Characterization and Modulation of Proteins Involved in Sulfur Mustard Vesication
Annual rept. 1 May 2005-30 Apr 2006
GEORGETOWN UNIV WASHINGTON DC
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In the current contracting period, we utilized specific and potent inhibitors of different caspases to determine the most effective means of preventing SM toxicity in vitro and vesication in vivo. We obtained tetrapeptide inhibitors to each of the caspases and treated KC for 30 min prior to SM exposure. In addition to determining total SM toxicity, we delineated the specific pathway for the activation of each of the caspases. When primary KC were treated for each of the caspase inhibitors, prior to SM exposure and assayed for activation of each caspase, IETD and LEHD caspase-8 and caspase-9 inhibitors were the most effective caspase inhibitors for human KCs in culture. These worked more effectively than the pan-caspase inhibitor, ZVAD. We have now utilized these inhibitors as compounds for testing in the in vivo human skin graft for their ability to block SM vesication. Primary human KC were used to establish a histologically and immunocytochemically normal epidermis grafted onto the back of nude mice. To test the effects of the peptide inhibitors of caspases on apoptosis and vesication in intact human epidermis, normal human skin was grafted onto the back of athymic mice, and 6-8 weeks after grafting, we inhibited the activity of caspases in vivo by topical application of the inhibitors at the graft site 30 min prior to SM exposure. These human grafts were then exposed to SM by vapor cup method, and showed a vesication response, utilizing an end point of micro or macro blisters. Frozen and fixed sections derived from graft sites of these animals were analyzed for apoptosis markers. Histological analysis of SM-exposed animals grafted with primary KC showed that SM microvesication can be reduced by topical application of zVAD-fmk. While there was no difference in the DMSO vehicle- treated and ZFA-treated control skin grafts, there was a notable decrease in the amount of microvesication in grafts treated with zVAD-fmk.
- Anatomy and Physiology
- Chemical, Biological and Radiological Warfare