This project is a strategic component of our long-range research goals, which are to identify the molecular mechanisms responsible for the contribution of endotoxin (LPS) to septicemia, and to use this new knowledge to develop efficacious treatments. The project has four objectives designed to test our central hypothesis that structurally unique lipid-A analogs based on the lipid-A of nitrogen-fixing rhizobial bacteria exert antagonistic effects on enteric LPS-induced production of proinflammatory mediators by interfering with the interaction between enteric LPS and CD14 receptors on human monocytes. To test this hypothesis, an initial series of twenty-seven uniquely hybrid lipid-A analogs having structural characteristics ranging from the classic lipid- A of E. coli to that of the rhizobial species will be obtained by organic synthesis. These well defined, pure analogs then will be tested for agonist activity on human monocytic cells, using the production of tumor necrosis factor (TNF) activity as the primary readout. These structure/function analyses will permit identification of individual structural alterations in lipid-A that result in the presence or lack of agonist activity. The analogs then will be tested for their ability to antagonize the pro-inflammatory effects of enteric LPS in classical pharmacologic antagonism studies and for their ability to prevent binding of radiolabeled enteric LPS to the monocytes. The latter studies will be performed to evaluate the involvement of CD14- dependent and CD14-independent pathways. Finally, studies will be performed to determine the relative roles of the NF B and MAP kinase pathways in tranducing the effects of the lipid-A analogs. Completion of these studies will provide the basis required to identify the structural features responsible for the deleterious effects of lipid-A and the beneficial effects of potentially novel LPS antagonists. On completion of this project, we will have important information about a potentially novel approach to prevent the proinflammatory effects of enteric LPS. The rationale for these studies is that until the molecular mechanisms responsible for LPS activation of mononuclear phagocytes are understood and the most efficacious antagonists are identified, it will not be possible to prevent LPS-induced complications of septicemia.
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