NUTRITIONAL COPPER DEFICIENCY AND THE IMMUNE RESPONSE

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The long-range objective of this research is to elucidate the biochemical roles of copper in immune function and to determine when during development copper is most critical to the immune system. Selected biochemical and immunological techniques will be used. To accomplish these goals and to test certain hypotheses, animal models will be employed. We will induce dietary copper deficiency during perinatal development in C57BL and C58 mice during several discrete periods from gestation to early adulthood. Repletion studies will be conducted. Copper status during depletion and following repletion will be assessed by determining blood hematocrit and plasma ceruloplasmin activity. Organ (spleen and thymus) and serum levels of total copper and iron will be measured by atomic absorption spectroscopy (AAS). Changes in functional copper pools will be evaluated by assaying the activity of copper-zinc superoxide dismutase or cytochrome c oxidase. Immunological evaluation will include investigating humoral immunity by determining the number of antibody-producing cells in spleen in response to sheep erythrocyte challenge. Cell-mediated immunity will be studied by quantifying mitogen reactivity of cultured thymocytes and splenocytes and by mixed lymphocyte reaction. Lymphocyte subpopulations from spleen will be determined by immuno-fluorescent procedures using monoclonal antibodies to surface antigens. Plant lectins (PNA and SBA) will be used to evaluate maturational development of lymphocytes from thymus, spleen and bone marrow. Serum IgM and IgG will be measured by ELISA techniques. Additional biochemical measurements will be conducted to elucidate the mechanisms of impaired immunity while testing specific hypotheses. Lymphocytes from spleen and thymus will be isolated, and the plasma membrane will be characterized for changes in lipid and protein composition using gas chromatography and gel electrophoresis, respectively. Antioxidant status will be evaluated in lymphoid organs by quantifying specific metabolites. Repletion studies will be conducted comparing cupric or ferrous gluconate and purified mouse ceruloplasmin or transferrin to determine the extent, if any, that altered immunity is iron-dependent. Plasma, brain and spleen levels of norepinephrine will be quantified by HPLC with electrochemical detection. Copper metabolism will be studied in developing lymphoid organs using 67Cu and AAS.