The objective of the research project is to study the pathogenesis of immunodeficiency diseases associated with inherited adenosine deaminase deficiency and urine nucleoside phosphorylase deficiency. Our hypothesis is that patients with adenosine deaminase deficiency accumulate deoxyadenosine as a result of phagocytosis of senescent cells by macrophages of the reticuloendothelial system. This hypothesis will be tested with mutant macrophage lines lacking adenosine deaminase. Deoxyadenosine excreted by the macrophages then causes killing of proliferating and non proliferating T-lymphocytes, resulting in immune deficiency. The mechanism of cytotoxicity of deoxyadenosine in proliferating lymphocytes will be studied with mutants of a mouse lymphoma cell line lacking adenosine deaminase and deoxyadenosine kinase. Its cytotoxic effects on non- proliferating T-lymphocytes will be investigated by using mitogen- stimulated mouse splenic T-lymphocytes, with emphasis on the early events, before the DNA synthesis begins. We are especially interested in blockade by deoxyadenosine of entry of the activated T-lymphocytes from G0/Gl into S phase. A hypothesis that the inhibition of c-myc and c-fos gene expression plays a role in such a blockade will be tested by the Northern hybridization technique. DNA strand breaks will be examined and a correlation, if any, between the DNA breaks and the production of a specific nuclease secondary to the deoxyadenosine-induced GO/Gl arrest will be made. Finally, we will isolate mouse embryonic stem cell mutants efficient in adenosine deaminase. This would be a first step toward the construction of an animal model for adenosine deaminase deficiency. Our research will enhance understanding of the pathogenesis of these two specific forms of immunodeficiency diseases, help clinicians in designing a biochemical treatment method for the diseases, and provide cell culture models and, eventually, an animal model for effectivee """"""""gene therapy"""""""" of hereditary adenosine deaminase deficiency.
