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Georgia Veterinary Scholars Program

GVSP Summer 2009 Scholars

Georgia Veterinary Scholar	Faculty Mentor
Denae LoBato University of Georgia Class of 2012	Dr. Benjamin Brainard Department of Small Animal Medicien & Surgery UGA College of Veterinary Medicine

Effect of different exposure times to TNF-α on cell receptor profiles in cultured canine endothelial cells

* LoBato, Denae N., Benjamin M. Brainard, and Elizabeth W. Howerth

Endothelial changes in response to inflammatory cytokines can lead to potentially harmful sequelae associated with systemic inflammation in veterinary patients. TNF-α activates endothelial cells (EC) and leads to the expression of various cell surface receptors (CSR); however, the sequence of CSR expression is not fully understood. Our objective was to determine how canine endothelial CSR profiles change when EC are exposed to TNF-α. Cultured canine ECs were incubated with human-derived TNF-α (100 ng/ml) for 2h, 4h, 6h, 16h, or 24h. Fluorescent-labeled antibodies to CD146—a constitutively expressed EC CSR—and to specific, inducible CSR associated with inflammation (CD62P, CD62E,CD54) were then incubated with the cells and assayed via flow cytometry. We hypothesize that CD62P will show strong expression on EC following short incubation times because it is among the first proteins to be released following EC activation. CD54 and CD62E, conversely, require several hours for expression following induction, but persist within vessels for up to 48h following exposure to TNF-α; thus, we predicted that cells exposed to TNF-α for longer periods of time would show an increased expression profile for these two markers. Binding of CD146 was not expected to change but served to identify cells as EC. Understanding the sequence of expression of EC CSR in response to inflammatory cytokines may allow for intervention in critical care situations. The use of flow cytometry to assay these receptors in clinical patients allows for a rapid and accurate profiling of the inflammatory response.