Effect of cholera toxin or forskolin on mouse bone marrow-derived dendritic cells
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ABSTRACT: Bone marrow-derived dendritic cells from C57BL/6 mice were treated with 1 ug/ml cholera toxin, 10 uM forskolin or control medium for 2 h. drug treatment groups
Project description:Bone marrow-derived dendritic cells from C57BL/6 mice were treated with 1 ug/ml cholera toxin, 10 uM forskolin or control medium for 2 h.
Project description:Oral cholera vaccines (OCVs) have become important components of strategies for cholera control, but the demand for OCVs has outstripped the supply2–4. There is a need for new cholera control measures for the one billion people living in cholera endemic regions5. The use of orally delivered single-domain antibodies has been proposed as a potential approach for the control of gastrointestinal pathogens6. Here, we describe the development of an orally deliverable bivalent VHH construct (BL3.2) that binds to the B-subunit of cholera toxin (CTXB). The epitope of CTXB for binding molecule BL3.2 was mapped by Hydrogen Deuterium Exchange HDX
Project description:The mechanisms by which dendritic cells (DCs) induce differentiation of naïve CD4+ T cells along the Th2 lineage is not well understood given that DCs themselves do not produce IL-4. In the present study, we undertook a microarray approach to identify genes involved in the induction of Th2 differentiation by DCs treated with a Th2-skewing adjuvant cholera toxin (CT). In the microarray analysis, murine bone marrow derived immature DCs were treated with CT. Of particular interest was tthe significant upregulation of the expression of c-kit. The upregulation of c-kit on DCs is critical for the induction of a Th2 response. Keywords: cholera toxin, bone marrow derived dendritic cells, gene expression array-based (RNA / spotted DNA/cDNA) Murine bone marrow cells were cultured in the presence of GM-CSF (10 ng/ml) for 6 days . On day 6 the cells were harvested and purified using magnetically labeled anti mouse CD11c+ beads . The DCs were stimulated with CT (1 ug/ml) for 24 hours and the RNA was isolated.
Project description:[original title] Gene expression analysis of leukemic samples derived from AF4-MLL- or AF4-MLL/MLL-AF4-transduced and transplanted hematopoietic stem/precursor cells in C57BL6 mice. We used microarrays to analyze the global gene expression in leukemic cells with three distinct immunophenotypes. We compared leukemic cells isolated from the bone marrow of diseased mice and compared these profiles with normal bone marrow.
Project description:The present study used microarray approach to identify the genomewide response to cholera toxin in the presence of nitrate. Considering that fact that the possibility of the existence of multiple Gα genes/proteins in plants has not been conclusively ruled out, analysis of the genomewide impact of RGA1 mutation in rice and GPA1 mutation in Arabidopsis reveal only those genes that are under their direct control. On the other hand, assuming that all those different Gα subunits in any given plant are regulated by cholera toxin, analysis of the genomwide response to cholera toxin could capture the entire G-protein responsive transcriptome, beyond what can be revealed by the mutant approach. This could reveal even those genes that respond to other, as yet unidentified Gα subunits, as well as reveal some genes that are non-specifically regulated by cholera toxin, independent of any G-proteins.
Project description:MDS is characterized by a disturbed function of the myeloid lineage of the hematopoietic system that may transform to AML, a malignant disease of the myeloid compartment. Epigenetic and genetic aberrations contribute to the initiation and progression of MDS/AML. GFI1 is a transcriptional repressor, which regulates expression of its target genes by, among other approaches, recruiting HDACs to its target genes to remove histone 3 lysine 9 (H3K9) acetylation, a marker for active gene expression. Low levels of GFI1 expression and deletion of one GFI1 allele contribute to MDS/AML development in human patients and are associated with a specific gene expression signature and inferior prognosis. To explore the mechanism behind this, we used a mouse strain, which expresses GFI1 only at 5-10% of the normal level (GFI1-Knock-down (KD)). Knock-down of GFI1 or loss of one murine Gfi1 allele reduced latency and increased incidence of AML in different murine models of human MDS/AML development. On the epigenetic level, KD of Gfi1 lead to increased amount of H3K9 acetylation, resulting in increased expression of genes involved in AML development. On a translational level both murine as well as human AML cells with low expression of GFI1 are resistant to standard epigenetic therapy. We show that treatment with histone acetyltransferase inhibitors might be a novel treatment approach for low Gfi1-expressing blast cells. GFI1 has a dose dependent role in myeloid malignancies and is a biomarker for therapeutic intervention. We used microarrays to detail the global programme of gene expression in bone marrow cells of Nup98HoxD13 leucemic mice and identified distinct classes of up-regulated genes during this process. Bone marrow cells of leukemic mice (from KI & KD Nup98HoxD13 mice) were collected for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain homogeneous populations of leukemic cell population, so we took samples of mice which had the same age.
Project description:The molecular mechanisms involved in host pathogen interactions at mucosal surfaces needs to be better understood in order to develop immune-mediated methods of protection against pathogens. Cholera toxin (CT) has the rare ability for a protein of inducing robust mucosal immunity in the gut and is therefore an excellent model with which to determine mechanisms of adjuvanticity and immunogenicity at intestinal mucosal surfaces. Jejunal epithelial cells are one of the first sites of antigen encounter. Therefore a porcine intestinal epithelial cell line, IPEC-J2, was cultured in 6-well transwell plates in the presence or absence of 50 ng/ml cholera toxin for up to 8 hours and the cell layer was harvested for gene expression analysis using the Affymetrix porcine genome array and real-time PCR analysis. Affymetrix analysis identified, and real-time PCR analysis of 15 genes confirmed, an increase in gene expression for 59 genes and a decrease in gene expression for 14 genes under CT treatment. An 8 hour time course of expression revealed that by 2-4 hours after CT treatment, all 10 upregulated genes were differentially expressed and by 4-6 hours after CT treatment 3 of the 5 downregulated genes were differentially expressed. These data suggest that the potent mucosal adjuvanticity and immunogenicity of CT derives from rapid alterations in gene expression at the site of first antigen encounter with the immune system. Characterization of early immune gene expression may elucidate potential biological mechanisms for mucosal immune induction leading to the development of effective vaccines against enteric pathogens. Experiment Overall Design: Confluent IPEC-J2 cell monolayers were treated with or without 50 ng/ml cholera toxin in transwell dishes for 8h. The experiment was repeated 3 times for a total of 6 chips, 3 cholera toxin treated and 3 untreated. Gene expression was compared between cholera toxin treated and untreated cells.
Project description:Human induced pluripotent stem cells provide an unlimited, scalable source of youthful tissue progenitors and secretome for regenerative therapies. The aim of our study was to assess the potential of conditioned medium (CM) derived from hiPSC-mesenchymal progenitors (hiPSC-MPs) to stimulate osteogenic differentiation of adult and aged human bone marrow-mesenchymal stromal cells (MSCs). In addition, we evaluated whether extended cultivation or osteogenic pre-differentiation of hiPSC-MPs could enhance the CM stimulatory activity.