ENDOTOXIN RESISTANCE AFTER ADENOVIRAL GENE TRANSFER

Temple University, Philadelphia PA

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Abstract

DESCRIPTION (Verbatim from the applicant's abstract) The interaction of the outer membrane components of bacteria with host cells in one form or another has been the subject of numerous investigations over the past decades. The lipopolysaccharide endotoxin (LPS) of Gram-negative bacteria is an example of an essential element of the outer membrane of these organisms that as an amphipathic molecule can bind to and stimulate various types of mammalian cells. The results of this interaction are multiple pathophysiological, pharmacological and immunological responses by the host. Over the preceding years, our long-term objective has been to gain a better understanding of how LPS endotoxin affects cells of the host. The knowledge that host responses are under genetic control is key to this understanding. Consequently, we have pursued the goal of isolating the LPS gene by the use of the C3H/HeJ mouse strain whose cells possess a specific defect for LPS. To this end, we have isolated a gene Lps/Ran by functional cDNA cloning, which encodes for Ran TC4 GTPase. We have also isolated the Ran Lps(d) cDNA from cells of C3H/HeJ mice, which has a point mutation at position 870 of the cDNA. This leads to profound changes in LPS endotoxin responses, which include down-modulation of TNFa production by macrophages, reduced B cell proliferation and mitogenicity, and resistance to endotoxin challenge. These differences in biological responses are the result of faster migration of the Lpsd/Ran protein into the nucleus. Our specific aims, which are a logical extension of these findings, as well as the recent exciting findings about the involvement of the toll-like receptors in innate immunity, include the studying of the in vivo biological effects of tlr4 and tlr2, with their dominant negative mutants, and compared these effects with Lps/Ran; the relationship between tlr2/tlr4 and Lps/Ran by endotoxin-induced lethality; the protective effect of Lpsd/Ran in several strains of inbred mice; the protective effect of Lpsd/Ran as a prophylactic; the types of tissues or cells highly expressing the Lpsd Ran gene in mice rendered resistant to endotoxin challenge; the examination if Lpsd/Ran could render C3H/HeOuJ mice resistant to, and if LpsD/Ran could render C3H/HeJ mice sensitive to endotoxin challenge; and the role of Lps/Ran in its resistance to Gram-negative bacterial infection. We believe these study will provide insightful information towards clinical application.