Intestinal infection
2004 – 2007 Dr Hadwen Trust Research Fellowship
Prof Y R Mahida and Dr K Solomon, Nottingham University
Establishment of model systems to investigate interactions between human intestinal mucosa and Clostridium difficile and its toxins
Dr Katie Solomon was supported by a Dr Hadwen Trust postdoctoral Research Fellowship for the duration of this project. The work was overseen by Prof Yashwant Mahida, Co-Director of the Institute of Infection, Immunity and Inflammation, Nottingham University.
Clostridium difficile is the commonest infectious cause of diarrhoea in hospitalised patients and usually follows the disruption of the normal bowel flora by broad-spectrum antibiotics [1]. Incidences of C. difficile-associated illness in the UK are rising, and with the recent emergence of a more virulent strain of infection, the development of new non-antibiotic therapies is desirable. Such treatments could also be used as a prophylaxis for high-risk patients such as the elderly.
The disease is mediated via two secreted bacterial toxins designated A and B. Therapeutic agents are designed to bind toxins A and B, inhibiting their interaction with intestinal epithelial and other mucosal cells. Newly developed agents are tested in animals, including hamsters, rats and mice, infected with C. difficile. However, these provide an unreliable source of information since the candidate toxin-binding receptor in other animal species is not present in humans [2].
Dr Hadwen Trust-funded research fellow, Dr Katie Solomon, has established novel in vitro model systems that can be used to investigate interactions between human intestinal mucosal cells and purified C. difficile toxins. These in vitro models provide biologically relevant information and can be used to replace animals in designing new therapies.
Building on earlier work [3], an in vitro model consisting of monolayer cultures of human colonic carcinoma (Caco-2) cells grown in a horizontal diffusion chamber system was developed for studying the effects of Clostridial toxins on monolayer integrity. Future exploitation of this model with co-cultures will shed further light on how toxins overcome intestinal cellular defence mechanisms.
Binding studies of fluorescently labelled toxin A were conducted with a range of human cell types from large bowel and terminal ileum specimens. Toxin A was able to bind to all human cell types studied, and immune cells bound toxin A with a different capacity to epithelial cells. Comparing subsets of immune cells, there were distinct differences between lymphocytes and macrophages, which may reflect their role in immune responses to infections. Variations in samples from different individuals possibly reflected patient-specific responses to infection.
Interruption of toxin binding could be a medically important target for therapeutic agents to prevent C. difficile-associated disease. In this research, a novel technique for quantifying and standardising the degree of toxin A binding to the cell surface was developed. This technique will enable the activity of potential therapeutic agents to be quantified and studied in more depth than is possible with current animal tests.
The precise molecular interactions and downstream consequences of toxin A binding to Caco-2 cells and progression towards cytotoxic cell death were also studied. Elucidating the important steps in toxin A-induced cytotoxicity could lead to the development of more efficient therapies.
Two potential therapeutic agents, the specific anti-toxin A monoclonal antibody (PCG-4) and a soluble anionic polymer, were investigated in vitro. PCG-4 successfully prevented toxin A binding to the surface of Caco-2 epithelial cells, and abolished cytotoxicity and loss of barrier function. Interestingly, the soluble anionic polymer prevented cytotoxicity but did not reduce binding of toxin A to the surface of Caco-2 cells. This suggests that potential therapeutic agents may not have to rely entirely on blocking binding of toxin A to cells.
With funding from the Dr Hadwen Trust new in vitro models for investigating human responses to C. difficile toxins have been developed. It is hoped that these reliable and relevant in vitro models will be increasingly used in place of animal studies in the search for new therapies, and provide an innovative approach to tacking an increasingly serious medical problem.
Summary
- Clostridium difficile infection of hospitalised patients is becoming more widespread and more difficult to treat.
- New treatments are being sought and tested in animals (hamsters, rats and mice), but species differences mean results from animals cannot be reliably translated to humans.
- In vitro models to study interactions between C. difficile toxins and human intestinal cells were developed to provide relevant information and replace animals in designing new therapies.
- Novel in vitro systems for investigating the effects of Clostridial toxin A on human intestinal cell barrier integrity and cell surface binding have been developed.
- Differences in the toxin A binding capacity of different human intestinal cell types may reflect the roles these cells play in C. difficile-associated disease.
- A new technique for quantifying toxin A binding to the cell surface has been devised, that will enable the activity of potential therapies to be studied in greater depth.
- The role of two potential therapeutic agents has been investigated in vitro, which has shed new light on the progression of toxin A-induced cytotoxicity.
- These human-relevant models will allow innovative approaches to studying C. difficile induced disease in place of animals and could help in the development of more efficient therapies.
References
1. Johal SS, Hammond J, Solomon K et al (2004). Clostridium difficile associated diarrhoea in hospitalised patients: onset in the community and hospital and role of flexible sigmoidoscopy. Gut 53:673-677.
2. Galili U (1989). Abnormal expression of alpha-galactosyl epitopes in man. A trigger for autoimmune processes? Lancet 2:358-361.
3. Johal SS, Solomon K, Dodson S et al (2004). Differential effects of varying concentrations of Clostridium difficile toxin A on epithelial barrier function and expression of cytokines. J Infect Dis 189:2110-9.