Endotoxic shock is a common clinical syndrome which has a high mortality despite appropriate medical management. The syndrome appears to be due, in large part, to the release of endotoxin (lipopolysaccharide, LPS), a heterogeneous mixture of ampipathic molecules located in the outer membrane of gram- negative organisms. Innovative approaches to the treatment of gram-negative septicemia and shock are urgently needed. Lipid A is a relatively constant domain of LPS and responsible for most of its toxicity. Recent advances in lipid A biochemistry have led to the isolation and purification of lipid A precursors. Lipid X, a non-toxic monosaccharide lipid A precursor has been shown to protect animals from endotoxin and serious infections. The mechanism of protection is unknown. In order to better understand the pathophysiology of gram-negative infections as well as the protection afforded by lipid X, cellular and molecular studies are necessary. As these studies are beyond the expertise of the candidate, a rigorous five year training program is proposed. The training program combines coursework and benchwork to teach the candidate cell culture techniques, lipid and receptor biochemistry, genetics and some aspects of molecular biology. Furthermore, the candidate will be working in a productive basic science setting under the close guidance of an accomplished investigator. In this proposal, the use of novel lipid A precursors as biochemical probes is emphasized for, unlike radiolabeled endotoxins, these probes are chemically defined, and can be highly purified without loss of biological activity. Three projects are outlined: 1) the use of radiolabeled lipid A precursors to study the metabolism of lipid A; 2) the use of purified radiolabeled tetraacyl 1,4'-bisphosphate as a ligand to help identify lipid A binding protein (s); 3) the development of a eukaryotic cell line deficient in lipid A binding (phase II). This proposal should provide excellent scientific training for the candidate as well as advance fundamental understanding of the pathophysiology of gram-negative septicemia.
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