Ten to 35 percent of the U.S. adult population has some form of venous disorder and more than 500,000 people suffer from venous stasis ulcers (2). Although the association between venous hypertension and ulcer formation, and prolonged wound healing remains ill-defined (1). Dermal infiltration of mononuclear cells with clinical disease progression, suggests that these cells play a pivotal role in the pathogenesis of CVI (3,4,5). Results of pilot studies performed during the applicant's research year of his vascular fellowship, indicate that with clinical progression of CVI, mononuclear cell proliferative responses to bacterial exotoxins, produced by normal skin flora, decrease. The applicant hypothesizes that loss of epidermal integrity exposes mononuclear cells to bacterial byproducts which stimulate the production of interferon (IFN- gamma), the interleukin-4 (IL-4), and interleukin. The former cytokines inhibit bacterial overgrowth and depress epidermal regeneration while the latter, stimulate epidermal and fibroblast ingrowth. The short term objectives of this research project are to determine the cellular alterations and molecular regulation of cytokine induction associated with CVI. To test the above hypothesis, the specific aims during the award are: 1) To determine the functional responses and intracellular signalling mechanisms of T-lymphocyte and monocytes from the systemic circulation and wounds of patients with CVI, as compared to normal, healthy, volunteers. Mononuclear cell function will be assessed by the degree of proliferation (measured a thymidine uptake into DNA) in response to antigenic challenges with tetanus toxoid and candida mitogenic challenges with staphylococcal enterotoxins (SEs) A, B, C1 , D, E and phytohemagglutinin (PHA). Differences in the relative number of T cells, B cells, natural killer cells, CD4+ cells, and CD8+ cells will determined phenotypically with flow cytometry. b) to determine the molecular regulation of cytokine induction at the transcriptional and post transcriptional level alter exposure to SEs, tetanus, candida, and PHA, as compared to normal health nitrols. Post transcriptional regulation will be determined at the protein and steady state mRNA levels. The supenatants of systemic and wound mononuclear cell cultures will be analyzed for cytokine production us commercially available ELISA kits. Steady state mRNA levels of interleukins 2,4, 6, TGF-alpha, beta-FGF and IFN-gamma will be determined using northern analysis and/or S1 nuclease protection assays. The S1 nuclease protection assay will only be used if northern blot analysis is not sensitive enough. If insufficient quantities of leukocytes are harvested from venous ulcers, cytokine mRNA amplification with RT-PCR will be utilized. Transcriptional regulation will be determined using nuclear run-off assays.
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