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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI019565-05
Application #
3128886
Study Section
Immunological Sciences Study Section (IMS)
Project Start
1983-09-01
Project End
1991-03-31
Budget Start
1988-04-01
Budget End
1989-03-31
Support Year
5
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Washington
Department
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Lynch, C M; Clowes, M M; Osborne, W R et al. (1992) Long-term expression of human adenosine deaminase in vascular smooth muscle cells of rats: a model for gene therapy. Proc Natl Acad Sci U S A 89:1138-42
Ramesh, N; Osborne, W R (1991) Assay of neomycin phosphotransferase activity in cell extracts. Anal Biochem 193:316-8
Stockschlaeder, M A; Storb, R; Osborne, W R et al. (1991) L-histidinol provides effective selection of retrovirus-vector-transduced keratinocytes without impairing their proliferative potential. Hum Gene Ther 2:33-9
Palmer, T D; Rosman, G J; Osborne, W R et al. (1991) Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes. Proc Natl Acad Sci U S A 88:1330-4
Adam, M A; Ramesh, N; Miller, A D et al. (1991) Internal initiation of translation in retroviral vectors carrying picornavirus 5' nontranslated regions. J Virol 65:4985-90
Kaleko, M; Garcia, J V; Osborne, W R et al. (1990) Expression of human adenosine deaminase in mice after transplantation of genetically-modified bone marrow. Blood 75:1733-41
Osborne, W R; Miller, A D (1988) Design of vectors for efficient expression of human purine nucleoside phosphorylase in skin fibroblasts from enzyme-deficient humans. Proc Natl Acad Sci U S A 85:6851-5
Osborne, W R (1986) Nucleoside kinases in T and B lymphoblasts distinguished by autoradiography. Proc Natl Acad Sci U S A 83:4030-4
Osborne, W R; Barton, R W (1986) A rat model of purine nucleoside phosphorylase deficiency. Immunology 59:63-7
Osborne, W R; Deeg, H J; Slichter, S J (1986) A canine model of induced purine nucleoside phosphorylase deficiency. Clin Exp Immunol 66:166-72

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