affect the future design of programmes, enhance measurement tools for Health Related Quality of Life and more appropriately support the specific needs of patients18.There are now specific guidelines and recommendations in the United Kingdom regarding Pulmonary Rehabilitation Programmes, including how to select patients, the timing and number of sessions, intensity and type of exercise, the key educational, psychological and behavioural components, oxygen supplementation and outcome assessment19. The purpose of consensus methods is to reach an agreement on a particular issue. Consensus methods can also mitigate some of the problems sometimes associated with group decision-making processes. In particular, where dominant views may lead the process and crowd out other perspectives19.Consensus methods are techniques used gain opinions and views from appropriate experts regarding the current position in a particular field. They provide a mechanism for assimilating and synthesising information, particularly where published information may be inadequate or non-existentet al. in the 1970s20. The Nominal Group Technique normally involves four main phases: a nominal phase, during which each individual silently considers the issues under deliberation; an item-generation phase, during which each individual discloses the results of their deliberation to the group; a discussion and clarification phase, during which the group assures itself that it has understood the items that have been advanced; and a voting phase, during which the items are evaluated and the issue is decided (e.g. a ranking exercise). Nominal Group Technique promotes individual contributions allowing each individual the opportunity to voice their opinions. Factors that would normally inhibit participation are therefore avoided and even the more reticent group members are encouraged to participate in all phases21.Nominal Group Technique is one of the commonly used consensus methods within healthcare and medical settings. The technique was first developed as an organisational planning technique by Delbecq22, we aimed to provide a picture of the perceived benefits and challenges of Pulmonary Rehabilitation Programmes for COPD patients. Here we report the quantitative analysis of the Nominal Group Technique activities.By adopting a mixed methods design, employing qualitative and quantitative methods during consultation with mixed stakeholder groups, and by including a modified Nominal Group Technique component as described previouslyFollowing regional ethics and research and development approval, a series of consultation workshops were held between January and December 2012, in a District General Hospital in Wales, United Kingdom, serving a mixture of urban and agricultural communities. The hospital delivers a regular Pulmonary Rehabilitation Programme which includes 18 sessions of outpatient multidisciplinary input from occupational therapists, physiotherapists, dietetics staff, physicians, specialist respiratory nurses, social workers and a smoking cessation counsellor. The content and timings of the Pulmonary Rehabilitation Programme is evidenced-based and is tailored to individual requirements and personalised goal setting.We recruited across the South West Wales Regional Health Board, United Kingdom that serves 385,000 people and included patient, professional and significant other groups, to ensure we included a wide range of views, experience and knowledge of COPD and Pulmonary Rehabilitation Programmes.Patients with COPD who were currently participating in or who had completed a Pulmonary Rehabilitation Programme within the last 2 years were approached to participate in the study, with most being approached in their last Pulmonary Rehabilitation Programme session. Information sheets were given to patients for their significant others  inviting them to contact the researcher if they wished to participate. Professionals who were identified as playing a significant role in the delivery of the Pulmonary Rehabilitation Programmes and the treatment of COPD patients  were also approached to participate in the study. All 20 participants  provided written informed consent.Our aim was to gain an understanding of the positive and challenging aspects of Pulmonary Rehabilitation Programmes for patients with COPD and to gain a consensus regarding what constitute the most important aspects of Pulmonary Rehabilitation Programmes.23.Nominal Group Technique consensus exercises were carried out as one aspect of a multi-layered, mixed-method consultation during three half-day workshops . Based on guidance in the literature for optimal numbers for qualitative group consultations, we aimed to recruit six participants to each of the three workshops22.Each workshop was made up of three parts. Part one began with a broad discussion that examined the nature and content of Pulmonary Rehabilitation Programmes through a semi-structured group interview. The second part involved more extensive discussion with participants. Having attended a Pulmonary Rehabilitation Programme, participants were encouraged, using personal examples to describe what the Programme meant to them. This included exploring their perceived views regarding the benefits and challenges of Pulmonary Rehabilitation Programmes and impact on patient Health Related Quality of Life. An adapted Nominal Group Technique exercise was employed in the final part of the workshop. The focus of this stage was to address the following question with participants: “what are the positive, and what are the challenging aspects of Pulmonary Rehabilitation Programmes for the treatment and rehabilitation of COPD patients?” During the Nominal Group Technique exercise, issues that were raised in the early parts of the workshop were refined and condensed into a list of approximately ten positive and ten challenging aspects. At the end of the workshop, participants were asked to rank these aspects in order of significance  scores. Dataset 2: NGT activity with patients- challenging aspects Final agreed list of challenging aspects of a Pulmonary Rehabilitation Programme for COPD patients. Data are illustrated for each of the patient participants and the generated aspects are ranked in order of importance according to their calculated median and inter-quartile range (IQR) scores. Dataset 3: NGT activity with significant others- positive aspects Final agreed list of positive aspects of a Pulmonary Rehabilitation Programme for COPD patients. Data are illustrated for each of the significant other (Sig oth) participants and the generated aspects are ranked in order of importance according to their calculated median and inter-quartile range (IQR) scores. Dataset 4: NGT activity with significant others- challenging aspects Final agreed list of challenging aspects of a Pulmonary Rehabilitation Programme for COPD patients. Data are illustrated for each of the significant other (Sig oth) participants and the generated aspects are ranked in order of importance according to their calculated median and inter-quartile range (IQR) scores. Dataset 5: NGT activity with professionals- positive aspects Final agreed list of positive aspects of a Pulmonary Rehabilitation Programme for COPD patients. Data are illustrated for each of the professional (Prof) participants and the generated aspects are ranked in order of importance according to their calculated median and inter-quartile range (IQR) scores. Dataset 6: NGT activity with professions- challenging aspects Final agreed list of challenging aspects of a Pulmonary Rehabilitation Programme for COPD patients. Data are illustrated for each of the professional (Prof) participants and the generated aspects are ranked in order of importance according to their calculated median and inter-quartile range (IQR) scores. Dataset 7: Thematic NGT activity Final agreed thematic consensus list of positive and challenging aspects of a Pulmonary Rehabilitation Programme for COPD patients. Data are illustrated for all the participants and the generated aspects are ranked in order of importance according to their calculated median and inter-quartile range (IQR) scores.Click here for additional data file.22 in order to include an additional multi-group ranking round  for each of the themes. A final consensus ranked thematic list was produced based on these median ranks Step 10,. This wa28. The detailed content relating to each theme was extracted from the individual transcripts and was built up to articulate fully the set of aspects that it contained and to clarify any anomalies or ambiguities29. The final output of the consultation workshop was a ‘thematic template’ that ranked each theme in order and that provided a qualitative in-depth elaboration of the content contained within each theme.Notes and audio recordings from the three consultation workshops were transcribed. These transcripts were subjected to thematic and summative analysis to extract relevant information related to each of the generated themesWe recruited a total of 20 participants across the three consultation workshops see. Thirty the patient,physical health,mental health,knowledge and education,the programme,professionals and significant others andthe future .the patient, followed byphysical health. Jointly ranked as third were:mental health andknowledge and education.The programme andprofessionals and significant others were jointly ranked as fifth, withthe future ranked as the least important theme  as well as negative aspects 13. It has been shown that attending a Pulmonary Rehabilitation Programme is associated with better management of breathlessness, which in turn has a positive impact on physical and social activity, coping strategies and patient confidence18.The final outcome of the Nominal Group Technique exercise was a ranked list of seven themes , with ‘t30, a larger study, employing more consultation workshops conducted over a larger geographical area is necessary to consider whether all the important aspects of Pulmonary Rehabilitation Programmes have been revealed, and whether the themes we identified within this study are generalisable.This study was carried out within one geographical location in South-West Wales, United Kingdom, and employed only three consultation workshops. Whilst we are confident that the methods adopted are transferable, in line with our extensive engagement with the methods in a range of community and primary care settingsThe adapted Nominal Group Technique exercise was a mechanism for distilling the important aspects of Pulmonary Rehabilitation Programmes in a mixed group of individuals, which allowed the views of all the participating groups to be considered as equal. The process of qualitative elaboration of these themes in terms of what they meant to patients, professionals and significant others, provided a more comprehensive picture than other studies have derived. Moreover, combining qualitative with quantitative assessments provides more information, and these approaches could be used to make recommendations to improve and develop Pulmonary Rehabilitation Programmes across health-care contexts.Figshare: Nominal Group Technique consultation of a Pulmonary Rehabilitation Programme Data Set, doi:10.6084/m9.figshare.92854031 All participants provided written informed consent. The article gives a clear introduction to the concept of Pulmonary Rehabilitation (PR) acknowledging the evidence supporting the intervention and the positive impact that is has on participants lives post programme. Most importantly, it seeks to gather the opinions of “significant others” and professionals within the clinical team, something that is often overlooked but that could be a key component in service development.There is a clear introduction to the use of the Nominal Group Technique and its usefulness in facilitating all participants’ views to be gathered and the aim of providing a rank of perceived benefits and challenges in pulmonary rehabilitation is clearly stated.     The authors have used the Nominal Group Technique successfully to provide a useful ranking of themes and challenges from the perspectives of patients, professionals and significant others. These are useful to challenge current assumptions within Pulmonary Rehabilitation Programmes, but the study would benefit from consideration of the socio-demographics of the group as these also impact on health related outcomes. The small sample size also makes it difficult to generalise to a wider population, something which the authors themselves acknowledge. There is also a strong male bias in the sample group, what affect did this have on the results?The data and techniques used are clearly described and well presented and are representative and reflective of findings in the clinical setting.In conclusion, going forwards further research is required to establish whether the rankings are concurrent across the country, and if so do they have a role in guiding PR and assisting patients and significant others in overcoming the perceived barriers. Many of the themes and issues identified are already known to those working within the clinical setting and are addressed through education and management within PR in the UK already. There are also assumptions in practice that patients “Learn” as is stated on page 6 in the Thematic Template generation. How is this evidenced? One finding that the authors also highlight is that significant others have more time for themselves as patients become more independent therefore supporting the self management of PR; this would benefit from further exploration.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. STUDY PURPOSE The purpose of the present study was to describe the perceived benefits and challenges of pulmonary rehabilitation program for patients with COPD from the point of view of patients, professionals, and significant others (carers).TITLE AND ABSTRACTThe title reflects the content of the article. The abstract provides a suitable summary of the work.  The conclusion would be strengthened by a main message.INTRODUCTIONThe introduction provides detailed information about the area of research, gaps in the literature, and techniques employed to answer the research question. The references to the relevant literature on the mixed method design were also provided in order to support the proposed methodology.METHODSThe design of the study was also described clearly, including thematic generation and consensus, as well as responsibilities for integration. However, the authors state that the focus is on the quantitative analysis rather than the qualitative analysis, yet the analysis described appears to be qualitative in nature, and the authors comment on the need to maintain qualitative rigour throughout the study. A ranking by median scores by the participants is still qualitative in nature.  If the intent is to undertake a quantitative analysis of the data, the selection of the median rank and interquartile ranges as appropriate statistical tests that is meant to answer the study research question seems insufficient. The authors might want to explore the differences between the different groups in regard to the perceived benefits and challenges of pulmonary rehabilitation program for patients with COPD. We consider that there is a need for clearly stating the hypothesis being tested and a motivation for selecting specific statistical analysis, should a quantitative analysis be intended. The individual data table would be better presented in the results section.RESULTSThe results section is appropriately explained, but the emphasis is again placed on the qualitative than quantitative research findings. In addition, the authors state that 20 participants were initially included in the study  and 12 participants were finally included in the analysis. Given the small sample size, there is also a need to specify the status of the participants who did not complete the study. This information may influence the interpretation of the study results. There is also a disagreement between the evaluable responses across the paper: 60% on the page 6 versus 70% on the page 9. The authors also state that COPD patients who were currently participating in or completed a pulmonary rehabilitation program within the last 2 years were approached to participate in the study. However, Table 1 shows 2005 as year of PR program for one COPD patient. It is also worth mentioning the weak participation of physiotherapists in this study, although the pulmonary rehabilitation program was the main component of this research and the physiotherapists play a significant role in the delivery of pulmonary rehabilitation program. The thematic template generation does not offer clear guidance to the reader on what are the clear benefits and challenges – the list is somewhat self-evident and simplistic, and appears to focus on the benefits of the pulmonary rehabilitation program, but the challenges are not clearly articulated.DISCUSSIONThe conclusions are generally balanced and justified, although special emphasis is placed on the qualitative findings. Some of the discussion would be better suited to the results. The main benefits and challenges of pulmonary rehabilitation for patients, carers, and professionals as presented in the themes are not specific enough to be of use for making recommendations for improving the pulmonary rehabilitation program.We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. This is an interesting and useful article that explores the implications and lived experience of people dealing with the challenges of a debilitating chronic disease (COPD). This is not a "sexy" area of health care research but it is an important one as COPD substantially limits life in both quantity and quality. As such it is a worthy area of investigation, as the aim of this article is to identify how to improve the delivery of service for a sometimes overlooked patient group. The article is well presented and reflects a high level of attention to detail in design and analysis. The research team has identified an appropriate method of investigation which is sufficiently novel to generate new knowledge that will potentially inform clinical practice. Details of the data collection and analysis processes are well presented and the data are discussed comprehensively. The research team acknowledges that there are limitations in the study in terms of the level of participants, and the level of geographical specificity of the study. Whilst this is a sensible approach such caveats should not detract from the potential value of the study for practitioners who deliver such programmers. Finally, the conclusions are well balanced and insightful with a clear pathway to future research identified.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Allergic rhinitis (AR) is a relevant risk factor for the development of asthma in children. We recruited a cohort of 104 children with AR and re-evaluated them after 5 years. We considered the ARIA classification. All patients, who had moderate to severe persistent AR at baseline, developed asthma symptoms. These results strongly indicate that the severity of AR may be an important factor that increases the risk of asthma development in children. Allergic rhinitis (AR) is a relevant risk factor for the development of asthma in children and is an important trigger factor for exacerbations in patients with asthma . MoreoveAR is presently classified as intermittent or persistent on the basis of the symptoms duration according to ARIA guidelines . In addiTherefore, this study aimed to investigate whether the ARIA classification of AR may be useful to predict the possible development of asthma in children with AR alone.One hundred four children  with AR were evaluated in this prospective study. Children were recruited and visited at the Institute of Pediatrics of the University of Perugia .Inclusion criteria were: age range between 6 and 14 years, documented and validated diagnosis of AR, and written informed consent signed by parents. Exclusion criteria were, absence of sensitization, history of asthma symptoms, impaired lung function, previous or current specific allergen immunotherapy, and chronic diseases. The review board approved the study. Patients were treated with medications alone on demand.Demographic and clinical data were recorded at baseline and after 5-year follow-up.At baseline, all patients were evaluated for sensitization to inhaled allergens by skin prick test (SPT), lung function, and AR was graded according to ARIA guidelines . After 5AR diagnosis was based on the consistency between nasal symptoms history and sensitization. Asthma diagnosis was performed on asthma symptoms history, impaired lung function, and functional testing (mainly bronchodilation).1 (97.1 ± 5% of predicted) and PEF (97.7 ± 5.1% of predicted). According to ARIA classification, patients were divided into 3 groups: 74 patients with moderate-severe intermittent AR (Group 1), 24 patients with mild persistent AR (Group 2), and 6 patients with moderate-to-severe persistent AR (Group 3), as reported in Figure At baseline, all patients were sensitized to aeroallergens , 10.5% were polysensitized. Lung function was normal in all patients, both concerning FEVAt the 5-year follow-up visit, 19 children developed asthma. Considering the ARIA classification: 5/74 (7%) patients of Group 1 developed intermittent asthma, 8/24 (33%) patients of Group 2 had asthma (7 intermittent and 1 mild persistent), and 6/6 (100%) patients with moderate-severe persistent AR presented asthma , as reported in Figure SPT evaluation after 5 years showed the presence of 42 new sensitizations in 34/104 patients , with no difference according to AR severity at baseline.1 (95.5 ± 6.3% of predicted) and PEF (94.2 ± 5.4% of predicted).Lung function evaluation, performed after 5 years in patients who developed asthma, showed a not significant changes both concerning FEVIt is well known that AR and asthma are closely linked, concerning clinical, functional, and immunological aspects. In fact, nasal inflammation is associated with bronchial airflow limitation , as wellThis study demonstrates that all children with moderate-severe persistent AR developed asthma as well as 33% of children with mild persistent AR. This finding underlines the concept that persistence of AR may be associated with progression from AR towards asthma. The possible explanation might be that persistent nasal inflammation may be associated with involvement of lower airways both concerning mucosal infiltration and lung function impairment ,8. ThereHowever, this study has the limitation that there was no control group without AR, even though the AR sub-classification according to ARIA criteria may allow to consider the relevance of AR severity on the progression of the allergic reaction from the nose to the bronchi.In conclusion, all patients, who presented moderate-to-severe persistent AR, developed asthma symptoms. These results strongly indicate that the persistence of AR may be an important factor that increases the risk of asthma development in children.


ISO inhibited the expression of the surface molecules CD86 and MHC-II, inhibited the stimulation of T lymphocyte proliferation by DC and TNF-α secretion, and promoted DC antigen uptake and IL-10 secretion. The effects of ISO on MHC-II expression, DC stimulation of T lymphocyte proliferation, and DC antigen uptake were mediated by β2-AR. Treatment with salbutamol ameliorated the severity of AA and histopathology of the joints and inhibited proliferation of thymus lymphocytes and FLS in vivo. β2-AR signaling was weaker in AA rats compared to the control. Elevated GRK2 and decreased β2-AR expression in DC cytomembranes were observed in AA and may have decreased the anti-inflammatory effect of β2-AR signaling. Decreased β2-AR signaling may be relevant to the exacerbation of arthritis inflammation.Rheumatoid arthritis (RA) is characterized by inflammation of the synovium, which leads to the progressive destruction of cartilage and bone. Adrenoreceptor (AR) signaling may play an important role in modulating dendritic cell (DC), which may be involved in the pathogenesis of RA. We examined the effect of the β-AR agonist isoprenaline (ISO) on DC function, the impact of the β Both knockdown of costimulatory factors such as CD80 and CD86 and expression of immunosuppressive molecules in DCs have been exploited to generate tolerogenic DCs. These tolerogenic DCs effectively suppress the onset of collagen-induced arthritis, produce IL-10, and induce T-cell tolerance via immunosuppressive cytokines8Dendritic cells (DCs) are essential regulators of both the innate and acquired arms of the immune system. DCs likely contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA) in several ways13RA is characterized by inflammation of the synovium, which leads to progressive destruction of cartilage and bone101213In vitro, NE reduces the ability of murine DCs to present antigen in a mixed lymphocyte reaction using an antigen-specific T cell cloneAR signaling may play an important role in modulating DC function during both the innate and adaptive immune responses172-AR agonist salbutamol on adjuvant-induced arthritic (AA) rats, and changes in β2-AR signaling in DCs from AA rats over the course of the disease. This research aims to elucidate the role of β2-AR signaling in RA pathogenesis and provide an experimental basis for the identification of new drug targets.What is the effect of β-ARs on the function of DCs? Is any such effect involved in the regulation of RA pathogenesis? In the present study, we investigated the effect of the β-AR agonist isoprenaline (ISO) on the function of DCs, the impact of the βMale Sprague Dawley (SD) rats weighing 150–180 g were purchased from the Experimental Animal Center of Anhui Medical University . The animals were housed in a room with a controlled ambient temperature (22 ± 2 °C) and humidity (50% ± 10%), with food and water ad libitum. All procedures were performed in accordance with the guidelines of the Animal Care and Use Committee of Anhui Medical University and were approved by the Ethics Committee of Anhui Medical University.2-AR, GRK2 and β-actin were from Abcam . Horseradish peroxidase (HRP)-labeled goat anti-rabbit and goat anti-mouse antibodies were acquired from Santa Cruz Biotechnology . LPS, FITC-dextran (40 kD), isoprenaline hydrochloride, CGP20712A, ICI118551 and CCK-8 were purchased from Sigma . Salbutamol sulfate was from Shanghai Xudong Haipu Pharmaceutical Co., Ltd. . ELISA kits for interleukin 10 (IL-10) and TNF-α were purchased from RayBiotech, Inc. .Recombinant rat interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were purchased from Peprotech . RPMI-1640 medium, Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were from Hyclone . Phycoerythrin (PE)-conjugated anti-CD80, -CD86, and -MHC-II, Alexa Fluor 647-CD103 and isotype control antibody were purchased from BioLegend . The antibodies against β6 cells/ml in 6-well plates. Three hours later, non-adherent cells were discarded, and new medium supplemented with IL-4 (10 ng/ml) and GM-CSF (10 ng/ml) was added. The cultures were fed fresh medium and cytokines every 3 days. On day 5, isoprenaline  was added; no isoprenaline was added to the control group. Simultaneously, LPS (100 ng/ml) was added to the BMDCs, except the LPS(−) group. Loosely adherent clusters were harvested on day 6–8 and used for experiments.Bone marrow cells were collected from the tibias and femurs of SD rats by flushing the bones. The cells were pipetted vigorously up and down several times to obtain single-cell suspensions and passed through a nylon cell strainer to remove small pieces of bone and debris. The cells were cultured in RPMI-1640 medium containing 10% FBS at a density of 5 × 106 cells) prepared above and spleen lymphocytes were acquired for each sample and stained for CD103 (Alexa Fluor 647), CD80 (PE), CD86 (PE), MHC-II (PE), or the corresponding isotype control for 30 min at 37 °C. Because CD103 is a specific marker for rat DCs, CD103+ cells were gated; within this population, the expression of CD80, CD86 and MHC-II on DCs was measured by flow cytometry. Data analysis was performed using FlowJo analysis software  and reported as the mean fluorescence intensity (MFI).The BMDCs , and the incorporation of FITC-dextran was analyzed by flow cytometry. The data are presented as mean fluorescence intensities (MFIs).5 cells/well) were collected through nylon wool and co-cultured with these BMDCs in 96-well plates at ratios of 10:1 for 48 h at 37 °C. Four hours before the end of the incubation, 20 μl of CCK-8 was added to each well, and the absorbance at 490 nm was determined using a multi-well plate reader . The experiments were conducted in triplicate for each condition.The BMDCs prepared above were harvested on day 7 and treated with mitomycin (25 μg/ml) at 37 °C for 30 min, then washed twice with PBS. Rat splenic T lymphocytes  into the right hind footpad. The day of CFA injection was designated day 0, and the secondary inflammatory reaction occurred after day 14 (d14).After the onset of arthritis on d14, the animals were randomly allocated to 6 groups: control, AA model, salbutamol , and MTX . The rats received medication from d15 to d28. The rats in the normal control and AA model groups received an equal volume of 0.5% sodium carboxymethylcellulose (CMC-Na) at the same time points.To evaluate the severity of arthritis, the secondary inflammatory paw (left hind) swelling of rats was evaluated at 0, 7, 14, 21, 28, 35 days using a Paw Volume MeterRats were sacrificed on day 28 to dissect the left hind knee joint. The joints were removed, fixed in formalin, decalcified in 10% ethylenediaminetetraacetic acid (EDTA) and embedded in paraffin for histopathological analysis. Serial paraffin sections were stained with hematoxylin and eosin (H&E).The severity of arthritis in the joint was graded from 0 to 4 according to the intensity of lining layer hyperplasia, mononuclear cell infiltration and pannus formation, as described previously 6) from each group were placed in 96-well plates with ConA (5 mg/l), and spleen cells  from each group were placed in 96-well plates with LPS (4 mg/l); all suspensions were prepared in triplicate and incubated at 37 °C in 5% CO2 for 48 h. Four hours before the end of the incubation, 20 μl of CCK-8 was added to each well. The absorbance was measured by a Multiskan Spectrum . The results are presented as the average of triplicate counts.Rats were sacrificed on d35 after immunization. The thymus and spleen were dislodged under sterile conditions. The cells were suspended in a lymphocyte separation medium and washed three times with PBS. Thymus cells . The results are presented as the average of triplicate counts.The rats were anesthetized and sacrificed on d35 after immunization, and the synovial tissues from the knees joints were excised. FLSs were isolated from individual tissues using a tissue transplantation method and cultured in DMEM supplemented with 20% fetal calf serum, penicillin (200 U/ml), and streptomycin (200 ng/ml) at 37 °C in 5% CO2-AR and GRK2 (1:1000) and mouse monoclonal anti-β-actin (1:500) at 4 °C overnight. Then, the membranes were incubated with secondary antibodies conjugated to HRP, and detection was achieved by measuring the chemiluminescence of the blotting agent after exposure of the filters to films. Finally, the densities of the bands were quantified with a computerized densitometer . Equivalent protein loading and transfer efficiency were verified by staining for β-actin.Rats were sacrificed on d0, d7, d14, d21, and d28 after immunization. BMDCs were isolated from each group and prepared as above, then lysed in cell lysis buffer with 1 mM PMSF, followed by centrifugation  for 60 min; the precipitates were diluted to 4 mg protein/ml and stored frozen at −80 °C until use. The precipitate mainly comprised cytomembrane proteins. A total of 50 μg of denatured protein was separated by 10% SDS-PAGE, transferred onto polyvinylidene fluoride membranes , and then incubated with primary antibodies to βData are expressed as the mean and standard deviation (SD). Analysis of variance (ANOVA) and Student’s t-test were performed to determine significant differences between groups. Calculations were performed using the SPSS version 11.5 statistical package. Values of P < 0.05 were considered significant.−5, 10−6, or 10−7 mol/L) and LPS (100 ng/ml) were added to BMDCs (except the LPS(−) control group), followed by staining (1 × 106 cells). Compared with the LPS(−) group, LPS significantly promoted the expression of the surface molecules CD80, CD86 and MHC-II. ISO (10−5 and 10−6 mol/l) significantly inhibited the expression of the surface molecules CD86 and MHC-II on DCs treated with LPS but had no significant effect on the expression of CD80  and LPS (100 ng/ml) were added to BMDCs (except the LPS(−) group), and FITC-dextran was then used as an antigen to evaluate the antigen uptake capability of BMDCs by flow cytometry. Background staining at 4 °C was used as a negative control. As shown in a representative experiment (−5 and 10−6 mol/l) significantly upregulated the antigen uptake capability of BMDCs treated with LPS.ISO  and LPS, and the levels of IL-10 and TNF-α were measured by ELISA. ISO (10−5 and 10−6 mol/l) significantly promoted IL-10 secretion and inhibited TNF-α secretion from the BMDCs treated with LPS  and LPS were mixed, and their ability to induce allogeneic T-cell proliferation was evaluated. ISO (10−5 mol/l) inhibited the stimulation of T lymphocyte proliferation by LPS treatment of BMDCs  and a β2-AR antagonist (ICI118551) were used to identify the role of β-AR subtypes in BMDCs treated with LPS.ISO inhibited the expression of the BMDC surface molecules CD86 and MHC−6 mol/l) significantly decreased CD86 expression, and the selective β1-AR antagonist CGP20712A (10−6 mol/l) antagonized the effect of ISO, whereas the selective β2-AR antagonist ICI 118551 (10−6 mol/l) had no obvious effect  significantly decreased MHC-II expression on BMDCs compared with the control group treated with LPS, and the selective β2-AR antagonist ICI 118551 (10−6 mol/l) antagonized the effect of ISO, whereas the selective β1-AR antagonist CGP20712A (10−6 mol/l) had no obvious effect (ISO (10s effect .−6 mol/l) significantly promoted antigen uptake capability. The selective β2-AR antagonist ICI 118551 (10−6 mol/l) antagonized the effect of ISO, whereas the selective β1-AR antagonist CGP20712A (10−6 mol/l) had no obvious effect  inhibited the stimulation of T lymphocyte proliferation in BMDCs treated with LPS. The selective β2-AR antagonist ICI 118551 (10−6 mol/l) antagonized the effect of ISO, whereas the selective β1-AR antagonist CGP20712A (10−6 mol/l) had no obvious effect  clearly attenuated the degree of paw swelling 14 days after administration (on d28), compared with the AA model .The ameliorating effect of salbutamol on AA was further confirmed by histopathological analysis of joints. Synoviocytes were in a monolayer, and there was no infiltration of inflammatory cells in the control rat knee joints. In the AA model group, synoviocytes proliferated over three layers with pannus formation, and articular cartilage and bone were eroded and infiltrated with inflammatory cells. Histopathological evaluation revealed that synovial hyperplasia, cell infiltration, pannus and bone erosion were significantly increased compared with normal controls. These abnormalities were significantly alleviated in AA rats after administration of salbutamol  .Proliferation was significantly increased in thymus lymphocytes, spleen lymphocytes and FLSs from AA rats compared with control rats. Treatment with salbutamol (3.0 mg/kg) significantly inhibited thymus lymphocyte and FLS proliferation compared with cells from AA rats, with no obvious effect on the proliferation of spleen lymphocytes .The expression of the surface molecules CD86 and MHC-II on DCs on spleen lymphocytes was significantly increased in AA rats compared with the control rats, and treatment with salbutamol (3.0 mg/kg) significantly inhibited the expression of MHC-II compared with AA rats, with no obvious effect on the expression of CD80 and CD86 .The expression of the surface molecules CD86 and MHC-II was significantly increased on BMDCs from AA rats, and treatment with salbutamol (3.0 mg/kg) significantly inhibited the expression of MHC-II and promoted antigen uptake capability compared with AA rats, with no obvious effect on the expression of CD80 and CD86, consistent with the results for DCs from spleen lymphocytes .−5, 10−6, or 10−7 mol/l) ex vivo. T cells were then mixed, and the ability to induce allogeneic T cell proliferation was assessed. Salbutamol (10−5 or 10−6 mol/l) significantly inhibited MLR of BMDCs from control rats, and only the highest concentration of salbutamol (10−5 mol/l) significantly inhibited MLR of BMDCs from AA rats. Salbutamol inhibited the stimulation of T lymphocyte proliferation in control and AA rats, but the inhibitory effect of salbutamol (10−5 mol/l) was stronger for cells from the control group compared to cells from the AA group. These results suggest that β2-AR signaling may be weaker in BMDCs from the AA model (BMDCs generated from control (non AA) and AA rats were treated with salbutamol  and β2-AR antagonist (ICI118551) were used. The results demonstrated that the effects of ISO on MHC-II expression, mixed lymphocyte reactions of BMDCs, and antigen uptake function were mediated by β2-AR.DCs are potent antigen-presenting cells (APCs) that play a major role in the regulation of immune responses to a variety of antigens. DCs first capture antigens via endocytosis and then present these antigens in the context of MHC-II molecules at the cell surface to activate antigen-specific CD42-AR signaling. Our results are consistent with studies have demonstrated that noradrenaline enhances DC antigen uptake27In the present study, we demonstrated that ISO promotes the antigen uptake capability of DCs via β2-AR signaling on MHC-II expression, the BMDC MLR results, and antigen uptake function, we further investigated the effect of the β2-AR selective agonist salbutamol on the inflammatory response of AA rats in vivo. We evaluated disease progression based on the degree of paw swelling degree and joint histopathology in AA rats. Treatment with salbutamol significantly ameliorated the severity of arthritis and abnormal joint histopathology. A related β2-AR agonist, terbutaline, produced similar results in collagen-induced arthritis and AA292-AR agonists on arthritis, Straub et al. observed that splenic IFN secretion is stimulated by NE via β-ARs, contributing to a proinflammatory effect after the onset of CIA. The reason for this effect is not clear but could be related to disease progressionin vivo. This finding is consistent with the known effects of β2-AR agonists on T lymphocyte proliferation and differentiation2-AR agonist terbutaline inhibits the proinflammatory effects of IL-7R+ B cells2-AR agonists compared with IL-7R+ B cells. How does the β2-AR selective agonist salbutamol play a therapeutic role in AA rats? Salbutamol significantly inhibited the expression of MHC-II in DCs from splenocytes of AA rats, and MHC-II is an important surface molecule in the activation of Ag-specific CD4+ T cells. Synovial fluid DCs in RA are more mature than normal: they express high levels of MHC-II molecules and potently stimulate a variety of T cell responses322-AR selective agonist salbutamol can ameliorate the severity of arthritis, perhaps by inhibiting the maturation of DCs and promoting their tolerance. This conclusion is consistent with a report by Cobelens and coworkers that salbutamol potentiates oral induction of tolerance, suppressing adjuvant arthritis and antigen-specific immunityConsidering the effect of β2-AR signaling activation can inhibit DC maturation and promote tolerance, we examined the potential differences in the effect of β2-AR signaling activation on DCs in normal and AA rats? The effects of ISO on BMDC MLRs were mediated by β2-AR. BMDCs from control and AA rats were treated with salbutamol and mixed with T cells, and the ability to induce allogeneic T-cell proliferation was compared. The inhibitory effect of salbutamol on the AA group was weaker than that on the control group. These suggest that β2-AR signaling may be weaker in BMDCs from the AA rat model. To further confirm β2-AR expression in DCs at different stages of AA, we detected the expression of β2-ARs and the negative regulatory protein GRK2 in the cytomembrane of DCs. β2-AR expression significantly decreased at d21 and d28, whereas GRK2 expression significantly increased at d21 and d28, consistent with peak disease. Reduction of β2-AR signaling may be responsible, at least in part, for DC dysfunction in AA.Because β2-AR induces the activation of the Gs/cAMP/protein kinase A (PKA) pathway. Agonist stimulation triggers β2-AR desensitization involving GRK phosphorylation, which in turn triggers arrestin binding, internalization, recycling and resensitization35372-AR, which diminished the anti-inflammatory effect of β2-AR signaling. β2-AR expression significantly decreased at the peak of disease, which may be relevant to inflammatory response exacerbation.Under normal physiological conditions, agonist stimulation by β2-AR signaling. Treatment with the β2-AR selective agonist salbutamol significantly ameliorated the severity of arthritis and abnormal histopathology of joints and inhibit thymus lymphocyte and FLS proliferation from AA rats in vivo. These effects may be mediated by inhibition of DC maturation and promotion of tolerance. However, elevated GRK2 and decreased β2-AR in DC cytomembranes were observed in the AA model and may have decreased the anti-inflammatory effect of β2-AR signaling. Abnormal β2-AR signaling may be relevant to the exacerbation of arthritic inflammation. GRK2 is an important molecule that induces a decrease in β2-AR; therefore, drug targeting of GRK2 may be a direction for future research on RA.In summary, the present study reports that the effects of ISO on MHC-II expression, mixed lymphocyte reactions, and the antigen uptake function of BMDCs are mediated by βHow to cite this article: Wu, H. et al. β2-adrenoceptor signaling reduction in dendritic cells is involved in the inflammatory response in adjuvant-induced arthritic rats. Sci. Rep.6, 24548; doi: 10.1038/srep24548 (2016).