Project description:Purpose:The purpose of this study is to detect activated or silenced genes during LPS-induced dendritic cell maturation. Gene expression differences between two samples could be found using transcriptome profiling (RNA-seq) analysis. Methods:Mouse dendritic cells were generated from bone marrow cells in RPMI-1640 medium with recombinant mouse GM-CSF and IL-4, mature DCs were obtained after LPS induced maturation. Immature DCs and mature DCs were sorted respectively based on maturation marker CD86 and Iab(MHCII) using flowcytrometer. DC mRNA profiles were generated by deep sequencing,using Illumina Results: We mapped about 10 million sequence reads per sample to the mouse genome, identified 1,300 upregulated genes and 1,475 dow regulated genes during dendritic cell maturation. DC mRNA profiles immature and mature moouse BMDCs were generated by deep sequencing
Project description:Purpose:The purpose of this study is to detect activated or silenced genes during LPS-induced dendritic cell. Gene expression differences between two samples could be found using transcriptome profiling (RNA-seq) analysis. Methods:Mouse dendritic cells were generated from bone marrow cells in RPMI-1640 medium with recombinant mouse GM-CSF and IL-4, immature DCs were obtained before or after LPS stimulation. Immature DCs were sorted respectively based on marker CD86 and Iab(MHCII) using flowcytrometer. DC mRNA profiles were generated by deep sequencing,using Illumina. Results: We mapped about 10 million sequence reads per sample to the mouse genome, identified hundreds of genes with significant mRNA variation during LPS stimulation. DC mRNA profiles immature BMDCs were generated by deep sequencing
Project description:MIF and its receptor, CD74, are pivotal regulators of the immune system. Here, we demonstrate for the first time that partial MHC class II constructs comprised of linked ?1?1 domains with covalently attached antigenic peptides (also referred to as recombinant T-cell receptor ligands - RTLs) can inhibit MIF activity by not only blocking the binding of rhMIF to immunopurified CD74, but also downregulating CD74 cell-surface expression. This bifunctional inhibition of MIF/CD74 interactions blocked downstream MIF effects, including enhanced secretion of proinflammatory cytokines, anti-apoptotic activity, and inhibition of random migration that all contribute to the reversal of clinical and histological signs of EAE. Moreover, we demonstrate that enhanced CD74 cell-surface expression on monocytes in mice with EAE and subjects with multiple sclerosis can be downregulated by humanized RTLs, resulting in reduced MIF binding to the cells. Thus, binding of partial MHC complexes to CD74 blocks both the accessibility and availability of CD74 for MIF binding and downstream inflammatory activity.
Project description:In a rat model of induced intestinal obstruction, a transcriptomic analysis was used to measure global gene expression. Rat fetuses small intestines of different stages of development (ED15, ED17, ED19 and ED21, were studied as non-operated controls and compared to upper and lower segments of rat fetuses small intestine with an induced obstruction (ligature at ED18).
Project description:In vitro maturation (IVM) of the oocytes is a routine method in bovine embryo production. The competence of bovine oocytes to develop into embryo after IVM and in vitro fertilization (IVF) is lower as compared to in vivo preovulatory oocytes. Cumulus cells (CC) that enclose an oocyte are involved in the acquisition of oocyte quality during maturation. Using transcriptomic approach we compared cumulus cells gene expression during IVM with that in vivo preovulatory period. Global transcriptional profiling was performed using cumulus cells collected from mature bovine oocytes (metaphase-II stage) after maturation performed either in vivo or in vitro. In vivo matured cumulus cells were collected from ovulatory follicles of Montbeliard adult cows by ovum pick-up in vivo (OPU, n=4). In vitro matured cumulus cells were recovered from the oocytes after 22h of in vitro culture of cumulus-oocyte complexes (50 COC per experiment) from 2-6 mm ovarian follicles of adult cows (MIV, n=4). Gene expression analysis was carried out between in vivo and in vitro matured cumulus representing a total of 8 slides (dye swap protocol)
Project description:Comparison of gene expression from subjects who resolved or formed pustules to H.ducreyi. In human inoculation experiments, the cutaneous immune response to Haemophilus ducreyi consists of serum, PMN, macrophages, T cells and myeloid DC. In reinfection experiments, some subjects form pustules twice (PP group) or resolve infection twice (RR group). Although pustule formation is associated with serum resistance and phagocytic failure, there are no differences in the ability of isolated phagocytes or serum obtained from PP and RR subjects to ingest or kill H. ducreyi. To identify the basis for differential host susceptibility to H. ducreyi, we used microarrays to profile gene expression in infected and uninfected tissue and monocyte-derived DC obtained from PP and RR subjects. In infected tissue, both groups had a core response to H. ducreyi. Many additional transcripts that signify active immune function were upregulated exclusively in RR tissue, while PP tissue exclusively contained differentially regulated transcripts consistent with immune dysregulation. The core response of infected DC from both groups was typical of a DC1 response. RR DC exclusively expressed many additional transcripts indicative of DC1 function, while PP DC uniquely expressed differentially regulated transcripts characteristic of both DC1 and DCreg. The data suggest that DC from PP and RR subjects are prewired to respond differentially to H. ducreyi. Experiment Overall Design: Six healthy adult volunteers (5 females, 1 male, 36 ± 13 yrs, mean age ± SD) who had been infected with H. ducreyi twice were inoculated a third time. Each volunteer was inoculated at 3 sites on the upper arm with live H. ducreyi 35000HP (a human passaged isolate of strain 35000) and at 1 site with sterile PBS. Forty-eight hours after inoculation, lesion size was measured and RNA was isolated from the infected site that had the largest diameter. RNA was also obtained from the PBS control site (uninfected site). We obtained peripheral blood 6 to 12 months after inoculation of the 6th subject and derived myeloid DC in vitro. In brief, CD14+ peripheral blood monocytes were isolated from PBMC by positive selection magnetic beads (Miltenyi Biotec, Auburn, CA) and grown in the presence of recombinant human (rh) IL-4 (1 ng/ml) and rhGM-CSF (0.2 ng/ml) (R&D Systems, Minneapolis, MN). The cells were HLA-DR+, CD86+ CD40+, CD3-, CD14-, and CD19- by flow cytometry. The experiment was done in pairs so that DC from 1 RR subject were exposed to the same inoculum as DC from 1 PP subject. DC were incubated with nonopsonized H. ducreyi for 90 minutes at an MOI of 30:1. The DC were washed to remove non-associated bacteria, and incubated an additional 22.5 hours. Cells were collected and used for microarray analysis. Supernatants were collected and analyzed for cytokines using the Human Th1/Th2 II Cytometric Bead Array Kit per manufacturerâs instructions (BD Biosciences).
Project description:SMILE, a molecule for which no information is available in the literature, is overexpressed in the blood of patients tolerating a kidney graft without treatment. We performed microarray analysis to determine cellular functions affected by SMILE silencing. Each condition of control and SMILE siRNA transfected cells was made in triplicates.
Project description:In this study, we focused on the changes of mRNA expression in bovine oviduct epithelial cells in vitro co-cultured with embryos during preimplantation development in order to identify genes, which might be involved in embryo-maternal communication. For this purpose, we used large-scale cDNA microarray hybridizations to identify the genes differentially regulated in bovine oviduct epithelial cells (Boec) during in vitro culture. The main objective was to identify genes which are differentially expressed due to the presence or absence of bovine embryos on cell monolayers. Global transcriptional profiling was performed using 13 days Bovine oviduct epithelial cells (Boec) cultured in vitro with SOF media RNA as control samples (BOEC_SOF_CTL, n=3) for comparison to the experimental samples taken at the same culture time (day 13) and stimulated by the presence of bovine embryos during the last 8 days of culture (BOEC_SOF+EMB, n=3).Gene expression analysis was carried out between Boec with or without stimulation of embryos representing a total of 6 slides (dye swap protocol).
Project description:F6 murine leukaemic cells were transfected either with MHC II or with GFP and transcriptionally profiled by RNA-seq. Triplicate samples for each genotype were sequenced. This is part of ongoing investigation of a possible cell-intrinsic role of MHC II B cells.
Project description:Determination of differentially expressed genes in the proximal colon and distal ileum tissue in MR1 and IL-17A deficiency at steady-state. Tissue from naïve mice was harvested, total RNA extracted and subjected to RNASeq analysis.