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Crohn's disease (CD) and colorectal cancer (CRC) are major disorders of the intestine. Inflammation in CD often precedes fibrosis and stricture formation, and is linked to increased cancer risk. Hypoxia is a common feature of inflammation and CRC that can severely compromise the effectiveness of current therapy regimes including chemo-radiotherapy and maintenance of remission in CD patients. MicroRNAs (miRNAs) are key regulatory molecules involved in cellular proliferation, apoptosis and fibrosis, which are all modulated by hypoxia. This thesis aims to understand the role of miRNAs in these two intestinal diseases. Microarray profiling identified differentially expressed miRNA in the intestinal mucosa overlying strictured and non-strictured CD tissue samples and in six CRC cell lines cultured in hypoxic conditions compared to normoxia. Validation experiments using qRT-PCR confirmed the differential expression of miR-29a, -29b, -29c, -34a, -493* and -708 in CD mucosa and miR-21, -210, -30d, -320a, -320b and -320c in CRC cell lines. Functionally, over-expression of miR-29b in CD intestinal fibroblasts modulated the down-regulation of collagen I and III transcripts and collagen III protein in a TGF-β-dependent manner. Furthermore, miR-29b induced indirectly the expression of the anti-apoptotic protein Mcl-1 via the cytokines IL-6 and IL-8. A positive correlation between miR-210 and the hypoxia marker CAIX was found in CRC tissue in vivo. Furthermore, HCT116 cells cultured under hypoxia were more resistant to the chemotherapy drug 5-FU than cells grown under normoxia. Knockdown of miR-21 or miR-30d under hypoxia may sensitise CRC cells to 5-FU. CRC cell lines grown under hypoxic conditions present an altered cellular metabolic profile compared to their normoxic counterparts. This thesis has showed that critical miRNAs have a functional role in the progression of two important diseases of the intestine. The work presented highlights the potential of miRNAs as biomarkers and therapeutic targets to improve the clinical management of patients with digestive diseases.

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Thesis (Ph. D.)--Ohio State University, 2005. / Document formatted into pages; contains xxi, 216 p. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 March 9.

Crohn's disease (CD) is an idiopathic, chronic, relapsing inflammatory disorder, which is most commonly involved terminal ileum and colon. The incidence and prevalence of CD has dramatically increased during the last 50 years; however, the etiology and mechanism of this disorder remain unveiled. Besides genetic susceptibility, recent integrated researches investigated the role of environmental triggers such as microflora, measles viruses and mycobacteria in the pathogenesis of CD. The association between M. avium subsp paratuberculosis (MAP) and CD has been heightened because of clinical resemblance to Johne's disease (JD), a granulomatous enteritis in ruminants caused by MAP. Isolation of MAP from tissue and milk samples from CD patients and from commercial pasteurized milk and dairy products from JD-infected animals implies a possible re-classification of CD as zoonotic disorder. Clinical signs and symptoms of CD are often non-specific and a challenge to distinguish it from other disorders. Current methods for inflammatory bowel disease diagnosis, especially for CD are highly invasive, distressing and expensive. In this study, the recombinant clone pB11 containing 1.1 kb insert, identified from a MAP genomic library constructed in E. coli, expressed a 20 kDa (p20) antigen encoded on 549 bp partial MAP gene with an ORF cloned in frame within pBAD/His cloning vector. Immunoreactivity of p20 was confirmed by Immunoblot. Purified p20 antigen was then used in the development of an enzyme-linked immunosorbent assay (ELISA) for possible serodiagnosis of Inflammatory Bowel Disease (IBD) associated with MAP infection. All variables associated with ELISA test with regard to concentrations, washes and incubations were optimized using hyper immune rabbit t-anti-MAP polyclonal IgG antibodies and sera from CD and non-CD subjects. The cut-off value for positive response was established as 0.3 following the analysis of statistically formulated samples from normal and non-CD subjects. The developed ELISA test was then used to test a blindly coded 2 17 clinical sera. All sera samples were tested in duplicates and in both p20-coated and uncoated micro titer plates. Consequently, 116/134 (87%) CD sera were positive compared to 24/83 (33%) non-CD sera (P<0.05). Specifically anti-MAP IgG was detected in 8/22 (36%) Ulcerative colitis and 16/61 (26%) non-IBD sera. p20-ORF encoding sequence was recloned (pB11/B6) and the expressed protein reactivity remained consistent. Moreover, the full length of the cloned gene was also identified through blast and alignment analysis and predicted to encode 346 amino acids with unknown function and no identity with other known proteins. The latter supports the clinical data, which reflect on the unique characteristics of this antigen. The result so far suggests that the recombinant clone and its subclone derivative may have potential role in serodiagnosis of CD

Crohn’s disease (CD) is an inflammatory bowel disease characterised by chronic inflammation with a complex pathophysiology involving host, environmental, and microbial factors. The intestinal microbiota is an important regulator of inflammation within the intestine, and a disruption of the interplay between gut bacteria and host immunity is a key factor in CD development. Intestinal inflammation itself is known to cause changes to the intestinal physiology that affect the ability of various bacteria to survive. Additionally, certain environmental risk factors for CD such as antibiotics are also known for their ability to impact the intestinal microbiota. CD is associated with various changes in the intestinal microbiome including increased colonisation with a group of bacteria known as adherent-invasive Escherichia coli (AIEC). The purpose of this study is to investigate how AIEC interact with antibiotics and intestinal inflammation in vivo. Multiple classes of antibiotics were found to increase the colonisation of AIEC and to increase its persistence. These antibiotics caused a loss diversity in the intestinal microbiome, but this did not explain the increased infectivity of AIEC. Antibiotic-induced inflammation was found to produce metabolites that benefitted AIEC growth in the intestine and similar results were found with chemically-induced inflammation. These results show that AIEC can benefit from both antibiotics and other sources of inflammation through inflammation-derived metabolites, which contributes to a greater understanding of the interactions between AIEC and CD. / Thesis / Doctor of Philosophy (PhD) / Crohn’s disease is a type of inflammatory bowel disease that affects many young adults in Canada. It causes a wide range of symptoms including nausea, pain, and diarrhea. While the disease can be treated with surgery and medications, it is considered incurable and affects most individuals for life. The exact cause of Crohn’s disease is not known, but it is thought to be caused by a combination of factors including genetics, environmental exposures, and changes in the number and types of bacterial species in the intestine. Intestinal bacteria play an important role in preventing inflammation in the intestine. An unusual strain of bacteria called adherent-invasive E. coli is found more commonly in Crohn’s disease patients than in healthy individuals. This strain does not cause the disease on its own, but it may interact with other environmental factors that are also associated with Crohn’s disease, such as taking antibiotics. Antibiotic use is a risk factor for developing Crohn’s disease later in life and antibiotics have previously been shown to promote the growth of other E. coli strains in the intestine. In a mouse model of Crohn’s disease, we found that antibiotics made mice more vulnerable to infection with this E. coli strain. This increased vulnerability was because the antibiotics caused inflammation, and we also found that other sources of inflammation benefitted this E. coli strain. These findings help us understand how gut bacteria and other Crohn’s disease risk factors might interact to cause the disease.

The inflammatory bowel diseases (IBD), encompassing Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory disorders of the gastrointestinal tract. Their aetiology is not fully understood but is thought to be a combination of the effect of environmental factors in a genetically susceptible person. The work presented is an examination of the phenotypic characteristics of CD in the Scottish population, and an investigation into genetic factors that may influence susceptibility and progression. An IBD cohort from Dundee was recruited (CD=367, UC=265), and extensive phenotypic information collected from these patients together with genomic DNA. Together with the Edinburgh CD cohort already established, the total CD population (n=1155) was examined for time to disease progression (stricturing and/or penetrating disease, according to the Montreal classification) and first resection; a multivariate analysis was performed for factors influencing these outcomes. In this Scottish CD population, the median time to disease progression and first resection was 14.2 years and 8.9 years respectively. The major factor influencing risk of resection and disease progression was disease location, with patients having pure ileal (L1) disease or ileocolonic (L3) disease being more susceptible than those with pure colonic (L2) disease. Compared with L2 disease, the hazards ratios (HR) for disease progression were 4.7 and 2.8, and risk of resection 5.2 and 2.6 for L1 and L3 disease respectively. Disease progression and risk of resection are surrogate markers of disease severity. To try to better understand the determinants of severe disease, a novel score for disease severity was developed and applied to the Dundee CD cohort. This composite score encompassed the variables of medical and surgical management, disease behaviour and location, nutritional status as well as hospitalisations, with a total score that could range from 1 to 16. A score of 7 or more was found to define the 50% of patients with the most severe disease. This cut-off was used to divide patients into less severe and more severe categories; phenotypic and genetic factors were examined for correlation with more severe disease. Genetic factors examined were the 32 most significant CD susceptibility single nucleotide polymorphisms (SNPs) uncovered by recent genome-wide association scans (GWAS). Factors correlated with more severe disease included disease location (L1, odds ratio (OR) 2.20, p=0.0025), age group at diagnosis (p=0.0004) and two CD susceptibility SNPs (rs9286879 and rs17582416; p=0.0085 and p=0.045 respectively). NOD2 was the first IBD susceptibility gene identified. In order to further define pathways involving NOD2, a yeast two-hybrid screen in our laboratory using NOD2 cDNA as the bait had already identified an interaction between NOD2 and UDP-Nacetyl- alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GALNT2). This enzyme is involved in O-glycosylation, important in the post translational modification of mucins. A GALNT2 genotype/phenotype analysis on the Edinburgh IBD population was completed, with the Dundee IBD population used as a replication cohort. In the Edinburgh IBD population, the GALNT2 tagging SNP rs7536663 was associated with CD susceptibility (OR 1.38, p=0.0008 vs controls), but replication was not achieved in the Dundee cohort (p=0.469). There was no association of any of the GALNT2 SNPs with UC. The GALNT2/NOD2 interaction was further investigated by completing coimmunoprecipitation between the two genes to characterise the level and type of interaction. An interaction between GALNT2 and NOD2 was confirmed in mammalian cells, with the interaction being at the N-terminal end of the NOD2 protein. GALNT2 expression in a cell line and biopsies was investigated by quantitative polymerase chain reaction and immunohistochemistry respectively. There were no statistically significant changes in GALNT2 or NOD2 mRNA expression in the LS174T cell line after stimulation with specific ligands for NOD2 and GALNT2. GALNT2 protein expression was characterised in intestinal biopsy samples to be predominantly in the lamina propria, with some expression in the enterocytes. To further define the contribution of mucin genes to IBD susceptibility, tagging SNPs across the MUC2, MUC3A and MUC19 genes were genotyped in the Edinburgh IBD cohort and examined for a link with IBD, CD and UC susceptibility, but associations were not found. In view of the strong association with CD susceptibility of a SNP near the MUC19 locus in a recent GWAS, tagging SNPs across the leucine rich repeat kinase-2 (LRRK2) gene, near the MUC19 gene, were also genotyped and examined in the Dundee cohort for an association with IBD, CD and UC susceptibility, but was also negative when corrected for multiple testing. The studies presented allow an improved understanding of the influence of phenotypic characteristics on disease progression, need for surgery and severity in CD. The role of disease location has been determined to be particularly critical, in keeping with other published studies. A detailed examination of the influence of specific genes on disease susceptibility has failed to definitely demonstrate an association between germline variation in GALNT2, MUC2, MUC3A, MUC19 or LRRK2 and IBD, CD or UC susceptibility. An interaction in mammalian cells between NOD2 and GALNT2 has been shown, but further work is required to demonstrate that this is a biologically relevant interaction.

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