Keywords

IBD, Crohn's disease, Ulcerative colitis, MAP, cellular immunology, gene expression

Abstract

Despite the chronic debate in the etiology of crohn's disease (cd), a debilitating inflammatory bowel disease (ibd) closely related to ulcerative colitis (uc), an emerging interest in a possible mycobacterial role has been marked. Granuloma and pathologic manifestations in cd resemble aspects found in tuberculosis, leprosy and paratuberculosis. The latter, a chronic enteritis in cattle, goat, sheep and primates, which is similar to human enteritis, also known as cd, is caused by a fastidious, slow growing mycobacterium avium subspecies paratuberculosis (map). Due to the similarities between cd and paratuberculosis, a mycobacterial cause in cd has been proposed. Recent discovery of a possible association between nod2/card15 mutations and risk of cd added support to microorganism-host interactions. In this study, a possible mycobacterial role in cd etiology has been evaluated by investigating the presence of map dna, the state of the cellular immune response and microarray gene expression profiling in peripheral blood and surgical tissue from cd, uc and healthy control subjects. Nested pcr detected map dna in tissue from 10/12(83%) cd patients compared to 1/6(17%) non-ibd subjects. Fluorescence in situ hybridization (fish) with the aid of confocal scanning laser microscopy (cslm) detected map dna in 8/12(67%) cd subjects compared to 0/6(0%) in non-ibd subjects. The detection of map dna by either technique in tissue from cd subjects is significant compared to non-ibd subjects (p < 0.05). Map dna was also detected in both inflamed and non-inflamed tissue from patients with cd suggesting map infiltration in human tissue. Correlation of possible map presence and the function of polymorphonuclear leukocytes (pmn) and peripheral blood mononuclear cells (pbmc) in 19 cd patients and 12 controls have been evaluated. Pmn phagocytosis of viable fitc-map was suppressed in 13/19(68%) cd patients compared to 0/12(0%) in healthy controls (p<0.05). Pbmc phagocytosis of viable fitc-map was suppressed in 5/19(26%) of cd patients compared to 0/12(0%) of healthy controls (p<0.05). The proliferative response of pbmc with t-cell majority from cd and controls subjects was evaluated against pha, candida albicans, pwm and map ppd. Dysfunctional proliferative response against pha was found in 8/19(42%) cd patients compared to 1/12(8.3%) in controls suggesting possible t-cell anergy. Pbmc from 11 cd subjects reacted normally to pha, 7/11(64%) reacted strongly to map ppd suggesting previous exposure to mycobacteria, and 3/11(27%) did not react with map ppd suggesting lack of pre-exposure to mycobacteria. From the seven mycobacterial pre-exposed samples, 6/7(86%) showed a normal ability to recall antigens by activated macrophages when exposed to c. Albicans, and all 7 samples had a normal pwm response. Finally, microarray-chip technology was employed to identify the expression profile of genes that have a role in the immune response of cd patients. Rna was isolated from fresh buffy coats from 8 healthy controls, 2 cd, and 1 uc patients. Chips with an estimated of 30,000 human genes were hybridized to cdna from these samples. We found that 17% of the total number of genes was differentially expressed. Over 200 genes were involved in the immune response, 7 genes where common to both forms of ibd (uc and cd), and 8 genes were found to be either downregulated in cd and upregulated in uc or viceversa. The ifngr1 gene, which encodes the ligand-binding chain of the ifn-gamma receptor, was found to be downregulated in 2/2(100%) of cd patients, but not in uc patients. It is known that defects in ifngr1 are a cause of atypical mycobacterial infection and bcg infection. Patients suffering from this deficiency have an immunologic defect predisposing them to infection with mycobacteria. This correlates with the proposed theory as map being the causative agent of cd. Furthermore, the results indicate a host susceptibility requirement for the establishment of mycobacterial infection in cd patients. Further characterization of ifngr1 using real-time pcr is underway. Collectively, detection of map dna in the majority of cd tissue and the alteration in pmn and pbmc to respond efficiently to map may be related to the fact that mycobacterial pathogens infect phagocytic cells of susceptible hosts and consequently the immune response is dysregulated. Furthermore, the fact that a gene linked to mycobacterial susceptibility was found to be downregulated in cd patients only, strengthens the mycobacterial etiology of cd. In general, the data suggest a possible role for a bacterial pathogen in cd pathogenesis.

Notes

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Graduation Date

2004

Semester

Fall

Advisor

Naser, Saleh

Degree

Doctor of Philosophy (Ph.D.)

College

Burnett College of Biomedical Sciences

Degree Program

Biomolecular Sciences: Ph.D.

Format

application/pdf

Identifier

CFE0000170

URL

http://purl.fcla.edu/fcla/etd/CFE0000170

Language

English

Release Date

January 2005

Length of Campus-only Access

None

Access Status

Doctoral Dissertation (Open Access)

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