Whole-genome expression analysis in IFNgamma stimulated bone marrow-derived macrophages from transgenic mice expressing only non-nuclear mutant Socs1
ABSTRACT: To study the role of SOCS1 in the cell nucleus in vivo, we generated a transgenic mouse model using a bacterial artificial chromosome (BAC) containing a mutated Socs1 locus (non-nuclear Socs1ΔNLS), GFP and firefly Luciferase, termed MGL. MGL transgenic mice expressing only non-nuclear mutant Socs1 (Socs1-/- MGLtg mice) survive the early lethal phenotype of Socs1-/- mice that die within 3 weeks due to excessive immune signaling, mainly depending on hyperresponsiveness to IFNgamma. Therefore, we suggested that classical IFNgamma signaling might still be efficiently regulated by SOCS1ΔNLS. To prove this hypothesis, bone marrow-derived macrophages (BMMs) from Socs1-/- MGLtg mice and the control group (Socs1+/- MGLtg mice) were stimulated with IFNgamma for 24 h and subjected to whole-genome expression analysis.
Project description:The study assessed the efficacy of R-flurbiprofen in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis in mice. R-flurbiprofen, also known as tarenflurbil, is the R-enantiomer of the cyclooxyygenase inhibitor S-flurbiprofen. It is ineffective in terms of cyclooxygenase inhibition and has no relevant toxicity in humans. Oral R-flurbiprofen prevented and attenuated primary progressive EAE in C57BL6/J mice and relapsing-remitting EAE in SJL mice, even if the treatment was initiated on or after the first flare of the disease. R-flurbiprofen reduced immune cell infiltration and microglia activation and inflammation in the spinal cord, brain and optic nerve and attenuated myelin destruction and EAE-evoked hyperalgesia. R-flurbiprofen treatment increased CD4+CD25+FoxP3+ regulatory T-cells, CTLA4+ inhibitory T-cells and interleukin-10, whereas the EAE-evoked upregulation of pro-inflammatory genes in the spinal cord was strongly reduced (Sentrix6 results). The effects were associated with an increase of plasma and cortical endocannabinoids but decreased spinal prostaglandins, the latter likely due to R- to S inversion. The promising results suggest potential efficacy of R-flurbiprofen in human MS. To assess effects of R-flurbiprofen on EAE evoked gene regulations in the spinal cord a genome wide expression analysis was performed using Illumina Sentrix 6 v2 BeadChips. For the microarray study female C57BL6/J mice were immunized according to a standard protocol using the Hooke Kit™ MOG35-55/CFA emulsion PTX (EK-2110, Hooke Labs, St Lawrence, MA), which contains 200 µg myelin oligodendrocyte glycoprotein (MOG) 35-55 emulsified in 200 µl Complete Freund’s Adjuvant (CFA). The emulsion was injected subcutaneously at two sites followed by two intraperitoneal (i.p.) injections of 200 ng pertussis toxin (PTX) in phosphate buffered saline (PBS), the first 1-2 h after MOG35-55, and the second 24 h after MOG35-55. Control mice received CFA without MOG35-55 (sham mice). Treatment with R-flurbiprofen or vehicle (n = 12 per group) was started 5 days after immunization and was administered continuously via the drinking water up to the end. Spinal cords were dissected out during the flare of the disease, day 16 after immunization. For microarray analysis, total RNA was extracted from homogenizedlumbar spinal cord tissue with Trizol reagent, followed by clean-up and DNase I treatment with QIAGEN RNeasy mini kit. RNA quality was checked (Nanodrop ND-1000, Agilent 2100 Bioanalyzer), and subsequently biotinylated and hybridized to Mouse Sentrix-6 V2 Expression BeadChips (Illumina). Each sample consisted of pooled lumbar spinal cord tissue from 3 animals and 3 replicate samples were analyzed per group, i.e. the analysis is based on 9 mice per group. Groups were CFA-control with vehicle treatment, CFA-control with R-flurbiprofen, EAE-vehicle and EAE-R-flurbiprofen treatment. Treatment was started 5 days after immunization. For dissection, pairs were matched according to the clinical scores. QC, labeling, hybridization and raw data evaluation and normalization were done according to standard protocols at the core facilities of the Deutsche Krebsforschungszentrum, Heidelberg, Germany.
Project description:The extent of intratumoral heterogeneity, the subclonal structures and the mechanisms of treatment-induced clonal selection by cisplatin was investigated in the squamous cell carcinoma cell line model FaDu. We picked 96 single cell-derived clones from the cisplatin-sensitive parental FaDu cell line. After expansion as separate cultures, these clones were tested for their sensitivity to CDDP. By this approach, we isolated individual cell clones that were primarily resistant (clones 5 & 78) and others that showed high sensitivity to CDDP (clones 46 & 54). Basal mRNA expression levels associated with CDDP sensitivity / resistance were determined in two independent microarray analyses.
Project description:Modification of Gene Expression of Skeletal Muscle in Response to postmenopause with or without Hormone Replacement Therapy. Even though menopause is often accompanied with first signs of age-associated changes in muscle structure and function, the effects of hormone replacement therapy (HRT) or menopause-related decline in estrogen production in the muscles of postmenopausal women is not well understood. We have used a randomized double-blinded study design together with an explorative microarray experiment to characterize possible effects of continuous, combined HRT and estrogen deprivation on the skeletal muscle of fifteen early postmenopausal women from which 10 used HRT and 5 used placebo for 12-months in a douple-blinded design. Keywords: time course analysis from HRT users and non-users comparison of gene expression in skeletal muscle of healthy postmenopausel women using HRT (n=10) vs not-using HRT (n=5)
Project description:Cardiovascular disease (CVD) is exceedingly severe in patients with chronic kidney disease (CKD) and further aggravated by peritoneal dialysis (PD), exposing the patients to excessive amounts of intraperitoneal glucose. Children are devoid of pre-existing CVD and give insight into specific uremia and PD induced pathomechanisms. Fat surrounded omental arterioles beyond PD fluid penetration level were microdissected from uremic children at time of first PD catheter insertion (n=8), children on PD (n=5), and age and gender matched non-uremic children as controls (n=6). Adjacent sections of 4 arterioles per patient were used for transcriptomic analyses.
Project description:This experiment describes the differential gene expression between parental gemcitabine-sensitive BxPC-3 cells and their resistant subclones, Bx-GEM. To select for the resistant subclones, parental BxPC-3 cells were treated with increasing concentrations of gemcitabine (10, 25, 50, 100 and 200 nM) for more than one year. Cells resistant at each stage of drug dosing were re-cultured in the subsequent dose, and their resistance confirmed via cell viability assays. Subclones resistant to 200 nM gemcitabine, in additon to the parental cells were used for gene profiling by array. Total RNA was extracted from the cells using the miRNeasy Mini Kit (Qiagen). The Illumina TotalPrep RNA Amplification Kit (life technologie) was used to generate single-stranded cRNA from input amounts of 500ng total RNA. 1,5 ug of cRNA were hybridized for 17 hr at 58°C on Illumina human HT12-Microarray using the Standard Illumina Hybridisation Protocol Part # 11322355 (Whole-Genome Gene Expression Direct Hybridization Assay Guide). Gene Expression Microarrays were scanned using the Illumina iScan-Scanner according to the Standard Illumina Scanning Protocol Part # 11322355 (Whole-Genome Gene Expression Direct Hybridization Assay Guide).
Project description:Brisbane Systems Genetics Study comprises of a total of 862 individuals from 374 families. Families consist of combinations of both MZ and DZ twin pairs, their siblings and for 72 families their parents. Whole blood for expression profiling was collected directly into PAXgene tubes and total RNA was extracted using the WB gene RNA purification kit. RNA from all samples was run on an Agilent Bioanalyzer to assess quality.
Project description:Resident human lamina propria immune cells serve as powerful effectors in host defense. Molecular events associated with the initiation of an intestinal inflammatory response in these cells are largely unknown. Here, we aimed to characterize phenotypic and functional changes induced in these cells at the onset of intestinal inflammation using a human intestinal organ culture model. In this model, healthy human colonic mucosa was depleted of epithelial cells by EDTA treatment. Following loss of the epithelial layer, expression of the inflammatory mediators IL-1β, IL-6, IL-8, IL-23p19, TNF-α, CXCL2 and the surface receptors CD14, TLR2, CD86, CD54 was rapidly induced in resident lamina propria cells in situ as determined by qRT-PCR and immunohistology. Gene microarray analysis of lamina propria cells obtained by laser-capture microdissection provided an overview of global changes in gene expression occurring during the initiation of an intestinal inflammatory response in these cells. Bioinformatic analysis gave insight into signalling pathways mediating this inflammatory response. Furthermore, comparison with published microarray datasets of inflamed mucosa in vivo (ulcerative colitis) revealed a significant overlap of differentially regulated genes underlining the in vivo relevance of the organ culture model. The organ culture model characterized may be useful to study molecular mechanisms underlying the initiation of an intestinal inflammatory response in normal mucosa as well as potential alterations of this response in inflammatory bowel disease. Gut specimens were derived from individuals undergoing resection for localized colon cancer. Microscopically normal colonic mucosa was dissected from the surgical specimen near the resection margin and immediately subjected to the experimental procedures. After extensive washing the mucus layer was removed by dithiothreitol (DTT) treatment. Subsequently, punches of defined surface area were prepared and denuded of epithelial cells by exposure to EDTA. Samples were collected prior to culturing and washing (control, t = 0 h, total mucosa, TM) as well as after loss of the epithelial layer (t = 5 h, mucosa after loss of epithelial layer, LEL-M) and snap frozen in liquid nitrogen. Lamina Propria (LP) was subsequently isolated via Laser Capture Microdissection (LMD) followed by RNA isolation. Microarray analysis of TM-LP (control) and LEL-LP samples was performed using the WG-DASL Human HT_12 V4 Expression BeadChip Assay from Illumina employing a minimum of 200 ng total RNA per sample. Four replicates from individual experiments were measured for each time point.
Project description:Regulatory T (Treg) cells are critical determinants of both immune responses and metabolic control. Here we show that systemic ablation of Treg cells compromised the adaptation of whole-body energy expenditure to cold exposure, correlating with impairment in thermogenic marker gene expression and massive invasion of pro-inflammatory macrophages in brown adipose tissue (BAT). Indeed, BAT harbored a unique sub-set of Treg cells characterized by a unique gene signature. As these Treg cells respond to BAT activation upon cold exposure, this study defines a BAT-specific Treg sub-set with direct implications for the regulation of energy homeostasis in response to environmental stress. We isolated regulatory and conventional T cells from brown-adipose tissue of warm-conditioned or cold-conditioned mice. As controls, we harvested spleen-Treg and Tconv cells from warm-treated mice. Cells were isolated pooled organs and target cells purified by FACS. RNA was extracted and gene expression measured.
Project description:Comparison of expression profiles of primary colorectal cancers with liver metastases of the same patient. Additionally, expression data of normal colon and liver tissue. Abstract of publication will be included upon publication Keywords: expression profiling, colorectal cancer, colon cancer, liver metastasis, normal colonic tissue, normal liver tissue RNA of 18 primary colorectal cancers, 18 matched liver metastases, 7 normal colon epithelium samples and 5 normal liver tissue samples hybridized on Human Sentrix-6 V2 (Illumina)