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This application tests two hypotheses regarding the binding and toxicity of Shiga toxin (Stx) of enterohemorrhagic Escherichia coli (EHEC) to renal and cerebral endothelial cells, a critical process in HUS/TTP pathogenesis: <ol>
<li> Metabolic regulation of globotriaosylceramide (Gb3) and galactosyl Gb3 (Gal Gb3), the glycolipid receptor(s) for Stx in endothelial cells, is central to HUS/TTP.</li>
<li> Binding to specific Gb3/GaI Gb3 molecular species is responsible for biological activity of Stx. </li></ol></p>
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Acute renal failure and central nervous system (CNS) injury are life-threatening complications of hemolytic uremic syndrome and thrombotic thrombocytopenic purpura (HUS/TTP). Survivors of HUS/TTP sometimes suffer irreversible damage. </p>
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Specific fatty acid moieties in the ceramide portions of Gb3/GaI Gb3 have been shown to influence Stx binding, and specific Gb3 species relate more strongly than total Gb3 to Stx-induced injury of endothelial cells from human saphenous veins. Using the cells most relevant to HUS/TTP, human glomerular endothelial cells (hGEC) and human cerebral endothelial cells (hCEC), investigations will:
<ol>
<li> Isolate and measure individual Gb3/GaI Gb3 species in hGEC and hCEC cultures incubated with and without TNF-alpha and IL1-beta. </li>
<li> Determine which Gb3 and Gal Gb3 species relate to Stx1 and Stx2 toxicity in cytotoxin-stimulated and unstimulated cultures and to Stx binding to cells. </li>
<li> Define the metabolic pathways that synthesize the individual molecular species of Gb3/GaI Gb3 in response to TNF-alpha or IL1-beta stimulation by selectively inhibiting previously described pathways. </li>
<li> Determine which UDPgalactose: lactosylceramide alpha1, 4 galactosyltransferase isozymes control this synthesis. </li></ol></p>
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Elucidation of the different sensitivities of renal and cerebral cells to Stx may explain differences in the pathogenesis of renal and CNS disease in HUS/TTP. The detailed description of the expression of these endothelial cell Stx receptors will potentially lead to development of precisely targeted therapies that can reduce Stx toxicity and these devastating effects of HUS/TTP.</p>