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Background The quantification of serum IgG4 is commonly performed during the diagnostic workup of IgG4-related diseases (IgG4-RD). According to recent literature, IgG4 values above 1.35 g/L are characteristic of IgG4-RD and support its diagnosis at initial presentation. The purpose of this study was to evaluate comparability and accuracy of the two main commercially available IgG4 assays (Siemens Healthineers and The Binding Site). Methods Method comparison was performed for IgG and IgG subclasses using a collective of selected samples with elevated serum IgG4. In addition, we assessed the accuracy of both assays using purified polyclonal and monoclonal IgG4 preparations. Results Our data show significant discrepancies between the two IgG subclass assays for the measurement of IgG4 and, to a lesser extent, IgG3. Conclusions The lack of standardization between the two main providers of commercially available IgG4 assays leads to significant inter-assay result discrepancies, which might potentially cause unnecessary clinical workup. We conclude that serum IgG4 assay-specific decision limits, and not an assay-independent single cut-off level for IgG4 (e.g. 1.35 g/L), should be used when assessing patients for IgG4-RD. An internationally recognized, certified reference material for IgG subclasses is urgently needed, and assay manufactures are encouraged to undertake steps toward standardization of measurements of IgG4 and other IgG subclasses.

Background: Rhinitis is a very common disease with allergies being the most frequent causative factor. It can co-occur together with asthma and eczema in atopic as well as in nonatopic patients., Objectives: To assess the prevalence of allergic sensitization within patient groups with rhinitis in consideration of the co-occurring disorders of asthma and eczema., Methods: Students of the third year of medical school completed an anonymous questionnaire on age, gender, and clinical symptoms, such as seasonal rhinitis, perennial rhinitis, asthma, and eczema, and underwent an ImmunoCAP Rapid test. We calculated the prevalence of sensitization within subgroups of patients reporting allergic disorders, such as rhinitis, asthma, and eczema., Results: Questionnaires and ImmunoCAP Rapid tests of 1513 medical students were analyzed. The participants' self-reported presence of seasonal/perennial rhinitis, asthma, and eczema was compared to the presence of sensitization. Data of 1467 subjects could be analyzed. Seasonal rhinitis was the most common symptom, followed by eczema, asthma, and perennial rhinitis. The participants were differentiated into 16 subgroups according to the combined clinical manifestations of the different symptoms and association to sensitization within subgroups. The prevalence of sensitization ranged from 18% in subjects reporting only eczema without any other symptom to 100% in those reporting to have asthma, seasonal/perennial rhinitis, and eczema together. In subjects reporting no sign or symptom at all, the prevalence of sensitization was 19%. Seasonal rhinitis was the strongest single predictor for sensitization with the highest proportion of sensitized participants in all symptom combinations (67%-100%), followed by perennial rhinitis (31%-100%), asthma (30%-100%), and eczema (18%-100%)., Conclusion: Rhinitis most often is associated with allergen sensitization, and the probability of sensitization is substantially enhanced by co-occurrence of asthma. A careful assessment of clinical signs and symptoms is important and enables the selection of patients in whom targeted diagnostic analysis and therapy is appropriate. Trial registration: retrospectively registered by the Cantonal Ethics Committee Zurich on 22.01.2016; Nr: 08-2016.

Background: Atopic allergy is a widespread disease with increasing prevalence in the second half of the twentieth century and is most often associated with clinical symptoms, like rhinoconjunctivitis, asthma or eczema. This study explored the prevalence of atopy and polysensitization in nine cohorts of Swiss medical students during the period of 2007-2015. Furthermore, the self-reported allergic symptoms, such as rhinoconjunctivitis, asthma and eczema, among students with and without atopy were assessed., Methods: Each cohort was assessed in the third study year. Students underwent an ImmunoCAP rapid test, a qualitative point-of-care test, and completed an anonymous questionnaire on age, gender and clinical symptoms including rhinoconjunctivitis, asthma and eczema. Statistical analyses assessed the overall prevalence of atopy in each group and estimated the average annual increase using a linear mixed model. We examined the frequency of occurrence of polysensitization and differences of reported symptoms among students with and without atopy., Results: Data of 1513 students (mean age 22.4-23.3 years across cohorts) in nine cohorts (median cohort size 215 interquartile range IQR 193-222) were available for analysis. Test results consistent with atopy were present in 39.9% of students. Average increase of atopy over the 9 years of observation was 2.25% (95% CI 0.18-4.31%; p = 0.037). Main drivers for this increase were the ubiquitously available allergens, house dust mite, timothy grass and birch pollen. Atopy and polysensitization were more pronounced in male students: Polysensitization also increased in the observation period. The clinical symptoms, rhinoconjunctivitis, asthma and eczema were reported by 463 (76.7%) atopic and by 141 (15.5%) non-atopic students., Conclusions: We observed a slight increase of atopy and polysensitization within 9 years of observation in Swiss medical students. The most frequent sensitization occurred with allergens with the highest chance of exposure. Rhinoconjunctivitis, asthma and eczema are a symptom complex associated with atopy but also found in non-atopic students. Trial registration retrospectively registered by the Cantonal Ethics Committee Zurich on 22.01.2016; Nr: 08-2016.

Background: Drug resistance is a major barrier to successful antiretroviral treatment (ART). Therefore, it is important to monitor time trends at a population level., Methods: We included 11 084 ART-experienced patients from the Swiss HIV Cohort Study (SHCS) between 1999 and 2013. The SHCS is highly representative and includes 72% of patients receiving ART in Switzerland. Drug resistance was defined as the presence of ≥1 major mutation in a genotypic resistance test. To estimate the prevalence of drug resistance, data for patients with no resistance test was imputed based on the patient's risk of harboring drug-resistant viruses., Results: The emergence of new drug resistance mutations declined dramatically from 401 to 23 patients between 1999 and 2013. The upper estimated prevalence limit of drug resistance among ART-experienced patients decreased from 57.0% in 1999 to 37.1% in 2013. The prevalence of 3-class resistance decreased from 9.0% to 4.4% and was always <0.4% for patients who initiated ART after 2006. Most patients actively participating in the SHCS in 2013 with drug-resistant viruses initiated ART before 1999 (59.8%). Nevertheless, in 2013, 94.5% of patients who initiated ART before 1999 had good remaining treatment options based on Stanford algorithm., Conclusions: Human immunodeficiency virus type 1 drug resistance among ART-experienced patients in Switzerland is a well-controlled relic from the era before combination ART. Emergence of drug resistance can be virtually stopped with new potent therapies and close monitoring., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

A history of infectious mononucleosis (IM), symptomatic primary infection with the Epstein Barr virus, is associated with the development of autoimmune diseases and increases the risk to develop multiple sclerosis. Here, we hypothesized that immune activation during IM triggers autoreactive immune responses. Antibody responses towards cellular antigens using a HEp-2 based indirect immunofluorescence assay and native myelin oligodendrocyte glycoprotein (MOG) using a flow cytometry-based assay were determined in 35 patients with IM and in 23 control subjects. We detected frequent immunoglobulin M (IgM) reactivity to vimentin, a major constituent of the intermediate filament family of proteins, in IM patients (27/35; 77%) but rarely in control subjects (2/23; 9%). IgG autoantibodies binding to HEp-2 cells were absent in both groups. In contrast, IgG responses to native MOG, present in up to 40% of children with inflammatory demyelinating diseases of the central nervous system (CNS), were detectable in 7/35 (20%) patients with IM but not in control subjects. Normalization of anti-vimentin IgM levels to increased total IgM concentrations during IM resulted in loss of significant differences for anti-vimentin IgM titers. Anti-MOG specific IgG responses were still detectable in a subset of three out of 35 patients with IM (9%), even after normalization to increased total IgG levels. Vimentin-specific IgM and MOG-specific IgG responses decreased following clinical resolution of acute IM symptoms. We conclude from our data that MOG-specific memory B cells are activated in subset of patients with IM.

Background: Reducing the fraction of transmissions during recent human immunodeficiency virus (HIV) infection is essential for the population-level success of "treatment as prevention"., Methods: A phylogenetic tree was constructed with 19 604 Swiss sequences and 90 994 non-Swiss background sequences. Swiss transmission pairs were identified using 104 combinations of genetic distance (1%-2.5%) and bootstrap (50%-100%) thresholds, to examine the effect of those criteria. Monophyletic pairs were classified as recent or chronic transmission based on the time interval between estimated seroconversion dates. Logistic regression with adjustment for clinical and demographic characteristics was used to identify risk factors associated with transmission during recent or chronic infection., Findings: Seroconversion dates were estimated for 4079 patients on the phylogeny, and comprised between 71 (distance, 1%; bootstrap, 100%) to 378 transmission pairs (distance, 2.5%; bootstrap, 50%). We found that 43.7% (range, 41%-56%) of the transmissions occurred during the first year of infection. Stricter phylogenetic definition of transmission pairs was associated with higher recent-phase transmission fraction. Chronic-phase viral load area under the curve (adjusted odds ratio, 3; 95% confidence interval, 1.64-5.48) and time to antiretroviral therapy (ART) start (adjusted odds ratio 1.4/y; 1.11-1.77) were associated with chronic-phase transmission as opposed to recent transmission. Importantly, at least 14% of the chronic-phase transmission events occurred after the transmitter had interrupted ART., Conclusions: We demonstrate a high fraction of transmission during recent HIV infection but also chronic transmissions after interruption of ART in Switzerland. Both represent key issues for treatment as prevention and underline the importance of early diagnosis and of early and continuous treatment., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Background: HIV surveillance requires monitoring of new HIV diagnoses and differentiation of incident and older infections. In 2008, Switzerland implemented a system for monitoring incident HIV infections based on the results of a line immunoassay (Inno-Lia) mandatorily conducted for HIV confirmation and type differentiation (HIV-1, HIV-2) of all newly diagnosed patients. Based on this system, we assessed the proportion of incident HIV infection among newly diagnosed cases in Switzerland during 2008-2013., Methods and Results: Inno-Lia antibody reaction patterns recorded in anonymous HIV notifications to the federal health authority were classified by 10 published algorithms into incident (up to 12 months) or older infections. Utilizing these data, annual incident infection estimates were obtained in two ways, (i) based on the diagnostic performance of the algorithms and utilizing the relationship 'incident = true incident + false incident', (ii) based on the window-periods of the algorithms and utilizing the relationship 'Prevalence = Incidence x Duration'. From 2008-2013, 3'851 HIV notifications were received. Adult HIV-1 infections amounted to 3'809 cases, and 3'636 of them (95.5%) contained Inno-Lia data. Incident infection totals calculated were similar for the performance- and window-based methods, amounting on average to 1'755 (95% confidence interval, 1588-1923) and 1'790 cases (95% CI, 1679-1900), respectively. More than half of these were among men who had sex with men. Both methods showed a continuous decline of annual incident infections 2008-2013, totaling -59.5% and -50.2%, respectively. The decline of incident infections continued even in 2012, when a 15% increase in HIV notifications had been observed. This increase was entirely due to older infections. Overall declines 2008-2013 were of similar extent among the major transmission groups., Conclusions: Inno-Lia based incident HIV-1 infection surveillance proved useful and reliable. It represents a free, additional public health benefit of the use of this relatively costly test for HIV confirmation and type differentiation.

Background.  Although acquired immune deficiency syndrome-associated morbidity has diminished due to excellent viral control, multimorbidity may be increasing among human immunodeficiency virus (HIV)-infected persons compared with the general population. Methods.  We assessed the prevalence of comorbidities and multimorbidity in participants of the Swiss HIV Cohort Study (SHCS) compared with the population-based CoLaus study and the primary care-based FIRE (Family Medicine ICPC-Research using Electronic Medical Records) records. The incidence of the respective endpoints were assessed among SHCS and CoLaus participants. Poisson regression models were adjusted for age, sex, body mass index, and smoking. Results.  Overall, 74 291 participants contributed data to prevalence analyses (3230 HIV-infected; 71 061 controls). In CoLaus, FIRE, and SHCS, multimorbidity was present among 26%, 13%, and 27% of participants. Compared with nonsmoking individuals from CoLaus, the incidence of cardiovascular disease was elevated among smoking individuals but independent of HIV status (HIV-negative smoking: incidence rate ratio [IRR] = 1.7, 95% confidence interval [CI] = 1.2-2.5; HIV-positive smoking: IRR = 1.7, 95% CI = 1.1-2.6; HIV-positive nonsmoking: IRR = 0.79, 95% CI = 0.44-1.4). Compared with nonsmoking HIV-negative persons, multivariable Poisson regression identified associations of HIV infection with hypertension (nonsmoking: IRR = 1.9, 95% CI = 1.5-2.4; smoking: IRR = 2.0, 95% CI = 1.6-2.4), kidney (nonsmoking: IRR = 2.7, 95% CI = 1.9-3.8; smoking: IRR = 2.6, 95% CI = 1.9-3.6), and liver disease (nonsmoking: IRR = 1.8, 95% CI = 1.4-2.4; smoking: IRR = 1.7, 95% CI = 1.4-2.2). No evidence was found for an association of HIV-infection or smoking with diabetes mellitus. Conclusions.  Multimorbidity is more prevalent and incident in HIV-positive compared with HIV-negative individuals. Smoking, but not HIV status, has a strong impact on cardiovascular risk and multimorbidity.

Unlabelled: Gene-engineered CD34(+) hematopoietic stem and progenitor cells (HSPCs) can be used to generate an HIV-1-resistant immune system. However, a certain threshold of transduced HSPCs might be required for transplantation into mice for creating an HIV-resistant immune system. In this study, we combined CCR5 knockdown by a highly efficient microRNA (miRNA) lentivector with pretransplantation selection of transduced HSPCs to obtain a rather pure population of gene engineered CD34(+) cells. Low-level transduction of HSPCs and subsequent sorting by flow cytometry yielded >70% transduced cells. Mice transplanted with these cells showed functional and persistent resistance to a CCR5-tropic HIV strain: viral load was significantly decreased over months, and human CD4(+) T cells were preserved. In one mouse, viral mutations, resulting presumably in a CXCR4-tropic strain, overcame HIV resistance. Our results suggest that HSPC-based CCR5 knockdown may lead to efficient control of HIV in vivo. We overcame a major limitation of previous HIV gene therapy in humanized mice in which only a proportion of the cells in chimeric mice in vivo are anti-HIV engineered. Our strategy underlines the promising future of gene engineering HIV-resistant CD34(+) cells that produce a constant supply of HIV-resistant progeny., Importance: Major issues in experimental long-term in vivo HIV gene therapy have been (i) low efficacy of cell transduction at the time of transplantation and (ii) transduction resulting in multiple copies of heterologous DNA in target cells. In this study, we demonstrated the efficacy of a transplantation approach with a selection step for transduced cells that allows transplantation of an enriched population of HSPCs expressing a single (low) copy of a CCR5 miRNA. Efficient maintenance of CD4(+) T cells and a low viral titer resulted only when at least 70% of the HIV target cells were genetically modified. These findings imply that clinical protocols of HIV gene therapy require a selective enrichment of genetically targeted cells because positive selection of modified cells is likely to be insufficient below this threshold. This selection approach may be beneficial not only for HIV patients but also for other patients requiring transplantation of genetically modified cells., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Detection of HIV-1 p24 antigen permits early identification of primary HIV infection and timely intervention to limit further spread of the infection. Principally, HIV screening should equally detect all viral variants, but reagents for a standardised test evaluation are limited. Therefore, we aimed to create an inexhaustible panel of diverse HIV-1 p24 antigens., Methods: We generated a panel of 43 recombinantly expressed virus-like particles (VLPs), containing the structural Gag proteins of HIV-1 subtypes A-H and circulating recombinant forms (CRF) CRF01&#95;AE, CRF02&#95;AG, CRF12&#95;BF, CRF20&#95;BG and group O. Eleven 4th generation antigen/antibody tests and five antigen-only tests were evaluated for their ability to detect VLPs diluted in human plasma to p24 concentrations equivalent to 50, 10 and 2 IU/ml of the WHO p24 standard. Three tests were also evaluated for their ability to detect p24 after heat-denaturation for immune-complex disruption, a pre-requisite for ultrasensitive p24 detection., Results: Our VLP panel exhibited an average intra-clade p24 diversity of 6.7%. Among the 4th generation tests, the Abbott Architect and Siemens Enzygnost Integral 4 had the highest sensitivity of 97.7% and 93%, respectively. Alere Determine Combo and BioRad Access were least sensitive with 10.1% and 40.3%, respectively. Antigen-only tests were slightly more sensitive than combination tests. Almost all tests detected the WHO HIV-1 p24 standard at a concentration of 2 IU/ml, but their ability to detect this input for different subtypes varied greatly. Heat-treatment lowered overall detectability of HIV-1 p24 in two of the three tests, but only few VLPs had a more than 3-fold loss in p24 detection., Conclusions: The HIV-1 Gag subtype panel has a broad diversity and proved useful for a standardised evaluation of the detection limit and breadth of subtype detection of p24 antigen-detecting tests. Several tests exhibited problems, particularly with non-B subtypes.

Background: While the numbers of hepatitis-C-virus (HCV) infections among men who have sex with men (MSM) who are co-infected with the human immunodeficiency virus (HIV) are on the rise, with vast evidence for sexual transmission of HCV in this population, concerns have also been raised regarding sexual HCV-transmission among MSM without HIV infection. Therefore, the aim of this study was to estimate the prevalence of hepatitis C among MSM without HIV diagnosis in Zurich (Switzerland)., Methods: Participants were recruited from a gay health centre and various locations such as dark rooms, saunas and cruising areas in Zurich. Participants self-completed a questionnaire assessing known and suspected risk factors for HCV-infection, and provided a blood sample for detection of past (antibodies) and present (core antigen, RNA) infections with HCV., Results: In total, 840 MSM aged 17-79 (median: 33 years) underwent HCV-testing and completed the questionnaire, among whom 19 reported living with HIV. Overall, seven tested positive for HCV-antibodies, and two were also positive for HCV core antigen and HCV-RNA-these two were immigrants, one from a country where HCV is endemic. None of the seven were aware of their infection. The seroprevalence of hepatitis C among the 821 non-HIV-diagnosed MSM was 0.37% (95%-CI: 0.12-1.69%), and one man harboured replicating virus (0.12%; 0.02-0.69%), resulting in a number needed to test of 821 to detect one active infection. Significant univariable associations of lifetime HCV-infection were found with known HIV-diagnosis (OR=72.7), being tattooed (OR=10.4), non-injection use of cocaine/amphetamines (OR=8.8), and non-Swiss origin (OR=8.5). For MSM without HIV-diagnosis, the only variable marginally associated with positive HCV-serostatus was being tattooed (OR=8.3). No significant associations were observed with reported injection drug use, unprotected anal intercourse, sexual practices that may lead to mucosal trauma, or proxy measures for group sex and lesion-prone STIs., Conclusions: Our findings suggest that in Switzerland, hepatitis C among MSM without diagnosed HIV is not more prevalent than in the general population. We found no evidence of elevated rates of sexual transmission of HCV among MSM without HIV-infection. Therefore, we currently see no reason for promoting HCV-testing among all MSM in Switzerland.

Anti-nuclear antibodies (ANA) are fundamental for the diagnosis of autoimmune diseases, and have been determined by indirect immunofluorescence assay (IIFA) for decades. As the demand for ANA testing increased, alternative techniques were developed challenging the classic IIFA. These alternative platforms differ in their antigen profiles, sensitivity and specificity, raising uncertainties regarding standardisation and interpretation of incongruent results. Therefore, an international group of experts has created recommendations for ANA testing by different methods. Two groups of experts participated in this initiative. The European autoimmunity standardization initiative representing 15 European countries and the International Union of Immunologic Societies/World Health Organization/Arthritis Foundation/Centers for Disease Control and Prevention autoantibody standardising committee. A three-step process followed by a Delphi exercise with closed voting was applied. Twenty-five recommendations for determining ANA (1-13), anti-double stranded DNA antibodies (14-18), specific antibodies (19-23) and validation of methods (24-25) were created. Significant differences between experts were observed regarding recommendations 24-25 (p<0.03). Here, we formulated recommendations for the assessment and interpretation of ANA and associated antibodies. Notably, the roles of IIFA as a reference method, and the importance of defining nuclear and cytoplasmic staining, were emphasised, while the need to incorporate alternative automated methods was acknowledged. Various approaches to overcome discrepancies between methods were suggested of which an improved bench-to-bedside communication is of the utmost importance. These recommendations are based on current knowledge and can enable harmonisation of local algorithms for testing and evaluation of ANA and related autoantibodies. Last but not least, new more appropriate terminologies have been suggested.

Background : Alongside hazel, alder and birch pollen allergies, ash pollen allergy is a relevant cause of hay fever during spring in the European region. For some considerable time, ash pollen allergy was not routinely investigated and its clinical relevance may well have been underestimated, particularly since ash and birch tree pollination times are largely the same. Ash pollen extracts are not yet well standardized and diagnosis is therefore sometimes unreliable. Olive pollen, on the other hand, is strongly cross-reactive with ash pollen and is apparently better standardized. Therefore, the main allergen of olive pollen, Ole e 1, has been postulated as a reliable alternative for the detection of ash pollen sensitization. Methods : To determine to what extent specific IgE against Ole e 1 in patients with ash pollen allergy is relevant, we included 183 subjects with ash pollen allergy displaying typical symptoms in March/April and positive skin prick test specific IgE against Ole e 1 (t224) and ash pollen (t25) and various birch allergens (Bet v 1, Bet v 2/v 4) in a retrospective study. Results : A significant correlation was seen between specific IgE against Ole e 1 and ash pollen, but also to a slightly lesser extent between IgE against Ole e 1 and skin prick test with ash pollen, the latter being even higher than IgE and skin prick test both with ash pollen. No relevant correlation was found with birch pollen allergens, demonstrating the very limited cross-reactivity between ash and birch pollen. Conclusion : It appears appropriate to determine specific IgE against Ole e 1 instead of IgE against ash pollen to detect persons with ash pollen allergy. Our findings may also support the idea of using possibly better standardized or more widely available olive pollen extracts instead of ash pollen extract for allergen-specific immunotherapy.

Background: Tests for recent infections (TRIs) are important for HIV surveillance. We have shown that a patient's antibody pattern in a confirmatory line immunoassay (Inno-Lia) also yields information on time since infection. We have published algorithms which, with a certain sensitivity and specificity, distinguish between incident (< = 12 months) and older infection. In order to use these algorithms like other TRIs, i.e., based on their windows, we now determined their window periods., Methods: We classified Inno-Lia results of 527 treatment-naïve patients with HIV-1 infection < = 12 months according to incidence by 25 algorithms. The time after which all infections were ruled older, i.e. the algorithm's window, was determined by linear regression of the proportion ruled incident in dependence of time since infection. Window-based incident infection rates (IIR) were determined utilizing the relationship 'Prevalence = Incidence x Duration' in four annual cohorts of HIV-1 notifications. Results were compared to performance-based IIR also derived from Inno-Lia results, but utilizing the relationship 'incident = true incident + false incident' and also to the IIR derived from the BED incidence assay., Results: Window periods varied between 45.8 and 130.1 days and correlated well with the algorithms' diagnostic sensitivity (R(2) = 0.962; P<0.0001). Among the 25 algorithms, the mean window-based IIR among the 748 notifications of 2005/06 was 0.457 compared to 0.453 obtained for performance-based IIR with a model not correcting for selection bias. Evaluation of BED results using a window of 153 days yielded an IIR of 0.669. Window-based IIR and performance-based IIR increased by 22.4% and respectively 30.6% in 2008, while 2009 and 2010 showed a return to baseline for both methods., Conclusions: IIR estimations by window- and performance-based evaluations of Inno-Lia algorithm results were similar and can be used together to assess IIR changes between annual HIV notification cohorts.

Background & Aims: Little is known about the pathogenic mechanisms of autoimmune pancreatitis (AIP), an increasingly recognized, immune-mediated form of chronic pancreatitis. Current treatment options are limited and disease relapse is frequent. We investigated factors that contribute to the development of AIP and new therapeutic strategies., Methods: We used quantitative polymerase chain reaction, immunohistochemical, and enzyme-linked immunosorbent analyses to measure the expression of cytokines and chemokines in tissue and serum samples from patients with and without AIP. We created a mouse model of human AIP by overexpressing lymphotoxin (LT)α and β specifically in acinar cells (Ela1-LTab mice)., Results: Messenger RNA levels of LTα and β were increased in pancreatic tissues from patients with AIP, compared with controls, and expression of chemokines (CXCL13, CCL19, CCL21, CCL1, and B-cell-activating factor) was increased in pancreatic and serum samples from patients. Up-regulation of these factors was not affected by corticosteroid treatment. Acinar-specific overexpression of LTαβ (Ela1-LTαβ) in mice led to an autoimmune disorder with various features of AIP. Chronic inflammation developed only in the pancreas but was sufficient to cause systemic autoimmunity. Acinar-specific overexpression of LTαβ did not cause autoimmunity in mice without lymphocytes (Ela1-LTab/Rag1(-/-)); moreover, lack of proinflammatory monocytes (Ela1-LTab/Ccr2(-/-)) failed to prevent AIP but prevented early pancreatic tissue damage. Administration of corticosteroids reduced pancreatitis but did not affect production of autoantibodies, such as antipancreatic secretory trypsin inhibitor in Ela1-LTab mice. In contrast, inhibition of LTβR signaling reduced chemokine expression, renal immune-complex deposition, and features of AIP in Ela1-LTab mice., Conclusions: Overexpression of LTαβ specifically in acinar cells of mice causes features of AIP. Reagents that neutralize LTβR ligands might be used to treat patients with AIP., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Serologic testing algorithms for recent HIV seroconversion (STARHS) provide important information for HIV surveillance. We have previously demonstrated that a patient's antibody reaction pattern in a confirmatory line immunoassay (INNO-LIA™ HIV I/II Score) provides information on the duration of infection, which is unaffected by clinical, immunological and viral variables. In this report we have set out to determine the diagnostic performance of Inno-Lia algorithms for identifying incident infections in patients with known duration of infection and evaluated the algorithms in annual cohorts of HIV notifications., Methods: Diagnostic sensitivity was determined in 527 treatment-naive patients infected for up to 12 months. Specificity was determined in 740 patients infected for longer than 12 months. Plasma was tested by Inno-Lia and classified as either incident (< = 12 m) or older infection by 26 different algorithms. Incident infection rates (IIR) were calculated based on diagnostic sensitivity and specificity of each algorithm and the rule that the total of incident results is the sum of true-incident and false-incident results, which can be calculated by means of the pre-determined sensitivity and specificity., Results: The 10 best algorithms had a mean raw sensitivity of 59.4% and a mean specificity of 95.1%. Adjustment for overrepresentation of patients in the first quarter year of infection further reduced the sensitivity. In the preferred model, the mean adjusted sensitivity was 37.4%. Application of the 10 best algorithms to four annual cohorts of HIV-1 notifications totalling 2'595 patients yielded a mean IIR of 0.35 in 2005/6 (baseline) and of 0.45, 0.42 and 0.35 in 2008, 2009 and 2010, respectively. The increase between baseline and 2008 and the ensuing decreases were highly significant. Other adjustment models yielded different absolute IIR, although the relative changes between the cohorts were identical for all models., Conclusions: The method can be used for comparing IIR in annual cohorts of HIV notifications. The use of several different algorithms in combination, each with its own sensitivity and specificity to detect incident infection, is advisable as this reduces the impact of individual imperfections stemming primarily from relatively low sensitivities and sampling bias.

Unlabelled: Genetic polymorphisms near IL28B are associated with spontaneous and treatment-induced clearance of hepatitis C virus (HCV), two processes that require the appropriate activation of the host immune responses. Intrahepatic inflammation is believed to mirror such activation, but its relationship with IL28B polymorphisms has yet to be fully appreciated. We analyzed the association of IL28B polymorphisms with histological and follow-up features in 2335 chronically HCV-infected Caucasian patients. Assessable phenotypes before any antiviral treatment included necroinflammatory activity (n = 1,098), fibrosis (n = 1,527), fibrosis progression rate (n = 1,312), and hepatocellular carcinoma development (n = 1,915). Associations of alleles with the phenotypes were evaluated by univariate analysis and multivariate logistic regression, accounting for all relevant covariates. The rare G allele at IL28B marker rs8099917-previously shown to be at risk of treatment failure-was associated with lower activity (P = 0.04), lower fibrosis (P = 0.02) with a trend toward lower fibrosis progression rate (P = 0.06). When stratified according to HCV genotype, most significant associations were observed in patients infected with non-1 genotypes (P = 0.003 for activity, P = 0.001 for fibrosis, and P = 0.02 for fibrosis progression rate), where the odds ratio of having necroinflammation or rapid fibrosis progression for patients with IL28B genotypes TG or GG versus TT were 0.48 (95% confidence intervals 0.30-0.78) and 0.56 (0.35-0.92), respectively. IL28B polymorphisms were not predictive of the development of hepatocellular carcinoma., Conclusion: In chronic hepatitis C, IL28B variants associated with poor response to interferon therapy may predict slower fibrosis progression, especially in patients infected with non-1 HCV genotypes., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Anti-Golgi antibodies are rare autoantibodies that have been described in systemic autoimmune diseases. Not all Golgi auto-antigens are known. The objective of this study was to identify a novel auto-antigen associated with anti-Golgi immune reactivity. Sera from a patient with Golgi immune reactivity and from a control individual were used for Western blotting after 2-dimensional gel separation of a rat Golgi-enriched extract. Betaine homocysteine S-methyltransferase 1 (BHMT1) was identified as an auto-antigen by MALDI-TOF/TOF mass spectrometry. Using human recombinant BHMT1, a strong positive blotting signal was obtained with serum from the patient but not from a control. Pre-absorption of the serum sample with reactivity to BHMT1 with recombinant human BHMT1 resulted in decreased reactivity on Western blotting and in disappearance of the Golgi-like pattern on indirect immunofluorescence. Using immunocytochemistry, we confirmed the subcellular localization of BHMT1 to the Golgi apparatus. Antibodies to BHMT1 were found in four of 80 samples with a Golgi-pattern on indirect immunofluorescence. The antibodies were not associated with a specific clinical condition. We identified BHMT1 as a novel auto-antigen associated with anti-Golgi immune reactivity., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Humanized mice generate a lymphoid system of human origin subsequent to transplantation of human CD34+ cells and thus are highly susceptible to HIV infection. Here we examined the efficacy of antiretroviral treatment (ART) when added to food pellets, and of long-acting (LA) antiretroviral compounds, either as monotherapy or in combination. These studies shall be inspiring for establishing a gold standard of ART, which is easy to administer and well supported by the mice, and for subsequent studies such as latency. Furthermore, they should disclose whether viral breakthrough and emergence of resistance occurs similar as in HIV-infected patients when ART is insufficient., Methods/principal Findings: NOD/shi-scid/γ(c)null (NOG) mice were used in all experimentations. We first performed pharmacokinetic studies of the drugs used, either added to food pellets (AZT, TDF, 3TC, RTV) or in a LA formulation that permitted once weekly subcutaneous administration (TMC278: non-nucleoside reverse transcriptase inhibitor, TMC181: protease inhibitor). A combination of 3TC, TDF and TMC278-LA or 3TC, TDF, TMC278-LA and TMC181-LA suppressed the viral load to undetectable levels in 15/19 (79%) and 14/14 (100%) mice, respectively. In successfully treated mice, subsequent monotherapy with TMC278-LA resulted in viral breakthrough; in contrast, the two LA compounds together prevented viral breakthrough. Resistance mutations matched the mutations most commonly observed in HIV patients failing therapy. Importantly, viral rebound after interruption of ART, presence of HIV DNA in successfully treated mice and in vitro reactivation of early HIV transcripts point to an existing latent HIV reservoir., Conclusions/significance: This report is a unique description of multiple aspects of HIV infection in humanized mice that comprised efficacy testing of various treatment regimens, including LA compounds, resistance mutation analysis as well as viral rebound after treatment interruption. Humanized mice will be highly valuable for exploring the antiviral potency of new compounds or compounds targeting the latent HIV reservoir.

Background: To perform a comprehensive study on the relationship between vitamin D metabolism and the response to interferon-α-based therapy of chronic hepatitis C., Methodology/principal Findings: Associations between a functionally relevant polymorphism in the gene encoding the vitamin D 1α-hydroxylase (CYP27B1-1260 rs10877012) and the response to treatment with pegylated interferon-α (PEG-IFN-α) and ribavirin were determined in 701 patients with chronic hepatitis C. In addition, associations between serum concentrations of 25-hydroxyvitamin D(3) (25[OH]D(3)) and treatment outcome were analysed. CYP27B1-1260 rs10877012 was found to be an independent predictor of sustained virologic response (SVR) in patients with poor-response IL28B genotypes (15% difference in SVR for rs10877012 genotype AA vs. CC, p = 0.02, OR = 1.52, 95% CI = 1.061-2.188), but not in patients with favourable IL28B genotype. Patients with chronic hepatitis C showed a high prevalence of vitamin D insufficiency (25[OH]D(3)<20 ng/mL) during all seasons, but 25(OH)D(3) serum levels were not associated with treatment outcome., Conclusions/significance: Our study suggests a role of bioactive vitamin D (1,25[OH](2)D(3), calcitriol) in the response to treatment of chronic hepatitis C. However, serum concentration of the calcitriol precursor 25(OH)D(3) is not a suitable predictor of treatment outcome.

Background: Serologic testing algorithms for recent HIV seroconversion (STARHS) provide important information for HIV surveillance. We have shown that a patient's antibody reaction in a confirmatory line immunoassay (INNO-LIA HIV I/II Score, Innogenetics) provides information on the duration of infection. Here, we sought to further investigate the diagnostic specificity of various Inno-Lia algorithms and to identify factors affecting it., Methods: Plasma samples of 714 selected patients of the Swiss HIV Cohort Study infected for longer than 12 months and representing all viral clades and stages of chronic HIV-1 infection were tested blindly by Inno-Lia and classified as either incident (up to 12 m) or older infection by 24 different algorithms. Of the total, 524 patients received HAART, 308 had HIV-1 RNA below 50 copies/mL, and 620 were infected by a HIV-1 non-B clade. Using logistic regression analysis we evaluated factors that might affect the specificity of these algorithms., Results: HIV-1 RNA < 50 copies/mL was associated with significantly lower reactivity to all five HIV-1 antigens of the Inno-Lia and impaired specificity of most algorithms. Among 412 patients either untreated or with HIV-1 RNA ≥ 50 copies/mL despite HAART, the median specificity of the algorithms was 96.5% (range 92.0-100%). The only factor that significantly promoted false-incident results in this group was age, with false-incident results increasing by a few percent per additional year. HIV-1 clade, HIV-1 RNA, CD4 percentage, sex, disease stage, and testing modalities exhibited no significance. Results were similar among 190 untreated patients., Conclusions: The specificity of most Inno-Lia algorithms was high and not affected by HIV-1 variability, advanced disease and other factors promoting false-recent results in other STARHS. Specificity should be good in any group of untreated HIV-1 patients.

Aims: Nanomagnets with metal cores have recently been shown to be promising candidates for magnetic drug delivery due to higher magnetic moments compared with commonly used metal oxides. Successful application strongly relies on a safe implementation that goes along with detailed knowledge of interactions and effects that nanomagnets might impart once entering the body., Materials & Methods: In this work, we put a particular focus on the interactions of ultra-strong metal nanomagnets (≥ three-times higher in magnetization compared with oxide nanoparticles) within the vascular compartment. Individual aspects of possible effects are addressed, including interactions with the coagulation cascade, the complement system, phagocytes and toxic or inflammatory reactions both by blood and endothelial cells in response to nanomagnet exposure., Results: We show that carbon-coated metal nanomagnets are well-tolerated by cells of the vascular compartment and have only minor effects on blood coagulation., Conclusion: These findings provide the fundament to initiate successful first in vivo evaluations opening metal nanomagnets with improved magnetic properties to fascinating applications in nanomedicine.

Background: Pulmonary-renal syndrome associated with anti-glomerular basement membrane (GBM) antibodies, also known as Goodpasture's syndrome, is a rare but acute and life-threatening condition. One third of patients presenting as anti-GBM antibody positive pulmonary-renal syndrome or rapidly progressive glomerulonephritis are also tested positive for anti-neutrophil cytoplasmic antibodies (ANCA). Whilst anti-GBM disease is considered a non-relapsing condition, the long-term course of double-positive patients is less predictable., Case Presentation: We report a patient with such dual positivity, who presented with pulmonary hemorrhage, crescentic glomerulonephritis and membranous nephropathy. Plasmapheresis in combination with immunosuppressive therapy led to a rapid remission but the disease relapsed after two years. The serum of the patient was tested positive for antibodies to human lysosomal membrane protein 2 (hLAMP2), a novel autoantigen in patients with active small-vessel vasculitis (SVV). The anti-hLAMP2 antibody levels correlated positively with clinical disease activity in this patient., Conclusion: We hypothesize that this antibody may indicate a clinical course similar to ANCA-associated vasculitis in double-positive patients. However, this needs to be confirmed on comprehensive patient cohorts.

Aortic dilatation/dissection (AD) can occur spontaneously or in association with genetic syndromes, such as Marfan syndrome (MFS; caused by FBN1 mutations), MFS type 2 and Loeys-Dietz syndrome (associated with TGFBR1/TGFBR2 mutations), and Ehlers-Danlos syndrome (EDS) vascular type (caused by COL3A1 mutations). Although mutations in FBN1 and TGFBR1/TGFBR2 account for the majority of AD cases referred to us for molecular genetic testing, we have obtained negative results for these genes in a large cohort of AD patients, suggesting the involvement of additional genes or acquired factors. In this study we assessed the effect of COL3A1 deletions/duplications in this cohort. Multiplex ligation-dependent probe amplification (MLPA) analysis of 100 unrelated patients identified one hemizygous deletion of the entire COL3A1 gene. Subsequent microarray analyses and sequencing of breakpoints revealed the deletion size of 3,408,306 bp at 2q32.1q32.3. This deletion affects not only COL3A1 but also 21 other known genes (GULP1, DIRC1, COL5A2, WDR75, SLC40A1, ASNSD1, ANKAR, OSGEPL1, ORMDL1, LOC100129592, PMS1, MSTN, C2orf88, HIBCH, INPP1, MFSD6, TMEM194B, NAB1, GLS, STAT1, and STAT4), mutations in three of which (COL5A2, SLC40A1, and MSTN) have also been associated with an autosomal dominant disorder (EDS classical type, hemochromatosis type 4, and muscle hypertrophy). Physical and laboratory examinations revealed that true haploinsufficiency of COL3A1, COL5A2, and MSTN, but not that of SLC40A1, leads to a clinical phenotype. Our data not only emphasize the impact/role of COL3A1 in AD patients but also extend the molecular etiology of several disorders by providing hitherto unreported evidence for true haploinsufficiency of the underlying gene.

The contribution of B cells to the pathology of Omenn syndrome and leaky severe combined immunodeficiency (SCID) has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a homozygous Rag1 S723C mutation that impairs, but does not abrogate, V(D)J recombination activity. In spite of a severe block at the pro-B cell stage and profound B cell lymphopenia, significant serum levels of immunoglobulin (Ig) G, IgM, IgA, and IgE and a high proportion of Ig-secreting cells were detected in mut/mut mice. Antibody responses to trinitrophenyl (TNP)-Ficoll and production of high-affinity antibodies to TNP-keyhole limpet hemocyanin were severely impaired, even after adoptive transfer of wild-type CD4(+) T cells. Mut/mut mice produced high amounts of low-affinity self-reactive antibodies and showed significant lymphocytic infiltrates in peripheral tissues. Autoantibody production was associated with impaired receptor editing and increased serum B cell-activating factor (BAFF) concentrations. Autoantibodies and elevated BAFF levels were also identified in patients with Omenn syndrome and leaky SCID as a result of hypomorphic RAG mutations. These data indicate that the stochastic generation of an autoreactive B cell repertoire, which is associated with defects in central and peripheral checkpoints of B cell tolerance, is an important, previously unrecognized, aspect of immunodeficiencies associated with hypomorphic RAG mutations.

Hypomorphic RAG mutations, leading to limited V(D)J rearrangements, cause Omenn syndrome (OS), a peculiar severe combined immunodeficiency associated with autoimmune-like manifestations. Whether B cells play a role in OS pathogenesis is so far unexplored. Here we report the detection of plasma cells in lymphoid organs of OS patients, in which circulating B cells are undetectable. Hypomorphic Rag2(R229Q) knock-in mice, which recapitulate OS, revealed, beyond severe B cell developmental arrest, a normal or even enlarged compartment of immunoglobulin-secreting cells (ISC). The size of this ISC compartment correlated with increased expression of Blimp1 and Xbp1, and these ISC were sustained by elevated levels of T cell derived homeostatic and effector cytokines. The detection of high affinity pathogenic autoantibodies toward target organs indicated defaults in B cell selection and tolerance induction. We hypothesize that impaired B cell receptor (BCR) editing and a serum B cell activating factor (BAFF) abundance might contribute toward the development of a pathogenic B cell repertoire in hypomorphic Rag2(R229Q) knock-in mice. BAFF-R blockade reduced serum levels of nucleic acid-specific autoantibodies and significantly ameliorated inflammatory tissue damage. These findings highlight a role for B cells in OS pathogenesis.

Bacterial translocation from the gut and subsequent immune activation are hallmarks of HIV infection and are thought to determine disease progression. Intestinal barrier integrity is impaired early in acute retroviral infection, but levels of plasma lipopolysaccharide (LPS), a marker of bacterial translocation, increase only later. We examined humanized mice infected with HIV to determine if disruption of the intestinal barrier alone is responsible for elevated levels of LPS and if bacterial translocation increases immune activation. Treating uninfected mice with dextran sodium sulfate (DSS) induced bacterial translocation, but did not result in elevated plasma LPS levels. DSS-induced translocation provoked LPS elevation only when phagocytic cells were depleted with clodronate liposomes (clodrolip). Macrophages of DSS-treated, HIV-negative mice phagocytosed more LPS ex vivo than those of control mice. In HIV-infected mice, however, LPS phagocytosis was insufficient to clear the translocated LPS. These conditions allowed higher levels of plasma LPS and CD8+ cell activation, which were associated with lower CD4+/CD8+ cell ratios and higher viral loads. LPS levels reflect both intestinal barrier and LPS clearance. Macrophages are essential in controlling systemic bacterial translocation, and this function might be hindered in chronic HIV infection.

Rectal transmission is one of the main routes of infection by human immunodeficiency virus type 1 (HIV-1). To efficiently study transmission mechanisms and prevention strategies, a small animal model permissive for rectal transmission of HIV is mandatory. We tested the susceptibility of RAG2(-/-)gamma(c)(-/-) mice transplanted with human cord blood hematopoietic stem cells to rectal infection with HIV. We rectally exposed these humanized mice to cell-free and cell-associated HIV. All mice remained HIV negative as assessed by plasma viral load. The same mice infected intraperitoneally showed high levels of HIV replication. In the gut-associated lymphatic tissue, we found disproportionately smaller numbers of human cells than in other lymphoid organs. This finding may explain the observed resistance to rectal transmission of HIV. To increase the numbers of local HIV target cells and the likelihood of HIV transmission, we treated mice with different proinflammatory stimuli: local application of interleukin-1beta, addition of seminal plasma to the inoculum, or induction of colitis with dextran sodium sulfate. These procedures attracted some human leukocytes, but the transmission rate was still very low. The humanized mice showed low levels of human engraftment in the intestinal tract and seem to be resistant to rectal transmission of HIV, and thus they are an unsuitable model for this application.

Autoantibodies play a key role in diagnostic laboratories as markers of autoimmune diseases. In addition to their role as markers they mediate diverse effects in vivo. Autoantibodies with protective effect have been described. Natural protective IgM autoantibodies against tumour-antigens of malignant cells or their precursors may contribute to increased survival rates of carcinoma patients. In a mouse model of systemic lupus erythematosus it has been shown that anti-dsDNA IgM autoantibodies protect from glomerular damage. In contrast, a direct pathogenic role of autoantibodies has been well established e.g. in myasthenia gravis or in Goodpasture syndrome. Similarly autoantibodies against SSA Ro52 are detrimental in neonatal lupus erythematosus with congenital heart block. Moreover, putatively protective autoantibodies may become pathogenic during the course of the disease such as the onconeuronal autoantibodies whose pathogenicity depends on their compartmentalisation. In patients with paraneoplastic syndromes tumour cells express proteins that are also naturally present in the brain. Anti-tumour autoantibodies which temporarily suppress tumour growth can provoke an autoimmune attack on neurons once having crossed the blood-brain barrier and cause specific neurological symptoms. Only a restricted number of autoantibodies are useful follow-up markers for the effectiveness of treatment in autoimmune diseases. Certain autoantibodies hold prognostic value and appear years or even decades before the diagnosis of disease such as the antimitochondrial antibodies in primary biliary cirrhosis or anti-citrullinated protein (CCP)-antibodies in rheumatoid arthritis. It is crucial to know whether the autoantibodies in question recognise linear or conformational epitopes in order to choose the appropriate detection methods. Indirect immunofluorescence microscopy remains a very useful tool for confirmation of results of commercially available immunoassays and for detection of special and rare autoantibodies that otherwise often remain undetected. Standardisation of autoimmune diagnostics is still underway and requires joint efforts by laboratories, clinicians and industry.

Many biomarkers are currently used to monitor patients in clinical studies. Technologies which evaluate, validate and monitor biomarkers in a cost effective and efficient manner are a necessity. Here we describe the development, validation and implementation of a protein microarray platform for the quantitative and simultaneous analysis of six proteins: IL-1beta, IL-1ra, IL-6, IL-8, MCP-1 and TNFalpha. The platform utilizes a 96-well plate as a solid support on which antibodies are immobilized using non-contact piezoelectric printing. The reaction is based on a sandwich ELISA and the signal is quantified by chemiluminescence with a CCD camera. The robustness and reproducibility of the methodology was investigated using the Food and Drug Administration (FDA) regulatory guidelines for pharmacokinetic assay validation, in which a spike-recovery validation test was elaborated and run over 3 days. The method was shown to be both quantitative and reproducible, with assay accuracy between 70% and 130%, and an assay precision of less than 30%. In addition, protein microarray performance was compared with the classical ELISA approach. Sera collected from a total of 78 individuals were assayed using both approaches. Correlation coefficients (R2) between the two technologies were calculated for each of the analytes: 0.90 for IL-1beta, 0.60 for IL-1ra, 0.93 for IL-6, 0.96 for IL-8, 0.94 for MCP-1 and 0.95 for TNFalpha. The results obtained demonstrate the applicability of this protein microarray for quantitative and simultaneous analysis of IL-1beta, IL-1ra, IL-6, IL-8, MCP-1 and TNFalpha in clinical samples.

Migration of smooth muscle cells from media to intima is critical for the development of neointimal thickening after balloon catheter injury of the rat carotid artery. The present experiments were designed to identify molecules expressed by smooth muscle cells migrating in vivo in the injured artery. Cell migration was maximized by infusing recombinant platelet-derived growth factor-BB (PDGF-BB) after a minimal filament denudation of the rat carotid artery, whereas cell proliferation was minimized by injecting an antibody against basic fibroblast growth factor (bFGF). This treatment caused an eightfold increase in smooth muscle cell migration into the intima but only a twofold increase in intimal smooth muscle cell replication rates. Differential display screening was used to isolate cDNAs that were overexpressed in the injured PDGF-BB-treated versus unmanipulated rat carotids. One of the clones isolated hybridized to a 4.2-kb mRNA species and shared 90% sequence homology to mouse alpha 1 type VIII collagen. Northern and Western blots confirmed overexpression of type VIII collagen in the injured PDGF-BB-treated vessels. In a separate series of experiments, we performed filament denudation injury and administered antibodies to inhibit the actions of endogenous bFGF and PDGF-BB, thereby decreasing smooth muscle cell migration, and found that type VIII collagen mRNA expression varied with migration. Using a different arterial injury model (balloon catheter injury), we showed that expression of type VIII collagen was maximal 2 to 4 days after injury, in coincidence with cell migration from the media to the intima. This molecule constitutes an important component of smooth muscle cell response to vessel injury and may play an important functional role in mediating migration.

Laminin, a major structural glycoprotein complex of basement membranes has been found to be modulated by angiotensin II in vitro and in vivo. In cultures of aortic organoids and vascular smooth muscle cells, expression of laminin was stimulated by exogenous vasoconstrictor peptide. Stimulation of laminin protein and mRNA expression was observed for both laminin B1/B2-chains and an unknown laminin heavy chain. Compared with PYS-2 cells, a mouse teratocarcinoma cell line which constitutively expresses a 10-kb mRNA transcript for 'classical' laminin A-chain, cultured vascular smooth muscle cells (VSMC) did not express a corresponding mRNA. However, cultured VSMC were found to express laminin A-chain-related mRNAs of approximately 1.8 kb and approximately 3.8 kb, respectively. The 1.8-kb species of transcript was expressed in a constitutive manner, whereas the 3.8-kb mRNA was found to be regulated by angiotensin II. Laminin complexes secreted by cultured cells contained a approximately 300 kD heavy chain which did not immunoreact with immunoreagents raised against either the classical laminin complex secreted by EHS tumor cells or the merosin heavy chain. The putative A-chain analogue possibly represents a new form of a tissue-specific laminin heavy chain, distinct from the A- and M-chains thus far described. Translation products encoded by the A-chain-related transcripts of cultured smooth muscle cells could not be specified using currently available antibodies. The putative protein(s) is speculated to contain the biological features of the N-terminus of the laminin A-chain, namely self-assembly and association with collagen type IV.

Endothelin-1 (ET-1), a vasoconstrictor peptide produced by endothelial and vascular smooth muscle cells (VSMC) might play a role in vascular remodelling. To investigate the proposed 'mitogenic' potential of ET-1, we examined the effects of chronic exposure of VSMC to ET-1 on cell cycle, growth/proliferation and differentiation under essentially mitogen-free culture conditions. Bulk cultures of thoracic aortic VSMC of spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats, although exhibiting genetically determined differences in growth/proliferation (due to shortened G1 and G2 phases in SHR VSMC), respond in a similar manner to ET-1 exposure: long-term exposure (12-15 days) of VSMC from both sources to ET-1 in nonmitogenic medium did not promote cycling of cells. On the contrary, ET-1 attenuated the cycling of VSMC which had already cycled beyond the S phase. For cells which had not cycled beyond the S phase, ET-1 interrupted progression through the cell cycle at the late G1/early S phase. The specific ability of SHR VSMC to grow in mitogen-free medium was abolished by ET-1, most likely via down-regulation of platelet-derived growth factor (PDGF)-alpha receptors. Subsequent to ET-1 exposure, VSMC expressed increased levels of mRNA and protein for smooth-muscle-specific alpha-actin. However, expression of smooth muscle alpha-actin did not predominate over beta-actin as observed for adult contractile VSMC in vivo. The ET-1-induced expression of smooth-muscle-specific alpha-actin mRNA was dose dependent (EC50 approx. 2 x 10(-9) M), and alpha-actin protein expressed was associated with organized actin fibers.

In a prospective randomized trial the effect of prolonged antithrombotic treatment with anticoagulants or antiplatelet drugs (50 mg aspirin + 400 mg dipyridamole daily) on late bypass-graft occlusion was studied. After 3 months active treatment was replaced by placebo in half of the patients. Between the angiographic checkups 2 weeks and 12 months postoperatively, 28/330 (8%) new graft occlusions had occurred on continued therapy, versus 44/319 (14%) on placebo (p = 0.03). This difference was most pronounced in individual grafts (6% vs 12%, p = 0.01), so that fewer patients with 12 months' active therapy had at least one occluded graft (22% versus 32%, p = 0.08). These findings suggest that antithrombotic treatment should not be halted 3 months after CABG surgery but should be continued for at least one year and possibly longer.

To validate a new portable scintillation probe (VEST) for determination of LV function, 68 patients were studied after standard radionuclide angiography (RNA). Reproducibility of VEST LV-ejection fraction was good (rest r = 0.93; exercise r = 0.96) with unchanged probe position, somewhat less good after probe repositioning (r = 0.84) and correlated with RNA (rest r = 0.79; exercise r = 0.83). Long term measurements in 7 patients showed valid data for up to 12 hours. Combined with simultaneous two lead ECG recording VEST seems suitable for detection of transient changes of LV function such as occur during ischemia or due to drug treatment.