Use of an in-vitro polarized cell culture model to study the translocation of Clostridium difficile toxins

Abstract

Clostridium difficile is the etiologic agent of antibiotic-associated diarrhea; the most common form of nosocomial infectious diarrhea. C. difficile produces two large molecular weight protein exotoxins; toxins A and B. In this study, a polarized tissue culture model employing Caco-2 cells grown on Transwell inserts was established to study the translocation of purified Clostridium difficile toxins A and B. Clostridium difficile toxins were $\sp{125}$I labeled and inoculated onto confluent polarized Caco-2 cell monolayers to study translocation dynamics. The ability of toxins A and B to enhance translocation of lipopolysaccharide was also assessed. Electrical resistance measurements were utilized to monitor monolayer confluence and tight junction integrity. Sera from patients afflicted with C. difficile-associated diarrhea were analyzed for circulating cytotoxin, as well as, serum neutralizing antibodies. Synsorb CD and Cholestyramine were also examined for their ability to inhibit cytotoxic effects on Caco-2 cells and Human foreskin fibroblasts. (Abstract shortened by UMI.)