Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): Develop immune reagents for veterinary animals of economic importance including cattle, swine, poultry, horses, and aquaculture species. Approach (from AD-416): Immune reagents will be developed for basic and applied studies in veterinary animal species. They will be developed into multiplex assays to measure cytokine and chemokine production during infections and host immune responses. As a member of a large consortium grant to develop immunological reagents for five different animal species, we will identify major cytokines and cell surface markers of poultry, express them in suitable expression vectors and validate their function using in vivo and in vitro assay systems. The main mission of this NIFA-funded AFRI consortium grant is to develop immunological reagents specific for poultry, ruminants, swine, equine and aquaculture species. During this reporting period, we have characterized a protein called CD83 that is primary found on the cell surface of mature chicken dendritic cells. The dendritic cell is an important immune cell type as it can stimulate an immune response from other immune cells, particularly lymphocytes, involved in the development of acquired immunity. To characterize chicken CD83 protein, we developed two distinct mouse antibodies that detect CD83. The antibodies identified other immune cells expressing CD83 and regions in tissues where CD83 positive cells, likely dendritic cells, are found. These antibodies will be useful for basic and applied research on chicken dendritic cells.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): Develop immune reagents for veterinary animals of economic importance including cattle, swine, poultry, horses, and aquaculture species. Approach (from AD-416): Immune reagents will be developed for basic and applied studies in veterinary animal species. They will be developed into multiplex assays to measure cytokine and chemokine production during infections and host immune responses. As a member of a large consortium grant to develop immunological reagents for five different animal species, we will identify major cytokines and cell surface markers of poultry, express them in suitable expression vectors and validate their function using in vivo and in vitro assay systems. This agreement was established as a 4-year consortium grant to advance veterinary immunology. The main mission is to develop immunological reagents specific for poultry, ruminants, swine, equine, and aquaculture species. During this reporting period, we have characterized chicken receptor, which binds to interleukin-2 (IL-2). Interleukin-2 is synthesized and secreted mainly by activated T-type lymphocytes and plays a pivotal role in the growth and differentiation of T- and B-type lymphocytes, monocytes, and natural killer cells. Three IL-2 receptor subunits form a trimeric complex consisting of alpha (CD25), beta (CD122), and gamma (CD132) chains. In chickens, limited information exists for CD25, a transmembrane glycoprotein that binds to IL-2 with high affinity and modulates T-cell proliferation. In this reporting period, ARS scientists developed a recombinant plasmid (chCD25/horse immunoglobulin G4) as an immunogen to induce mouse hybridomas to produce monoclonal antibodies against chicken CD25. These monoclonal antibodies detected CD25 cells in chicken tissues such as spleen, bursa, and duodenum, as well as several chicken cell lines, and a 55 kilodalton protein of chicken spleen blast cell. These results show that chCD25 is an important immunological tool for investigating biological functions of chicken regulatory T cells.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) Develop immune reagents for veterinary animals of economic importance including cattle, swine, poultry, horses, and aquaculture species. Approach (from AD-416) Immune reagents will be developed for basic and applied studies in veterinary animal species. They will be developed into multiplex assays to measure cytokine and chemokine production during infections and host immune responses. As a member of a large consortium grant to develop immunological reagents for five different animal species, we will identify major cytokines and cell surface markers of poultry, express them in suitable expression vectors and validate their function using in vivo and in vitro assay systems. This is a reimbursable agreement with the University of Massachusetts to advance veterinary immunology. The main goal of this agreement is to develop immunological reagents specific for poultry, ruminants, swine, equine and aquaculture species. Veterinary immunological reagents that will be developed in this grant period include monoclonal antibodies (mAb) and polyclonal antibodies which can identify the major leukocyte subpopulations. Sequence of 5 genes (IL1R1, IL7R, IL10R-beta, IL17R, and IL21R) were verified and these genes were sent to Cornell Univ. for the construction of chimeric proteins with equine IgG1 heavy chain constant region. Eleven chicken cDNA homologs (CXCL4, GM-CSF, IFN-gamma, IL12p40, IL17A, IL17D, IL21, IL22, LITAF, Lymphotactin, and TNFSF) were amplified from Con-A stimulated chicken spleen cell RNA using gene-specific primers and sent to the Kingfisher Laboratory for protein expression. Chicken IL4, IL12, GM-CSF, IFN-gamma, and TNFSF, and turkey IGF-1 and IGF-2 were biologically active. CD25 and CD80 expressed at Univ. of Mass and IL18 expressed at ARS were biologically active. Four hybridomas (IL-17A, IL17D, IFN-gamma, and TL1A) have been developed against each chicken cytokines, showed preferential binding to its respective immunizing antigen, and were single cell cloned. Twelve Polyclonal Abs (Ch IL2, IL4, IL8, IL17A, IL17D, TGF-�4, TL1A, cMIF, EaMIF, Net B, a-toxin, and Gam82) were developed. This agreement is monitored by monthly teleconferences, annual progress reports, and bi-annual meetings among the participating scientists. This research relates to NP103, C2, P.S.2c
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