The goals of the research program are to characterize hematopoietic growth factors which influence the proliferation of early human progenitors, both normal and neoplastic. Four areas will be emphasized. First, we will continue studies of the regulation of growth factors release form mesenchymal cells. This will include Northern analyses of the kinds of growth factors released from cells such as lymphocytes, fibroblasts, endothelial cells, and smooth muscle cells. Probes for human granulocyte /macrophage (GM), granulocyte (G), macrophage (M) and multi- (interleukin-3) colony-stimulating factors (CSFs) will be employed. The effects of various mediators of immune and inflammatory responses will be studied and will include interleukin1, lipoppolysaccharide, tumor necrosis factor, lymphotoxin, and thrombin. Relative transcription rates of the genes for the CSFs will be measured to determine if they are differentially expressed by the various mediators of inflammation. Second, we will explore the structure/function relationship of human GM- and G-CSF at the protein and molecular levels. For studies at the protein level, we will generate monoclonal antibodies to human GM- and G-CSF by purifying them from mammalian cells transfected with cDNA for these factors. At the molecular level, sitespecific mutagenesis will be carried out to determine which element of the gene are important for biological function of the intact protein. Third, we will study the effect of purified recombinant GM- and G-CSF, alone and together, on progenitor cells obtained from the peripheral blood and marrow of normal volunteers and patients with myeloproliferative disorders. To assess the nature of the interaction of the growth factors with the progenitor cells, the cells will be grown at low concentrations under serumfree conditions. Fourth, we will seek to clone the cDNA for a potentially novel growth factor produced by feline leukemia virusinfected embtyonic cat fibroblasts. The strategy will involve subtraction hybridization to enrich mRNA for the growth factor, generation of a cDNA library in an expression vector, and identification of useful clones by biological assays.
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