Infections with certain agents, such as HIV, can be exacerbated by the presence of opiates or opioids, which may act as cofactors in the pathogenesis of infections. Cells of the immune system express opioids and are functionally modified by both opioids and opiates, most likely through opioid receptors similar to those in the brain. The mechanisms by which these compounds affect immune cell function remain unclear. We have been investigating the role of proenkephalin A (PEA) mRNA and enkephalins in thymocyte activation and maturation. The present application seeks to analyze the role enkephalins play in the growth and differentiation of fetal CD4+ thymocytes. In addition, the effect of an opiate, morphine, on these parameters will be studied. I. We will study the expression of PEA mRNA and enkephalins, as well as delta opioid receptor mRNA, in fetal thymocytes by asking: (i) In which subset of double-negative fetal thymocytes are PEA mRNA and enkephalins expressed? (ii) Is PEA mRNA inducible in CD4+ fetal thymocytes? (iii) Is there a developmental connection between a subset of double-negative thymocytes and a subset of CD4+ thymocytes that can be defined by the ability of these cells to express PEA mRNA? (iv) We will also quantitate by PCR the amount of selected cytokine mRNAs present during gestation and analyze their relationship to the expression of PEA mRNA and enkephalins. In addition, we will (v) quantitate by PCR the amount of delta opioid receptor mRNA expressed during gestation; (vi) identify the cell subset(s) in which it is expressed; (vii) analyze its regulation, and (viii) relate its expression to functional studies of PEA mRNA and enkephalins in fetal thymocytes using delta opioid receptor agonists and antagonists. II. We will study the function of PEA mRNA and enkephalins in fetal thymoctye maturation by asking: (i) What are the mechanisms by which enkephalins inhibit fetal thymocyte proliferation? and (ii) What are the effects of blocking enkephalin function or expression in fetal thymic organ cultures on thymocyte differentiation? III. We will also study the effect of an opiate, morphine, on thymocyte maturation by analyzing its effects on PEA mRNA and enkephalin expression in vitro in fetal thymocytes and on aspects of fetal thymocyte maturation as measured in fetal thymic organ cultures. These studies will elucidate a role for endogenous enkephalins in fetal thymocyte maturation, as well as provide information on the mechanisms by which a drug of abuse, such as morphine, might affect both thymocyte maturation and, ultimately, T lymphocyte development and the establishment of cell-mediated immune responses. Such data could lead to greater understanding of the role of opioids and opiates as cofactors in the pathogenesis of infectious agents, such as HIV.