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Differential Binding and Internalization of Clostridium difficile Toxin A by Human Peripheral Blood Monocytes, Neutrophils and Lymphocytes
Author(s) -
Modi N.,
Gulati N.,
Solomon K.,
Monaghan T.,
Robins A.,
Sewell H. F.,
Mahida Y. R.
Publication year - 2011
Publication title -
scandinavian journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.934
H-Index - 88
eISSN - 1365-3083
pISSN - 0300-9475
DOI - 10.1111/j.1365-3083.2011.02578.x
Subject(s) - toxin , trypan blue , clostridium difficile toxin a , peripheral blood mononuclear cell , microbiology and biotechnology , monocyte , biology , clostridium difficile , alexa fluor , immunology , cell , in vitro , fluorescence , biochemistry , physics , quantum mechanics , antibiotics
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.