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62 PROSTAGLANDIN E2 DISRUPTS HUMAN COLONIC EPITHELIAL CELL TIGHT JUNCTION AND BARRIER FUNCTION M Lejeune, K Chadee Entamoeba histolytica is an enteric dwelling protozoan parasite and the causative agent of amoebiasis, accounting for more than 100,000 deaths per year. Studies on the pathogenesis of amebiasis are intriguing yet little is known on how the parasite overcomes epithelial barrier functions. Although a contact dependent cytolysis of intestinal epithelial cells facilitated by parasitic virulent factors, such as Gal/GalNac adherence lectin, cysteine proteinase and amoebapore has been well characterized, there are also reports on contact independent roles of stimulating inflammatory responses in intestinal epithelial cells, which can facilitate pathogenesis. The virulent components responsible for contact independent roles in the pathogenesis of intestinal amebiasis are not well characterized. We recently showed that E histolytica produces prostaglandin E2 (PGE2) that may play a major role in disrupting epithelial barrier function. As PGE2 alters secretory functions in colonic epithelial cells, in this study we determine if PGE2 disrupts tight junction (TJ) proteins and transepithelial electrical resistance (TER). Monolayers of T84 human colonic epithelial cells treated with purified PGE2 showed a concentration (8 nM and 1 µM) and temporal dependent decrease in TER from 15 min to 300 min. Colonic cells exposed to 1 µM PGE2 caused a significant decrease in the levels of Zonula Occludens 1 (ZO-1), a 225 KDa cytoplasmic plaque protein as early as 30 min. When exposed to 8 nM PGE2 and probed with antibodies for Occludin, a transmembrane TJ protein, a distinct cleaved band at 18 kDa from the 64 kDa protein at 12 h was evident. Similarly, the level of active protein kinase C
Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta
Supported by CIHR