All authors small molecule library screening declare no conflicts of interest. “
“Colitis due to Clostridium difficile infection is mediated by secreted toxins A and B and is characterized by infiltration by cells from the systemic circulation. The aim of our study was to investigate interactions between fluorescently labelled toxin A and peripheral blood monocytes, neutrophils and lymphocytes. Purified toxin A was labelled with Alexa Fluor® 488 (toxin A488) and incubated with isolated human peripheral blood mononuclear cells or washed whole blood cells for varying time intervals at either 37 or 4 °C/ice. The ability of trypan blue to quench cell surface–associated (but not cytoplasmic) fluorescence was also investigated. At 37 °C, toxin
A488-associated fluorescence in monocytes peaked at 1 h (majority internalized), with subsequent loss associated with cell death. In contrast to monocytes, binding of toxin A488 in neutrophils was greater on ice than at 37 °C. Studies using trypan
blue suggested that over 3 h at 37 °C, most of the toxin A488-associated fluorescence in neutrophils remained at the cell surface. Over 48 h (37 °C and ice/4 °C), there was minimal toxin A488-associated fluorescence in lymphocytes. These studies suggest major differences in interactions between toxin A and circulating cells that infiltrate the mucosa during Hydroxychloroquine manufacturer colonic inflammation in C. difficile infection. Clostridium difficile, an anaerobic gram-positive bacterium, is the most common cause of nosocomial diarrhoea and the aetiological agent of antibiotic-associated pseudomembranous colitis, a severe form of the disease that is often characterized histologically by focal inflammation associated
with epithelial ulceration [1–3]. Infection due to C. difficile is a significant clinical problem, especially in hospitalized patients on antibiotics. Studies in hamsters [4, 5] and rabbits [6–8] have shown that the intestinal disease is mediated see more by secreted toxins A and B. In vitro studies using human intestinal epithelial cell lines suggest an essential role of toxin A in the disruption of epithelial barrier function [9, 10]. Toxins A and B are among the largest toxins known and consist of three major domains: the N-terminal region with glucosyltransferase activity, a hydrophobic central region (thought to be required for translocation across cell membranes), and a highly repetitive C-terminal region, which is believed to be responsible for binding to cell surface receptors [11, 12] and is required in entirety for binding-induced endocytosis [13]. Monoclonal antibody PCG-4 recognizes epitopes within the C-terminal region and in animal studies has been shown to be capable of neutralizing enterotoxic activity of toxin A [14]. Moreover, this antibody has been shown to block toxin A-induced disruption of epithelial barrier function in vitro [9, 10]. Previous studies have also demonstrated carbohydrate-specific recognition by toxin A [15–17]. During the initial phase of C.