Peritoneal flap for lymphocele prophylaxis following robotic-assisted laparoscopic radical prostatectomy with pelvic lymph node dissection: study protocol and trial update for the randomized controlled PELYCAN study.

BACKGROUND: Data from interventional studies suggest that a peritoneal flap after pelvic lymph node dissection (LND) during laparoscopic, robotic-assisted radical prostatectomy (RARP) may reduce the rate of symptomatic lymphoceles in transperitoneal approach. However, most of these studies are not conducted in a randomized controlled fashion, thus limiting their scientific value. A recent prospective, randomized, controlled trial (RCT) did not show superiority of a peritoneal flap while further trials are lacking. Therefore, the aim of the presented RCT will be to show that creating a peritoneal flap decreases the rate of symptomatic lymphoceles compared to the current standard procedure without creation of a flap. METHODS/DESIGN: PELYCAN is a parallel-group, patient- and assessor-blinded, phase III, adaptive randomized controlled superiority trial. Men with histologically confirmed prostate cancer who undergo transperitoneal RARP with pelvic LND will be randomly assigned in a 1:1 ratio to two groups-either with creating a peritoneal flap (PELYCAN) or without creating a peritoneal flap (control). Sample size calculation yielded a sample size of 300 with a planned interim analysis after 120 patients, which will be performed by an independent statistician. This provides a possibility for early stopping or sample size recalculation. Patients will be stratified for contributing factors for the development of postoperative lymphoceles. The primary outcome measure will be the rate of symptomatic lymphoceles in both groups within 6 months postoperatively. Patients and assessors will be blinded for the intervention until the end of the follow-up period of 6 months. The surgeon will be informed about the randomization result after performance of vesicourethral anastomosis. Secondary outcome measures include asymptomatic lymphoceles at the time of discharge and within 6 months of follow-up, postoperative complications, mortality, re-admission rate, and quality of life assessed by the EORTC QLQ-C30 questionnaire. DISCUSSION: The PELYCAN study is designed to assess whether the application of a peritoneal flap during RARP reduces the rate of symptomatic lymphoceles, as compared with the standard operation technique. In case of superiority of the intervention, this peritoneal flap may be suggested as a new standard of care. TRIAL REGISTRATION: German Clinical Trials Register DRKS00016794 . Registered on 14 May 2019.

[Urological research in Germany : A retrospective, longitudinal observational study]. / Urologische Forschung in Deutschland : Eine retrospektive, longitudinale Beobachtungsstudie.

BACKGROUND AND OBJECTIVES: The congress of the German Society of Urology reflects urologic research in German-speaking countries. The objective was to identify trends by analyzing the congress' abstracts and following full publications longitudinally. MATERIALS AND METHODS: The abstracts of the 2016 congress were systematically analyzed regarding content, study design, cooperation, following full publications and journals which they were published in. Thereafter, the 2016 congress was compared to the 2002 and 2009 congresses. Statistical analysis included χ2-, Mann-Whitney U-, Cochran-Armitage-, and Kruskal-Wallis test. RESULTS: A total of 1073 abstracts were presented at the 2002, 2009, and 2016 congresses. We found an increase in abstracts regarding prostate disease (24.2%, 29.7%, and 34.0%; p = 0.0043), oncological abstracts (50.6%, 57.9%, and 61.7%; p = 0.003), multicenter studies (18.3, 28.6, and 34.3%; p < 0.0001) and cooperation (55.6%, 62.9%, and 70.5%, p < 0.0001). Experimental (29.0%, 33.2%, and 22.8%; p = 0.009) and prospective studies (62.1%, 42.0%, and 36.0%; p < 0.0001) declined. Abstracts including statistical analysis (18.4%, 14.7%, and 41.2%; p < 0.0001) and the impact factor of following full publications (2.08, 3.42, 4.42; p < 0.0001) rose. In 2016, 11.2% of those full publications were published Open Access. The publication rates of the presented abstracts were 49.1%, 56.3%, and 52.3%, respectively (p = 0.15). CONCLUSIONS: National and international networking of the urological research community has increased. Presentation of prospective studies has declined. The rate of peer-reviewed full publications following the DGU abstracts remains at a stable high level over the three congresses. The publication rate in Open Access journals is low.

Effect of mycophenolic acid on inosine monophosphate dehydrogenase (IMPDH) activity in liver transplant patients.

BACKGROUND: Due to the development of immunosuppressants, the focus in transplanted patients has shifted from short-term to long-term survival as well as a better adjustment of these drugs in order to prevent over- and under-immunosuppression. Mycophenolic acid (MPA) is a noncompetitive inhibitor of inosine monophosphate dehydrogenase (IMPDH) and approved for prophylaxis of acute rejection after kidney, heart, and liver transplantation, where it has become a part of the standard therapy. Targeting inosine monophosphate IMPDH activity as a surrogate pharmacodynamic marker of MPA-induced immunosuppression may allow a more accurate assessment of efficacy and aid in limiting toxicity in liver transplanted patients. AIM: Assess IMPDH-inhibition in liver transplant recipients and its impact on biliary/infectious complications, acute cellular rejection (ACR) and liver dependent survival. METHODS: This observational cohort study comprises 117 liver transplanted patients that were treated with mycophenolate mofetil (MMF) for at least 3 months. Blood samples (BS) were collected and MPA serum level and IMPDH activity were measured before (t(0)), 30minutes (t(30)) and 2h after (t(120)) MMF morning dose administration. Regarding MPA, we assessed the area under the curve (AUC). Patients were prospectively followed up for one year and assessed for infectious and biliary complications, episodes of ACR and liver dependent survival. RESULTS: The MPA levels showed a broad interindividual variability at t(0) (2.0±1.8ng/ml), t(30) (12.7±9.0ng/ml) and t(120) (7.5±4.3ng/ml). Corresponding IMPDH activity was at t(o) (23.2±9.5 nmol/h/mg), at t(30) (16.3±8.8 nmol/h/mg) and t(120) (18.2±8.7 nmol/h/mg). With regard to MPA level we found no correlation with infectious or biliary complications within the follow-up period. Patients with baseline IMPDH(a) below the median had significant more viral infections (6 (10.2%) vs. 17 (29.3%); P=0.009) with especially more cytomegalovirus (CMV) infections (1 (3.4%) vs. 6 (21.4%); P=0.03)). Furthermore, patients with baseline IMPDH(a) above the median developed more often non-anastomotic biliary strictures (8 (13.6%) vs. 1 (1.7%), P=0.03). We found the group reaching the combined clinical endpoint of death and re-transplantation showing significantly lower MPA baseline values (t(0) 0.9±0.7 vs. 2.1±1.8µg/ml Mann-Whitney-U: P=0.02). We calculated a simplified MPA(AUC) with the MPA level at baseline, 30 and 120minutes after MPA administration. Whereas we found no differences with regard to baseline characteristics at entry into the study patients with MPA (AUC) below the median experienced significantly more often the combined clinical endpoint (12.1% (7/58) vs. 0.0% (0/57); P=0.002) and had a reduced actuarial re-transplantation-free survival (1.0 year vs. 0.58 years; Log-rank: P=0.007) during the prospective one-year follow-up period. In univariate and multivariate analysis including gender, age, BMI, ACR, MPA (AUC) and IMPDH(a) only BMI, MPA (AUC) and IMPDH(a) were independently associated with reduced actuarial re-transplantation-free survival. CONCLUSION: MPA-levels and IMPDH-activity in liver transplanted patients allows individual risk assessment. Patients with higher IMPDH inhibition acquire more often viral infections. Insufficient IMPDH inhibition is associated with development of non-anastomotic bile duct strictures and reduced re-transplantation-free survival.

Chronic hemodialysis in pediatric patients: technical and practical aspects of use.

Chronic kidney disease (CKD) is a continuum of progressive reduction in kidney function lasting for more than three months, due to either structural and/or functional renal abnormalities that may lead to irreversible kidney damage. The term "renal supportive therapy" (RST) generally characterizes the spectrum of dialysis therapies available to support existing renal function in patients with CKD during progression to end-stage renal disease (ESRD) and/or renal transplantation. Chronic RST modalities include conventional hemodialysis, peritoneal dialysis and home hemodialysis therapies. The modality chosen to deliver RST in the pediatric patient is often guided by a variety of factors including institutional resources, local expertise, patient characteristics, treatment goals, and physician preference. Chronic RST in a pediatric population requires the flexible utilization of multiple delivery modalities for effective care across infancy into adulthood and is not typically initiated until GFR declines to between 15-30 mL/min per 1.73 m2, although thresholds for initiation of RST will vary between patients. This review will provide an overview of current approaches to management and technical approaches to pediatric patients requiring chronic hemodialysis.

Characterizing the performance of two optical particle counters (Grimm OPC1.108 and OPC1.109) under urban aerosol conditions.

The performance of Grimm optical particle counters (OPC, models 1.108 and 1.109) was characterized under urban aerosol conditions. Number concentrations were well correlated. The different lower cut-off diameters (0.25 and 0.3 µm) give an average difference of 23.5%. Both detect less than 10% of the total particle concentration (0.01-1 µm; Differential Mobility Analyzer), but in the respective size ranges, differences are <10%. OPC number size distributions were converted to mass concentrations using instrument-specific factors given by the manufacturer. Mass concentrations for OPC1.108 were 60% higher than for OPC1.109 and (in case of OPC1.109) much lower than those measured with an impactor in the relevant size range or a TSP filter. Using the C-factor correction suggested by the manufacturer, OPC1.109 underestimated mass concentrations by 21% (impactor) and by about 36% (TSP filter), which is in the range of comparability of co-located different mass concentration methods (Hitzenberger, Berner, Maenhaut, Cafmeyer, Schwarz, & Mueller et al., 2004).

Gas cooking is associated with small reductions in lung function in children.

Inconsistent effects of gas cooking on lung function have been reported. In a previous study from Austria, we demonstrated a significant, though small, reduction of lung function parameters in children living in homes with gas stoves. We used a larger international database to check if this finding can be generalised. To study the relative impact of cooking with gas on lung function parameters of primary school children in a wide range of geographical settings, we analysed flow and volume data of approximately 24,000 children (aged 6-12 yrs) from nine countries in Europe and North America. Exposure information was obtained by comparable questionnaires and spirometry according to an American Thoracic Society/European Respiratory Society protocol. Linear regressions were used, controlling for individual risk factors and study area. Heterogeneity between study-specific results and mean effects were estimated using meta-analytical tools. On average, gas cooking reduced lung function parameters. Overall effects were small (-0.1-0.7%) and only significant for forced vital capacity and forced expiratory volume in 1 s. There was some indication that allergic children were more affected by gas cooking. Under current housing conditions, gas cooking is associated with only small reductions in lung function.

Italy and Austria before and after study: second-hand smoke exposure in hospitality premises before and after 2 years from the introduction of the Italian smoking ban.

UNLABELLED: The aim of this paper was to compare nicotine concentration in 28 hospitality premises (HPs) in Florence and Belluno, Italy, where a smoking ban was introduced in 2005, and in 19 HPs in Vienna, Austria, where no anti-smoking law entered into force up to now. Airborne nicotine concentrations were measured in the same HPs in winter 2002 or 2004 (pre-ban measurements) and winter 2007 (post-ban measurements). In Florence and Belluno, medians decreased significantly (P < 0.001) from 8.86 [interquartile range (IQR): 2.41-45.07)] before the ban to 0.01 microg/m3 (IQR: 0.01-0.41) afterwards. In Austria (no smoking ban) the medians collected in winters 2004 and 2007 were, respectively, 11.00 (IQR: 2.53-30.38) and 15.76 microg/m3 (IQR: 2.22-31.93), with no significant differences. Measurements collected in winter 2007 in 28 HPs located in Naples, Turin, Milan (0.01 microg/m3; IQR: 0.01-0.16) confirmed post-ban results in Florence and Belluno. The medians of nicotine concentrations in Italy and Austria before the Italian ban translates, using the risk model of Repace and Lowery, into a lifetime excess lung cancer mortality risk for hospitality workers of 11.81 and 14.67 per 10,000, respectively. Lifetime excess lung cancer mortality risks for bar and disco-pub workers were 10-20 times higher than that calculated for restaurant workers, both in Italy and Austria. In winter 2007, it dropped to 0.01 per 10,000 in Italy, whereas in Austria it remained at the same levels. The drop of second-hand smoke exposure indicates a substantial improvement in air quality in Italian HPs even after 2 years from the ban. PRACTICAL IMPLICATIONS: The nation-wide smoking ban introduced in Italy on January 10, 2005, resulted in a drop in second-hand smoke exposure in hospitality premises, whereas in Austria, where there is no similar nation-wide smoking ban, the exposure to second-hand smoke in hospitality premises remains high. Given that second-hand smoke is considered a group 1 carcinogen according to the International Agency for Research on Cancer classification, the World Health Organization Framework Convention on Tobacco Control strongly recommends the implementation of nation-wide smoke-free policies in order to improve the indoor air quality of hospitality premises and workplaces. Results from our study strongly supports this recommendation.

Long term success of short smoking cessation seminars supported by occupational health care.

The objective of this longitudinal (3 year) study was to determine predictors of abstinence in 515 employees of a steel plant (28% female, age 18-67 years) after participation in Allen Carr seminars (intensive group counselling in a single session of 6 h). Answers given in computer aided phone interviews were analysed by stepwise and Cox regression. Of 510 responding persons 262 (51.4%) reported continuing abstinence. In a random sample of 61 respondents cotinine concentration in urine was measured, showing high agreement with smoking history. Social support increased abstinence, which was more persistent in males and office workers. Also in female participants the non-smoking spouse was a significant predictor for abstinence while a higher body weight predicted relapse. Relapsed female smokers did not show a sustainable reduction of cigarette consumption. Compared to cessation clinics higher population coverage would be achievable by workplace seminars. Every second smoker motivated to participate seems to be able to quit even without medication and to stay abstinent. Especially in females these seminars should be followed by physical exercise and continued support of gender specific occupational health care.

NO2 and children's respiratory symptoms in the PATY study.

OBJECTIVES: NO2)is a major urban air pollutant. Previously reported associations between ambient NO2)and children's respiratory health have been inconsistent, and independent effects of correlated pollutants hard to assess. The authors examined effects of NO2 on a spectrum of 11 respiratory symptoms, controlling for PM10 and SO2, using a large pooled dataset. METHODS: Cross sectional studies were conducted in Russia, Austria, Italy, Switzerland, and the Netherlands, during 1993-99, contributing in total 23 955 children. Study-specific odds ratios for associations with ambient NO2 are estimated using logistic regressions with area-level random effects. Heterogeneity between study-specific results, and mean estimates (allowing for heterogeneity) are calculated. RESULTS: Long term average NO2 concentrations were unrelated to prevalences of bronchitis or asthma. Associations were found for sensitivity to inhaled allergens and allergy to pets, with mean odds ratios around 1.14 per 10 microg/m3 NO2. SO2 had little confounding effect, but an initial association between NO2 and morning cough was reduced after controlling for PM10. Associations with reported allergy were not reduced by adjustment for the other pollutants. Odds ratios for allergic symptoms tended to be higher for the 9-12 year old children compared with the 6-8 year old children. CONCLUSIONS: Evidence for associations between NO2 and respiratory symptoms was robust only for inhalation allergies. NO2 most likely is acting as an indicator of traffic related air pollutants, though its direct effect cannot be ruled out. This remains important, as policies to reduce traffic related air pollution will not result in rapid reductions.

Low levels of air pollution induce changes of lung function in a panel of schoolchildren.

In search of sensitive screening parameters for assessing acute effects of ambient air pollutants in young schoolchildren, the impact of 8-h average air pollution before lung function testing was investigated by oscillatory measurements of resistance and spirometry with flow-volume loops. At a central elementary school in Linz, the capital of Upper Austria, 163 children aged 7-10 yrs underwent repeated examinations at the same time of day during 1 school year, yielding a total of 11-12 lung function tests per child. Associations to mass concentrations of particulate matter and nitrogen dioxide (NO(2)) measured continuously at a nearby monitoring station were tested, applying the Generalised Estimating Equations model. Reductions per 10 microg.m(-3) (both for particles and for NO(2)) were in the magnitude of 1% for most lung function parameters. The most sensitive indicator for acute effects of combustion-related pollutants was a change in maximal expiratory flow in small airways. NO(2) at concentrations below current standards reduced (in the multipollutant model) the forced expiratory volume in one second by 1.01%, maximal instantaneous forced flow when 50% of the forced vital capacity remains to be exhaled (MEF(50%)) by 1.99% and MEF(25%) by 1.96%. Peripheral resistance increased by 1.03% per 10 microg.m(-3) of particulate matter with a 50% cut-off aerodynamic diameter of 2.5 mum (PM(2.5)). Resistance is less influenced by the child's cooperation and should be utilised more often in environmental epidemiology when screening for early signs of small airway dysfunction from urban air pollution, but cannot replace the measurement of MEF(50%) and MEF(25%). In the basic model, the reduction of these parameters per 10 microg.m(-3) was highest for NO(2), followed by PM(1), PM(2.5) and PM(10), while exposure to coarse dust (PM(10)-PM(2.5)) did not change end-expiratory flow significantly. All acute effects of urban air pollution found on the lung function of healthy pupils were evident at levels below current European limit values for nitrogen dioxide. Thus, planned reduction of nitrogen dioxide emission (Euro 5; vehicles that comply with the emission limits as defined in Directive 99/96/EC) of 20% in 2010 would seem to be insufficient.

Smoking cessation at the workplace: 1 year success of short seminars.

OBJECTIVES: In search of less time-consuming methods of smoking cessation Allen Carr' seminars performed at workplaces in Austria were evaluated. METHODS: Of all the 357 smokers attending a seminar in summer 2002, 308 (86%), consented to participate in a repeated health survey, including the SF-36 questionnaire. After 3 months 268 (87%) gave computer-aided telephone interviews and 223 (72%) after 1 year. Analysis by logistic regression was done separately for males and females. RESULTS: The 1-year quit rate was 40% (worst case assumption) to 55% (best estimate). In 96% of quitters an intensive counseling for 6 h without pharmaceutical aid thereafter was sufficient to maintain abstinence for 12 months. A long smoking history or many earlier unsuccessful attempts to quit did not predict failure. The risk of relapse was found higher in young men with a high number of pack-years and in women with good physical fitness but high Fagerstroem score and financial reasons for the intention to quit smoking. While an average weight gain of 3 kg in males was not associated with failure to quit smoking, we found the highest weight gain (4 kg) in females in the group abstinent at 3 months but smoking again at 12 months. In all participants subjective life quality scored by SF-36 improved. In quitters perception of general health improved more. CONCLUSIONS: Group counseling at the workplace was found to be an efficient method of smoking cessation, capable of increasing subjective life quality and health and to smooth the way to smoke-free enterprises.

Environmental tobacco smoke exposure in public places of European cities.

BACKGROUND: Exposure to environmental tobacco smoke (ETS) has important public health implications. The results of the first European multi-centre study that measured ETS exposure in a range of public places (transport, educational settings, and leisure facilities such as bars and restaurants) are presented. METHOD: Nicotine vapour phase was measured using ETS passive samplers containing a filter treated with sodium bisulfate. RESULTS: Bars and discos are the places with the highest concentrations of nicotine from ETS, median ranging from 19 to 122 microg/m(3). Restaurants had the next highest values. Concentrations of nicotine generally range from 0.1-5 microg/m(3) in airports, and from 0.5-10 microg/m(3) in train stations. Nicotine was also found in schools and universities, yet schools tended to have the lowest concentrations compared to all the other public places sampled. In hospitals levels were generally below 5 microg/m(3). CONCLUSIONS: Although there is some variability between cities, this study shows that tobacco smoke is present in most of the studied public places. The study also showed that in areas where smoking is prohibited, concentrations of nicotine are lower than in areas where smoking is allowed but they are not zero. The results of this study indicate that policies should be implemented that would effectively reduce levels of tobacco smoke in public areas.

Lung cancer and dust exposure: results of a prospective cohort study following 3260 workers for 50 years.

AIMS: To study the lasting health impact of occupational dust exposure on life expectancy and specific causes of death. METHODS: Male Viennese workers, selected at age > or =40 (mean 54) years during preventive check-ups between 1950 and 1960, were followed prospectively until death. Half of them (1630) were exposed at work to (non-fibrous) particulates, while the non-exposed workers were matched for year, age, and smoking status at the start of observation. RESULTS: Average life expectancy of those exposed was 1.6 years less than that of those non-exposed. Only a small part of this decrease in life expectancy (hazards ratios in brackets) was related to acknowledged occupational diseases such as silicosis and silicotuberculosis (67.12). Chronic obstructive lung disease (1.82) and cancer of the lung (1.42) and stomach (1.77) were found more frequently among those exposed. CONCLUSIONS: Results support the hypothesis that high exposure to insoluble particulates such as silica in the metal, glass, ceramics, and stone industries promotes bronchial cancer and chronic obstructive pulmonary disease. The finding of an increased incidence of stomach cancer might be related to particles swallowed after clearance from the airways.

Production of antigen-specific human monoclonal antibodies: comparison of mice carrying IgH/kappa or IgH/kappa/lambda transloci.

Here we compare human monoclonal antibody (MAb) production from mouse strains that carry disruptions of their endogenous mouse IgH/IgK loci and harbor human IgM + Igkappa(BABkappa) or human IgM + Igkappa + IgA transloci (BABkappa,lambda). We found that whereas both strains proved effective for the isolation of antigen-specific IgM antibodies, many of the IgM MAbs elicited from BABkappa comprise human mu chains that are associated with mouse lambda chains. In contrast, BABkappa,lambda mice gave rise to fully functional, polymeric human IgM antibodies comprising both human IgH and human IgL chains. Therefore, the inclusion of a human Iglambda translocus (in addition to the human IgH + Igkappa transloci) not only diminishes problems of endogenous mouse Iglambda expression but also provides a strain of mice that yields fully human MAbs to a wide range of antigens, as witnessed by the isolation of MAbs to human blood cells, tumor cell lines, and an immunoglobulin idiotype.

Moulds in housing: visual inspection and spore counts comparison--implications for future strategies in the public health setting.

OBJECTIVES: Among the problems of health related to living conditions presented to a public consulting centre for environmental medicine in Vienna, the main point at issue was damp housing and mould growth. On answering this demand indoor exposure to fungal spores was identified by visual semi-quantitative assessment of fungal growth on the one hand and quantitative measurement of viable spores on the other hand. The validity and practicability of this approach was investigated. METHOD: We applied these two simple methods in a standardised form and compared the results in a field study, which has been conducted between 1995 and 1999. In 197 rooms in Viennese flats (183 of which were claimed to be "mouldy") fungal spore concentrations were measured by RCS-impactor and Rose-Bengal-agar. Outdoor measurements were taken simultaneously outside the buildings and were used for reference. RESULTS: Independent classification of visible growth of mould in flats correlated significantly (r = 0.635; p < 0.0001) with the ratio of indoor/ outdoor concentration of fungal spores. CONCLUSIONS: It is usually possible to classify the severity of the problem just by local inspection. Visible plaques should always be an indication for sanitary measures. However, spore counting is indicated for quantifying health relevant exposures, for scientific comparisons and in documentation and follow up such as for litigation purposes. Quantitative data bear more credibility and so help to prove the need of sanitation.

Novartis Medal Lecture. Antibodies: a paradigm for the evolution of molecular recognition.

Novel proteins have been elaborated over evolutionary time by an iterative alternation of mutation and selection. In a similar way, the humoral immune system also uses an iterative alternation of mutation and selection to generate novel antibodies that display a high affinity for their cognate antigen -- but this is achieved in a matter of a days. Gene rearrangement is used to produce a primary repertoire of antibodies and, on entering the body, antigen triggers the clonal expansion of those B lymphocytes that express a cognate antibody, albeit one of low affinity. Rapid and specific affinity maturation is then achieved by subjecting the immunoglobulin genes in the rapidly expanding B cells to a period of intense mutation. The intensity of this mutational assault is tolerated because it is targeted specifically to the immunoglobulin genes, causing relatively little damage to other loci. Antigen-mediated selection then allows the preferential expansion of those mutants expressing antibodies displaying improved binding characteristics. Here, studies are described that have been performed to glean insight into the mechanisms of the hypermutation and selection processes. Experiments are also described in which an attempt has been made to recapitulate aspects of physiological antibody generation in vitro, allowing the development of novel approaches to the generation of proteins with high-affinity binding sites.

Switch junction sequences in PMS2-deficient mice reveal a microhomology-mediated mechanism of Ig class switch recombination.

Isotype switching involves a region-specific, nonhomologous recombinational deletion that has been suggested to occur by nonhomologous joining of broken DNA ends. Here, we find increased donor/acceptor homology at switch junctions from PMS2-deficient mice and propose that class switching can occur by microhomology-mediated end-joining. Interestingly, although isotype switching and somatic hypermutation show many parallels, we confirm that PMS2 deficiency has no major effect on the pattern of nucleotide substitutions generated during somatic hypermutation. This finding is in contrast to MSH2 deficiency. With MSH2, the altered pattern of switch recombination and hypermutation suggests parallels in the mechanics of the two processes, whereas the fact that PMS2 deficiency affects only switch recombination may reflect differences in the pathways of break resolution.

Epstein-Barr virus and the somatic hypermutation of immunoglobulin genes in Burkitt's lymphoma cells.

It has been suggested that Epstein-Barr virus (EBV) might suppress antibody maturation either by facilitating bypass of the germinal center reaction or by inhibiting hypermutation directly. However, by infecting the Burkitt's lymphoma (BL) cell line Ramos, which hypermutates constitutively and can be considered a transformed analogue of a germinal center B cell, with EBV as well as by transfecting it with selected EBV latency genes, we demonstrate that expression of EBV gene products does not lead to an inhibition of hypermutation. Moreover, we have identified two natural EBV-positive BL cell lines (ELI-BL and BL16) that hypermutate constitutively. Thus, contrary to expectations, EBV gene products do not appear to affect somatic hypermutation.

Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation.

After gene rearrangement, immunoglobulin V genes are further diversified by either somatic hypermutation or gene conversion. Hypermutation (in man and mouse) occurs by the fixation of individual, non-templated nucleotide substitutions. Gene conversion (in chicken) is templated by a set of upstream V pseudogenes. Here we show that if the RAD51 paralogues XRCC2, XRCC3 or RAD51B are ablated the pattern of diversification of the immunoglobulin V gene in the chicken DT40 B-cell lymphoma line exhibits a marked shift from one of gene conversion to one of somatic hypermutation. Non-templated, single-nucleotide substitutions are incorporated at high frequency specifically into the V domain, largely at G/C and with a marked hotspot preference. These mutant DT40 cell lines provide a tractable model for the genetic dissection of immunoglobulin hypermutation and the results support the idea that gene conversion and somatic hypermutation constitute distinct pathways for processing a common lesion in the immunoglobulin V gene. The marked induction of somatic hypermutation that is achieved by ablating the RAD51 paralogues is probably a consequence of modifying the recombination-mediated repair of such initiating lesions.

B cells acquire antigen from target cells after synapse formation.

Soluble antigen binds to the B-cell antigen receptor and is internalized for subsequent processing and the presentation of antigen-derived peptides to T cells. Many antigens are not soluble, however, but are integral components of membrane; furthermore, soluble antigens will usually be encountered in vivo in a membrane-anchored form, tethered by Fc or complement receptors. Here we show that B-cell interaction with antigens that are immobilized on the surface of a target cell leads to the formation of a synapse and the acquisition, even, of membrane-integral antigens from the target. B-cell antigen receptor accumulates at the synapse, segregated from the CD45 co-receptor which is excluded from the synapse, and there is a corresponding polarization of cytoplasmic effectors in the B cell. B-cell antigen receptor mediates the gathering of antigen into the synapse and its subsequent acquisition, thereby potentiating antigen processing and presentation to T cells with high efficacy. Synapse formation and antigen acquisition will probably enhance the activation of B cells at low antigen concentration, allow context-dependent antigen recognition and enhance the linking of B- and T-cell epitopes.