Systemic lupus erythematosus (SLE) is a multisystemic autoimmunune disorder of unknown etiology. Many patients afflicted with SLE develop a severe nephritis mediated by the localization of immune complexes within glomeruli. Current treatments of this nephritis and other symptoms of the disease center on the use of steroids or cytotoxic immunosuppressive agents. While effective in some patients, these agents are non-specific and can cause serious side effects that often force discontinuation of treatment. We have identified a benzodiazepine (1) that does not bind to the central benzodiazepine receptor, which shows significant efficacy in treating the glomerulonephritis and other symptoms of disease in both (NZB x NZW)F1 and MRL/MpJ-lpr/lpr mice, the two most clinically relevant animal modes of human SLE. Both in vitro and in vivo experiments demonstrate that this benzodiazepine selectively induces apoptosis in lymphocytes directly responsible for autoimmune (B and T cell) mediated inflammation. At therapeutic doses, treatment suppresses the autoimmune response without altering normal immune function or evidence of side effects. Further development of 1 into a clinically useful agent requires a better understanding of both how this molecule induces cell death, and by what means apoptosis of lymphocytes results in disease improvement. To address this need, we propose to characterize the effects 1 has on cellular immune function by determining the phenotype of lymphoid cells killed by 1 in vivo and in vitro. Phenotypic analysis will be based on plasma membrane determinants and cytokine expression. Next, to uncover the mechanism by which 1 functions, we will identify the sub-cellular systems (e.g. caspases, mitochondria and other components of death response) triggered to execute 1-mediated cell death. Lastly, we will isolate the receptor of 1 based on yeast three-hybrid screening. If 1 is lethal to yeast, we will pursue an alternative strategy of mutagenesis followed by classical yeast genetics and functional cloning to generate resistant strains and identify molecules necessary for 1-induced death. The resistant strains developed will then serve as host strains for the three-hybrid system. These experiments will complement on-going efforts to identify the receptors(s) for 1 using more traditional affinity chromatography methods.
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