Alterations in cellular immunity and cytokine (lymphokine) production are features of many human diseases, both neoplastic and non-neoplastic. Therapeutic manipulation of this important element of the immune response requires a more complete biochemical understanding of cytokine structure and function than is currently available. T cell fibronectin (FN), a lymphokine produced by antigen and mitogen activated T ells, is involved in the initiation of delayed hypersensitivity inflammatory reactions. During the past project period we have found that monoclonal antibodies able to neutralize the monocyte agglutinating activity of T cell FN in vitro inhibit the expression of delayed hypersensitivity vascular responses in vivo. The FN are a family of closely related, high molecular weight glycoproteins found in plasma and tissues. Different FN arise by cell- specific alternative splicing of mRNA derived from a primary transcript of a single complex gene. Although T cell FN shares many properties with other FN, its distinctly smaller molecular weight and extraordinarily high activity relative to other cellular FN documented during the past project period suggests that it differs from them in primary sequence. The project is divided into two subprojects. Subproject 1 will define differences between T cell and other FN at the primary sequence level. Segments of T cell FN cDNA synthesized from T cell FN mRNA by reverse transcriptase will be amplified by the polymerase chain reaction, and their size compared with size predicted from the known sequence of the FN gene and observed after amplification of other cDNA derived from other cellular FN. Segments differing in length from the predicted value will be sequenced to determine which domains are present in T cell FN and how this choice of domains differs from that seen in other FN. Subproject 2 will study the mechanism of action of T cell FN and its interaction with other lymphokines in the initiation of DH inflammatory reactions. Studies in vitro will employ a model based on a two-compartment culture system separated by a confluent endothelial cell monolayer and assays for T cell FN activities (monocyte agglutination, matrix-driven translocation). Lymphokines, vasodilating arachidonic acid metabolites, peptides based on T cell FN sequences and expressed sequences of T cell FN (from materials generated in subproject 1) that are able to modify release of T cell FN or its activities in vitro will be studied for their effects on in vivo delayed hypersensitivity reactions in order to clarify the role of T cell FN in these cellular immune reactions.
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