3R-Project 48-96

Utilization of a model of culture of human intestinal cell lines for the study of the pathogenicity of different strains of Clostridium difficile

Jean Pierre Kraehenbuhl
ISREC - Institut Suisse de Recherches Expérimentales sur le Cancer, 1066 Epalinges, Switzerland
jean-pierre.kraehenbuhl@isrec.unil.ch

Keywords: gastrointestinal tract; inflammation; reduction; diagnostic approaches: microorganisms

Duration: 1 year Project Completion: 1998

Background and Aim
Clostridium difficile is recognized as the major etiologic agent of pseudomembranous colitis in humans. The microorganism produces two lethal toxins: toxin A and toxin B. The development of an in vitro model to assess the cellular cytotoxicity of both toxins will greatly reduce the use of animals which serve as models for the disease. These include mice, hamsters or rabbits. The in vitro model can be used further to evaluate the potency of chemicals and polyclonal or monoclonal antibodies as therapeutic agents.

Method and Results
We tested the direct effects of the two exotoxins A and B on intestinal permeability and epithelial integrity using human colonic T84 cells grown as monolayers on permeable substrates. When applied from the lumenal side at concentrations at which toxin A completely abolished transepithelial resistance, toxin B did not alter epithelial permeability nor morphological integrity. When added together there was a slight additive effect. In contrast, both toxins induced drastic and rapid epithelial alterations from the basolateral side. IgG and dimeric IgA monoclonal antibodies specific for toxin A were sufficient to prevent toxin-induced epithelial damages when added to the lumena compartment. In contrast, IgG did not block the action of toxin A when added basolaterally.These data establish that efficient immune protection against C. difficile infection requires the presence of secretory antibodies specific to toxin A in the intestinal lumen and serum antibodies directed against both toxins in the scrosal compartments. The results provide new molecular clues which should facilitate the design of an efficient mucosal vaccine and minimize the use of animal trials to determine the optimal conditions for an efficient vaccine.

Conclusions and Relevance for 3R
The development of an epithelial culture system base allowed to assess the cytotoxicity of two Clostridium difficile toxins and to establish the sidedness of their action. It is now clear that a vaccine has to elict a mucosal antibody response against toxin A and a systemic response against toxin B. Neutralization of toxin A is sufficient for protection since toxin A allows toxin B to act inside the mucosal barriers. This work can now be extended to other pathogens.

References
1. Stubbe H., Berdoz J., Kraehenbuhl J.P., Corthésy B. (2000), Polymeric IgA is superior to monomeric IgA and IgG carrying the same variable domain in preventing Clostridium difficile Toxin A damaging of T84 monolayers, The Journal of Immunology, 164: 1952-1960.