Spore germination and intestinal colonization byC. of the hamsters in the treated group survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized withC. difficilefor up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients. == INTRODUCTION == Clostridium difficileis an anaerobic, Gram-positive, endospore-forming gastrointestinal pathogen and the leading cause of antibiotic-associated diarrhea (C. difficile-associated disease [CDAD]) in developed nations. The bacterium is transmitted as a spore through the fecal-oral route, and asymptomatic carriage is found in 4 to 20% of the adult population (1). Onset of the disease follows disruption of the endogenous gastrointestinal flora, commonly caused by use of broad-spectrum antibiotics for treatment of a primary condition permitting germination and colonization ofC. difficilein the colon (2). Every year, 1 to 3% of all hospitalized North American patients receiving antibiotics as part of their treatment subsequently become infected withC. difficile, making it the most prominent nosocomial infection (3). Clinical symptoms of CDAD range from mild self-limiting to severe diarrhea, with up to 25% of affected patients experiencing recurrent infections (4). Severe cases of CDAD can lead to pseudomembranous colitis and progress further to toxic megacolon, with a fatal ending in approximately one-third of cases (5). The toxicity ofC. difficilearises primarily from two virulence factors, toxin A (TcdA; 308 kDa) and toxin B (TcdB; 269 kDa), both of which are large, single-subunit exotoxins which share extensive homology (for a review, see reference6). Both have a modular domain structure with an N-terminal enzymatic domain, a central translocation domain, and a C-terminal Entasobulin receptor binding domain (see Fig. S3 in the supplemental material). The binding domain is thought to be responsible for initial binding to epithelial cells and Entasobulin induces toxin uptake through receptor-mediated endocytosis. Upon lowering of the endosomal pH, the central domain exposes a hydrophobic membrane insertion domain that inserts and translocates the Entasobulin N-terminal catalytic domain from the endosome to the cytosol. The N-terminal enzymatic domain carries a cysteine protease that, through autocatalytic cleavage, releases the domain from the endosome into the cytosol. The released N-terminal glucosyltransferase domain glucosylates the Rho-GTPases in the cytosol, blocking the Rho signaling pathway and leading to cellular shutdown and a loss of cellular barrier function. The causative roles of both TcdA and TcdB have been well established for CDAD, with both toxins inducing epithelial tissue damage and extended colonic inflammation in infected hosts. The precise role of each toxin in CDAD has been debated, but recent experimental evidence with toxin deletion strains points to TcdB being the dominant virulence factor (7,8). Recently, with the emergence of new hypervirulent strains, both the severity and mortality ofC. difficileoutbreaks have risen significantly. The increased virulence was initially identified in the North American isolate BI/NAP1/027 (9) and was manifested in epidemic outbreaks in North American hospitals that subsequently were mirrored on other continents (10,11). The hypervirulence has been connected with resistance to fluoroquinolones (12) and increased cytotoxicity and highlights the need for new and better treatment Rabbit polyclonal to ALS2CL strategies for the management ofC. difficileinfections (CDI). The primary treatment against CDAD is antibiotics, with metronidazole and vancomycin being the most commonly used ones (13). Although it is effective, the treatment may lead to emergence of resistant strains, and there are Entasobulin concerns that antibiotics inhibit reestablishment of the endogenous bacterial biota, potentially prolonging susceptibility to reinfection at the end of therapy. With Entasobulin the pressing need for improved therapies for CDAD, two alternative treatment strategies currently showing promise are reconstitution of the gastrointestinal flora by fecal transplantation and antibody-based toxin neutralization (1416). The use of antibody-based therapies stems from the observation that patients with low antitoxin IgG titers suffer from more severe effects of CDAD and more frequently experience recurrent infections (17,18). Both intravenous and oral routes of delivery of toxin-neutralizing antibodies have been explored with positive results, but the majority of studies have been conducted in animal models. In humans, intravenous therapy with combined anti-TcdA and -TcdB human monoclonal antibodies (hMAbs) has been shown to significantly reduce the rate of recurrent infections (16). Oral delivery of hyperimmune bovine colostrum (HBC) from cows immunized withC. difficileculture.