Scientific Abstract

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 immunoregulatory diseases of the intestine of unknown etiology. Current research data support the belief that these diseases are triggered by bacterial flora in a susceptible host. We, along with others, have studied patients with Crohn’s disease and have found their intestinal permeability is increased. This increase in permeability is associated with increased immune activation, in particular the CD45RO CD20 population in Crohn’s patients. Work by others has shown that increased permeability may be one of the initiating events triggering IBD. We believe that the passage of bacterial products through a permeable gut triggers a damaging immune response in a susceptible host. We have studied the contributing factors in inflammatory bowel disease in human and animal models. Our group and others have used the HLA-B27 transgenic rat model of intestinal inflammation to study two important aspects of the disease, the environmental and genetic contributions.

To study the development of permeability in the rat gut, we have used sugar molecules that are absorbed in different parts of the intestine and therefore will be able to measure permeability changes in different anatomic locations in this animal model. Although other investigators have measured the colonic permeability of the HLA -B27 rat, they have focused on the colon or the whole intestinal tract and not been able to study the parts of the intestinal tract due to the nature of their permeability probe molecules. As increased permeability may provide access for bacterial antigens through the small bowel just as a colonic permeability can, the ability to measure small bowel permeability simultaneously and independently with colon is an important issue. Using this method, we have identified increased permeability in the small bowel mucosa using lactulose/mannitol permeability.  This is a novel finding, as the small intestine is only minimally or not affected at all in this model and the permeability occurred before colonic permeability.

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.