This proposal involves the study of biochemical mechanisms and purine metabolizing enzymes involved in purine nucleoside phosphorylase (PNP) deficiency and a comprehensive immunological and biochemical study of canine models of induced PNP deficiency. The distribution of deoxynucleoside kinases in isolated lymphocytes will be determined by autoradiography and catalytic assay and the kinetics of phosphorylation of deoxyguanosine by purified enzymes. Mechanisms including catabolism of nucleotides, inosine mediated inhibition of T cell function and intracellular cyclic nucleotides will be studied. We will investigate in vitro the proliferation and metabolism of deoxyguanosine in isolated dog, rat, and human lymphocyte sub- populations. We will determine if 8-aminoguanosine is a specific PNP inhibitor. We will study the immunosuppression produced by 8-aminoguanosine treatment in three model canine systems: the prevention of platelet immunization to allogeneic platelet transfusions; the prolongation of skin graft survival in dogs receiving grafts from DLA nonidentical unrelated donor dogs; the prevention of graft-versus-host disease in allogeneic bone marrow transplantation. In these studies the phenotypic characterization of B and T cells and T cell subsets will be assayed by specific monoclonal antibodies. Immunological responses in vivo will be monitored by immunization with sheep red blood cells and keyhole limpet hemocyanin, and in vitro by mitogen stimulation and mixed leukocyte culture. To monitor the efficacy of PNP inhibition we will use HPLC to assay the bioactive inhibitor 8-aminoguanine and purine metabolites in plasma, and nucleotides in red and white cells. This research provides a novel approach to the specific regulation of T cell function; the results may be directly applicable to platelet transfusion therapy in the support of cancer patients, and may benefit organ and marrow transplantation and treatment of T cell malignancies.
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