The long-term objectives and specific aims of this application are to understand the pathogenesis of Rickettsia rickettsii, and other members of the spotted fever group of rickettsiae by better studying the processes involved in causing cell injury. The investigators are interested in the interaction of R. rickettsii (Rocky Mountain spotted fever) with the endothelial cell, the target cell in human infection, and the mechanism(s) of cell injury caused by this obligate intracellular bacterium. It has been determined by the Principal Investigator's laboratory that cell injury appears to be initiated by reactive oxygen species produced during internalization and intracellular rickettsial growth. With an interest in abrogating endothelial cell injury, they have incorporated antioxidants into infected cell culture systems. One antioxidant, (alpha-lipoic acid, was able to effectively reverse the leading predictors of cell injury due to reactive oxygen species and to substantially prolong the viability of infected cell cultures. Studies to date have been carried out both in human umbilical vein endothelial cells and the permanent human endothelial cell line, EA.hy926. The investigators will continue to carry out experiments in these cells to characterize changes in the cytoskeleton of endothelial cells following R. rickettsii infection and to evaluate the link between these changes and increases in endothelial permeability. They will determine whether R. rickettsii-induced oxidative injury is responsible for the alterations in the actin pools and whether these changes can be prevented (controlled) by the use of antioxidants. They believe that it is also important to test our in vitro observations on oxidant-mediated injury in a relevant animal model to determine whether oxidant injury occurs and if antioxidants are effective in altering the course of disease. The most suitable model for these studies is the Rickettsia conorii-C3H/HeN mouse model which has been extensively studied by Walker and his colleagues. R. conorii is closely related to R. rickettsii, causes Mediterranean spotted fever, a syndrome clinically similar to RMSF, and as they have demonstrated, results in the same dramatic morphological manifestations of cell injury as R. rickettsii in human endothelial cells. These preliminary in vitro studies and those in the mouse model, indicate that this rickettsia is also able to cause oxidative stress. They propose to test the hypothesis that oxidant injury contributes to the pathologic changes within the animal. They will determine whether oxidative injury correlates with the local presence of rickettsiae within particular organs and tissues and whether the antioxidant, alpha-lipoic acid, can ameliorate oxidative injury as they have shown in vitro.
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