The complement system is a prime mediator of tissue inflamation. The present grant proposal seeks to characterize the complement system in both normal and inflamed corneas and aqueous humor and elucidate the role of activated complement in anterior segment inflammation.
The specific aims of the grant include the following: 1) Alternative pathway. Little is known about the alternative pathway in the cornea and aqueous humor. We propose to measure levels of functional factor B and D in normal and inflamed human corneas and aqueous humor using hemolytic diffusion plates. Also, we plan to demonstrate the presence of an intact functional alternative pathway of complement in both normal and inflamed human corneas and aqueous humor using hemolysis of unsensitized rabbit red blood cells. 2) Production of C3 by corneal fibroblasts. Human corneal fibroblasts will be grown in tissue culture to detemine if they have the ability to synthesize and secrete C3, the most important complement compotent and pivotal component of the classical and alternative pathways. 3) Regulatory proteins. We will attempt to identify the important regulatory proteins of the complement system (C1 inactivator, C3b inactivator and beta 1H) in both normal and inflamed human corneas and aqueous humor using gel double diffusion. In addition, we propose to quantitate levels of C1 inactivator and C3b inactivator using functional assays depending on the ability of C1 inactivator to inhibit hemolytic activity of purified C1 and C3b inactivator to inhibit immune adherence. 4) Role of complement in host defense against bacteria. Using systemic decomplementation with cobra venom factor, we plan to assess the role of complement as a host defense mechanism of guinea pigs in bacterial keratitis and endophthalmitis caused by S. aureus or P. aeruginosa. Severity of infection in control and decomplemented guinea pigs will by assessed by bacterial counts in cornea and vitreous. 5) Activated complement. We plan to identify activated complement in guinea pig and human corneas and aqueous humor. Immunoelectrophoresis and crossed immunoelectrophoresis will be used to detect cleavage products of C3 in guinea pigs and C3, C4 and Factor B in humans. The extremely sensitive technique of radioimmunoassay will be used to measure levels of C3a in human corneas and aqueous humor. These techiques should allow a direct assessment of complement activation in anterior segment inflammation.
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