RICKETTSIA RICKETTSII IN HUMAN ENDOTHELIAL CELLS

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Rocky Mountain spotted fever (RMSF), caused by Rickettsia rickettsii, is considered the most severe of the human rickettsioses. The putative target cells of infection are the endothelial cell and occasionally smooth muscle cells of small blood vessels. The primary purpose of this investigation is to determine the specific mechanism of cell injury to human vascular endothelial cells cause by R. rickettsii, and to examine the potential biological ramifications of platelet adherence to infected endothelial cells. Understanding of the mechanism of cell injury by R. rickettsii and the consequences of platelet adherence to a normally non-thrombogenic surface should contribute considerably to a better undertanding of the pathogenesis of RMSF, and could provide for more specific therapeutic management of severe forms of the disease. This study will be carried out exclusively in human endothelial cells derived from the umbilical vein using combined biochemical and electron microscopic techniques. The hypothesis that endothelial cell injury caused by R. rickettsii is the direct result of intracellular rickettsial metabolism, will be tested. Specifically, that lipid peroxidation of intracellular membranes by toxic free radicals leads to dilatation and disorganization of the ER and eventually to lysis of the cell. Analysis of levels of malonaldehyde, the primary degradation product of peroxidation, and superoxide anion and superoxide dismutase will be carried out as well as the activity of two ER membrane markers, glucose-6-phosphatase and cytochrome P-450 which are destroyed as a result of peroxidation. Steroids and the antitoxidants glutathione and vitamin E will be examined for their protective capacity as free radical scavengers. The effects of superoxide dismutase incorporated into unilamellar liposomes and added to R. rickettsii-infected endothelial cells will be studied to determine whether cell injury caused by the organisms can be modified. Studies on platelet adherence using [3H] adenine and scanning electron microscopy, and the activation status of adherent platelets using [3H] serotonin will also be studied. Lastly, as a prelude to a future study, an attempt will be made to isolate and characterize plasmid DNA from R. rickettsii.