TY - JOUR A1 - Dembek, Marcin A1 - Barquist, Lars A1 - Boinett, Christine J. A1 - Cain, Amy K. A1 - Mayho, Matthew A1 - Lawley, Trevor D. A1 - Fairweather, Neil F. A1 - Fagan, Robert P. T1 - High-throughput analysis of gene essentiality and sporulation in Clostridium difficile JF - mBio N2 - Clostridium difficile is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with C. difficile requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely relies on a small number of broad-spectrum antibiotics, which further exacerbate intestinal dysbiosis and leave the patient acutely sensitive to reinfection. Development of novel targeted therapeutic interventions will require a detailed knowledge of essential cellular processes, which represent attractive targets, and species-specific processes, such as bacterial sporulation. Our knowledge of the genetic basis of C. difficile infection has been hampered by a lack of genetic tools, although recent developments have made some headway in addressing this limitation. Here we describe the development of a method for rapidly generating large numbers of transposon mutants in clinically important strains of C. difficile. We validated our transposon mutagenesis approach in a model strain of C. difficile and then generated a comprehensive transposon library in the highly virulent epidemic strain R20291 (027/BI/NAP1) containing more than 70,000 unique mutants. Using transposon-directed insertion site sequencing (TraDIS), we have identified a core set of 404 essential genes, required for growth in vitro. We then applied this technique to the process of sporulation, an absolute requirement for C. difficile transmission and pathogenesis, identifying 798 genes that are likely to impact spore production. The data generated in this study will form a valuable resource for the community and inform future research on this important human pathogen. KW - Bacillus subtilis KW - expression KW - spores KW - toxin KW - transcription KW - germination KW - transposition KW - metabolism KW - infection KW - in vitro Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143745 VL - 6 IS - 2 ER - TY - JOUR A1 - Becker, Philip P. A1 - Rau, Monika A1 - Schmitt, Johannes A1 - Malsch, Carolin A1 - Hammer, Christian A1 - Bantel, Heike A1 - Müllhaupt, Beat A1 - Geier, Andreas T1 - Performance of serum microRNAs -122, -192 and -21 as biomarkers in patients with non-alcoholic steatohepatitis JF - PLoS ONE N2 - Objectives Liver biopsies are the current gold standard in non-alcoholic steatohepatitis (NASH) diagnosis. Their invasive nature, however, still carries an increased risk for patients' health. The development of non-invasive diagnostic tools to differentiate between bland steatosis (NAFL) and NASH remains crucial. The aim of this study is the evaluation of investigated circulating microRNAs in combination with new targets in order to optimize the discrimination of NASH patients by non-invasive serum biomarkers. Methods Serum profiles of four microRNAs were evaluated in two cohorts consisting of 137 NAFLD patients and 61 healthy controls. In a binary logistic regression model microRNAs of relevance were detected. Correlation of microRNA appearance with known biomarkers like ALT and CK18-Asp396 was evaluated. A simplified scoring model was developed, combining the levels of microRNA in circulation and CK18-Asp396 fragments. Receiver operating characteristics were used to evaluate the potential of discriminating NASH. Results The new finding of our study is the different profile of circulating miR-21 in NASH patients (p<0.0001). Also, it validates recently published results of miR-122 and miR-192 to be differentially regulated in NAFL and NASH. Combined microRNA expression profiles with CK18-Asp396 fragment level scoring model had a higher potential of NASH prediction compared to other risk biomarkers (AUROC = 0.83, 95% CI = 0.754-0.908; p<0.001). Evaluation of score model for NAFL (Score = 0) and NASH (Score = 4) had shown high rates of sensitivity (91%) and specificity (83%). Conclusions Our study defines candidates for a combined model of miRNAs and CK18-Asp396 levels relevant as a promising expansion for diagnosis and in turn treatment of NASH. KW - fatty liver disease KW - independent marker KW - expression KW - injury KW - NAFLD KW - circulating micrornas KW - caspase activation KW - fibrosis KW - miR-122 KW - apoptosis Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-145147 VL - 10 IS - 11 ER - TY - JOUR A1 - Kumar, Praveen A1 - Naumann, Ulrike A1 - Aigner, Ludwig A1 - Wischhusen, Joerg A1 - Beier, Christoph P A1 - Beier, Dagmar T1 - Impaired TGF-β induced growth inhibition contributes to the increased proliferation rate of neural stem cells harboring mutant p53 JF - American Journal of Cancer Research N2 - Gliomas have been classified according to their histological properties. However, their respective cells of origin are still unknown. Neural progenitor cells (NPC) from the subventricular zone (SVZ) can initiate tumors in murine models of glioma and are likely cells of origin in the human disease. In both, p53 signaling is often functionally impaired which may contribute to tumor formation. Also, TGF-beta, which under physiological conditions exerts a strong control on the proliferation of NPCs in the SVZ, is a potent mitogen on glioma cells. Here, we approach on the crosstalk between p53 and TGF-beta by loss of function experiments using NPCs derived from p53 mutant mice, as well as pharmacological inhibition of TGF-beta signaling using TGF-beta receptor inhibitors. NPC derived from p53 mutant mice showed increased clonogenicity and more rapid proliferation than their wildtype counterparts. Further, NPC derived from p53\(^{mut/mut}\) mice were insensitive to TGF-beta induced growth arrest. Still, the canonical TGF-beta signaling pathway remained functional in the absence of p53 signaling and expression of key proteins as well as phosphorylation and nuclear translocation of SMAD2 were unaltered. TGF-beta-induced p21 expression could, in contrast, only be detected in p53\(^{wt/wt}\) but not in p53\(^{mut/mut}\) NPC. Conversely, inhibition of TGF-beta signaling using SB431542 increased proliferation of p53\(^{wt/wt}\) but not of p53\(^{mut/mut}\) NPC. In conclusion, our data suggest that the TGF-beta induced growth arrest in NPC depends on functional p53. Mutational inactivation of p53 hence contributes to increased proliferation of NPC and likely to the formation of hyperplasia of the SVZ observed in p53 deficient mice in vivo. KW - mouse brain KW - tumors KW - cancer KW - TGF-beta KW - glioblastoma stem cell KW - pathways KW - expression KW - astrocytoma KW - glioblastoma KW - transforming growth factor-beta-1 KW - neurogenesis KW - gliomas KW - neural stem cell KW - p53 KW - subventricular zone KW - premalignant lesion Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144262 VL - 5 IS - 11 ER - TY - JOUR A1 - Stepniak, Beata A1 - Kästner, Anne A1 - Poggi, Giulia A1 - Mitjans, Marina A1 - Begemann, Martin A1 - Hartmann, Annette A1 - Van der Auwera, Sandra A1 - Sananbenesi, Farahnaz A1 - Krüger-Burg, Dilja A1 - Matuszko, Gabriela A1 - Brosi, Cornelia A1 - Homuth, Georg A1 - Völzke, Henry A1 - Benseler, Fritz A1 - Bagni, Claudia A1 - Fischer, Utz A1 - Dityatev, Alexander A1 - Grabe, Hans-Jörgen A1 - Rujescu, Dan A1 - Fischer, Andre A1 - Ehrenreich, Hannelore T1 - Accumulated common variants in the broader fragile X gene family modulate autistic phenotypes JF - EMBO Molecular Medicine N2 - Fragile X syndrome (FXS) is mostly caused by a CGG triplet expansion in the fragile X mental retardation 1 gene (FMR1). Up to 60% of affected males fulfill criteria for autism spectrum disorder (ASD), making FXS the most frequent monogenetic cause of syndromic ASD. It is unknown, however, whether normal variants (independent of mutations) in the fragile X gene family (FMR1, FXR1, FXR2) and in FMR2 modulate autistic features. Here, we report an accumulation model of 8 SNPs in these genes, associated with autistic traits in a discovery sample of male patients with schizophrenia (N = 692) and three independent replicate samples: patients with schizophrenia (N = 626), patients with other psychiatric diagnoses (N = 111) and a general population sample (N = 2005). For first mechanistic insight, we contrasted microRNA expression in peripheral blood mononuclear cells of selected extreme group subjects with high-versus low-risk constellation regarding the accumulation model. Thereby, the brain-expressed miR-181 species emerged as potential "umbrella regulator", with several seed matches across the fragile X gene family and FMR2. To conclude, normal variation in these genes contributes to the continuum of autistic phenotypes. KW - permutation KW - miR-181 KW - PGAS KW - FXR2 KW - FXR1 KW - FMR2 KW - FMR1 KW - identification KW - protein KW - fraxe mental retardation KW - CGG repeat KW - CPG Island KW - schizophrenia KW - expression KW - males Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-136893 VL - 7 IS - 12 ER - TY - JOUR A1 - Ramachandran, Sarada Devi A1 - Schirmer, Katharina A1 - Münst, Bernhard A1 - Heinz, Stefan A1 - Ghafoory, Shahrouz A1 - Wölfl, Stefan A1 - Simon-Keller, Katja A1 - Marx, Alexander A1 - Øie, Cristina Ionica A1 - Ebert, Matthias P. A1 - Walles, Heike A1 - Braspenning, Joris A1 - Breitkopf-Heinlein, Katja T1 - In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells JF - PLoS One N2 - In this study we used differentiated adult human upcyte (R) cells for the in vitro generation of liver organoids. Upcyte (R) cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte (R) process). Proliferating upcyte (R) cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte (R) cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel\(^{TM}\), they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions. KW - adults KW - enzyme metabolism KW - albumins KW - primary cells KW - induction KW - expression KW - human heptocytes KW - mesenchymal stem cells KW - oragnoids KW - heptaocytes KW - drug metabolism Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-139552 VL - 10 IS - 10 ER - TY - JOUR A1 - Harter, Patrick N. A1 - Bernatz, Simon A1 - Scholz, Alexander A1 - Zeiner, Pia S. A1 - Zinke, Jenny A1 - Kiyose, Makoto A1 - Blasel, Stella A1 - Beschorner, Rudi A1 - Senft, Christian A1 - Bender, Benjamin A1 - Ronellenfitsch, Michael W. A1 - Wikman, Harriet A1 - Glatzel, Markus A1 - Meinhardt, Matthias A1 - Juratli, Tareq A. A1 - Steinbach, Joachim P. A1 - Plate, Karl H. A1 - Wischhusen, Jörg A1 - Weide, Benjamin A1 - Mittelbronn, Michel T1 - Distribution and prognostic relevance of tumor-infiltrating lymphocytes (TILs) and PD-1/PD-L1 immune checkpoints in human brain metastases JF - Oncotarget N2 - The activation of immune cells by targeting checkpoint inhibitors showed promising results with increased patient survival in distinct primary cancers. Since only limited data exist for human brain metastases, we aimed at characterizing tumor infiltrating lymphocytes (TILs) and expression of immune checkpoints in the respective tumors. Two brain metastases cohorts, a mixed entity cohort (n = 252) and a breast carcinoma validation cohort (n = 96) were analyzed for CD3+, CD8+, FOXP3+, PD-1+ lymphocytes and PD-L1+ tumor cells by immunohistochemistry. Analyses for association with clinico-epidemiological and neuroradiological parameters such as patient survival or tumor size were performed. TILs infiltrated brain metastases in three different patterns (stromal, peritumoral, diffuse). While carcinomas often show a strong stromal infiltration, TILs in melanomas often diffusely infiltrate the tumors. Highest levels of CD3+ and CD8+ lymphocytes were seen in renal cell carcinomas (RCC) and strongest PD-1 levels on RCCs and melanomas. High amounts of TILs, high ratios of PD-1+/CD8+ cells and high levels of PD-L1 were negatively correlated with brain metastases size, indicating that in smaller brain metastases CD8+ immune response might get blocked. PD-L1 expression strongly correlated with TILs and FOXP3 expression. No significant association of patient survival with TILs was observed, while high levels of PD-L1 showed a strong trend towards better survival in melanoma brain metastases (Log-Rank p = 0.0537). In summary, melanomas and RCCs seem to be the most immunogenic entities. Differences in immunotherapeutic response between tumor entities regarding brain metastases might be attributable to this finding and need further investigation in larger patient cohorts. KW - B7-H1 KW - PD-L1 KW - immunoresistance KW - immunosurveillance KW - safety KW - survival KW - expression KW - melanoma KW - breast cancer KW - PC-1 blockade KW - cell lung cancer KW - tumor-infiltrating lymphocytes KW - brain metastases KW - PD-1 Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-137107 VL - 6 IS - 38 SP - 40836 EP - 40849 ER - TY - JOUR A1 - Ruck, Tobias A1 - Bittner, Stefan A1 - Afzali, Ali Maisam A1 - Göbel, Kerstin A1 - Glumm, Sarah A1 - Kraft, Peter A1 - Sommer, Claudia A1 - Kleinschnitz, Christoph A1 - Preusse, Corinna A1 - Stenzel, Werner A1 - Wiendl, Heinz A1 - Meuth, Sven G. T1 - The NKG2D-IL-15 signaling pathway contributes to T-cell mediated pathology in inflammatory myopathies JF - Oncotarget N2 - NKG2D is an activating receptor on T cells, which has been implicated in the pathogenesis of autoimmune diseases. T cells are critically involved in idiopathic inflammatory myopathies (IIM) and have been proposed as specific therapeutic targets. However, the mechanisms underlying T cell-mediated progressive muscle destruction in IIM remain to be elucidated. We here determined the involvement of the NKG2D - IL-15 signaling pathway. Primary human myoblasts expressed NKG2D ligands, which were further upregulated upon inflammatory stimuli. In parallel, shedding of the soluble NKG2D ligand MICA (sMICA) decreased upon inflammation potentially diminishing inhibition of NKG2D signaling. Membrane-related expression of IL-15 by myoblasts induced differentiation of naive CD8\(^+\) T cells into highly activated, cytotoxic \(CD8^+NKG2D^{high}\) T cells demonstrating NKG2D-dependent lysis of myoblasts in vitro. \(CD8^+NKG2D^{high}\) T cell frequencies were increased in the peripheral blood of polymyositis (PM) patients and correlated with serum creatinine kinase concentrations, while serum sMICA levels were not significantly changed. In muscle biopsy specimens from PM patients expression of the NKG2D ligand MICA/B was upregulated, IL-15 was expressed by muscle cells, CD68\(^+\) macrophages as well as CD4\(^+\) T cells, and \(CD8^+NKG2D^+\) cells were frequently detected within inflammatory infiltrates arguing for a local signaling circuit in the inflammatory muscle milieu. In conclusion, the NKG2D - IL-15 signaling pathway contributes to progressive muscle destruction in IIM potentially opening new therapeutic avenues. KW - MIC ligands KW - pathology section KW - T cell activation KW - idiopathic inflammatory myopathies KW - polymyositis KW - IL-15 KW - NKG2D KW - receptor KW - expression KW - lymphokine-activated killer KW - human muscle-cells KW - multiple sclerosis KW - celiac disease KW - tumor immunity KW - NKG2D ligands KW - cutting edge Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-136047 VL - 6 IS - 41 ER - TY - JOUR A1 - Rovituso, Damiano M. A1 - Duffy, Catharina E. A1 - Schroeter, Michael A1 - Kaiser, Claudia C. A1 - Kleinschnitz, Christoph A1 - Bayas, Antonios A1 - Elsner, Rebecca A1 - Kuerten, Stefanie T1 - The brain antigen-specific B cell response correlates with glatiramer acetate responsiveness in relapsing-remitting multiple sclerosis patients JF - Scientific Reports N2 - B cells have only recently begun to attract attention in the immunopathology of multiple sclerosis (MS). Suitable markers for the prediction of treatment success with immunomodulatory drugs are still missing. Here we evaluated the B cell response to brain antigens in n = 34 relapsing-remitting MS (RRMS) patients treated with glatiramer acetate (GA) using the enzyme-linked immunospot technique (ELISPOT). Our data demonstrate that patients can be subdivided into responders that show brain-specific B cell reactivity in the blood and patients without this reactivity. Only in patients that classified as B cell responders, there was a significant positive correlation between treatment duration and the time since last relapse in our study. This correlation was GA-specific because it was absent in a control group that consisted of interferon-\(\beta\) (IFN-\(\beta\))-treated RRMS patients (n = 23). These data suggest that GA has an effect on brain-reactive B cells in a subset of patients and that only this subset benefits from treatment. The detection of brain-reactive B cells is likely to be a suitable tool to identify drug responders. KW - cortical pathology KW - natural history KW - disability KW - expression KW - antibodies KW - disease KW - lesions KW - trial KW - multiple sclerosis Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148172 VL - 5 IS - 14265 ER - TY - JOUR A1 - Neuhaus, Winfried A1 - Schlundt, Marian A1 - Fehrholz, Markus A1 - Ehrke, Alexander A1 - Kunzmann, Steffen A1 - Liebner, Stefan A1 - Speer, Christian P. A1 - Förster, Carola Y. T1 - Multiple antenatal dexamethasone treatment alters brain vessel differentiation in newborn mouse pups JF - PLoS ONE N2 - Antenatal steroid treatment decreases morbidity and mortality in premature infants through the maturation of lung tissue, which enables sufficient breathing performance. However, clinical and animal studies have shown that repeated doses of glucocorticoids such as dexamethasone and betamethasone lead to long-term adverse effects on brain development. Therefore, we established a mouse model for antenatal dexamethasone treatment to investigate the effects of dexamethasone on brain vessel differentiation towards the blood-brain barrier (BBB) phenotype, focusing on molecular marker analysis. The major findings were that in total brains on postnatal day (PN) 4 triple antenatal dexamethasone treatment significantly downregulated the tight junction protein claudin-5, the endothelial marker Pecam-1/CD31, the glucocorticoid receptor, the NR1 subunit of the N-methyl-D-aspartate receptor, and Abc transporters (Abcb1a, Abcg2 Abcc4). Less pronounced effects were found after single antenatal dexamethasone treatment and in PN10 samples. Comparisons of total brain samples with isolated brain endothelial cells together with the stainings for Pecam-1/CD31 and claudin-5 led to the assumption that the morphology of brain vessels is affected by antenatal dexamethasone treatment at PN4. On the mRNA level markers for angiogenesis, the sonic hedgehog and the Wnt pathway were downregulated in PN4 samples, suggesting fundamental changes in brain vascularization and/or differentiation. In conclusion, we provided a first comprehensive molecular basis for the adverse effects of multiple antenatal dexamethasone treatment on brain vessel differentiation. KW - preterm birth KW - fetal lung KW - corticosteroids KW - glucocorticoids KW - exposure KW - endothelial cells KW - in vitro KW - barrier KW - expression KW - rat Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148268 VL - 10 IS - 8 ER - TY - JOUR A1 - Ebert, Regina A1 - Benisch, Peggy A1 - Krug, Melanie A1 - Zeck, Sabine A1 - Meißner-Weigl, Jutta A1 - Steinert, Andre A1 - Rauner, Martina A1 - Hofbauer, Lorenz A1 - Jakob, Franz T1 - Acute phase serum amyloid A induces proinflammatory cytokines and mineralization via toll-like receptor 4 in mesenchymal stem cells JF - Stem Cell Research N2 - The role of serum amyloid A (SAA) proteins, which are ligands for toll-like receptors, was analyzed in human bone marrow-derived mesenchymal stem cells (hMSCs) and their osteogenic offspring with a focus on senescence, differentiation andmineralization. In vitro aged hMSC developed a senescence-associated secretory phenotype (SASP), resulting in enhanced SAA1/2, TLR2/4 and proinflammatory cytokine (IL6, IL8, IL1\(\beta\), CXCL1, CXCL2) expression before entering replicative senescence. Recombinant human SAA1 (rhSAA1) induced SASP-related genes and proteins in MSC, which could be abolished by cotreatment with the TLR4-inhibitor CLI-095. The same pattern of SASP-resembling genes was stimulated upon induction of osteogenic differentiation, which is accompanied by autocrine SAA1/2 expression. In this context additional rhSAA1 enhanced the SASP-like phenotype, accelerated the proinflammatory phase of osteogenic differentiation and enhanced mineralization. Autocrine/paracrine and rhSAA1 via TLR4 stimulate a proinflammatory phenotype that is both part of the early phase of osteogenic differentiation and the development of senescence. This signaling cascade is tightly involved in bone formation and mineralization, but may also propagate pathological extraosseous calcification conditions such as calcifying inflammation and atherosclerosis. KW - human atherosclerotic lesions KW - senescence KW - expression KW - toll-like receptor KW - mineralization KW - osteogenic differentiation KW - serum amyloid A KW - inflammation KW - mesenchymal stem cells KW - WNT5A KW - model KW - lines KW - stromal cells KW - RT-PCR Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148491 VL - 15 ER -