Project description: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 Socs1ÎNLS), 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 SOCS1ÎNLS. 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:Atherosclerosis is a chronic inflammatory disease. Lesion progression is primarily mediated by cells of the monocyte/macrophage lineage. Interleukin-17A is a pro-inflammatory cytokine, which modulates immune cell trafficking and is involved inflammation in (auto)immune and infectious diseases. But the role of IL-17A still remains controversial. In the current study we investigated effects of IL-17A on advanced murine and human atherosclerosis, the common disease phenotype in clinical care. 26-weeks old apolipoprotein E-deficient (Apoe-/-) mice were fed a standard chow diet and treated either with IL-17A mAb (n=15) or irrelevant immunoglobulin (n=10) for 16 weeks. Furthermore, essential mechanisms of IL-17A in atherogenesis were studied in vitro. Inhibition of IL-17A markedly prevented atherosclerotic lesion progression (P=0.001) by reducing inflammatory burden and cellular infiltration (P=0.01) and improved lesion stability (P=0.01). In vitro experiments showed that IL-17A plays a role in chemoattractance, monocyte adhesion, sensitization of antigen-presenting cells toward pathogen-derived TLR4 ligands. Also, IL-17A induced a unique transcriptome pattern in monocyte-derived macrophages distinct from known macrophage types. Stimulation of human carotid plaque tissue ex vivo with IL-17A induced a pro-inflammatory milieu and up-regulation of molecules expressed by the IL-17A-induced macrophage subtype. We here show for the first time that functional blockade of IL-17A prevents atherosclerotic lesion progression and induces plaque stabilization in advanced lesions in Apoe-/- mice. The underlying mechanisms involve reduced inflammation and distinct effects of IL-17A on monocyte / macrophage lineage. In addition, translational experiments underline the relevance for the human system. Effects of IL-17A on human monocyte-derived macrophages were assessed (n=2 per group).
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 KitM-bM-^DM-" MOG35-55/CFA emulsion PTX (EK-2110, Hooke Labs, St Lawrence, MA), which contains 200 M-BM-5g myelin oligodendrocyte glycoprotein (MOG) 35-55 emulsified in 200 M-BM-5l Complete FreundM-bM-^@M-^Ys 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:Background: Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in adults. Radiotherapy is together with surgery the mainstay treatment in management of patients with GBM, however, infiltrative growth pattern and radioresistance of glioma cells impedes therapeutic effects. Our study sought to investigate 1) whether GBM invasive cells render them resistant to radiotherapy and 2) what are the potential molecular mechanisms underlying the radioresistance via gene expression profiling. <br>Procedures: In-vitro selection of invasive cells using matrigel-coated transwell assay: U87 cells were starved 8h prior to seeding. 5x105 cells in 2 mL serum-free DMEM medium were plated in the upper transwell inserts of a 6-well Boydem chamber. 10% FCS was added in the medium of the lower chamber. After 20h incubation, non-invasive cells on the surface of the membrane were scratched and recovered. Invasive cells migrating through matrigel due to the gradient of attractant were recovered by trypsin/EDTA after the membrane was washed with PBS twice and swabbed by cotton tips. Evaluate the invasiveness of selected phenotype of U87 cells by time-lapse wound-healing assay: 5x104 cells in 100μL serum-free DMEM medium were seeded in a 96-well ImageLock plate with each well was pre-coated with matrigel. Wounds were scratched using a 96-pin WoundMaker and gently washed with 2xPBS. After cooling equilibration, the wounds were coated with a second layer of matrigel. The microplate was incubated at 37oC and 5% CO2 for 30min. Cell invasion was recorded by living-image microscopy over 48h. Microarray: after confirmation of enhanced invasive ability, invasive and non-invasive cell populations were used for RNA isolation, cDNA transcription, and chip hybridization.
Project description:New targets and drugs for treatment of breast cancer are needed. We tested the effects of the TKI258 (Novartis) FGFR-inhibitor on tumor formation in mice. 4T1 mouse cell line, which had been derived from BALB/c mice, was injected into tail veins of BALB/c mice (syngraft model). Seven days later, mice were treated with 0, 15 or 40 milligram per kilogram (dose) TKI258 inhibitor (compound). Tumors were resected after different time points (time) and effects of drug treatment on global gene expression was monitored.
Project description:Analysis of the role of Hipk2 in regulating gene expression in medullary thymic epithelial cells. The whole genome gene signatures of purified mTEC subsets (CD80 low, CD80 high) from TEC-specific Hipk2 knockout mice were compared to mating wildtype control mice (floxed, Cre-). Results provide the up- or down-regulated genes, affected by the Hipk2 gene knockout. Total RNA obtained from isolated CD80 low and CD80 high mTECs of TEC-specific Hipk2 knockout mice compared to control mice mTEC subsets
Project description:Analysis of gene co-expression patterns in TRA-specific medullary thymic epithelial cell (mTEC) subsets. The whole genome gene signatures of purified mTEC subsets respectively positive for the TRAs Gp2, Pdpn, Cea1, Gad1, Ins2, Tspan8 were compared to their corresponding TRA-negative mTEC subset control. Results provide the enriched and depleted gene expressions in the different subsets. Total RNA obtained from FACS isolated mTECs positive for the respective TRAs were compared to their TRA-negative mTEC subsets using specific antibodies (Pdpn, Gp2, Cea1, Tspan8) or reporter mice (Gad1, Ins2).
Project description:Comparative gene expression profiling of thymocytes at the DP, CD4 SP and CD8 SP stage derived from FoxN1-Gpr177 mice (FoxN1-Cre mediated deletion of (Exon3 of) Gpr177/Wtls) or C57Bl/6N mice as comparison. Objective was to test the influence of TEC-secreted Wnt ligands on the transcriptome of thymocytes at the respective developmental stages. Total RNA extracted from FACS-sorted primary mouse thymocytes. CD4/8 double positive (DP) thymocytes, CD4 single positive (CD4 SP) thymocytes and CD8 single positive (CD8 SP) thymocytes were FACS-sorted from conditional knock-out mice (FoxN1-Gpr177) and C57Bl/6N mice as comparison.
Project description:Siomycin A treatment in prostate cancer cell lines resulted in a concurrent inhibition of specific cell cycle genes and strongly impaired cell viability. Illumina microarray experiments were done using PC3 and LNCaP cell lines treated with 0.5 microM Siomaycin A either 24h or 48h in triplicates.
Project description:In order to address the putative role of MELK and UBE2C in prostate cancer development and progression, we performed functional analysis upon siRNA-based knockdown, and searched for downstream genes and processes by microarray experiments. RNAi-based inhibition of MELK and UBE2C was efficient in PC3 prostate cancer cells and decreased transcriptional level down to about 30% remaining expression level. Illumina microarray experiments were done upon siRNA based knockdown 48h after transfection of PC3 cells in triplicates.