The major emphasis of the research project concerns the characterization of mast cell mediators, in particular the neutral proteases, as to their role in mast cell-mediated events. The rationale for assuming an important role for mast cell proteases is based on their abundant presence (accounting for 15-30% of total cell protein) and their selective presence in mast cells and mast cell subsets. Mast cells obtained from rat serosal surfaces and those derived from mouse hematopoietic tissue and maintained in culture, both of which can be obtained regularly in greatest abundance and purity, and from human pulmonary tissue, which are of greatest relevance to human biology, will be utilized.
Specific aims with rodent mast cells include determination of the mechanism by which exogenous chymase activates rat serosal mast cells and its relation to immunologic activation; the role of released chymase in the mast cell-mediated inflammatory response; purification and characterization of the chymotryptic and tryptic murine mast cell proteases; and studies of the synthesis of neutral protease, histamine, and proteoglycan and their incorporation into secretory granules. Projects with human mast cells include development of conditions to improve the yield and purity of lung mast cells; production of monoclonal and monospecific antibodies against human mast cell tryptase to be used for immunoassays, localization of tryptase in tissues and cells, and immunoaffinity chromatography of tryptase; determination of the enzymatic properties of tryptase on synthetic and biologic substrates and characterization of the stabilization of tryptase by heparin and the effect of neutrophils on tryptase. Biologic activities of tryptase on the coagulation, kinin, and complement pathways, connective tissue components and on production of chemotactic activities will be examined. Tryptase appears to be a specific marker for mast cells; preliminary results indicate that the specific and sensitive immunoassay for tryptase recently developed detects it in biologic tissues and fluids and indicates mast cell activation. This unique ability to determine mast cell involvement in human disease will assist with the diagnosis and eventual treatment of allergic diseases and in a better understanding of the biology of mast cells.
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