Project description:Analysis genes which alter after anti-dsDNA antibodies stimulation in human mesangial cells. The hypothesis is anti-dsDNA antibodies might affected a pro-inflammatory cytokine gene or genes in cell proliferation and apoptosis pathways
Project description:This study contains a series of ChIP-seq experiments performed on nuclear material similar to what was used in Tissue- and Stage-specific Capture-C for selected TSS and CRM (BioStudies/ArrayExpress collection E-MTAB-9310). ChIP-seq with chromatin isolated from nuclei isolated from 2-3h whole embryos samples or Mef2-/Elav-sorted nuclei from 6-8h and 10-12h embryo samples was performed using anti-H3K27ac (Anti-Histone H3 (acetyl K27) antibody, Abcam #ab4729, purified polyclonal), anti-CTCF (rabbit anti Drosophila CTCF antibody, Reinkawitz lab, purified polyclonal), anti-Beaf (mouse anti Drosophila BEAF antibody, DSHB #1553420, monoclonal) and anti-Su(Hw) (goat anti Drosophila Su(Hw) antibody, Geyer lab, purified polyclonal) antibodies.
Project description:To further understand differential phenotypes between C57BL/6 and Ackr4-deficient B cells, we have employed whole genome microarray expression profiling as a discovery platform to identify genes which are differentially expressed in the two genotypes. Murine B cells from spleens of C57BL/6 and Ackr4-deficient mice were purified by negative magnetic activated cell sorting (MACS) and activated in vitro with 10 µg/mL polyclonal anti-IgM and10 µg/mL anti-CD40 (clone FGK4.5) antibodies for three days in culture (96well plate, 37°C, 5%CO2). On day three, living B cells were sorted and RNA was extracted.
Project description:ChIP on chip assay of AbrC3 to determine its binding sites in S. coelicolor M145 genome using purified anti-AbrC3 polyclonal antibodies. Samples from nutrient broth (NB) cultures were collected at 36, 48 and 60 hours, cross-linked, and finally hybridized to DNA 104K microarrays designed by University of Surrey.
Project description:Methylation profiling of immortalized human prostate epithelial cells after CTCF knockdown by short hairpin RNA. Methylated DNA was immunoprecipitated with anti-methylcytosine antibodies from genomic DNA and detected by Affymetrix CytoScanHD chips. Methylated DNA was purified by 5-mC antibodies and Affymetrix CytoScanHD arrays were performed according to the manufacturer's protocols.
Project description:we performed genome-wide transcriptome profiling in mesangial cells and tubular epithelial cells (TECs), which were stimulated by high glucose (HG) and detected the expression of inflammation associated genes. HG increased the mRNA expression of oxidative stress, inflammasome and mammalian target of rapamycin (mTOR) related genes in mesangial cells. Pro-inflammatory/Th1 gene expression was upregulated, but Th2 related gene expression was downregulated in mesangial cells. In TECs, HG stimulation increased pro-inflammatory/Th1/Th2 gene expression. mRNA profiles of human mesangial cells and tubular epithelial cells, which were stimuated HG for 24 hours or mannitol contorol.
Project description:Purified primary human CD8 T cells were stimulated in vitro in parallel using a magnetic bead conjugated to either anti-CD3/28 or anti-CD2/3/28 antibodies. Cells were cultured for 6 days in complete RPMI1640 and 10ng/ml IL2 and were harvested on day 6 and RNA was isolated for further labelling and hybridisation.
Project description:Blood taken from a healthy human donor was used to isolate CD4CD45RO T-cells. Purified cells were stimulated with either interleukin-2 (IL-2), Interleukin-6 (IL-6) and TNF-alpha and harvested at 24 hours, 72 hours (3 days) and 144 hours (6 days) or anti-CD3 antibodies and harvested at 24 hours. These were compared to unstimulated purified CD4CD45RO T-cells harvested at 0 hours.
Project description:Anti-dsDNA antibodies are a hallmark of systemic lupus erythematosus and are highly associated with its exacerbation. Cumulative evidence has suggested that somatic hypermutation contributes to the high-affinity reactivity of anti-dsDNA antibodies. Our previous study demonstrated that these antibodies are generated from germline precursors with low-affinity ssDNA reactivity through affinity maturation and clonal expansion in patients with acute lupus. This raised the question of whether such precursors could be subject to immune tolerance. To address this, we generated a site-directed knock-in (KI) mouse line, G9gl, which carries germline-reverted sequences of the VH–DH–JH and Vκ–Jκ regions of patient-derived, high-affinity anti-dsDNA antibodies. G9gl heterozygous mice had a reduced number of peripheral B cells, only 27% of which expressed G9gl B cell receptor (BCR). The remaining B cells harbored non-KI allele-derived immunoglobulin heavy (IgH) chains or fusion products of upstream mouse VH and the KI gene, suggesting that receptor editing through VH replacement occurred in a large proportion of B cells in the KI mice. G9gl BCR-expressing B cells responded to ssDNA but not dsDNA, and exhibited several anergic phenotypes, including reduced surface BCR and shortened life span. Further, G9gl B cells were excluded from germinal centers (GCs) induced by several conditions. In particular, following immunization with methylated bovine serum albumin-conjugated bacterial DNA, G9gl B cells occurred at a high frequency in memory B cells but not GC B cells or plasmablasts. Thus, multiple tolerance checkpoints prevented low-affinity precursors of pathogenic anti-dsDNA B cells from undergoing clonal expansion and affinity maturation in GCs.
