Lay Summary

Proposal No.   IBD-0061
Principal Investigator:  Bruce R. Yacyshyn, M.D.
Applicant Organization:  Case Western Reserve University (Cleveland, Ohio, U.S.A.)
Project Title: Exposure to bacterial flora and gut permeability: a mechanistic correlation in the HLA-B27 animal model of inflammatory bowel disease
Period of Award:  July 1, 2003 - June 30, 2005

Crohn’s disease and ulcerative colitis are diseases of the intestine characterized by inflammation.  The causes of this inflammation are unknown, but research supports the theory that bacteria in the gut together with a genetic susceptibility together are required to trigger disease.  We speculate that the bacteria get access to the immune system of the patient by microscopic defects in the gut itself, causing the gut to act porous to bacterial particles.  This porosity may itself be genetically determined, and may result in susceptible persons triggering gut inflammation.

We have studied this theory in an animal model of gut inflammation, the HLA-B27 transgenic rat model of inflammation.  These rats develop inflammation of the gut only when they are exposed to bacteria.  That is, when they are housed in a bacteria-free environment they do not develop inflammation.  We believe that certain bacteria may increase the porosity of the intestine, further increasing the inflammation.  This model has two of the major criteria of inflammation.  They have a genetic risk and need bacteria to develop disease.  We have identified changes in the porosity of the intestine in relationship to the onset of inflammation of the gut.  Although this has been studied by others, we have used sugar molecules to study this porosity differently in various parts of the intestinal tract.  For instance, we can study stomach, small bowel and colon independently.  By using this model, we can determine the location of porous tissues and then identify the bacteria present in different parts of the gut different ages of development.  We can identify gut bacteria using a new molecular technique that allows us to distinguish them by their individual DNA types.  This technology offers many advantages to conventional technology of culturing gut contents, which does not identify most gut bacteria, because their growth conditions are unknown.  By using this molecular technique, we can then study the effects of interventions to modify the bacteria in the gut, including non-absorbable carbohydrate molecules.

In preliminary studies, we have found that some of these molecules given orally to our study animals can change the types of bacteria found in the gut of diseased animals and begin to decrease the inflammation.  It is our hope that these techniques may allow us to identify substances that will change the gut bacteria of diseased patients and improve the inflammation and perhaps reduce the risk of recurrence of disease in these people.

We have been able to study intestinal resident flora using a molecular technique in order to assess the species of bacteria. Briefly, after extraction of total DNA from the fecal contents of specific intestinal locations, the 16S rRNA bacterial gene is amplified by PCR. We then separate the PCR products using urea denaturing gradient gel electrophoresis (DGGE). This next step allows for the identification of individual 16S rRNA bacterial genes from both aerobic and anaerobic bacteria present in the intestine. As was shown in the work of others, this allowed for the identification of many more species present in the samples than standard analysis by culturing.  Using this technique, we can follow the development and colonization of bacteria in diseased and non-diseased animals and correlate the presence, absence or quantitative differences of particular bands (bacterial species) with permeability changes. Preliminarily, we have generated data showing obvious differences in chronically diseased and non-diseased rats.

Using the PCR/DGGE technique, we have been able to change the gut flora in chronically diseased rats using the broad-spectrum antibiotics, vancomycin and imipemen. These antibiotics have been shown to decrease the inflammatory process in the model. Probiotics have been show to alter flora in the intestine. We have altered the gut flora with a non-absorbable carbohydrate C-methylcellulose (CMC) or prebiotic. The oral gavage of this compound produced a profound effect on the gut flora of chronically diseased animals. Furthermore, we can demonstrate that both the antibiotic therapy and CMC therapy altered the inflammation and ameliorated the cecal crypt abscesses present in untreated chronically inflamed animals. By developing this system in the HLA-B27 transgenic rat model of inflammation, we anticipate that this work will be applied to humans.