Airway narrowing associated with chronic asthma results in the transmission of injurious compressive forces to the bronchial epithelium and promotes the release of pro-inflammatory mediators and the denudation of the bronchial epithelium. While the individual effects of compression or denudation are well characterized, there is no data to elucidate how these cells respond to the application of mechanical compression in the presence of a compromised epithelial layer.2O), and 4) 6 hours of static compression after a scrape wound. Following treatment, wound closure rate was recorded, media was assayed for mediator content and the cytoskeletal network was fluorescently labeled.Accordingly, differentiated normal human bronchial epithelial cells were exposed to one of four conditions: 1) unperturbed control cells, 2) single scrape wound only, 3) static compression (6 hours of 30 cmH2 media content. Compression stimulated depolymerization of the actin cytoskeleton and significantly attenuated wound healing. Closure rate was partially restored with the addition of exogenous PGE2, but not EGF.We found that mechanical compression and scrape injury increase TGF-β2 and endothelin-1 secretion, while EGF content in the media is attenuated with both injury modes. The application of compression after a pre-existing scrape wound augmented these observations, and also decreased PGE2 and a compromised cytoskeleton.Our results suggest that mechanical compression reduces the capacity of the bronchial epithelium to close wounds, and is, in part, mediated by PGE Asthma is a chronic inflammatory disease marked by recurrent episodes of reversible airway narrowing resulting in dramatically increased airway resistance, work of breathing and extreme risk to the health of a patient . As a reThe airway epithelium serves primarily as a protective barrier between the external environment and the interior of the lung. Numerous studies have demonstrated significant denudation of the airway epithelium in the small airways in lung biopsies of severe asthmatics ,8. The c2O, directly to the airway epithelium. This phenomenon has recently garnered significant attention in the literature, and it has been demonstrated that forces of this magnitude can induce the release of inflammatory and pro-fibrotic mediators [Additionally, the lung as a whole is a remarkably active mechanical environment which necessarily intersects with the chemical mediators of inflammation and wound healing. While mechanical strain as a result of lung expansion is thought to be the predominant force associated with pulmonary mechanics it is often overlooked that during airway constriction entire airways can close completely. Furthermore, it has been demonstrated through in vivo measurement , and supediators -15. It hWhile there has been significant work to characterize the response of the airway epithelium to denudation and mechanical compression independently, they are closely linked both temporally and spatially in vivo ,16. A si2 media content declined significantly only in the setting of both injury types. Mechanical compression significantly attenuated wound healing in culture; however, this effect was partially ameliorated with the addition of exogenous PGE2, but not EGF. These results suggest that airway constriction may retard epithelial migration and wound healing in the airway, thus promoting a pro-fibrotic state during chronic asthma.We found that mechanical compression and scrape injury increase TGF-β2 and endothelin-1 (ET-1) secretion in culture, while EGF content in the media is attenuated with both injury modes. The application of compression after a pre-existing scrape wound had a synergistic effect on these observations, and also stimulated a significant depolymerization of the f-actin network. Interestingly, PGE2 on uncoated Costar Transwells® inserts with 0.4 μm pore size  as previously described [® kits (Lonza) and retinoic acid (50 nM). The experiments were replicated in two separate donors (lot numbers 4F1430 and 4F1624) of NHBE cells acquired from Lonza and were qualitatively reproducible and similar. In the interest of brevity, the data from only one donor (4F1624) is included.Passage 3 normal human bronchial epithelial cells (NHBE)  were seeded at 150,000 cells/mmescribed . Followi2O compression for 6 hours (see description below). A six hour exposure to compression is similar to the range (1–8 hours) from previous studies [2 or EGF . Three additional groups of cells were exposed to mechanical compression with pretreatment with 900 pg/ml PGE2, 150 pg/ml additional EGF or both, and imaged at the respective time points.Following differentiation, cells were randomized into one of four groups (n = 6): 1) untreated control cells, 2) scrape wound only, 3) scrape and compression wounds, and 4) compression wound only. Media was changed 16 hours prior to the beginning of the experiment, and at time zero cells were exposed to their respective injury. A schematic of the experimental protocol is shown in Fig.  studies ,18, and 2 and 100% humidity.The method for applying mechanical compression to cells in culture is based on systems described elsewhere . BrieflyScrape wounds were imaged with a 4× objective using an Olympus 1X51 microscope  and a QImaging QICAM 12 bit camera with Qcapture Pro acquisition software. Each well was imaged along the entire length of the scrape wound. From each image, three representative widths were measured from the top, middle and lower third of the scrape and averaged using Image J software . Examples of measurements are shown in Fig. At assigned time points, cells slated for f-actin labeling were fixed in 4% formaldehyde for 20 minutes, and those slated for the labeling of microtubules were fixed in -20°C methanol for 2 minutes. Both groups were subsequently permeabilized with 0.5% Triton X-100 for 10 minutes, and nonspecific binding was blocked by incubation in 10% goat serum for one hour. Cells were rinsed with PBS and incubated in either 5 μg of Alexafluor 568 phalloidin  in 200 μl PBS or monocolonal anti-a-tubulin  1:250 dilution in PBS for two hours followed by an overnight incubation in Alexafluor 488 anti-mouse secondary antibody (Invitrogen) diluted in PBS 1:500. Following staining, cells were rinsed with 0.1% Triton X-100 four times and the Transwell membranes were carefully removed with a scalpel and mounted on a microscope slide. Cells were then imaged with a 20× or 40× objective.All data is presented as mean ± standard error. Each image was treated as a separate sample and all data were analyzed with SigmaStat software package  by one way ANOVA, bonferroni test. Differences between groups were considered statistically significant for p < 0.05.Two examples of the progression of wound healing for cells that underwent a scrape wound (left) and those that experienced both scrape and 6 hours of mechanical compression (right) are shown in Fig. rd column showing a group of cells that underwent cytochalasin-D treatment to chemically depolymerize the f-actin network. By 24 hours partial recovery in the f-actin network is evident, and essentially complete by 48 hours at which time the wound is nearly closed. Surprisingly, the microtubule network demonstrated no significant reorganization as a result of mechanical compression across all time points,  and mechanical compression and scrape (right) on the cytoskeletal components, actin network (20×) and microtubule network (40×), can be seen in Fig. The population averages for wound closure are presented in Fig. 2 media content at 24 and 48 hours for the scrape only, compression only, and scrape and compression treated cells are shown in Fig. Active TGF-β2, ET-1, EGF and PGEThe results for media analysis for EGF Fig  appear t2 media content is shown in Fig. 2 content as a result of compression or scrape alone; however, in combination, there was a decrease in content at 24 hours that approached significance (p = 0.057) and a significant decline at 48 hours. It should be noted that at both time points these declines were also significant in comparison to scrape only wounds.PGE2 was responsible for the attenuation in wound healing in the presence of mechanical compression the experiment was repeated in the presence of exogenously added PGE2 (900 pg/ml) or EGF (150 pg/ml) to offset the effects of mechanical compression on mediator content in the media. The results are shown in Fig. 2 supplementation resulted in a significant improvement in the rate of wound healing at 24 and 48 hours in the compressed cells, and a slight, however insignificant, improvement in scraped cells not exposed to compression. Additionally, treatment with Indomethacin, a Cox-1 and -2 inhibitor that suppresses PGE2 synthesis, resulted in a significant reduction in the rate of wound healing at all time points following the scrape injury  mechanical compression results in significant depolymerization of the actin network in these cells. We further suggest that this degradation of the actin cytoskeleton also exerts a negative influence on cell motility and wound healing. Mechanical compression and scrape wound have a similar influence on inflammatory mediator release, and the simultaneous application of these injuries has an even more exaggerated effect. However, it is obvious that mechanical compression has a dramatic effect on actin structure not seen with scrape injury. This observation may influence cell behavior beyond wound healing not considered in this investigation.The primary objective of this study was to directly test in cell culture the effects of mechanical compression on the rate of epithelial wound healing following denudation. Both conditions are present simultaneously in the setting of diseases such as chronic asthma. Although utilization of a scrape wound or mechanical compression are utilized as means to simulate epithelial injury thought to arise in vivo during chronic asthma, a direct comparison of these injury models and the interdependence has yet to be presented. The principal findings of this study are three-fold: 1) mechanical compression significantly attenuates epithelial migration and wound healing in NHBE cells in culture; 2) the underlying mechanism is, in part, PGE2 have been shown to have a significant role in lung epithelial wound healing [EGF and PGE healing ,21, whil healing ,22. Inte2, in the airway epithelium [2 is a key factor in regulating wound closure in the airway epithelium [2 synthesis with inhibition of epithelial repair as a result of strain. While this group utilized strain rather than mechanical compression, there are parallels with our current study. We did not examine PGE2 synthesis specifically, but we did demonstrate that PGE2 media content is significantly decreased as a consequence of mechanical compression. In light of these previous reports, we believe that this observation is a result of reduced secretion of PGE2 into the extracellular space, as opposed to increased consumption. This is supported by our observation, that replenishing PGE2 in the media partially recovered the rate of wound healing.Tissue culture techniques have been utilized to characterize the effects of epithelial denudation on the inflammatory response and wound healing of the airway epithelium. Thompson et al.  co-cultuithelium , and theithelium , althougAn interesting observation is that EGF media content decreased as a result of a scrape wound, mechanical compression, and even more so when both injury types were applied. Puddicombe and colleagues  have demIn order to achieve a properly differentiated phenotype of airway cells reflective of what is observed in vivo , the NHBE cells must be grown at an air liquid interface with specialized media. One necessary growth factor to obtain proper cellular characteristics is EGF, present in properly formulated media at approximately 550 pg/ml. It has been widely demonstrated that EGF expression is upregulated as a result of both mechanical compression  as well Cytoskeletal remodeling is a vital process in the progression of wound healing. Accordingly, we examined the effects of mechanical compression on the two major cytoskeletal structural protein networks, and more specifically, the relation of these structures to wound healing. We observed a nearly complete depolymerization of the f-actin network following 6 hours of mechanical compression which should significantly reduce cell motility and wound healing. We utilized an additional Transwell of differentiated NHBE cells and applied a scrape wound in the presence of 10 μg/ml of cytochaliasin-D. Not surprisingly, wound healing was nearly completely retarded in this case (data not shown).The cells that were injured with only a scrape wound appeared to heal in a manner well documented in other studies of wound healing in epithelial cells. In these cases a pronounced band of actin is present at the wound edge. This purse string formation was documented by Lotz et al.  and is ain-vivo wound healing and other tissue culture models of wound healing that was not directly examined in this study is cell proliferation. Fully differentiated confluent NHBE do not proliferate significantly in culture once confluent, and wound closure following a scrape injury has been shown to be independent of cell proliferation [Another key process present during feration . Accordi2 regulation and maintenance of the actin network is a distinct possibility; however the limited evidence in the literature suggests that actin depolymerization results in an upregulation of PGE2 synthesis [2 regulation, if any, certainly merits further investigation, and it is quite likely there are other factors involved not specifically addressed in this investigation.Here we present the novel fining that mechanical compression results in significant f-actin depolymerization however the mechanism is not abundantly clear. One possibility is that mechanotransduction of these forces results in an intercellular signaling cascade that ultimately leads to cell directed depolymerization of the network. If this is the case, the exact nature of this cascade is beyond the scope of this study; however the biological advantage to this type of response is not obvious. A more likely option is that the physical force imposed by compression physically altered the structure of the network or interfered with the dynamic nature of the actin networks preventing f-actin polymerization from g-actin monomers. A link between PGEynthesis . This wo2-mediated and it is highly likely that mechanical compression induced f-actin depolymerization also interferes with wound healing in this model. Our findings suggest that airway constriction further aggravates the pre-existing, pro-inflammatory state of the denuded airway epithelium. The accompanied increase in inflammatory mediator release in conjunction with a compromised barrier function of the airway epithelium likely serves to accelerate the progression of airway remodeling and the deleterious consequences on lung function during chronic asthma.In conclusion, we have demonstrated that mechanical compression attenuates epithelial wound healing under conditions thought to arise as a result of airway constriction during chronic asthma. These observations are, in part, PGEThe authors declare that they have no competing interests.SA designed, planned, and performed all of the experiments, and wrote significant sections of the manuscript; NKM assisted in SAEC culture and data interpretation; SCG provided overall guidance for the study, assisted in the experimental design, analysis and interpretation of the data, and writing of the manuscript. All authors have read and approved the final manuscript.


Ultrasound speckle tracking from grey scale images allows the assessment of regional strain derived from 2D regardless of angle intonation, and it is highly reproducible. The study aimed to evaluate regional left ventricular functional reserve in elite soccer players.50 subjects (25 elite athletes and 25 sedentary controls), aged 26 ± 3.5, were submitted to an echo exam, at rest and after the Hand Grip (HG) test. Both standard echo parameters and strain were evaluated.Ejection fraction was similar in athletes and controls both at rest (athletes 58 ± 2 vs controls 57 ± 4 p ns) and after HG (athletes 60 ± 2 vs controls 58 ± 3 p ns). Basal  segments showed similar strain values in athletes and controls both at rest  and after HG . Medium-apical segments showed similar strain values at rest , but higher values in athletes after HG In athletes with physiological myocardial hypertrophy, a brief isometric effort produces enhancement of the strain in medium-apical left ventricular segments, suggesting the presence of a higher regional function reserve which can be elicited with an inotropic challenge and suitable methods of radial function quantification such as 2D-derived strain. Regional myocardial function is currently evaluated by with 2D echocardiography in the four-chamber view at rest In athletes, where ischemia is an uncommon event, the main information on myocardial performance is related to global contractility, normally expressed by left ventricular Ejection Fraction (EF),4.Traditionally, a 2D echocardiographic exam is used in the four-chamber view for analysing the motion of the segments in the LV near the apex ; howeverThe segmental contributions of myocardial systolic function can be evaluated using non-invasive quantification of myocardial strain . Strain For reasons of homogeneity, all the subjects studied were men. Twenty-five elite soccer players  aged 26 ± 3.5, regularly trained for two hours at approximately 80% of heart rate five days a week for 10 months a year for 5 years or more, and played one game a week during the regular season. All were matched for age, weight and body surface with 25 controls who perform occasional sporting activity  stress. The subject held the dynamometer in one hand, squeezing the handle at 30% of their previously-established maximal grip strength for 3 minutes. The choice of employing a short, acute effort such as HG was made in order to create pressure load enhancement in the left ventricle chamber and thus in the deformation of the myocardial wall.At rest and at the end of the HG test, heart rate (HR), systolic and diastolic blood pressure  and standard systolic and diastolic echocardiographic parameters were measured. HG was considered efficient if the mean systolic and diastolic pressure values increased significantly during handgrip as compared to the resting state (p < 0.05).2), [Echographic exams were performed at rest and during HG with MyLab 50  equipped with a 2.5 MHz transducer. In accordance with guidelines , the tra2),  and EjecThis software employs the speckle tracking method to automatically evaluate myocardial dynamic properties from two-dimensional B-mode echocardiographic clips ,8,10 Fi.The software, based on a grey scale and without any angle dependence, offers the possibility of detecting strain in the basal and medium-apical segments of the left ventricle myocardial walls. Frame rate was kept between 50 and 80 Hz to optimise the application of speckle tracking for calculating Longitudinal Peak Systolic Strain (LPSS %), and LV (cm/sec) in left ventricle basal and medium-apical segments. We considered the LPSS and the LV values as the means of the values obtained at the basal segments of the interventricular septum and lateral wall and medium-apical sections of the same regions Fig , 2.Data is presented as means ± standard deviation. The comparisons between athletes and controls at rest and at peak stress were performed with Student's two-tailed unpaired test (p < 0.05), while the comparison between basal (resting) state and Hand Grip for each group was performed with Student's paired "t" test. Statistical and power analysis were performed using Statview and STATA Stata – Corp 2003 software.2 athletes vs 92.100 g/m2 controls with p < 0.05 Table Basal -20.99, CMI 101.54, R = 0.09, LPSS Medium-Apical -24.47, CMI 101.54, R = 0,13).All the subjects were healthy, with no statistically significant differences among the general parameters Table . In bothInterest has recently grown ,13 in evIn athletes, where a major curvature of the walls is present ,4 as a cThis investigation, aimed to evaluate, using LPSS, the contribution of the regional contractility of left ventricle medium-apical segments to overall heart performance in the athlete with physiological myocardial hypertrophy.The results demonstrate the applicability of the new speckle tracking method to the calculation of strain in all sections of the heart, also in the medium -apical segments, with results which come within the normal and validated range  A particThese results point to the importance of considering myocardial deformation parameters in LV segments adjacent to the apex, particularly in athletes where regular training produces increased myocardial contractility. We must remember that the actual performance of the apical segments is not correctly assessable with standard 2D; also the current application of the method of strain estimation by TDI is normally limited to basal and medial wall regions due to its angle dependence.In this study, the measurements of myocardial fibre velocity values in the same segments where strain was determined were made with the speckle tracking method, thus overcoming the limitations of aligning the beam with the scan line.Our results show a physiological gradient with a decrease in values from basal to apical segments in the two groups, confirming the literature dataIn conclusion, the significant increase in LV longitudinal strain in athletes suggests the importance of evaluating deformation parameters in order to more thoroughly assess those morphological and functional modifications in the heart which cannot be discovered by traditional echo measurements.In agreement with the normal values recently reported in the literature , these rIn athletes, this evaluation might be extended in future to the investigation of the strain of the two ventricles contracting in real time, in order to determine a the different behaviour of value of these two chambers that normally work at different load pressures.This investigation was performed in athletes with high-quality myocardial echo-images and no further information is available about the possibility of obtaining the same results with high reproducibility in other conditions where image resolution is poor. The endocardial border needs to be optimal, with a clear delineation of the myocardial tissue and extracardiac structures.We must also remember that myocardial contractility is a complex mechanism, recently addressed with new sophisticated software evaluating all components of wall deformation, like radial strain and torsion. In this study the evaluation of the deformation of segments around the myocardial apex, which is normally difficult from the standard 2D echocardiographic four-chamber view can howeAlthough the contribution of the contractility of left ventricle medium apical segments was found to be higher than in the basal ones in soccer players submitted to an HG test, no more information about the impact of a different kind of effort, different sport or different training level is yet available. For this reason, this study must be considered as a partial investigation, which however signals the usefulness of further studies to complete performance evaluation in the athlete's heartThe author(s) declare that they have no competing interests.The initial concept of the study was of GG and LS, who designed the study. LS, GP, LT performed all the measurements and statistical analyses. LS wrote the manuscript and all the authors contributed to, read, and approved the final version.


The factors affecting long-term survival following oesophagectomy for oesophageal cancer are poorly understood. We examined the significance of microscopic tumour involvement at the circumferential resection margin (CRM) on postoperative survival following oesophagectomy. The case notes of 329 patients who underwent a potentially curative oesophagectomy for squamous or adenocarcinoma were reviewed retrospectively. As part of the procedure, all patients underwent an en-bloc resection of their perioesophageal tissue. The presence of tumour either at, or within, 1 mm of the CRM was recorded and correlated with their TNM and survival data. A total of 67 patients (20%) were noted to have a positive CRM, of which 40 cases (12%) had tumour at the resection margin and the remainder had tumour within 1 mm of the margin. Univariate analysis showed no statistically significant association between survival and either category of CRM involvement. Multivariate analysis showed that only T-stage, nodal status and tumour grade were prognostic markers. In conclusion, the presence of microscopic tumour at the CRM following an en-bloc oesophagectomy is not a significant prognostic marker. Despite advances in the management of oesophageal cancer, survival following oesophagectomy remains poor with a 5-year survival of approximately 25% . A numbeThe case notes of 431 patients who underwent an oesophagectomy for squamous or adenocarcinoma between January 1987 and July 1996 at Nottingham City Hospital were reviewed retrospectively. We then excluded all cases of surgical mortality (defined as death occurring within 30 days of operation), incomplete excision , primary gastric carcinomas, synchronous tumours and tumours with distant metastases. In addition, we excluded all cases of T4 tumours. These were defined as cases where macroscopic invasion of adjacent structures was noted at operation, as well as all cases where there was microscopic invasion of structures that were resected along with the oesophagus . Following these exclusions, the results of the remaining 329 potentially curative oesophagectomies were analysed in detail. The case notes and operation notes of these patients were reviewed and their survival data were recorded. Patients were followed up for life following their operation, with outpatient clinic review every 3 months for the first year, every 6 months for the next 4 years and yearly thereafter. There was a 100% follow-up rate with a minimum follow-up period among the survivors of 5 years.All operations were performed by the same team of three thoracic surgeons, who used similar surgical techniques and uniform preoperative management. None of these patients underwent any pre- or postoperative chemotherapy or radiotherapy. All patients underwent a two-field en-bloc oesophagectomy. This entailed local mediastinal dissection with mobilisation of the intrathoracic portion of the oesophagus using diathermy, followed by excision of all perioesophageal tissue with the subcarinal and parahiatal lymph nodes; both parietal pleura overlying the oesophagus and the aortic adventitia. Tumours at the level of the diaphragm were excised together with a cuff of diaphragm. In the abdomen, the lymph nodes from the left gastric artery pedicle were routinely excised, and flush ligation of the left gastric pedicle achieved by application of a vascular stapler.Pathological analysis was performed by the same team of three histopathologists. On receipt in the laboratory, the surgical specimens were opened avoiding, where possible, the tumour bearing portion of the oesophagus. The specimens were then pinned under gentle tension to a cork board with the serosal surface of the specimen facing upwards. This surface was marked with Indian ink and the specimens were fixed in formalin. The tumours were serially sectioned in the transverse plane and the section with maximal lateral spread of tumour was identified. This section was then embedded and multiple blocks were examined to measure the shortest distance from the outermost part of the tumour to the nearest inked margin. Cases where this distance was less than 1 mm were deemed to have an involved CRM (positive). In addition, a further distinction was made between those cases where the tumour was within 1 mm of the CRM and those where the tumour was present at the margin itself. It should be noted that any case where tumour at the circumferential margin was also within 1 mm of the proximal or distal excision margin was deemed to be a noncurative oesophagectomy and excluded as previously stated. Further analysis was then carried out to determine the histological subtype, T-stage and nodal status of the tumours using the UICC TNM classification . The tumχ-test to establish any relation between these factors and 5-year survival. Factors that achieved statistical significance (P<0.05) on univariate analysis were entered into a multivariate analysis using Cox proportional hazards model to identify independent predictors of survival. Having identified these factors, the effect of CRM involvement on 5-year survival within individual prognostic subgroups was reassessed using a χ-test.Statistical analysis was performed using commercially available software packages. Survival curves for patients with and without CRM involvement were analysed by the Kaplan–Meier method, with differences in survival rates assessed using the log-rank test. The patients' sex, histological tumour type, T-stage, N-stage, tumour grade and CRM involvement were then categorised and a univariate analysis was performed using a P=0.19). The proportion surviving 5 years in the CRM-positive group was 22%  and in the CRM-negative group 29% (23–35%). The difference was not significant (P=0.25).Of the 329 patients, 218 were male and 111 female with a mean age 65 years (range 28–84). The histological characteristics of the specimens are shown in P=0.57).Of the 67 CRM-positive cases, 40 were noted to have tumour at the CRM, and 27 had the presence of tumour within 1 mm of the CRM, but not at the CRM. Where the tumour was at the CRM, the 5-year survival was 20% (8–32%) and within 1 mm it was 26% (9–43%). The difference was not significant , nodal involvement  and tumour grade  were all independent predictors of survival.Multivariate analysis revealed that T-stage .No cases of CRM involvement were seen in any patients with T1 or T2 tumours. In view of this, a comparison was made between the 5-year survival of T3 cases with and without CRM involvement. The survival rate for CRM-positive cases was 22% (12–32%) and without CRM involvement it was 24% (18–30%). There was no significant difference between the two groups (P=0.84) or N1 (P=0.52) disease.Having established that CRM-positive cases occur only in T3 tumours, an analysis of the survival of T3N0 and T3N1 tumours with and without CRM involvement was performed. The survival data are shown in P=0.72) or the poorly differentiated group (P=0.58).The potential prognostic effect of CRM involvement was then reassessed by comparing the survival of T3 well/moderately and T3 poorly differentiated tumours with and without CRM involvement. The survival data of these four groups are shown in et al showed that CRM involvement was associated with decreased median survival, while Dexter et al showed that the presence of tumour in the circumferential margin was not only an adverse prognostic factor, but also an independent predictor of survival. The results of our study, however, appear to contradict these findings. Analysis of the survival curves of our CRM-positive and CRM-negative patients showed no statistically significant difference in the short- or long-term outcome. Moreover, this comparison does not even take into account the fact that all our cases of CRM involvement occurred in patients with T3 tumours, a factor which our results show to be an independent predictor of poor prognosis. When this bias is eliminated by analysing the effect of CRM involvement in T3 tumours alone, the situation is even more clear-cut. Within the T3 subgroup, the 5-year survival rates of patients with and without CRM involvement are very similar . Moreover, we failed to find any statistically significant difference in survival between those patients with tumour at the CRM and those with tumour within 1 mm of the CRM. In addition, we found that CRM involvement did not confer a survival detriment in any prognostic subgroup, unlike Dexter et al who found CRM involvement was of particular prognostic significance in patients with N0 disease.Although the presence of tumour at the CRM has long been suggested as a potential predictor of survival following oesophagectomy , this difference would not, in itself, account for our contrasting results. A partial explanation may lie in our surgical technique of en-bloc oesophagectomy. Although Dexter et al do not explicitly state what surgical technique they utilised when performing oesophagectomies, the fact that they reported cases of tumour at the CRM in T2 tumours (where tumour is confined to the muscularis layer) does suggest that they did not perform extensive mediastinal dissection. In addition, their comparatively high rate of CRM-positive cases (64% of their T3 tumours had a positive CRM in comparison to our rate of 25%) further suggests that our patients underwent a more complete resection of their perioesophageal tissue. If this is indeed true, then this raises some interesting questions about the whole question of en-bloc resections. Previous studies (et al hypothesised that this survival benefit may be a result of improved tumour clearance at the CRM. Although our data would support the notion that en-bloc oesophagectomy reduces the incidence of CRM involvement, we have not shown this reduction to be of any prognostic significance. Interestingly, in the case of rectal tumours, there is some evidence that the prognostic effect of CRM involvement is lessened following radical resections (The reason for our differing results is not clear. Although our study differed from that of Dexter It should be noted that the whole concept of the superiority of en-bloc oesophagectomy over more limited resections is by no means certain, and has been an issue of some controversy (et al who found that CRM and nodal status were the only significant prognostic determinants. Indeed, the dichotomy between our results and those of Dexter et al reflects the somewhat confused nature of the literature on the topic of prognostic markers in oesophageal cancer. Although a large number of studies have been conducted on this subject, there is conflicting evidence as to the prognostic importance of many factors such as T-stage (Within our cohort of patients, T-stage, nodal involvement and tumour grade were all independent prognostic factors. This once again contrasts with the results of Dexter These disparities highlight the importance of utilising standardised preoperative, operative and postoperative techniques on a large population in order to assess prognostic factors – criteria that our study, unlike most previous studies, does satisfy.In summary, we have shown that following a potentially curative oesophagectomy, the presence of microscopic tumour either at or within 1 mm of the CRM is not a significant prognostic variable. Given the prognostic irrelevance of CRM status, we would question whether this factor should be considered as an essential part of pathology reporting for oesophageal cancer.


COMT gene may therefore contribute to differences in pain sensitivity and response to analgesics. It is shown that a polymorphism in the COMT gene, Rs4680 , influence pain sensitivity in human experimental pain and the efficacy for morphine in cancer pain treatment. In this study we wanted to investigate if variability in other regions in the COMT gene also contributes to interindividual variability in morphine efficacy.Genetic variation contributes to differences in pain sensitivity and response to different analgesics. Catecholamines are involved in the modulation of pain and are partly metabolized by the catechol-O-methyltransferase (COMT) enzyme. Genetic variability in the COMT gene, and constructed haplotypes from these 11 SNPs, which were in Hardy-Weinberg equilibrium. We compared both genotypes and haplotypes against pharmacological, demographical and patient symptoms measurements in a Caucasian cancer patient cohort (n = 197) receiving oral morphine treatment for cancer pain. There were two frequent haplotypes (34.5% and 17.8%) in our cohort. Multivariate analyses showed that patients carrying the most frequent haplotype (34.5%) needed lower morphine doses than patients not carrying the haplotype, with a reduction factor of 0.71 (p = 0.005). On the allele level, carriers of alleles for six of the SNPs show weak associations in respect to morphine dose and the alleles associated with the lowest morphine doses constitute part of the most frequent haplotype.We genotyped 11 single nucleotide polymorphisms (SNPs) throughout the COMT gene influence the efficacy of morphine in cancer patients with pain, and that increased understanding of this variability is reached by expanding from analyses of single SNPs to haplotype construction and analyses.This study suggests that genetic variability in the  This polymorphism causes a substitution from a valine  to a methionine (Met) at amino acid position 158, leading to a three- to four-fold reduced activity of the COMT enzyme [et al., demonstrated that individuals with the Met/Met genotype had higher sensory and affective ratings of pain and a higher regional density of mu opioid receptors in the brain [One of the genes in which variability is believed to contribute to differences in pain sensitivity and response to analgesics is the MT) gene -3. The CT enzyme , severalhe brain . Resultshe brain , the incCOMT gene and shown that other regions of the gene may also contribute to pain perception [et al., identified three genetic variants (haplotypes) in the COMT gene and designated them as low pain sensitivity (LPS), average pain sensitivity (APS) and high pain sensitivity (HPS) haplotypes. The Rs4680  polymorphism was one of four SNPs included in their haplotype analyses. The authors argue that the Rs4680  SNP cannot account for the observed variations in pain perception alone, since both the LPS and HPS haplotypes possess the G allele that codes for the more stable Val variant of the COMT enzyme [et al., analysed 13 SNPs in the COMT gene and their association to acute post-surgical pain in humans [et al., found that a SNP in intron 1 (Rs740603) and a haplotype, defined by SNPs in the promoter region and intron 1, were significantly associated with drowsiness and confusion or hallucinations in a cancer patient cohort treated with morphine. In the study by Ross et al., the Rs4680  SNP did not influence the risk for morphine induced adverse effect [Other researchers have investigated other SNPs across the rception ,8 and inrception . DiatcheT enzyme . Kim et e effect .COMT gene have either pain perception or the risk for opioid adverse effects as the primary endpoint in the study [COMT gene are important for the analgesic efficacy of morphine. Therefore, in a patient cohort in which we have previously shown that the Rs4680  polymorphism influences the efficacy of morphine for cancer pain [COMT gene also contribute to interindividual variability in morphine efficacy. In addition to examining the effect from each individual SNP we constructed long haplotypes in order to study composite effect from combinations of 11 SNPs along the gene.All the three studies cited above that have investigated multiple SNPs in the he study ,8,9. No cer pain , we inveDNA from 197 patients receiving oral morphine treatment for cancer pain was analysed in this study.COMT gene is shown in Figure COMT gene are shown in Table A schematic presentation of the 11 SNPs analysed in the The pharmacological observations for genotype groups and allele carriage are shown in Table We observed that carriers of haplotype 1, the most frequent haplotype in this Caucasian population Table , needed Patient symptoms including average pain, fatigue, nausea and vomiting, dyspnea, sleep, appetite, constipation and cognitive function were similar for carriers and non-carriers of haplotype 1 Table . Also thIn a multivariate stepwise linear regression analysis the variables "time since morphine treatment started" and haplotype 1 were shown to influence the morphine dose . The carriers of haplotype 1 are also carriers of the A allele for the Rs4680  polymorphism, which is in agreement with our earlier observation that carriers of the Met variant of the enzyme  need lower morphine doses than carriers of the Val variant of the COMT enzyme [We have identified a frequent haplotype (haplotype 1) in the T enzyme . HoweverCOMT gene because the valine  to methionine (Met) substitution leads to a three-to four-fold reduced activity of the COMT enzyme [COMT gene on the effect of opioids in pain treatment.The Rs4680  polymorphism is the most studied SNP in the T enzyme . Pain seT enzyme . Taken tWe also observed that carriers of alleles for six of the SNPs analysed, the Rs5746849 and Rs740603 polymorphism in intron 1, the Rs6269 polymorphism in intron 2, the Rs2239393 polymorphism in intron 3 and the Rs4818 and Rs4680  polymorphisms in exon 4 were weakly associated to morphine dose  and as a consequence limiting the gene distance to which SNPs categorize into haplotypes. According to literature the COMT gene consists of at least three haploblocks in Caucasians [In the paper we have constructed long haplotypes across the entire ucasians ,14 and t10) of the 24 hour morphine dose as the dependent variable. Therefore, for each month of morphine treatment, the predicted 24 hour morphine dose increases by a factor of 10(b × months) which translates to that the dose on average increases by 43% every 12 months (100.013 × 12). Patient carrying haplotype 1 is predicted to need less morphine to relieve pain than a patient not carrying haplotype 1, with a reduction factor of 10(b) = 10(-0.147) = 0.713. In other words, if a patient, not carrying haplotype 1 need 100 mg of morphine to relieve pain, a patient carrying haplotype 1 is predicted to need 71 mg of morphine to relieve similar pain. The difference we observe in the median morphine dose between non-carriers and carriers of haplotype 1 is of similar order of magnitude, 100 mg versus 60 mg of the 24 hour morphine dose respectively  polymorphism and central side effects of morphine [et al., and haplotype 1 in our study are related as 7 of 10 possible SNP positions from the Rs5746849 polymorphism in intron 1 to the UTR' region carry the same allele and both haplotypes carry the A allele at the Rs4680  polymorphism. An explanation for the discrepancy of the haplotypes might be that efficacy for pain relief and risks of adverse effects have different relationships to genotypes.Ross cination . SNPs inmorphine . HaplotyThe need for morphine is a result of both the efficacy of morphine and influenced by the patients' pain perception. Patients can experience variable pain from a given nociceptive stimuli. Therefore genetic variability related to opioid efficacy as studied in the present study is closely linked to genetic variability related to pain perception.et al., have investigated COMT gene variability and association to pain responses [COMT gene strongly associated to pain sensitivity and they designated the different haplotypes as low pain sensitivity (LPS), average pain sensitivity (APS) and high pain sensitivity (HPS) haplotypes. Four SNPs  constitute these haplotypes, of which three of the SNPs  are included in our analyses. However, Diatchenko et al., did not include the region in intron 1 or the promoter regions, the region which the study by Ross and co-authors [COMT gene. A direct comparison with our study is difficult because we have included 11 SNPs in our haplotype analyses while Diatchenko et al., focused on four SNPs. Kim et al., have also investigated COMT gene variability and association to pain responses and found that the Rs740603 SNP was associated with maximum post-operative ratings of pain. Even though the comparison between Diatchenko et al., [et al., [et al., [et al., [Diatchenko esponses . They idIn the present study the serum concentrations of morphine, morphine-6-glucuoride (M6G) and morphine-3-glucuronide (M3G) reflected the different morphine doses between haplotypes Table , but didCOMT gene and 22 of the most frequent SNPs have been analysed regarding different aspects of pain and opioid responses [COMT gene. However, as many SNPs are tightly linked within haploblocks, most genetic variability is captured if the selections of SNPs are chosen to represent the different haploblocks building the entire gene [Numerous SNPs have been detected in the esponses ,8,9, so ire gene , which iCOMT gene, a conservative multiple test correction lead to missing real differences [In the present study we have carried out several comparisons. Multiple test correction, as the Bonferroni, is used when tests are independent and is therefore highly conservative. In a genetic association study where SNPs usually are partly linked to each other, as is the case for the ferences ,17. AlsoCOMT gene influence the efficacy of morphine in cancer pain patients, and that increased understanding of this variability is reached by expanding from analyses of single SNPs to haplotype constructions and analyses.This study suggest that genetic variability in the The study was carried out in accordance to the principles of the Helsinki declaration. The Regional Committee for Medical Research Ethics, Health Region IV, Norway, approved the study. All patients gave their oral and written informed consent before inclusion in the study.et al., [We investigated the same cohort as previously described by Rakvag et al., . Two hunPain was measured using the item "average pain" during the last 24 hours in the Brief Pain Inventory (BPI) questionnaire. The patients rated pain on an 11-point numeric scale, where 0 represents "no pain" and 10 represents "pain as bad as you can imagine". The BPI is developed for the use in cancer pain patients, validated in Norwegian, and recommended by the European Association of Palliative Care for use in clinical studies -20. The Collection of blood samples and determination of serum concentration of morphine and its metabolites (morphine-6-glucuronide and morphine-3-glucuronide) were done as described in a previous work from our group . CreatinThe genotyping was performed at the Clinical Genomics Group, Imperial College in London, UK. The selection of SNPs for this study and primer sequences for sequence specific polymerase chain reaction (SSP-PCR) are described in a study by Ross and co-authors investigating another cohort and another primary outcome . The sel2 in 0.5% tris borate EDTA buffer . Products were visualised with a UV illuminator and photographed with a Polaroid camera. The presence of an allele specific band of the expected size, in conjunction with a control band was used to identify an allele.Genomic DNA was isolated from 50 to 200 μL EDTA blood on a MagNA Pure LC  using the MagNA Pure LC DNA Isolation Kit I applying the manufacturers high performance protocol. Purified genomic DNA was eluted in 100 μL antiseptic water and stored at -20°C. Genotypes were determined using sequence specific primers in a polymerase chain reaction (SSP-PCR) . A seque[Genotype and allele frequencies and allele carriage were calculated and checked for Hardy-Weinberg equilibrium. Haplotypes were constructed from genotype information from each patient using the computer program Phase ,28.COMT alleles are expected to be codominant with respect to the Rs4680  polymorphism and COMT enzyme activity, we used the Jonckheere-Terpstra test for comparison between genotype groups, working with a hypothesis that μ1 ≤ μ2 ≤ μ3 (or the opposite μ1 ≥ μ2 ≥ μ3) [10) of the 24 hour morphine dose as the dependent variable in the regression analyses because the 24 hour morphine dose, as expected, did not display a normal distribution. The analysis was a stepwise enter linear regression with a criterion for removal of a variable of p > 0.1. The variables included in the regression analysis as independent variables were: haplotype 1, age, gender, tumour diagnosis, Karnofsky performance status, creatinine and albumin serum concentration, time since diagnosis, time since morphine treatment started, survival time after study, BPI average pain score, EORTC score for fatigue, nausea and vomiting, dyspnea, sleep, appetite and constipation, and finally the sum score for the Mini mental examination measuring cognitive function.The statistical software SPSS for windows v. 14.0 was used to run non-parametric tests and to run a stepwise linear regression analysis. Because the μ2 ≥ μ3) . For allInterpretation of p values in this study is done with caution considering the multiplicity of tests carried out.The authors declare that they have no competing interests.PK, SK, FS and TTR conceived of the study and PK, FS and SK collected the experimental data. TTR, JRR and HS carried out the molecular genetics and statistical analysis. All authors drafted the manuscript and approved the final version.


Sir,Olopatadine hydrochloride is one of the second-generation nonsedating antihistamines that is used for treating allergic disorders such as urticaria, rhinitis, and atopic dermatitis. OlopatadOlopatadine hydrochloride was tested in patients with chronic idiopathic urticaria (CIU). CIU is defined by the almost daily presence of urticaria for at least six weeks without an identifiable cause. Aim of this study was to investigate the efficacy and side effects of olopatadine hydrochloride in CIU patients.Twenty CIU patients in the age range of 14–70 years  were included in the study . DuratioP<0.05). Two patients (10%) complained of headache and sedation after initiation of the therapy. Of eight patients (6 men and 2 women) with more than 36 weeks of urticaria, one patient did not respond to therapy and administration of steroid became necessary to control the condition. Olopatadine therapy was well tolerated without major side effects by most patients in this study.Average UAS was 3.7 at 0 weeks which went down to 1.95 at the end of two weeks. At the end of four weeks, UAS further lessened to 1.2. Difference in UAS at 0 week and at 4 weeks was statistically significant (Olopatadine, a newly developed histamine H1-receptor antagonist, was compared with cetirizine in its suppressive effects on histamine-induced wheal and flare reaction using an iontophoresis technique in a double-blind, cross-over, placebo-controlled study. As a result, olopatadine was found to have effects comparable to cetirizine. In anothOlopatadine is an effective treatment for CIU with sedation being a side effect in 10% of patients. Further studies in this direction with larger study groups are required to vindicate above findings.


Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are clinically distinct neurodevelopmental genetic disorders that map to 15q11-q13. The primary phenotypes are attributable to loss of expression of imprinted genes within this region which can arise by means of a number of mechanisms. The most sensitive single approach to diagnosing both PWS and AS is to study methylation patterns within 15q11-q13; however many techniques exist for this purpose. Given the diversity of techniques available, there is a need for consensus testing and reporting guidelines.Testing and reporting guidelines have been drawn up and agreed in accordance with the procedures of the UK Clinical Molecular Genetics Society and the European Molecular Genetics Quality Network.A practical set of molecular genetic testing and reporting guidelines has been developed for these two disorders. In addition, advice is given on appropriate reporting policies, including advice on test sensitivity and recurrence risks. In considering test sensitivity, the possibility of differential diagnoses is discussed.An agreed set of practice guidelines has been developed for the diagnostic molecular genetic testing of PWS and AS. Prader-Willi syndrome  is characterised by severe hypotonia and feeding difficulties in early infancy, followed in later infancy or early childhood by excessive eating and gradual development of morbid obesity (unless eating is controlled by dietary restriction or behaviour modification). Motor milestones and language development are delayed. All individuals have some degree of cognitive impairment, although some will have an IQ within the normal range. A distinctive behavioral phenotype  is common. Hypogonadism is present in both males and females, and manifests as genital hypoplasia, incomplete pubertal development, and in most, infertility. Short stature is common; characteristic facial features, strabismus, and scoliosis are often present, and non-insulin-dependent diabetes mellitus often occurs in obese individuals. Consensus diagnostic clinical criteria for PWS have been developed [Angelman syndrome  is characterised by severe developmental delay, absent or severely limited speech, gait ataxia and/or tremulousness of the limbs, and a unique behavior with a happy demeanor that includes frequent and sometimes inappropriate laughter, smiling, and excitability. In addition, microcephaly and seizures are common. Affected individuals usually have a characteristic electroencephalography (EEG) appearance with striking high voltage activity. Developmental delay is first noted at around six months of age; however, the unique clinical features of AS may not manifest until after one year of age, and it can take several years before the correct clinical diagnosis is obvious. The diagnosis of AS rests upon a combination of clinical features as well as molecular genetic testing and/or cytogenetic analysis. Consensus clinical diagnostic criteria for AS have been developed .de novo deletion of this region, maternal uniparental disomy (UPD) of chromosome 15 or the silencing of the paternal alleles by an imprinting defect on the paternal chromosome. So far, several genes preferentially or exclusively expressed from the paternal chromosome have been described: MKRN3, MAGEL2, NDN, PWRN1, C15orf2, SNURF-SNRPN and several C/D box small nucleolar RNA (snoRNA) genes , the two snoRNA genes SNORD116 and SNORD115 are present in 24 and 47 gene copies, respectively. It has been recently demonstrated that deficiency of SNORD116 (previously HBII-85) snoRNAs causes the key characteristics of the PWS phenotype [The proximal long arm of human chromosome 15 (15q11-q13) contains a cluster of imprinted genes, which are under the control of a bipartite imprinting centre [de novo maternally derived deletion of 15q11-q13, paternal UPD or an imprinting defect affecting the maternal chromosome. In addition point mutations in the E6-AP ubiquitin-protein ligase gene (UBE3A) are known to cause AS [UBE3A expression is restricted to brain cells where expression is exclusively from the maternal chromosome and disruption of expression of this gene is now considered to be the major cause of the disease. There is one further gene in 15q11-q13 that is preferentially expressed from the maternal chromosome in brain and fibroblasts; ATP10C [In AS the major disease mechanism is either a cause AS ,9, ImpriMicrodeletions in a small number of patients with PWS or AS have helped define an imprinting centre (IC), which has two critical regions, the AS-SRO (shortest region of deletion overlap) and the PWS-SRO . By analUBE3A sequence analysis detects mutations in approximately a further 10% of individuals with AS, however UBE3A analysis is not considered further in this article.There are a number of cytogenetic and molecular approaches to the confirmation of these two disorders. The most common is DNA-based methylation testing to detect abnormal parent-specific methylation within the PWS and AS critical region. This will detect more than 99% of individuals with PWS and approximately 80% of individuals with AS. th May, 2008 and 6th January, 2010 and heads of the constituent laboratories were invited to comment. In the light of feedback amendments were made and the final document was ratified by the CMGS Executive Committee on 15th January, 2010. In addition they were approved by the European Molecular Genetics Quality Network (EMQN) Steering Group on 22nd January, 2010.Current practice in the molecular analysis and reporting of PWS and AS was assessed by consideration of the external quality assessment returns submitted to the European Molecular Genetics Quality Network (EMQN) and the United Kingdom External Quality Assessment Scheme (UKNEQAS) over a five year period. The guidelines in this article were posted on the web-site of the UK Clinical Molecular Genetics Society (CMGS) for consultation and amendment between 19The approach to the laboratory diagnosis of AS and PWS depends on many factors, including the availability of samples, the arrangement of laboratory services and the patterns of referral. The most sensitive single approach to diagnosing PWS and AS is to study methylation patterns within 15q11-q13 using molecular genetic techniques. These will detect deletions, UPD and imprinting defects by establishing either a solely maternal methylated imprint (PWS) or paternal methylated imprint (AS). Broadly speaking, methylation studies take one of two forms:SNRPN locus by use of methylation specific PCR (MS-PCR) or Southern blot analysis. This approach will confirm a diagnosis but will provide no further information regarding the disease mechanism necessitating follow up studies .(i) The detection of methylation status solely at the (ii) The simultaneous assessment of methylation status and genomic dosage at numerous sites across the 15q11-q13 region, by the use of methylation sensitive multiplex ligation-dependent probe amplification (MS-MLPA). This approach will confirm the diagnosis and further identify the presence of a causative deletion. However, in the absence of a deletion follow up studies  are required to distinguish between UPD and an imprinting defect.It is essential to note that approach (i) will not distinguish between the two classes of imprinting defect (ie. between one with no detectable deletion in the IC region and one due to a microdeletion of the IC). MS-MLPA however will give some limited information in this regard (see below). It is reasonable to request a standard karyotype analysis on the proband at the same time as a molecular analysis in order to investigate possible alternative explanations for the clinical presentation (if the PWS/AS investigations turn out to be negative). Upon the confirmation of a diagnosis, cytogenetic analysis of parental samples is recommended to investigate the possibility of balanced rearrangements, and genetic counseling should be offered to the family if the referral is not from a specialist in clinical genetics. Figure SNRPN exon 1/promoter regions. Two approaches have been shown to work reliably in interlaboratory comparisons: Kubota et al [et al [SNRPN exon 1/promoter region is highly conserved but contains a few very rare single nucleotide polymorphisms (SNPs) within the primer binding sites . A rare restriction fragment length polymorphism affecting a NotI site in intron 1 can result in a 3.9 kb fragment for the unmethylated allele and can lead to false positive results in short run gels where the 4.05 and the 3.9 kb bands are not resolved , one is from the completely unmethylated promoter regions of UBE3A, and another one is from the completely methylated SNORD116 snoRNA gene cluster region. The kit contains two additional methylation sensitive control probes for completely unmethylated sequences from other chromosomes that will indicate complete digestion by the HhaI enzyme.MS-MLPA provides a means to simultaneously detect copy number changes and DNA methylation within 15q11-q13 in a semi-quantitative manner. Most laboratories using this approach make use of a commercially available MS-MLPA kit available from MRC Holland APBA2. Another advantage of the ME028-B1 MLPA kit is that small deletions affecting the UBE3A gene or the SNORD116 gene region can be detected.Dosage analysis by MS-MLPA offers the opportunity to detect large deletions, the most frequent molecular lesions in patients with PWS and AS. In rare cases, a larger deletion can extend telemetric and include the probe for MS-MLPA has become the method of choice in many diagnostic laboratories as it investigates methylation status at several loci, thereby reducing the risk of a false positive or false negative result due to SNPs; in addition, if one probe fails there are four remaining probes with which to assess methylation status.SNRPN exon 1/promoter/intron 1, a region which represents the smallest region of deletion overlap in patients with PWS (PWS-SRO) and an IC deletion. Furthermore, two probes for SNRPN exon 3 and 7 are also included, which are deleted in most cases with an IC deletion. There are two probes which can be used to detect IC deletions in patients with AS and an imprinting defect. Both lie in the smallest region of deletion overlap found in patients with AS and an imprinting defect (AS-SRO).MS-MLPA (ME028-B1) will also identify IC deletions in PWS and AS cases with an imprinting defect. The kit contains four probes for the There appears to be some naturally occurring variation in dosage and methylation status which must be taken into account when interpreting MS-MLPA results;NIPA1 and TUBGCP5, copy number variation has been observed in healthy individuals. This has been attributed to deletions and duplications encompassing these two probes, and this complicates the definition of class 1 and class II deletions. This copy number variation has been observed in healthy individuals and seems to be non-pathogenic however it cannot be excluded as contributing to the phenotype in some individuals. Variation in the hybridization efficiency due to a copy number variation is also observed for the SNRPN exon u1B probe.(i) For the two most centromeric probes, (ii) SNPs under hybridisation binding sites can influence probe signals. Consequently laboratories must exercise extreme caution when interpreting results from a single probe.NDN locus is frequently observed.(iii) Normal variation in the degree of methylation at the NDN locus and also for the SNRD116 probe (12273-L13798), which in blood DNA is completely methylated . These results suggest that the methylation of the NDN locus is not fully established in chorionic tissue. It is therefore recommended that only the methylation status of the SNRPN locus is considered in the context of prenatal diagnosis.For prenatal diagnosis it should be noted that chorionic villi samples show a significant hypomethylation at the It should be noted that the current commercially available MS-MLPA kits are not certified for diagnostic use and must be fully validated in individual laboratories prior to implementation. We recommend that recurrent variation observed in the MRC MS-MLPA kit is reported to the manufacturer to facilitate future kit development.When a diagnosis of AS or PWS is confirmed with any of the above techniques microsatellite analysis will often be required to distinguish between the various disease mechanisms. There are many microsatellites suitable for this purpose -27, and D15S113 (LS6-1/2). This is a CA repeat from within the critical region. The presence of null alleles  have been observed with this marker and can complicate the analysis of AS and PWS cases. Under certain conditions, a non-amplification allele can be misinterpreted as a small deletion. The frequency of these alleles in families without AS has been estimated to be around 4%. Alternative primers can be designed; however, this marker is best avoided.• D15S817. The presence of three alleles has been observed with this marker with certain primer sets due to complex duplications in the region where the marker is located. Since there is a low frequency/density of useful markers for the more centromeric PWS/AS region, the use of the following primer pair  can resolve this problem.• Once the diagnosis of PWS or AS has been confirmed using methylation analysis, the interpretation of the microsatellite results is as follows:In this case the disease is due to a deletion of the critical region. In rare cases, microsatellites may be used to confirm a smaller deletion within the critical region, however, laboratories must not interpret results from a single informative microsatellite without supporting evidence.In this case, the disease is due to uniparental disomy. It is important to note that AS and PWS can be caused by either chromosomal isodisomy or heterodisomy. Further, heterodisomy or isodisomy at a single locus does not necessarily reflect the disomy status along the entire chromosome depending on the rate and level of crossing over and the meiotic stage at which non-segregation occurred.In this case the disease is presumed to be due to an imprinting defect.UBE3A analysis as mutations in this gene will have a recurrence risk of up to 50%, depending on the carrier status of the mother.A normal methylation pattern rules out PWS on the basis of most known cases to date and around 70-75% of AS cases. Despite early reports of possible deletion mosaicism in PWS ,29 the cde novo interstitial deletion of chromosome 15, del(15)(q11-q13), which includes the entire imprinted domain plus several non-imprinted genes extends approximately 6 Mb and is found in the majority of patients with PWS and AS. In both syndromes, the same region is affected, but in PWS the deletion is always on the paternal chromosome, whereas in AS it is always on the maternal chromosome. The deletion occurs at a frequency of about 1/15,000 newborns and is probably one of the most common pathogenic deletions observed in humans. In a few patients, the region is deleted as the result of an unbalanced translocation. At the molecular level, two classes of deletions (class I and II) can be distinguished. In both classes, the distal breakpoints are close to, but telomeric to the P gene  cytogenetic rearrangements in the appropriate parent (father for PWS and mother for AS) that may predispose to a deletion.The UBE3A is silent on the paternal chromosome, so upd(15)pat is associated with a complete loss of function of this gene in this tissue. Uniparental disomy arises in most cases of PWS as a result of a combination of meiotic and mitotic errors in female meiosis. During meoisis, the diploid set of chromosomes (n = 46) is reduced to a haploid set (n = 23). Nondisjunction of the homologous chromosomes 15 during female meiosis I or nondisjunction of the two sister chromatids during female meiosis II results in an oocyte with two chromosomes 15 or no chromosome 15. In these cases, fertilisation by a sperm with one chromosome 15 will result in a zygote which is trisomic or monosomic for chromosome 15 respectively. These conditions are not compatible with normal development, but can be 'rescued' by loss of one chromosome 15 from a trisomic cell or duplication of the paternal chromosome 15 in a monosomic cell. With trisomy rescue, in two-thirds of cases, one of the two maternal chromosomes will be lost from the trisomic cell, resulting in a normal set of chromosomes. If, however, the paternal chromosome is lost, the cell is left with two maternal chromosomes 15 (upd(15)mat). Duplication of the paternal chromosome 15 in a monosomic cell will lead to upd(15)pat.The second most frequent finding in PWS is maternal UPD (upd(15)mat) of chromosome 15. These patients have two maternal copies of chromosome 15 and lack a paternal copy. As the PWS genes are silent on the maternal chromosome, upd(15)mat is associated with a complete loss of function of these genes. The reciprocal finding is made in some patients with AS. These patients have two paternal copies of chromosome 15 and lack a maternal copy (upd(15)pat). In brain cells Alternative mechanisms for UPD such as complementation involving both a nullisomic and disomic gamete or rescued paternal 15 trisomy have also been reported, however are considered rare .There are a small number of patients with either PWS (1%) or AS (2-4%) that show biparental inheritance for chromosome 15 markers, both inside and outside the critical region, but have an abnormal methylation pattern, characteristic for the syndrome. These patients are presumed to have an imprinting defect.de novo or a consequence of germ line mosaicism in the father or the mother. In these families, the recurrence risk ranges from 0-50%, depending on the degree of the mosaicism in the germ line [The majority of patients with an imprinting defect are sporadic cases without any detectable mutations in the IC region at the DNA sequence level. However in 10-15% of cases, the imprinting defect is caused by a microdeletion of the imprinting centre (IC). In most cases, the IC deletion is a familial mutation associated with a 50% recurrence risk, however in some cases the IC deletion is erm line . An IC derm line .Few laboratories are equipped to fully analyse these rare cases; however, it is important they are referred on to a specialist laboratory in order to confirm the nature of the imprinting defect and better understand the recurrence risk.et al. 1997 Eur J Hum Genet, 5:94-99", or "ME028 MRC Holland http://www.mrc-holland.com)". It is essential that, where relevant, reports should inform as to the likelihood of recurrence, and, if the referral originates from a non-genetics specialist, genetic counselling is offered to all families where a diagnosis of AS or PWS is confirmed. In these cases cytogenetic analysis of the proband and father (for PWS) or mother (for AS) must be considered to rule out the remote possibility of cytogenetic rearrangements. More specific recommendations are as follows:It is recommended that laboratories do not use joint PWS/AS report templates. It is essential to state clearly on the report the method(s) used to carry out genetic analysis, with an appropriate reference; for example, "Zeschnigk Normal methylation result. This result excludes paternal deletion, uniparental disomy and an imprinting defect. The result makes a diagnosis of PWS highly unlikely. The sensitivity of the test should be stated on the report.(i) NORMAL RESULT. Absence of paternal allele at 15q11-q13 by methylation analysis. This result confirms the diagnosis of PWS. There is a common usage of terms such as "normal" and "abnormal" methylation pattern of the maternal or paternal allele/chromosome, however it is not appropriate to use these terms when the etiology is unknown. It is only appropriate to describe the imprinting pattern as "Abnormal" in patients with imprinting defect where it has been demonstrated that the paternal chromosome is aberrantly methylated. In deletion cases one allele is not present and therefore is not abnormally methylated. The same is true for maternal UPD, where the paternal allele is missing. With MS-PCR and Southern analysis it is best to describe what is observed; that is absence or presence of the relevant parental band and not simply to state that a methylation pattern typical of PWS is present. The laboratory should state that these approaches cannot determine the molecular cause of the result; and should request that parental blood samples be collected for microsatellite analysis in order to determine the mutational mechanism and recurrence risk.(ii) DIAGNOSIS CONFIRMED: Normal methylation and normal dosage at 15q11-q13. This result excludes paternal deletion, uniparental disomy and imprinting defect. A diagnosis of PWS is highly unlikely. The sensitivity of the test should be mentioned on the report.(i) NORMAL RESULT: Absence of paternal allele at 15q11-q13. This result confirms a diagnosis of PWS. The molecular cause of PWS is due to 15q11-q13 deletion.(ii) DIAGNOSIS CONFIRMED - DELETION IDENTIFIED: Absence of a paternal allele at 15q11-q13 by MLPA, normal dosage at 15q11-q13. This result confirms the diagnosis of PWS. The molecular cause of PWS may be due to maternal UPD or an imprinting defect. Laboratories should recommend microsatellite studies on the family to help confirm or exclude UPD.(iii) DIAGNOSIS CONFIRMED - NO DELETION IDENTIFIED: Absence of paternal allele for SNRPN exon 1/intron 1 probes and possibly SNRPN u1B probe. IC. deletion detected. This result confirms the diagnosis of PWS. IC deletions are associated with a recurrence risk of up to 50%. In case of an IC deletion the father should be investigated for the presence of the deletion since familial IC deletion may have consequences for other family members.(iv) DIAGNOSIS CONFIRMED - DELETION OF THE IMPRINTING CENTRE IDENTIFIED: Normal methylation. This result excludes maternal deletion, uniparental disomy and imprinting defects. The diagnosis of AS can not be confirmed in this patient. This result does not exclude a diagnosis of AS. See also the comments regarding reporting "abnormal mathylation patterns" in PWS (above). It may be appropriate to offer UBE3A analysis after a clinical re-assessment of the patient.(i) NORMAL RESULT: Absence of maternal allele at 15q11-q13 by methylation analysis. This result confirms the diagnosis of AS. This may be due to a de novo deletion, uniparental disomy or an imprinting defect. Collection of blood samples from the proband and parents is essential to determine the molecular cause and recurrence risk.(ii) DIAGNOSIS CONFIRMED: Normal methylation and, normal dosage at 15q11-q13. This result excludes maternal deletion, uniparental disomy and imprinting defect. This result does not exclude a diagnosis of AS. It may be appropriate to offer UBE3A analysis after a clinical re-assessment of the patient.(i) NORMAL RESULT: Absence of maternal allele at 15q11-q13 by methylation analysis. This result confirms a diagnosis of AS. The molecular cause of AS is due to 15q11-q13 deletion.(ii) DIAGNOSIS CONFIRMED - DELETION IDENTIFIED: Absence of maternal allele at 15q11-q13 by methylation analysis. This result confirms the diagnosis of AS. The molecular cause of AS may be due to paternal UPD or an imprinting defect. Recommend microsatellite analysis to confirm/exclude UPD.(iii) DIAGNOSIS CONFIRMED - NO DELETION IDENTIFIED: de novo deletions or disomy the recurrence risk is very low, however prenatal testing can be offered for reassurance. For cases of familial imprinting mutations due to an inherited deletion of the imprinting centre prenatal diagnosis should be offered as the recurrence risk is high (50%). Methylation status at SNRPN exon 1 is established early in embryonic development and testing DNA extracted from both amniotic cells and chorionic villi has been shown to give reliable results [in-situ hybridisation (FISH) and/or microsatellite analysis can be used where appropriate to support the methylation results.When the diagnosis has been confirmed and the molecular cause has been established in the index case, prenatal diagnosis can be performed. For cases of  results . Fluoresde novo or a consequence of germ line mosaicism in the parent, the recurrence risk is difficult to predict but may be as high as 50%. In cases of imprinting defects with no detectable mutation, the recurrence risk appears to be low, however since the possibility of recurrence cannot be excluded a prenatal diagnosis should be offered. For these latter two scenarios prenatal diagnosis is performed by methylation analysis only.For cases of imprinting mutations due to deletions of the imprinting centre that are either DLK1/GTL2 locus on 14q32 as well as upd(14)mat [Diagnoses which need to be considered in infants with hypotonia include congenital myopathies, the congenital form of myotonic dystrophy and type 1 spinal muscular atrophy. Peroxisomal disorders should also be ruled out if chromosome 15 methylation is normal . A furthd(14)mat ,39.When considering the differential diagnosis of older children with learning disability and obesity, Cohen syndrome, Borjesson-Forssman-Lehman syndrome , Bardet-Biedl syndrome and Alstrom syndrome along with chromosomal disorders including, diploid/triploid mosaicism and 1p36 microdeletion syndrome, should be considered .CDKL5 gene should be considered [ZFHX1B gene on chromosome 2, is associated with severe learning disability, limited speech, seizures and characteristic facial features that resemble those of AS. In addition Hirschsprung disease, congenital cardiac defects and agenesis of the corpus callosum may be associated with mutations in ZFHX1B. A strong indication is the characteristic appearance of the ear lobes which are upturned and look like "shell pasta". Pitt-Hopkins Syndrome (PHS) is a sporadic condition caused by mutations or deletions of the TCF4 gene on chromosome 18q; patients present with absent speech, seizures and facial features resembling AS, together with a sociable personality. The facial appearance in PHS coarsens with age and the lips in particular become prominent. Episodes of hyperventilation and apnoea may develop [et al. reported several patients with autosomal recessive PHS caused by deletions or point mutations in the NRXN1 and CTNAP2 genes. Breathing anomalies, epilepsy and autistic features were prominent features in these cases [SLC9A6 gene has been reported [MECP2 gene may also present with a phenotype suggestive of AS in males [MBD5 and a clinical and behavioural phenotype reminiscent of Angelman syndrome were reported [MTHFR) deficiency and adenylosuccinate lyase deficiency have been reported as presenting with learning disbility, ataxia, seizures, autistic features and excessive laughter [Around 10% of patients with a clinical diagnosis of Angelman syndrome have no demonstrable abnormality at 15q11-q13 using the techniques described here. In rare cases, these patients may be mosaic for an imprinting defect. However, it is more likely that there is an alternative clinical diagnosis and a careful review of the patient's history, clinical features and EEG findings is recommended. One diagnosis which should be considered in girls is Rett syndrome . It is extremely difficult to distinguish between Rett syndrome and AS during infancy when both can present with acquired microcephaly, ataxia and frequent smiling. Later, Rett syndrome may be distinguished by the presence of a history of developmental regression, the emergence of stereotypic hand-wringing movements, bouts of hyperventilation and the presence of vasomotor disturbance. If there is a very early onset of seizures, within the first few months, mutations within the nsidered . Mowat-W develop . Recentlse cases . An X-lireported . Specifiin males . Profounin males . Recentlreported . Seizurelaughter ,49. WithA practical set of molecular genetic testing guidelines has been developed for PWS and AS. In addition advice is given on appropriate reporting policies including advice on test sensitivity and recurrence risks. Feedback has been obtained from participants of the 2007 PWS/AS EMQN external quality assessment scheme (49 laboratories from 20 countries) and the heads of the constituent laboratories of the CMGS (46 laboratories from the UK). All comments received were minor; largely typographic corrections and some points of clarity. There was no disagreement on the recommendations made. All comments have been incorporated into this final document.All authors contributed equally to the content of this manuscript. SR drafted the manuscript and coordinated feedback in accordance with the procedures of the UK Clinical Molecular Genetics Society and the European Molecular Genetics Quality Network to ensure that consensus was agreed. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/11/70/prepubSNRPN exon 1/promoter region before (upper black line) and after (lower red line) bisulphite treatment . Single nucleotide polymorphisms (SNPs) in primer binding sites are boxed. In the bisulphite converted DNA sequence the X represents cytosines on methylated alleles. Primer binding sites for different PCR assays are shown as arrows. The binding site of two MLPA probes in the SNRPN exon 1/intron 1 region is given in blue. MF, forward primer for the maternal methylated allele; PF, forward primer for the paternal unmethylated allele; MR, reverse primer for the maternal methylated allele; PR, reverse primer for the unmethylated paternal allele.Sequence of the Click here for file


Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1) vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration (NeutNet) involving 18 independent participants was organized to compare different assays.Each laboratory evaluated four neutralizing reagents  at a given range of concentrations against a panel of 11 viruses representing a wide range of genetic subtypes and phenotypes. A total of 16 different assays were compared. The assays utilized either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) , or their Env-pseudotyped (gp160) derivatives produced in 293T cells (PSV assays) from molecular clones or uncloned virus. Target cells included PBMC and genetically-engineered cell lines in either a single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs that included extracellular or intracellular p24 antigen detection, RNA quantification and luciferase and beta-galactosidase reporter gene expression.PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus and inhibitor used. For example, for TriMab, the mean IC50 was always lower in PSV than in VI assays. However, with 4E10 or sCD4 some viruses were neutralized with a lower IC50 in VI assays than in the PSV assays. Inter-laboratory concordance was slightly better for PSV than for VI assays with some viruses, but for other viruses agreement between laboratories was limited and depended on both the virus and the neutralizing reagent.in vitro assay correlates with in vivo protection, a range of neutralization assays is recommended for vaccine evaluation.The NeutNet project demonstrated clear differences in assay sensitivity that were dependent on both the neutralizing reagent and the virus. No single assay was capable of detecting the entire spectrum of neutralizing activities. Since it is not known which  It is well established that neutralizing antibodies play a pivotal role in mediating protection against a range of virus infections including polio, measles, hepatitis and influenza Over the years a wide range of HIV-1 neutralization assays and variants thereof have been developed and described in the literature. It became apparent by the early 1990s that HIV-1 neutralization assays and reagents should be compared and evaluated and this was best done by international networks As described in a recent minireview www.europrise.org. The conventional PBMC based assay env sequences were amplified either from culture supernatants or from cloned plasmid.The methodologies used in this study are listed in Mabs and soluble sCD4 were distributed by the Programme EVA Centre for AIDS Reagents (CFAR) NIBSC, UK. Mabs 2F5, 2G12 and 4E10 were kindly provided by Dr. D Katinger, Polymun Scientific GmbH, Austria, b12 by Dr D Burton, The Scripps Research Institute, USA and 447-52D by Dr S Zolla-Pazner, New York University Medical Center, USA. The sCD4 was purchased from Progenics Pharmaceuticals Inc, USA. Three monoclonal antibody reagents were tested including TriMab, an equal mixture of the three Mabs IgG1b12, 2G12 and 2F5; Mabs 447-52D and 4E10. The Mabs and sCD4 were tested at starting concentrations of 25 µg/ml and 10 µg/ml, respectively, followed by five 2-fold dilutions. Mab IgG1b12 is directed to the CD4-binding domain of HIV-1 envelope gp120 Twelve HIV-1 isolates and/or their clonal derivatives were used see . The virAnalysis was based on the raw assay data returned by participating laboratories. Each laboratory was requested to perform twice the assays according to their standard protocol, with all dilutions tested at least in duplicates. The 50%, 75% and 90% inhibitory concentrations  were calculated with a linear interpolation method, using the mean of the duplicate responses.The linear interpolation method was implemented in an Excel spreadsheet, allowing consistent calculation of the IC50 values that was across laboratories. The assay readout equivalent to the IC50 was calculated as half the assay readout with no antibody present (similarly for IC75 ad IC90). The dilution interval containing the IC50 was identified, with assay readout for adjacent dilutions being above and below the 50% readout. The assay readouts for the dilutions above and below the IC50 were joined with a straight line, plotted against the log concentration of antibody. The position where the line crossed the 50% assay readout was taken as the estimate of IC50. Where the IC value was outside the range of concentrations tested, it was recorded as either greater than the highest concentration used, or less than the lowest concentration, as appropriate. Where the assay data were variable, and the observed dose-response crossed the relevant percentage inhibition level (e.g. 50% inhibition for IC50) more than once, no IC value was calculated. Absence of a calculated IC value may therefore be due to a laboratory not testing a particular combination of virus and antibody, or to the resulting assay data being too variable to allow a calculation. The variable data quality precluded the use of more sophisticated curve-fitting models for calculation of IC values.For each laboratory, a geometric mean IC value for the repeat tests was calculated. For each virus and antibody combination, an overall geometric mean of the individual laboratory means was calculated, along with the minimum, maximum, and range between laboratories. To allow calculations of the geometric means, any IC value that was greater than the highest concentration used were taken as equal to the next two-fold dilution step, so results recorded as >25 were taken as equal to 50. Similarly, IC values that were below the lowest concentration were taken as the next two-fold dilution step. IC values that were below 0.78 are recorded as <0.78, even if a lower IC value could be calculated from an extended dilution series. Although this may not fully reflect the differences in sensitivities of different assays, it does allow a consistent comparison between different laboratories and groups of assay methods, without introducing any bias from the range of concentrations included in the assays.The majority of assays had IC90 values above the highest concentration of inhibitor used. The IC75s were also above the highest concentration tested for many assays, particularly for 447-52D and sCD4. The calculated IC50s were used for all subsequent analyses.The overall pattern of neutralization of the different viruses by the four reagents is shown in TriMab, a mixture of three Mabs, was the most effective and broadly neutralizing of the four reagents used. Given that Mab 447-52D is specific for viruses that carry the GPGR motif at the tip of the V3 loop As illustrated in The aim of the present study was to compare the performance of a wide variety of HIV-1 neutralization assays as performed in different laboratories . The virDirect comparison of the PSV and VI assays was possible with 10 viruses. The results of the fusion assay were not included in these calculations. The PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus as well as the neutralizing reagent.vs. 4E10 lower, vs. sCD4 similar; whereas VI191 vs. 4E10 similar and vs. sCD4 lower IC50s in VI assays than in PSV assays; vs. VI assay comparison  was compared to all other plasmid-based assays . The results largely depended on the virus used . For TriPBMC-based assays with different readouts were compared. Four laboratories used extracellular p24 (EC-p24) detection , while intracellular p24 (IC-p24) or RNA viral load by real-time PCR comprised the other group (laboratories 8 and 11). No general trend between assay types was evident, since the observed range was affected by the cut-off of 25 (or 10) µg/ml as the highest concentration of reagent tested.There are several variables in the preparation of PBMC and in neutralization assay protocols , which mThe standard IC-p24 assay is based on PBMC infection, but it can also be adapted to measure neutralization using other target cells such as immature dendritic cells (DC) and monocyte-derived macrophages (MDM). Indeed, neutralizing Mabs had lower ICs on DC and even still lower ICs on MDM (CD4-T-cells<DC<MDM) For IC50 comparisons with the other assays the GHOST(3) cells expressing CCR5 or CXCR4 were used. In general, results of the plaque reduction assay fit in the middle range of IC50s of the other VI assays; better for some neutralizing reagents and viruses, worse for other neutralizing reagents and viruses  and U87.CD4, for assaying HIV-1 neutralization sensitivity. This comparison was done at one single reagent concentration (5 µg/ml) and showed a close agreement of the results on the two cell types .The fusion assay almost invariably showed the highest threshold for the detection of neutralization, except for sCD4. Strikingly, when mouse 3T3 cells were used as target cells the anti-gp41 Mab 4E10 did not neutralize any of the viruses  endogenously expressing CXCR4 and engineered to express human CD4 and CCR5. The individual components of TriMab were tested separately to see whether anti-gp41 MAbs inhibited neutralization. The results of parallel experiments carried out with two viruses, one R5 and one X4 , showed that the sensitivity of the fusion assay to detect neutralization was greatly increased when using HeLa cells as target cells . While Qenv gp160 gene and an HIV-1 env vector expressing luciferase allowed the comparison of plasmid and culture supernatant derived virus populations .An assay based on recombinant viruses produced by co-infection of HEK-293 cells with a cloned in vitro assay best correlates with biological (in vivo) protection, a panel of neutralization assays has to be recommended for vaccine evaluation.The primary aim of this study was to co-ordinate activities aimed at comparing methods for the measurement of neutralizing antibodies to HIV-1 for use in HIV-1 research as well as in human clinical trials of candidate HIV/AIDS vaccines. The comparisons led us to the conclusion that at present no single assay can be recommended for the measurement of HIV-1 neutralization because the assay results vary significantly depending on both the virus and the reagent used. Different assays vary in their sensitivities for measuring of neutralization with the different virus-reagent combinations. Both the virus and the reagent used for neutralization influence the assay sensitivity. Since it is not known at present which In the present study IC50 values were used for the comparisons. We also considered the use of IC75 or even IC90, but many of the assays would have been excluded from the comparison as 90% or even 75% neutralization was often not achieved in most of the assays used. In conventional VI assays with polyclonal reagents, such as patient sera or plasma, IC90 values are the parameters usually considered for measuring neutralization as this increased stringency parallels increased specificity. In the present study, however, we used defined reagents with known specificities, which allows comparisons at the level of IC50s. It is important to emphasize that in future comparisons of the assays with polyclonal reagents it may be mandatory to compare higher ICs during evaluation.in vivo in the infected individual. This problem might be overcome, at least in part, by using a pool of amplified clones rather than single clones, as occurred in the tests performed by partner 4 and 12. One may also consider that the envelope spike density and stability of pseudoviruses is different from those of primary isolates and this may account for a higher sensitivity to neutralization. Continued careful comparison of the assays is therefore very important.Although with some combinations of reagents PSV assays appeared to be more sensitive for the detection of neutralizing activity than VI assays, the use of molecularly-cloned pseudoviruses has the caveat of testing single clones and not a complex viral quasispecies as in the case of uncloned viral isolates. Single clones may give different results and may not be representative of the prevalent viruses in the quasispecies present in the corresponding isolate, and even less of the virus population circulating in vitro approximation to the in vivo situation. However, assays using PBMC from different donors isolated on different days show great variability in sensitivity as clearly reflected by our inter-laboratory comparisons. In addition, the number of donors included in each test, the time of incubation with the virus and the readout of the assay were all major variables amongst the different laboratories. Of all the variables, the length of time during which the inhibitor was present correlated best with the sensitivity of the assay. Again, however, this relationship seemed to be true for one virus (SF162), while other viruses did not follow this pattern. These data reinforce the conviction that there is a great need to standardize the PBMC assays for HIV-1 neutralization, an activity that is currently in progress in our network and other groups worldwide.The use of PBMC as target cells in neutralization assays is the closest An encouraging result emerging from our study was that the plaque reduction assays gave similar results to the PBMC-based assays. Using cell lines engineered to express the HIV-1 co-receptors may provide useful model systems for HIV-1 neutralization. PBMC-produced cytokines and chemokines may add to the existing variables in a virus-dependent manner, relating to the sensitivity or resistance of the virus to inhibition by cytokines and/or chemokines in vivo need to be investigated. It has also been described that the level of CCR5 but not CD4 expression on the cell surface determines neutralization by certain MAbs. For example, neutralization by 4E10 was less effective in HeLa cells engineered to express CCR5 at high levels as compared to low levels (comparable to those on CD4+ T lymphocytes) Another aspect related to the nature of the target cells used in HIV-1-neutralization assays has emerged in connection with the fusion assay. We found that mouse 3T3 cells supported neutralization by sCD4 but not by Mabs. However, when we employed human HeLa cells as targets the Mabs acted with similar efficiency as in other assays. Of note, efficient neutralization by Mabs was also observed using another human cell line of B-lymphoid origin, 721–22 (not shown), suggesting that the sensitivity to Mab-mediated neutralization in the fusion assay depends on the specific characteristics of the cell lines used as targets, with human cells showing a greater sensitivity than murine cells. In contrast to the results obtained in the fusion assay with murine target cells, Mabs showed an increased inhibitory activity when macrophages were used as target cells instead of PBMC in VI assays. The macrophage-based assay detected neutralization mediated by the Fab as well as the Fcγ fragment of the immunoglobulin. Conceivably, the antibodies induced endocytosis and degradation of immune complexes via binding to FcγR1 receptor (CD64) on macrophages The present results provided the necessary starting platform for designing the future strategies of the NeutNet programme. In the next step, we plan to assay plasma, sera and purified IgG from human and animal sources in order to make comparison of the assays more complete. A reduced virus/clone panel of 8 isolates will be proposed, and a number of polyclonal human plasma preparations will be evaluated using different assays optimized according to the data so far obtained. Taken together, the comparison of 16 different HIV-1 neutralization assays within the framework of an international network, NeutNet, comprised of 15 laboratories, led us to the conclusion that at present no single assay can be recommended for use alone as a potential correlate of vaccine efficacy. Both the virus and the reagent used for neutralization  contribute to the outcome of assay sensitivity, which in certain combinations is also influenced by the target cell used. Until we gather more precise information on which assays may correlate with protective immunity, a panel of neutralization assays must be recommended for vaccine evaluation.Figure S1Characteristics of the pseudotyped virus based assays. Assays are grouped as in (2.51 MB TIF)Click here for additional data file.Table S1Characteristics of the PBMC based assays Footnote to Supplementary Table S1: 1 Stimulated prior freezing 2 number of donors used 3number of cells/ml of medium 4 only in medium of the neutralization assay 5 incubation time (hr) virus and inhibitory reagent of the neutralization assay 6 absorption time (hr) virus/inhibitory reagent mix on the cells before wash step 7 3 washes in case patient's sera/plasma are used 8 incubation time before virus infectivity is measured 9 culture period is 2 days for real-time PCR and 8 days for p24 antigen(0.02 MB XLS)Click here for additional data file.