The broad long-tern objective of this proposal is to understand the role of superoxide in osteoclast-mediated bone resorption. Our relationship between osteoclastic superoxide generation within the ruffled border space and bone resorption; 2. to determine if the effect(s) of superoxide produced in bone is due to superoxide generated within the osteoclastic cellular membrane or outside or both; 3. to determine if a cytokine, Interferon Gamma, known too stimulate superoxide generation in leukocytes from patients with chromic granulomatous disease, enhances osteoclast- mediated bone resorption in and osteopetrotic mouse mutant and if so, to localize the site of action. In these experiments, the effects of superoxide scavengers will be monitored in cultures of isolated osteoclasts and calvaria. Comparing a scavenger which is excluded from cellular membranes by size to a low molecular weight scavenger which permeates membranes, the site of superoxide's effects upon bone resorption will be sought. By using neonatal animals of different ages, we shall assess the importance of the rate of osteoclasts formation upon the mechanism by which oxygen radicals affect bone resorption. NBT staining will be used to measure and localize changes in oxygen radical production in individual osteoclasts. Interferon Gamma will be used to stimulate superoxide generation in osteoclasts from osteopetrotic misc. If Interferon Gamma stimulates osteoclast-mediated bone resorption in tissues cultured from osteopetrotic animals, superoxide scavengers will be added to identical cultures to determine the contribution of superoxide generation to the increased bone resorptive capacity. These experiments have already provides the basis for a phase I/II trial of Interferon Gamma to treat osteopetrotic patients.
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