Final Progress Report

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

Summary of project aims:

The main emphasis and final aim of this project was to study the potential of Carboxy methylcellose, a non-adsorbable carbohydrate, in decreasing inflammation of the intestinal tract in the HLA-B27+ transgenic rat model of IBD (aim 3). For background we also studied, the temporal changes in permeability of the small and large intestine in diseased HLA-B27 and non-diseased littermates (aim1). As well we studied the changing bacterial patterns during development as well as before and after treatment (aim 2).


Accomplishments:

Aim 1: We completed a temporal study of permeability changes in both the small and large intestine of diseased and non-diseased littermates from ages 4-16 weeks. Briefly, we detected an earlier increase in permeability in the small intestine, followed by increased and sustained greater permeability in the colon. Results can be seen in figure 1A and 1B.





Figure 1:  Colonic permeabiliyt was measured  in the HLA-B27+ (  ) and HLA-B27-  (  ) by the fractional excretion (f.e.) of Surcralose (A) and the small intestinal permeability was measured by the fractional excretion (f.e.) of  the lactulose / mannitol ratio (B). In total 4 sugars were given by gavage to the rats.  Urine was collected for 24 hours after the gavage.  The fractional excretion of each sugar found in the urine was determined using HPLC. The (*) represents significant differences.  * .01<p<.05;  ** .001<p< .009;  *** .0001<p<.0009


Aim 2. We molecularly examined colonic flora in our laboratory using denaturing gradient gel electrophoresis (DGGE). We sent a technician down to Dr. Gerald. Tannock’s laboratory in New Zealand, to verify our use of the technique and performed all DGGE in our laboratory at the Cleveland VAMC. His laboratory and work is the gold standard for the DGGE technique in the study of IBD. We studied temporal changes in colonic bacteria from ages 3 – 12 weeks of age. During normal development there was constant changing of bacterial profiles before 7 weeks of age in the same animal. A more consistent molecular bacterial profile emerged after 8 weeks. Most importantly we found that different litters had different bacterial profiles. Therefore experiments needed to carried out and analyzed within the same litter if possible. Because there were constant differences between litters and in early ages this method of analysis did not demonstrate any visually consistent difference in DGGE profiles that could be correlated with permeability changes. However, CMC did affect the DGGE profiles.

Aim 3. We were able to study and document changes after ingestion of the non-absorbable carbohydrate carboxy methyl cellulose (CMC) in: 1) mucosal inflammation, (as measured by mucosal thickness and a severity of inflammation index); 2) permeability (as measured by the fractional excretion of orally gavaged sugars); 3) bacterial DGGE profiles and in 4) the detection of mRNA of CEACAM1.

CEACAM 1 (carcinoembryonic cellular adhesion molecule 1) has two main isoforms in the rat, a long (519 bp) and short (458bp) isoform. We are the first group to detect differences in this important and multifunctional cell surface glycoprotein in the development of intestinal inflammation in HLA-B27 rat model of IBD. CMC treatment increases the presence of detectable mRNA in the 11 week old B27+ rat, where there was complete absence of this long isoform in untreated inflammed 11 week B27+ rats.

We treated 3 ages of rats; 4-6, 7-11 and 15-19 weeks of age. Our baseline in untreated age matched animals (shown in figure 2 a-d) demonstrated that the 11 week age group showed the most dramatic changes in the establishment of mucosal inflammation and increased colonic permeability.









C. List of significant positive results when CMC was given orally between 7-11 weeks of age.

1. CMC significantly decreased colonic mucosal inflammation wh (figures 3a and 3b).

2. CMC significantly decreased colonic permeability (figure 4).

3. CMC altered overall colonic flora as measured by DGGE (figure 5).

4. CMC altered E.coli flora pattern as measured by DGGE (figure 6).

5. CMC increased the CEACAM1 long isoform (519 bp). This isoform is involved in signal transduction which can regulate cell adhesion, proliferation, and cellular function which can result in activation of NF B and AP-1 and cytokine release (figure 7a and 7b)







In Figure 5, the DGGE profile, several conclusions can be drawn:

1. The flora patterns appear more stable between 9-11 weeks in the untreated controls (B16, B17, B14 and B1.22g)
2. The flora is still more similar within one litter (B16, B17, B14 compared to B1.22 series).
3. Black arrows (without circles) point out lower band that remains more intense in 11week B27- littermates, untreated or CMC treated. The red arrows point to the band in the positive treated littermates (B1.22 series)
4. Black arrows with circles point to a band, which appear to be more intense in the negative CMC, treated samples as compared to the untreated littermate (B1.22g) or treated positive littermates (red arrows with circles).
5. The encircled area focuses attention on an area in which the band patterns stay the same in the negative littermate rats (either untreated or CMC treated), but change after the CMC treatment in the positive littermate rats (red circled area)




In Figure 6, if the rats were untreated the flora profiles remain fairly similar at 11 weeks of age, although the intensity of bands change. However, the CMC treatment altered the E.coli flora profiles. The arrow in figure 6 points to a doublet that is present after CMC treatment of B27- rats, but not B27+ rats.



Figure 6: PCR was carried out with E.coli species- specific primers and fecal samples from 11-week rats, both CMC  treated and untreated.


In Figure 7a and 7b there is a clear difference between treated and untreated rats.



At the 19 weeks of age endpoint we also saw a decrease in the SIS scores (which looks at the cellularity of inflammation, but treatment did not decrease mucosal width or colonic permeability at the CMC concentration given.
 

List of negative results:

1. In the 4-6 week old treated rats, both HLA-B27+ and HLA-B27-, CMC treatment did decrease the colonic permeability. However, as seen in figure 1b, colonic permeability is still developmentally increased for both the HLA-B27- and HLA-B27+ littermates and CMC treatment increased mucosal inflammation. Suggesting that CMC should not be used in younger rats and altering permeability at this time can cause inflammation both the B27- and B27+ rats and had a negative treatment effect.
 

Lay Summary:

A current hypothesis regarding the causes of inflammatory bowel disease suggests that in genetically susceptible individuals, an unregulated reaction to one or more bacterium may result in disease.   The studies funded by this grant have tested one bacteria, CBir1, and have found that it is expressed by a large portion of patients with Crohn’s disease and that the level of that expression may be associated with the severity of disease.