title: Differential binding and internalization of Clostridium difficile toxin A by human peripheral blood monocytes, neutrophils and lymphocytes.
abstract: 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 A(488)) 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 A(488) -associated fluorescence in monocytes peaked at 1 h (majority internalized), with subsequent loss associated with cell death. In contrast to monocytes, binding of toxin A(488) 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 A(488)-associated fluorescence in neutrophils remained at the cell surface. Over 48 h (37 °C and ice/4 °C), there was minimal toxin A(488)-associated fluorescence in lymphocytes. These studies suggest major differences in interactions between toxin A and circulating cells that infiltrate the mucosa during colonic inflammation in C. difficile infection.
authors: Institute of Infection, Immunity and Inflammation, University of Nottingham, Nottingham, UK.
authors: N
authors: N
authors: Modi
authors: Institute of Infection, Immunity and Inflammation, University of Nottingham, Nottingham, UK.
authors: N
authors: N
authors: Gulati
authors: Institute of Infection, Immunity and Inflammation, University of Nottingham, Nottingham, UK.
authors: K
authors: K
authors: Solomon
authors: Institute of Infection, Immunity and Inflammation, University of Nottingham, Nottingham, UK.
authors: T
authors: T
authors: Monaghan
authors: Institute of Infection, Immunity and Inflammation, University of Nottingham, Nottingham, UK.
authors: A
authors: A
authors: Robins
authors: Institute of Infection, Immunity and Inflammation, University of Nottingham, Nottingham, UK.
authors: H F
authors: HF
authors: Sewell
authors: Institute of Infection, Immunity and Inflammation, University of Nottingham, Nottingham, UK.
authors: Y R
authors: YR
authors: Mahida