Medizin - Open Access LMU - Teil 15/22 Archiv

Background: Restless legs syndrome (RLS) is associated with common variants in three intronic and intergenic regions in MEIS1, BTBD9, and MAP2K5/LBXCOR1 on chromosomes 2p, 6p and 15q.Methods: Our study investigated these variants in 649 RLS patients and 1230 controls from the Czech Republic (290 cases and 450 controls), Austria (269 cases and 611 controls) and Finland (90 cases and 169 controls). Ten single nucleotide polymorphisms (SNPs) within the three genomic regions were selected according to the results of previous genome-wide scans. Samples were genotyped using Sequenom platforms.Results: We replicated associations for all loci in the combined samples set (rs2300478 in MEIS1, p = 1.26×10-5, odds ratio (OR) = 1.47, rs3923809 in BTBD9, p = 4.11×10-5, OR = 1.58 and rs6494696 in MAP2K5/LBXCOR1, p = 0.04764, OR = 1.27). Analysing only familial cases against all controls, all three loci were significantly associated. Using sporadic cases only, we could confirm the association only with BTBD9.Conclusion: Our study shows that variants in these three loci confer consistent disease risks in patients of European descent. Among the known loci, BTBD9 seems to be the most consistent in its effect on RLS across populations and is also most independent of familial clustering.

Background: Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterised by accumulation of activated (myo)fibroblasts and excessive extracellular matrix deposition. The enhanced accumulation of (myo)fibroblasts may be attributed, in part, to the process of transforming growth factor \textgreekb1 (TGF\textgreekb1)-induced epithelial--mesenchymal transition (EMT), the phenotypic switching of epithelial to fibroblast-like cells. Although alveolar epithelial type II (ATII) cells have been shown to undergo EMT, the precise mediators and mechanisms remain to be resolved. The objective of this study is to investigate the role of SNAI transcription factors in the process of EMT and in IPF.Methods: Using quantitative reverse transcription-PCR (RT-PCR), immunofluorescence, immunohistochemistry, western blotting, as well as gain- and loss-of-function studies and functional assays, the role of SNAI1 and SNAI2 in TGF\textgreekb1-induced EMT in ATII cells in vitro was assessed; and the expression of SNAI transcription factors was analysed in experimental and human IPF in vivo.Results: TGF\textgreekb1 treatment increased the expression and nuclear accumulation of SNAI1 and SNAI2, in concert with induction of EMT in ATII cells. SNAI overexpression was sufficient to induce EMT, and small interfering RNA (siRNA)-mediated SNAI depletion attenuated TGF\textgreekb1-induced ATII cell migration and EMT. SNAI expression was elevated in experimental and human IPF and localised to hyperplastic ATII cells in vivo.Conclusions: The results demonstrate that TGF\textgreekb1-induced EMT in ATII cells is essentially controlled by the expression and nuclear translocation of SNAI transcription factors. Increased SNAI1 and SNAI2 expression in experimental and human IPF in vivo suggests that SNAI-mediated EMT may contribute to the fibroblast pool in idiopathic pulmonary fibrosis.

Background: Little is known about the asthma candidate gene neuropeptide S receptor 1 (NPSR1) in relation to environmental exposures, but recent evidences suggest its role as an effect modifier.Objectives: To explore the interaction between NPSR1 polymorphisms and environmental exposures related to farming lifestyle and to study the in vitro effects of lipopolysaccharide (LPS) stimulation on NPSR1 expression levels.Methods: We studied 3113 children from PARSIFAL, a European cross-sectional study on environmental/lifestyle factors and childhood allergy, partly focused on children brought up on a farm. Information on exposures and outcomes was primarily obtained from parental questionnaires. Seven tagging polymorphisms were analysed in a conserved haplotype block of NPSR1. Multivariate logistic regression was used to evaluate a multiplicative model of interaction. NPSR1 protein and messenger RNA (mRNA) levels in monocytes were measured after LPS stimulation by fluorescence activated cell sorting (FACS) and quantitative real-time polymerase chain reaction (PCR).Results: A strong interaction was seen between current regular contact to farm animals and several NPSR1 polymorphisms, particularly rs323922 and rs324377 (p

Germany has been an immigration country since the early 1950s. In December 2007, 6.7 million non-German citizens lived in the country. However, the total number of citizens with a migration background is 15–20 million, about 9 million of whom come from countries where sickle cell disease and thalassaemias are frequent. In a country with 82 million inhabitants health authorities are not worried by the presence of probably 1000–1500 sickle cell and 450 transfusion-dependent thalassaemia patients, and therefore no screening or preventive measures have been taken so far on a national scale. There are plans for a pilot project (1 year) to screen all newborns for sickle cell disease in obstetric hospitals in 4–5 cities with more than 20% migrants. Funding and lack of an infrastructure to provide counselling are major problems.

Traditionally, Crohn's disease has been associated with a Th1 cytokine profile, while Th2 cytokines are modulators of ulcerative colitis. This concept has been challenged by the description of tolerising regulatory T cells (Treg) and by proinflammatory Th17 cells, a novel T cell population characterised by the master transcription factor ROR\textgreekgt, the surface markers IL23R and CCR6, and by production of the proinflammatory cytokines IL17A, IL17F, IL21, IL22 and IL26, and the chemokine CCL20. Th17 cells differentiate under the influence of IL1\textgreekb, IL6, IL21 and IL23. Recent studies indicate that TGF\textgreekb is essential not only for the development of murine Th17 cells but also for differentiation of human Th17 cells. TGF\textgreekb reciprocally regulates the differentiation of inflammatory Th17 cells and suppressive Treg subsets, with the concomitant presence of proinflammatory cytokines favouring Th17 cell differentiation. Several studies demonstrated an important role of Th17 cells in intestinal inflammation, particularly in Crohn's disease. Genome-wide association studies indicate that IL23R and five additional genes involved in Th17 differentiation (IL12B, JAK2, STAT3, CCR6 and TNFSF15) are associated with susceptibility to Crohn's disease and partly also to ulcerative colitis. Taken together, both Th1 and Th17 cells are important mediators of inflammation in Crohn's disease, although activities previously ascribed to IL12 may be mediated by IL23. Anti-IL12/IL23p40 antibody therapy, which targets both Th1 and Th17 cells, is effective in Crohn's disease. However, the complex relationship between Th1 and Th17 cells has not been completely analysed. This will be of great importance to delineate the specific contributions of these cells to Crohn's disease and other autoimmune diseases.

Background and aims: Interleukin 26 (IL-26), a novel IL-10-like cytokine without a murine homologue, is expressed in T helper 1 (Th1) and Th17 cells. Currently, its function in human disease is completely unknown. The aim of this study was to analyse its role in intestinal inflammation.Methods: Expression studies were performed by reverse transcription-PCR (RT-PCR), quantitative PCR, western blot and immunohistochemistry. Signal transduction was analysed by western blot experiments and ELISA. Cell proliferation was measured by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. IL-26 serum levels were determined by an immunoluminometric assay (ILMA).Results: All examined intestinal epithelial cell (IEC) lines express both IL-26 receptor subunits IL-20R1 and IL-10R2. IL-26 activates extracellular signal-related kinase (ERK)-1/2 and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) mitogen-activated protein (MAP) kinases, Akt and signal transducers and activators of transcription (STAT) 1/3. IL-26 stimulation increases the mRNA expression of proinflammatory cytokines but decreases cell proliferation. In inflamed colonic lesions of patients with Crohn's disease, an elevated IL-26 mRNA expression was found that correlated highly with the IL-8 and IL-22 expression. Immunohistochemical analysis demonstrated IL-26 protein expression in colonic T cells including Th17 cells expressing the orphan nuclear receptor ROR\textgreekgt, with an increased number of colonic IL-26-expressing cells in active Crohn's disease.Conclusion: Intestinal cells express the functional IL-26 receptor complex. IL-26 modulates IEC proliferation and proinflammatory gene expression and its expression is upregulated in active Crohn's disease, indicating a role for this cytokine system in the innate host cell response during intestinal inflammation. For the first time, IL-26 expression is demonstrated in colonic ROR\textgreekgt-expressing Th17 cells in situ, supporting a role for this cell type in the pathogenesis of Crohn's disease.

Background: Previous cross-sectional studies have shown that job change due to breathing problems at the workplace (respiratory work disability) is common among adults of working age. That research indicated that occupational exposure to gases, dust and fumes was associated with job change due to breathing problems, although causal inferences have been tempered by the cross-sectional nature of previously available data. There is a need for general population-based prospective studies to assess the incidence of respiratory work disability and to delineate better the roles of potential predictors of respiratory work disability.Methods: A prospective general population cohort study was performed in 25 centres in 11 European countries and one centre in the USA. A longitudinal analysis was undertaken of the European Community Respiratory Health Survey including all participants employed at any point since the baseline survey, 6659 subjects randomly sampled and 779 subjects comprising all subjects reporting physician-diagnosed asthma. The main outcome measure was new-onset respiratory work disability, defined as a reported job change during follow-up attributed to breathing problems. Exposure to dusts (biological or mineral), gases or fumes during follow-up was recorded using a job-exposure matrix. Cox proportional hazard regression modelling was used to analyse such exposure as a predictor of time until job change due to breathing problems.Results: The incidence rate of respiratory work disability was 1.2/1000 person-years of observation in the random sample (95% CI 1.0 to 1.5) and 5.7/1000 person-years in the asthma cohort (95% CI 4.1 to 7.8). In the random population sample, as well as in the asthma cohort, high occupational exposure to biological dust, mineral dust or gases or fumes predicted increased risk of respiratory work disability. In the random sample, sex was not associated with increased risk of work disability while, in the asthma cohort, female sex was associated with an increased disability risk (hazard ratio 2.8, 95% CI 1.3 to 5.9).Conclusions: Respiratory work disability is common overall. It is associated with workplace exposures that could be controlled through preventive measures.

Aim: To demonstrate that interference microscopy of flat mounted internal limiting membrane specimens clearly delineates cellular proliferations at the vitreomacular interface. Methods: ILM specimens harvested during vitrectomy were fixed in glutaraldehyde 0.05% and paraformaldehyde 2% for 24 h (pH 7.4). In addition to interference microscopy, immunocytochemistry using antibodies against glial fibrillar acidic protein (GFAP) and neurofilament (NF) was performed. After washing in phosphatebuffered saline 0.1 M, the specimens were flat-mounted on glass slides without sectioning, embedding or any other technique of conventional light microscopy. A cover slide and 49,6-diamidino-2-phenylindole (DAPI) medium were added to stain the cell nuclei. Results: Interference microscopy clearly delineates cellular proliferations at the ILM. DAPI stained the cell nuclei. Areas of cellular proliferation can be easily distinguished from ILM areas without cells. Immunocytochemistry can be performed without changing the protocols used in conventional microscopy. Conclusion: Interference microscopy of flat mounted ILM specimens gives new insights into the distribution of cellular proliferations at the vitreomacular interface and allows for determination of the cell density at the ILM. Given that the entire ILM peeled is seen en face, the techniques described offer a more reliable method to investigate the vitreoretinal interface in terms of cellular distribution compared with conventional microscopy.

Background: Pancreatic infiltration by leucocytes represents a hallmark in acute pancreatitis. Although leucocytes play an active role in the pathophysiology of this disease, the relation between leucocyte activation, microvascular injury and haemorrhage has not been adequately addressed.Methods: We investigated intrapancreatic leucocyte migration, leucocyte extravasation and pancreatic microperfusion in different models of oedematous and necrotising acute pancreatitis in lys-EGFP-ki mice using fluorescent imaging and time-lapse intravital microscopy.Results: In contrast to the current paradigm of leucocyte recruitment, the initial event of leucocyte activation in acute pancreatitis was represented through a dose- and time-dependent occlusion of pancreatic capillaries by intraluminally migrating leucocytes. Intracapillary leucocyte accumulation (ILA) resulted in dense filling of almost all capillaries close to the area of inflammation and preceded transvenular leucocyte extravasation. ILA was also initiated by isolated exposure of the pancreas to interleukin 8 or fMLP, demonstrating the causal role of chemotactic stimuli in the induction of ILA. The onset of intracapillary leucocyte accumulation was strongly inhibited in LFA-1-/- and ICAM-1-/- mice, but not in Mac-1-/- mice. Moreover, prevention of intracapillary leucocyte accumulation led to the development of massive capillary haemorrhages and transformed mild pancreatitis into lethal haemorrhagic disease.Conclusions: ILA represents a novel protective and potentially lifesaving mechanism of haemostasis in acute pancreatitis. This process depends on expression of LFA-1 and ICAM-1 and precedes the classical steps of the leucocyte recruitment cascade.