After anaerobic culture (at 37C) of a single colony ofC. purified toxin A. Sepharose bead-conjugated ATAA was consequently used to investigate the contribution of toxin A in epithelial injury mediated byC. difficilesupernatant samples (containing toxins A, B and additional products). Loss of barrier function mediated by apical software of supernatant samples of research and epidemic 027 strains ofC. difficilewas abrogated by neutralization of toxin A. However, this was not the case when the supernatant samples were applied to the basal surface of epithelial monolayers. In conclusion, our studies have shown that (i) sepharose bead-conjugated ATAA is more effective in neutralizing toxin A than free antibody and (ii) when the apical (luminal) surface of epithelial monolayers is definitely exposed to the secretory products of research and 027 strains ofC. difficile, toxin A is required for the initial injury ARQ 621 that leads to loss of barrier function. Keywords:anti-toxin antibody, ARQ 621 colitis, paracellular permeability == Introduction == ToxigenicClostridium difficileis a Gram-positive anaerobic bacillus that is a major cause of diarrhoea and colitis (pseudomembranous colitis) in hospitalized patients. It secretes two toxins, A and B, which are Mouse monoclonal to EphA1 responsible for colonic inflammation and disease. Intestinal epithelial cells are believed to be the first host cells that interact withC. difficiletoxins and responses by these mucosal cells may determine the development and nature of the colonic disease. Early effects ofC. difficiletoxins include loss of epithelial barrier function and expression of proinflammatory cytokines, followed by programmed cell death [15]. Inhibition of epithelial-toxin interactions via secreted antibody and brokers that bind the toxins [6] are therefore likely to be protective. Indeed, studies suggest that impaired antibody-mediated protection may be responsible for the development of disease and its recurrence [79]. Orally administered antibodies toC. difficiletoxins may be therapeutically beneficial [10] and their efficacy is likely to be related to their ability to inhibit the toxin-mediated effects on epithelial cells described above. Both toxins A and B express cytopathic and cytotoxic activities in cultured cellsin vitrobut studies in animals have shown that toxin A plays an essential role in inducing intestinal disease. Thus, intragastric administration of purified toxin A has been reported to induce intestinal inflammation similar to that seen following contamination with toxigenicC. difficile[11]. By contrast, intragastric administration of purified toxin B did not cause any disease unless there was prior mucosal damage to the intestinal mucosa. Moreover, purified toxin A, but not toxin B, induced inflammation when injected into rabbit ileal and colonic loops [12,13]. However, studies using mucosal samples have shown that toxin B is usually cytotoxic to human colonic epithelial cells [14,15]. Toxin A-negative, toxin B-positive strains ofC. difficilehave also been reported to be capable of inducing disease [16]. However, the majority of patients withC. difficile-associated disease are infected with bacterial strains that express both toxin A and B. The recent increases in the number of cases of severe disease and also outbreaks have been attributed to a virulent strain (designated PCR ribotype 027/NAP1; [1719]) that produces greater amounts (compared with other toxigenic strain) of toxin A and Bin vitro[20]. The 027 strain also secretes binary toxin, whose role in disease pathogenesis remains to be decided. To date,in vitrostudies to investigate their effects on intestinal epithelial cells have involved the use of purified preparations ofC. difficiletoxins A and B. Such preparations may not necessarily reflect the relative importance of each toxin in initiating epithelial injury, especially when the contributions of other ARQ 621 secreted products (such as binary toxin) have not been fully characterized. We have therefore used supernatant samples of cultured toxigenicC. difficileand a specific monoclonal antibody to investigate the contribution of toxin A in inducing loss of epithelial barrier function. We show that during apical (but not basolateral) exposure to supernatant samples of reference and epidemic strains ofC. difficile, toxin A is required to induce loss of epithelial barrier function. Additionally, we show that anti-toxin A antibody has a greater capacity (compared with free antibody) to neutralize toxin A when it ARQ 621 is conjugated to sepharose beads. == Materials and methods == == Cells == The human intestinal epithelial cell lines Caco-2, T84 and Vero cells (derived from African Green.