ABSTRACT: To evaluate geneexpression profile in developing joints vs adjacent growth plate in control and TGF-beta type II receptor conditional knock-out in limb mesenchyjme We obtained E14.5 autopods from two normal controls and two Tgfbr2Prx1KO littermate embryos. Samples were frozen in OTC and cryosectioned and subjected to laser-capture microdissection for RNA sampling for interzone, growth plate chondrocytes and interdigital tissue. RNas were subjected to microarray analysis
Project description:The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells are essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the sequential loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the chromatin regulator gene level. Similarly, Tranforming Growth Factor-β1 (TGF-β1) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a sequential process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation. C57BL/6JxDBA/2 F1 female mice were subjected to lethal irradiation and intravenously injected with bone marrow cells harvested from C57BL/6J-βactinEGFPxDBA/2 F1 male mice. The livers of female mice were injured with CCl4. Liver individual cells were laser captured from frozen sections. Bone marrow derived hepatocytes (BMDH), hematopoietic cells, and hepatocytes, were analyzed individually.
Project description:The immune system cellular response to tissue damage and infection requires the recruitment of blood leukocytes to the target tissue. This process is mediated through a classical multistep mechanism which involves transient rolling on the endothelium and recognition of inflammation followed by extravasation. We show here, by direct examination of blood monocyte functions in vivo, that resident monocytes monitor the endothelium of healthy tissues through patrolling, a new mechanism which allows extravasation in the absence of rolling.Patrolling depends on the integrin LFA1 and the chemokine receptor CX3CR1, and is required for rapid tissue invasion and initiation of an early immune response by monocytes that differentiate into macrophages at the site of tissue damage and infection. The main goal of the experiment was to compare expression levels of genes in Gr1- monocytes in the blood and after recruitment in the peritoneum during experimental infection with Listeria monocytogenes (time course analysis). Experiment Overall Design: Six-weeks old (C57Bl6, Cx3cr1gfp/+) mice were intraperitonealy infected with a low number (1.104) of L. monocytogenes (EGDe strain) in exponential growth phase (bacteria were grown in BHI at 108/ml, and diluted 10.000x in PBS immediately before injection). Group of three mice were euthanized, before infection (time “0”) and 2 and 8 hours after infection (time “2” and “8”). Peripheral blood cells were recovered at time “0”, and peritoneal cells were recovered at time “0”, “2h”, and “8h” by peritoneal lavage. Cells from individual mice were stained with antibodies to CD11b (PECy7), Gr1 (APC), NK1.1, B220 and CD3 (PE), and F4/80 (biotin-conjugated followed by streptavidin–pacific blue) for sorting. Gr1- monocytes were purified as NK1.1- CD3- B220- CD11b+ F4/80low Gr1-, gfphigh; Gr1+ monocytes were purified as NK1.1- CD3- B220- CD11b+ F4/80low Gr1+, gfpint; and polymorphonuclear cells were purified as NK1.1- CD3- B220- CD11b+ F4/80- Gr1high, gfp-. 1.103 cells from each mice, time point, and phenotype were purified by facs sorting according to their phenotype. Samples were kept at 4°C before and during the sort. Cells were directly sorted in the SuperAmp Lysis Buffer (Miltenyi Biotec, Bergisch Gladbach, Germany) using a FACS Aria cell-sorter (BD biosciences).
Project description:Background: A specific subset of regulatory IL-10 producing B cells has been extensively studied in autoimmune and inflammatory pathologies. These cells are able to constrain exacerbated inflammation by inhibiting T cell mediated responses and maturation of antigen presenting cells. In allergic diseases, observations that increase of regulatory B cells is necessary for allergen tolerance suggest that development of allergic asthma would be associated with a defect in the regulatory B cells compartment. Objective: We sought to (i) characterize regulatory IL-10+ regulatory B cell subset in Balb/c mice by microarray and flow cytometry and (ii) investigate their regulatory capacity in vivo in a house dust mite model of allergic asthma. Results: We identified an IL-10 producing B cells subset able to control T cell proliferation in vitro in both control and asthmatic mice. This subset is decreased in allergic mice. IL-10+ Breg cells express high levels of CD9 and upregulate CD70 and CD73 after activation. Expression of CD9 allows identifying more than 50% of Bregs. Interestingly CD9+ B cells inhibit TH2-TH17 allergic airway inflammation in vivo after adoptive transfer in an IL-10 dependent manner. Conclusions: Herein, we demonstrate that induction of allergic asthma dampens the generation of Bregs contributing to exacerbated airway inflammation. We identified a distinct CD9+ Breg-cell population decreased in lung of HDM mice and able to control asthma and allergic airway inflammation by producing IL-10 after adoptive transfer. This study points B cells as an interesting therapeutic target in allergic asthma. IL-10+ B cells (n=3) and 3 IL-10- B cells (n=3) in control mice + IL-10+ B cells (n=3) and 3 IL-10- B cells (n=3) from asthmatic allergic (HDM) mice
Project description:Considerable information has accumulated about components of bone marrow that regulate survival, self-renewal and differentiation of hematopoietic cells. We have now studied Wnt signaling in that context, assessing influences on human and murine hematopoiesis. Microarray, PCR and staining experiments revealed that OP9 acquired osteoblastic characteristics while down-regulating some features associated with mesenchymal stem cells. This included down-regulation of Angiopoietin 1, c-Kit ligand and VCAM-1 expression. In contrast, production of decorin, tenascins and fibromodulin markedly increased. At least one of these extracellular matrix components, decorin is a regulator of hematopoiesis. That is, addition of the protein to OP9 co-cultures causes changes similar to Wnt3a. Two control samples (LZR1 & LZR2) were analyzed from OP9 mouse stromal cells transfected with vector only, and two experimental samples (W3A1 & W3A2) were analyzed from OP9 cells transfected with vector containing the Wnt3A gene.
Project description:Foxp3+ regulatory T cells (Treg) play a central role for tolerance against self and innocuous environmental antigens. However, the role of antigen-specificity for Treg-mediated tolerance is only incompletely understood. Here we show by direct ex vivo characterization of human CD4+ T cells, that the response against innocuous airborne antigens, such as plant pollen or fungal spores, is dominated by memory-like antigen-specific Treg. Surprisingly, breakdown of tolerance in atopic donors was not accompanied by a quantitatively or qualitatively altered Treg response, but instead correlated with a striking dichotomy of Treg versus Th2 target specificity. Allergenic proteins, are selectively targeted by Th2 cells, but not Treg. Thus human Treg specific for airborne antigens maintain tolerance at mucosal sites and the failure to generate specific Treg against a subgroup of antigens provides a window of opportunity for allergy development. PBMCs from sex and age matched birch pollen allergic patients and healthy controls, were stimulated (7h) with airborne fungal (A. fumigatus) or birch pollen antigen (birch) and sorted into antigen specific conventional and regulatory T cells according to their expression of CD154+ and CD137+ on CD4+ T cells, respectively. Number of samples per group in parentheses: Healthy controls stimulated with A. fumigatus (n=5), allergic patients stimulated with A. fumigatus (n=6), healthy controls stimulated with birch (n=6), allergic patients stimulated with birch (n=4).
Project description:Microarray and miRNA analysis of CD133(+), CD34(+)CD133(-) and CD133(-)CD34(-) cells The goal of the experiment was the comparison of expression levels of mRNA and miRNA in CD133(+) and CD34(+)CD133(-) cells The RNA of CD133(+), CD34(+)CD133(-) and CD133(-)CD34(-) bone marrow cells was amplified by using the µMACSTM SuperAmpTM Kit (Miltenyi Biotec).
Project description:Transcriptional profiling of subventricular zone (SVZ) progenitors comparing control healthy mice to mice induced to develop an autoimmune demyelination (EAE model). Goal was to unveil genes involved in demyelination-induced reactivity of SVZ progenitors. Two-condition experiment, healthy vs. EAE derived SVZ progenitors. Biological replicates: 2 control replicates, 2 EAE replicates. SVZ progenitors were sorted in two cell populations: neuronal progenitors (PSA-NCAM magnetic sorting) and glial progenitors (NG2 magnetic sorting). Progenitors from healthy mice are reference samples.
Project description:Purified CD4 T cells isolated from the cord blood were divided in subsets according to IL2R, IL7R and chemokine receptors (CCR6, CXCR3 and CCR4) and compared by whole genome microarray analysis. CD4 T cell memory subsets and Naive T cell collected from 3 donors (#480, #487, #488)
Project description:In vivo antigen (Ag)-induced differentiation of B lymphocytes into plasma cells (PCs) takes place in extra-follicular foci and germinal centers of the secondary lymphoid organs (SLOs). Most of these SLO PCs are short-living and only relatively few PCs, characterized by secreting high-affinity antibodies (Ab), travel through the circulation and finally home in specialized survival niches of the bone marrow (BM) and, at a lesser extent, in the SLOs, where they become long-living Ab-secreting PCs. We have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to distinguish between human circulating Ag-induced PCs in comparison with tonsil and BM PCs distinctively regulate genes involved in cell proliferation. Gene expressions in human plasma cells isolated from tonsil (7 samples), blood (6 samples) and bone marrow (7samples) were measured. Each sample was isolated from a different donor.
Project description:Analysis of Hoechst dye 33342-effluxing side population (SP) cells from B-CLL peripheral blood mononuclear cells. 9 biological replicates from B-CLL patients sorted into CD5+CD19+ SP and non-SP subsets. Two color comparative gene expression using Agilent microarrays.