Inflammatory bowel disease (IBD) is the second most commonly occurring chronic inflammatory disease found in the U. S. population. Although various therapeutics are currently available to downregulate inflammatio in general, the need remains to develop more effective and specifically targeted therapies for the treatment of IBD. There is increasing evidence that cytokines play an essential role in mediating and regulating intestinal inflammation. Therefore, the overall objective of the present research proposal is to further understand the mechanisms of IBD, at both the cellular and molecular levels, by examining the in vivo regulation of inflammatory cytokines during intestinal inflammation.
Th specific aims of this proposal are: (1) To investigate the regulation of endogenous IL-1ra during intestinal inflammation. We will investigat the in vivo regulation of intestinal IL-1 and IL-1ra in healthy rabbits injected with factors previously shown to modulate the in vitro production of IL-1 and IL-1ra mRNA and protein synthesis; these factors include sub-lethal doses of endotoxin as well as anti-inflammatory cytokines, such as IL-4, IL-10, and IL-13. The in vivo regulation of th different molecular forms of rabbit IL-1ra, (intracellular and secreted) following experimental manipulations will also be investigated by Wester blot analysis and quantitative PCR techniques. (2) To examine the effects of IL-1ra neutralization and overexpression. The neutralization of endogenously produced IL-1ra by anti-IL-1ra treatment with purified polyclonal and monoclonal antibodies against rabbit IL-1ra, will be achieved during experimental-induced colitis. Determination of disease outcome and inflammatory cytokine profiles will be evaluated in models of both acute and chronic experimental colitides. In contrast, the effects of rabbit IL-1ra overexpression in healthy rabbits, as well as in rabbits with experimentally-induced colitis will be determined. We will use cutting-edge gene therapy techniques, including a recently developed cellular transplantation system which has the ability to produce high levels of circulating systemic protein, as well as infectio protocols using retroviral vectors which are able to deliver specific proteins to a targeted organ. Targeted overexpression of IL-ra may provide a novel and extremely effective therapy for the treatment of IBD (3) To determine the role of TNF and IL-8 in mediating intestinal inflammation. We will measure rabbit TNF and IL-8 mRNA and protein levels, and determine the effects of specific TNF and IL-8 blockade, in acute and chronic models of intestinal inflammation. Based on these results, development of specific pharmacological and gene therapy treatments will be assessed in order to neutralize the activity of these pro-inflammatory cytokines. (4). To evaluate the role other anti- inflammatory cytokines (IL-4, IL-10 and IL-13) may play in regulating intestinal inflammation. We will clone rabbit IL-4, IL-10 and IL-13, an express their corresponding recombinant proteins. We will then develop specific cDNA probes and RIAs needed to determine the intestinal expression of IL-4, IL-10 and IL-13 in models of acute and chronic rabbi colitides. Based on the results, gene therapy techniques may also be developed to increase the potency of these anti-inflammatory cytokines during intestinal inflammation. The ultimate goal of the present research proposal is to approach the treatment of IBD through the regulation and manipulation of intestinal cytokines.
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