@article{StepniakKaestnerPoggietal.2015, author = {Stepniak, Beata and K{\"a}stner, Anne and Poggi, Giulia and Mitjans, Marina and Begemann, Martin and Hartmann, Annette and Van der Auwera, Sandra and Sananbenesi, Farahnaz and Kr{\"u}ger-Burg, Dilja and Matuszko, Gabriela and Brosi, Cornelia and Homuth, Georg and V{\"o}lzke, Henry and Benseler, Fritz and Bagni, Claudia and Fischer, Utz and Dityatev, Alexander and Grabe, Hans-J{\"o}rgen and Rujescu, Dan and Fischer, Andre and Ehrenreich, Hannelore}, title = {Accumulated common variants in the broader fragile X gene family modulate autistic phenotypes}, series = {EMBO Molecular Medicine}, volume = {7}, journal = {EMBO Molecular Medicine}, number = {12}, doi = {10.15252/emmm.201505696}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-136893}, pages = {1565-1579}, year = {2015}, abstract = {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.}, language = {en} } @article{EbertBenischKrugetal.2015, author = {Ebert, Regina and Benisch, Peggy and Krug, Melanie and Zeck, Sabine and Meißner-Weigl, Jutta and Steinert, Andre and Rauner, Martina and Hofbauer, Lorenz and Jakob, Franz}, title = {Acute phase serum amyloid A induces proinflammatory cytokines and mineralization via toll-like receptor 4 in mesenchymal stem cells}, series = {Stem Cell Research}, volume = {15}, journal = {Stem Cell Research}, doi = {10.1016/j.scr.2015.06.008}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148491}, pages = {231-239}, year = {2015}, abstract = {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.}, language = {en} } @article{SchuetzJurastowBaderetal.2015, author = {Sch{\"u}tz, Burkhard and Jurastow, Innokentij and Bader, Sandra and Ringer, Cornelia and Engelhardt, Jakob von and Chubanov, Vladimir and Gudermann, Thomas and Diener, Martin and Kummer, Wolfgang and Krasteva-Christ, Gabriela and Weihe, Eberhard}, title = {Chemical coding and chemosensory properties of cholinergic brush cells in the mouse gastrointestinal and biliary tract}, series = {Frontiers in Physiology}, volume = {6}, journal = {Frontiers in Physiology}, number = {87}, doi = {10.3389/fphys.2015.00087}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143550}, year = {2015}, abstract = {The mouse gastro-intestinal and biliary tract mucosal epithelia harbor choline acetyltransferase (ChAT)-positive brush cells with taste cell-like traits. With the aid of two transgenic mouse lines that express green fluorescent protein (EGFP) under the control of the ChAT promoter (EGFP\(^{ChAT}\)) and by using in situ hybridization and immunohistochemistry we found that EGFP\(^{ChAT}\) cells were clustered in the epithelium lining the gastric groove. EGFP\(^{ChAT}\) cells were numerous in the gall bladder and bile duct, and found scattered as solitary cells along the small and large intestine. While all EGFP\(^{ChAT}\) cells were also ChAT-positive, expression of the high-affinity choline transporter (ChT1) was never detected. Except for the proximal colon, EGFP\(^{ChAT}\) cells also lacked detectable expression of the vesicular acetylcholine transporter (VAChT). EGFP\(^{ChAT}\) cells were found to be separate from enteroendocrine cells, however they were all immunoreactive for cytokeratin 18 (CK18), transient receptor potential melastatin-like subtype 5 channel (TRPM5), and for cyclooxygenases 1 (COX1) and 2 (COX2). The ex vivo stimulation of colonic EGFP\(^{ChAT}\) cells with the bitter substance denatonium resulted in a strong increase in intracellular calcium, while in other epithelial cells such an increase was significantly weaker and also timely delayed. Subsequent stimulation with cycloheximide was ineffective in both cell populations. Given their chemical coding and chemosensory properties, EGFP\(^{ChAT}\) brush cells thus may have integrative functions and participate in induction of protective reflexes and inflammatory events by utilizing ACh and prostaglandins for paracrine signaling.}, language = {en} } @article{ReinholdBattiBilbaoetal.2015, author = {Reinhold, A. K. and Batti, L. and Bilbao, D. and Buness, A. and Rittner, H. L. and Heppenstall, P. A.}, title = {Differential Transcriptional Profiling of Damaged and Intact Adjacent Dorsal Root Ganglia Neurons in Neuropathic Pain}, series = {PLoS ONE}, volume = {10}, journal = {PLoS ONE}, number = {4}, doi = {10.1371/journal.pone.0123342}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143290}, pages = {e0123342}, year = {2015}, abstract = {Neuropathic pain, caused by a lesion in the somatosensory system, is a severely impairing mostly chronic disease. While its underlying molecular mechanisms are not thoroughly understood, neuroimmune interactions as well as changes in the pain pathway such as sensitization of nociceptors have been implicated. It has been shown that not only are different cell types involved in generation and maintenance of neuropathic pain, like neurons, immune and glial cells, but, also, intact adjacent neurons are relevant to the process. Here, we describe an experimental approach to discriminate damaged from intact adjacent neurons in the same dorsal root ganglion (DRG) using differential fluorescent neuronal labelling and fluorescence-activated cell sorting (FACS). Two fluorescent tracers, Fluoroemerald (FE) and 1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI), were used, whose properties allow us to distinguish between damaged and intact neurons. Subsequent sorting permitted transcriptional analysis of both groups. Results and qPCR validation show a strong regulation in damaged neurons versus contralateral controls as well as a moderate regulation in adjacent neurons. Data for damaged neurons reveal an mRNA expression pattern consistent with established upregulated genes like galanin, which supports our approach. Moreover, novel genes were found strongly regulated such as corticotropinreleasing hormone (CRH), providing novel targets for further research. Differential fluorescent neuronal labelling and sorting allows for a clear distinction between primarily damaged neuropathic neurons and "bystanders," thereby facilitating a more detailed understanding of their respective roles in neuropathic processes in the DRG.}, language = {en} } @article{HarterBernatzScholzetal.2015, author = {Harter, Patrick N. and Bernatz, Simon and Scholz, Alexander and Zeiner, Pia S. and Zinke, Jenny and Kiyose, Makoto and Blasel, Stella and Beschorner, Rudi and Senft, Christian and Bender, Benjamin and Ronellenfitsch, Michael W. and Wikman, Harriet and Glatzel, Markus and Meinhardt, Matthias and Juratli, Tareq A. and Steinbach, Joachim P. and Plate, Karl H. and Wischhusen, J{\"o}rg and Weide, Benjamin and Mittelbronn, Michel}, title = {Distribution and prognostic relevance of tumor-infiltrating lymphocytes (TILs) and PD-1/PD-L1 immune checkpoints in human brain metastases}, series = {Oncotarget}, volume = {6}, journal = {Oncotarget}, number = {38}, doi = {10.18632/oncotarget.5696}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-137107}, pages = {40836 -- 40849}, year = {2015}, abstract = {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.}, language = {en} } @article{WeiderWegenerSchmittetal.2015, author = {Weider, Matthias and Wegener, Am{\´e}lie and Schmitt, Christian and K{\"u}spert, Melanie and Hillg{\"a}rtner, Simone and B{\"o}sl, Michael R. and Hermans-Borgmeyer, Irm and Nait-Oumesmar, Brahim and Wegner, Michael}, title = {Elevated in vivo levels of a single transcription factor directly convert satellite glia into oligodendrocyte-like cells}, series = {PLoS Genetics}, volume = {11}, journal = {PLoS Genetics}, number = {2}, doi = {10.1371/journal.pgen.1005008}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144123}, pages = {e1005008}, year = {2015}, abstract = {Oligodendrocytes are the myelinating glia of the central nervous system and ensure rapid saltatory conduction. Shortage or loss of these cells leads to severe malfunctions as observed in human leukodystrophies and multiple sclerosis, and their replenishment by reprogramming or cell conversion strategies is an important research aim. Using a transgenic approach we increased levels of the transcription factor Sox10 throughout the mouse embryo and thereby prompted Fabp7-positive glial cells in dorsal root ganglia of the peripheral nervous system to convert into cells with oligodendrocyte characteristics including myelin gene expression. These rarely studied and poorly characterized satellite glia did not go through a classic oligodendrocyte precursor cell stage. Instead, Sox10 directly induced key elements of the regulatory network of differentiating oligodendrocytes, including Olig2, Olig1, Nkx2.2 and Myrf. An upstream enhancer mediated the direct induction of the Olig2 gene. Unlike Sox10, Olig2 was not capable of generating oligodendrocyte-like cells in dorsal root ganglia. Our findings provide proof-of-concept that Sox10 can convert conducive cells into oligodendrocyte-like cells in vivo and delineates options for future therapeutic strategies.}, language = {en} } @article{DembekBarquistBoinettetal.2015, author = {Dembek, Marcin and Barquist, Lars and Boinett, Christine J. and Cain, Amy K. and Mayho, Matthew and Lawley, Trevor D. and Fairweather, Neil F. and Fagan, Robert P.}, title = {High-throughput analysis of gene essentiality and sporulation in Clostridium difficile}, series = {mBio}, volume = {6}, journal = {mBio}, number = {2}, doi = {10.1128/mBio.02383-14}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143745}, pages = {e02383-14}, year = {2015}, abstract = {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.}, language = {en} } @article{KumarNaumannAigneretal.2015, author = {Kumar, Praveen and Naumann, Ulrike and Aigner, Ludwig and Wischhusen, Joerg and Beier, Christoph P and Beier, Dagmar}, title = {Impaired TGF-β induced growth inhibition contributes to the increased proliferation rate of neural stem cells harboring mutant p53}, series = {American Journal of Cancer Research}, volume = {5}, journal = {American Journal of Cancer Research}, number = {11}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144262}, pages = {3436-3445}, year = {2015}, abstract = {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.}, language = {en} } @article{RamachandranSchirmerMuenstetal.2015, author = {Ramachandran, Sarada Devi and Schirmer, Katharina and M{\"u}nst, Bernhard and Heinz, Stefan and Ghafoory, Shahrouz and W{\"o}lfl, Stefan and Simon-Keller, Katja and Marx, Alexander and {\O}ie, Cristina Ionica and Ebert, Matthias P. and Walles, Heike and Braspenning, Joris and Breitkopf-Heinlein, Katja}, title = {In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells}, series = {PLoS One}, volume = {10}, journal = {PLoS One}, number = {10}, doi = {10.1371/journal.pone.0139345}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139552}, pages = {e0139345}, year = {2015}, abstract = {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.}, language = {en} } @article{NeuhausSchlundtFehrholzetal.2015, author = {Neuhaus, Winfried and Schlundt, Marian and Fehrholz, Markus and Ehrke, Alexander and Kunzmann, Steffen and Liebner, Stefan and Speer, Christian P. and F{\"o}rster, Carola Y.}, title = {Multiple antenatal dexamethasone treatment alters brain vessel differentiation in newborn mouse pups}, series = {PLoS ONE}, volume = {10}, journal = {PLoS ONE}, number = {8}, doi = {10.1371/journal.pone.0136221}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148268}, pages = {e0136221}, year = {2015}, abstract = {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.}, language = {en} }