Comparison of TLR4-stimulated mouse microglia from wildtype and CD14ko strains
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ABSTRACT: The analysis reveals the importance of CD14 in microglial responses to pathogen- and damage-associated molecular patterns, based on stimulations with lipopolysaccharide (LPS) and fiibronectin (FN). The data show a dose- and agonist-dependent involvement of CD14. Lack of CD14 impairs responses to low-dose challenegs with LPS, whereas high-dose challenegs are affected to a much lesser degree. For FN, CD14 deficiency causes a major impairment of the response capacity. Mouse microglia from wt and cd14-/- strains were stimulated with 0.1 and 10 ng/ml of LPS or with 100 µg/ml of mouse FN for 3 h. Untreated cells served as the controls. Each condition was based on three independent cell preparations as wells a processing.
Project description:In pediatric glioma cell lines, treatment with ICG-001 had no inhibitory effect on canonical Wnt-target genes but induced significant up regulation of various known β-catenin target genes in both cell lines and top 20 GO-annotations of down-regulated genes by ICG-001 were associated with biosynthetic and metabolic processes and cell cycle division processes. Pediatric cell lines were treated with ICG-001 or DMSO for 48h
Project description:Multiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system with marked heterogeneity in several aspects including pathological processes. Four histopathological patterns of MS have been described. Pattern II is characterized by infiltrating macrophages and T-cells and by antibody and complement deposition. Transcriptome analysis of three patern II demyelinating brain lesions from a multiple sclerosis patient using RNA sequencing demonstrated the presence of mRNA transcripts for genes specific of activated macrophages, T and B cells as well as genes coding for immunoglobulins, complement proteins and some pattern II associated proteins, providing additional evidence supporting pattern II demyelination. Examination of 3 different demyelinating lesions identified by Immunohistopathology.
Project description:The analysis reveals the importance of CD14 in microglial responses to pathogen- and damage-associated molecular patterns, based on stimulations with lipopolysaccharide (LPS) and fiibronectin (FN). The data show a dose- and agonist-dependent involvement of CD14. Lack of CD14 impairs responses to low-dose challenegs with LPS, whereas high-dose challenegs are affected to a much lesser degree. For FN, CD14 deficiency causes a major impairment of the response capacity.
Project description:3’,5’-cyclic adenosine monophosphate (cAMP) is an ubiquitous second messenger which regulates multiple physiological functions by acting in distinct subcellular microdomains. Although several targeted cAMP biosensors have been developed and used in cell lines or neonatal cardiomyocytes, it is unclear whether such biosensors can be successfully used in vivo, especially in the context of disease. Here, we generated the first transgenic mouse model expressing a targeted cAMP sensor (Epac1-PLN) and analyzed microdomain-specific second messenger dynamics in the vicinity of the sarcoplasmic reticulum calcium ATPase (SERCA). We demonstrate the biocompatibility of this targeted sensor and its potential for real-time monitoring of compartmentalized cAMP signaling in adult cardiomyocytes isolated from healthy mouse heart and from in vivo cardiac disease model. Finally, we used gene arrays to verify that the mRNA expression levels of the main players involved in cAMP signaling such as beta-ARs, G-protein, adenylyl cyclases, PKAs, PDEs, and Epac were not significantly changed between wildtype and transgenic Epac1-PLN expressing cardiomyocytes. To compare cardiac specific mRNA expression between wildtype and Epac1-PLN transgenic mice, cardiomyocyte RNA from total of 5 individual wildtype and 5 Epac1-PLN transgenic mice was isolated and submitted for the Illumina gene array analysis to the Göttingen transcriptome analysis laboratory (TAL).
Project description:The analysis compares primary fibroblasts initially used for reprogramming, established marmoset ES cells and a marmoset iPS cell line which was generated witha non-viral approach using a six-factor-in-one-vector approach Cells were grown under standard conditions for marmoset pluripotent stem cells and primary fibroblasts without additional treatment. For each cell type at least four independent cell preparations were used.
Project description:Remodeling of the tricarboxylic acid (TCA) cycle is a metabolic adaptation mechanism accompanying inflammatory macrophage activation. During this process, endogenous metabolites can adopt regulatory roles that govern specific aspects of inflammatory response, as recently shown for succinate, which regulates the downstream pro-inflammatory IL-1β-HIF1a axis. Itaconate is one of the most highly induced metabolites in activated macrophages, yet its functional significance remains unknown. Here, we show that itaconate modulates macrophage metabolism and effector functions via its effect on succinate dehydrogenase, by inhibiting conversion of succinate to fumarate. Through this action, itaconate exerts anti-inflammatory effects when administered in vitro and in vivo during macrophage activation and ischemia-reperfusion injury. Using newly generated Irg1-/- mice, which lack the ability to produce itaconate, we show that endogenous itaconate regulates succinate levels and function, changes in mitochondrial respiration, and inflammatory cytokine production during macrophage activation. These studies highlight itaconate as a major physiological regulator of the global metabolic rewiring and effector functions of inflammatory macrophages. Experiment 1: mature WT BMDM were treated for 12h with 0.25 mM dimethyl itaconate (DI) or vehicle (Unst) and then stimulated with LPS (E. coli 0111:B4; 100 ng/ml, 4h) (DI+LPS; LPS); Experiment 2: mature Irg1-/- BMDM were stimulated with LPS (E. coli 0111:B4; 100 ng/ml) and murine recombinant IFNg (50 ng/ml) for 24h.
Project description:We report the generation and characterization of four novel marmoset monkey ES cell lines, derived from natural preimplantation embryos. Two cell lines were obtained from non-compacted morulae (DPZcjESC1 and 2), while the other two cell lines were derived from a compacted morula (DPZcjESC3) and an expanded blastocyst (DPZcjESC4), respectively. DPZcjESC1 and 2 showed trisomies for one and two chromosomes respectively, while DPZcjESC3 and 4 are euploid. We detected no obvious differences between morula- and blastocyst-derived cell lines. However, quantitative chromosome-wise transcriptome analyses precisely reflected the trisomies. We also show that the female ES cell lines exhibit different states of X-inactivation which is also impressively reflected by the abundance of the lncRNA X inactive-specific transcript (XIST). mRNA profiles of four different ES cell lines were generated by deep sequencing, in duplicate, using an Illumina HiSeq2000.
Project description:CD14+ Monocytes from healthy volunteers were purified by MACS (negative selection) and FACSorting and either left untreated or stimulated for 24h and 48h with LPS. THP-1 cells were stimulated for 4h, 24h and 48h with LPS. Glycoproteins were captured with hydrazide chemistry and tryptic and PNGase F-released peptide fractions analyzed by MS/MS. Quantitative assessment revealed differential glycoprotein expression in activated/LPS-tolerized monocytes and naïve monocytes and THP-1 cells.