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Crohn’s disease is a common inflammatory bowel disease affecting approximately 1 in 1000 of the population in the UK. Surgery for Crohn’s disease is common with the majority of patients requiring surgery at some point in the course of the disease. Both genetic and environmental factors influence Crohn’s disease. Smoking significantly influences the disease course in Crohn’s disease with more frequent relapses and increased need for surgery. Recent research had concentrated on the genes predisposing to the development of Crohn’s disease. In 2001 the CARD15 (NOD2) gene was identified and since then ~30 genes have been found to be associated with Crohn’s disease. This thesis aimed to investigate the influence of CARD15 (NOD2) on the time to second operation in terminal ileal Crohn’s disease. Crohn’s disease affects all parts of the gastrointestinal tract but particularly the terminal ileum. A particular cell type, the Paneth cell, has been implicated in the pathogenesis of Crohn’s disease. Paneth cells are located at the base of Crypts of Liberkühn throughout the small intestine but are found in greatest numbers in the terminal ileum. Paneth cells contain and secrete antimicrobial peptides in response to bacterial products. They have been found to express CARD15 (NOD2). The expression of antimicrobial peptides and the CARD15 (NOD2) genotype were investigated using the technique of in situ hibridization. There are well characterized histological features of Crohn’s disease. The are no known histological features of Crohn’s disease associated with the CARD15 (NOD2) genotype. These features were investigated. Osteoporosis is an important complication of Crohn’s disease and it’s treatment. Predictors of those at risk for the development of Crohn’s disease would be clinically useful in targeting therapy. Genetic influences on bone mineral density and Crohn’s disease were investigated. Recent publications have furthered our knowledge of the genetic factors influencing the development of Crohn’s disease. The Newcastle cohort of patients contributed to this knowledge. The aim of this study was to investigate survival to second operation in terminal ileal Crohn’s disease. The effect of environmental factors including smoking and exposure to thiopurines was investigated. In particular the effect of CARD15(NOD2) genotype and carriage of the 5q31 haplotype was studied.

The presence and replication of E.coli within Crohn’s Disease (CD) tissue has been confirmed by multiple authors and is hypothesised to be pivotal in the development of CD. In this thesis, quantification of E.coli bacteria within endoscopic biopsies has been achieved with high efficiency, sensitivity and reproducibility using PCR plasmid technology, and the technique’s utility demonstrated by quantifying E.coli within the biopsies of CD patients in clinical relapse and remission as well as within the first lesions present in CD relapse, aphthous ulcers. These studies showed some interesting correlations with clinical, macroscopic, and histological data, obtained during a clinical trial of soluble plantain fibre supplementation for prevention of relapse in CD. These included increased quantities of E.coli in tissues from the mucosa of patients in clinical relapse whose ileum was macroscopically normal yet histologically inflamed, and significant falls in the quantities of E.coli over time in the biopsy tissues of patients who were in clinical remission. The ability of a specific group of E.coli, the adherent invasive E. coli (AIEC), to replicate within macrophages, is increasingly perceived to be fundamental in CD pathogenesis. The replication of E.coli within the phagolysosomes of macrophages is implicated in the release of pro-inflammatory cytokines, granuloma formation, and the consequent mucosal injury seen in CD. The pharmacological inhibition of AIEC replication in this work within murine macrophage tissue in vitro by the immunosuppressive azathioprine and its active metabolite 6-thioguanine as well as by the antibiotic ciprofloxacin at clinically relevant concentrations is supportive of this central hypothesis, and also suggests new treatment combinations that might be trialled in active CD. Interestingly and unexpectedly the steroid hydrocortisone also inhibited AIEC replication within macrophages, suggesting that sepsis-related complications seen with steroid use clinically may be related to its effects on neutrophil recruitment, rather than on phagocytic killing of bacteria. The diarrhoeal pathogens EPEC, ETEC, and C. difficile represent a large disease burden in terms of infantile, travellers’, and antibiotic-associated diarrhoea respectively with C. difficile also sometimes implicated in the exacerbation of CD. Plantain (banana) non starch polysaccharide (NSP) has previously been demonstrated to inhibit AIEC adhesion to mucosal tissues, and this work demonstrates the inhibition of the adherence of ETEC and C. difficile to intestinal cells in vitro with two different modalities, using concentrations of soluble plantain fibre that are readily achievable in the distal intestinal lumen. Previous work on AIEC inhibition with plantain was confirmed, whilst oat fibre and apple pectin did not significantly inhibit ETEC adherence. EPEC was not inhibited by plantain fibre, which may be due to its unique epithelial interaction. These foodstuffs, if prepared as suitable supplements, may offer new prophylactic therapeutic interventions for global and institutional diarrhoeal illness following appropriate clinical trials.

Introduction: Mutations in the NOD2 gene are associated with the development of Crohn's disease, an inflammatory disorder of the gastrointestinal tract. The NOD2 protein induces cellular activation in response to the bacterial antigen muramyl dipeptide (MDP). The NOD2 gene is mainly expressed by circulating blood monocytes although NOD2-associated Crohn's disease involves mainly the terminal ileum. Paneth cells, which are most numerous in the terminal ileum, are specialised intestinal epithelial cells that secrete antimicrobial peptides in response to bacterial products and are critically important in enteric antibacterial defence. I hypothesised that Paneth cells express the NOD2 gene: this thesis describes the expression and quantification of the NOD2 gene in Paneth cells in inflammatory diseases such as Crohn's disease and necrotizing enterocolitis. I also identified factors that regulate NOD2 gene expression in intestinal epithelial cells and peripheral blood mononuclear cells (PBMC).;Methods: In situ hybridisation and immunohistochemistry were used to localize NOD2 mRNA and protein expression in intestinal tissue. Laser capture microdissection (LCM), and calcium chelation followed by mechanical disruption were used to isolate intestinal crypt and villus epithelial cells. Real-time RT-PCR was used to determine NOD2 gene expression in intestinal epithelial cells and PBMC.;Results: NOD2 mRNA and protein expression was readily detected in Paneth cells in normal and Crohn's disease affected terminal ileum NOD2 was also expressed by monocytes, but not by mature macrophages in the lamina propria or within granulomas. NOD2 mRNA levels were enriched in isolated crypt compared to villous epithelial cells, and NOD2 expression was mainly detected in LCM-acquired Paneth cells but not villous epithelial cells. In vitro, tumour necrosis factor alpha (TNFalpha) up-regulates NOD2 gene expression in intestinal epithelial cells. There is a possibility that Paneth cell antimicrobials are reduced in patients with NOD2-related Crohn's disease.;Conclusions: Paneth cells express NOD2 and could therefore play an important and hitherto unrecognized role in the development of NOD2-associated Crohn's disease.

Most theories concerning the primary cause of Crohn's disease focus on over-activation of the immune response. Paradoxically, the defect may instead relate to diminished acute inflammation. Neutrophil accumulation to sites of dermal trauma has been shown to be reduced. Were the same phenomenon to occur in the gut, it might impair bacterial clearance thus provoking granuloma formation. In this thesis, a novel technique demonstrated attenuated neutrophil accumulation following trauma to the bowel. A modified skin window technique linked this failure of migration to defective IL-8 production. Polymorphisms in CARD15, associated with susceptibility to Crohn's disease, compounded the problem by abrogating the normal pro-inflammatory action of the protein but did not underlie the basic phenotype. Consequently, the response of macrophages to other inflammatory agonists was examined. IL-8 production was also impaired in Crohn's disease after stimulation with wound fluid, C5a or TNF-a. The response of Crohn's patients to gut bacteria was assessed directly in vivo by subcutaneous injection of killed Escherichia coli. This elicited substantial local inflammation in controls, manifested by an NO-mediated increase in blood flow. The response was considerably lower in Crohn's patients, particular those with colonic disease. In contrast, acute phase reactants were highest in the latter, supporting the hypothesis that an impaired local response can drive a systemic pro inflammatory state. The demonstration of attenuated acute inflammation in Crohn's disease may have important implications for understanding its pathogenesis and targeting novel therapies.

Volatile organic compounds (VOCs) and lactoferrin have been studied as potential faecal biomarkers for ISO. Changes in VOC patterns in stool reflect both physiological and pathological processes within the gut. Lactoferrin, a protein present in neutrophils can be quantified in stool. This thesis studies the potential and efficacy of both urine and faecal VOCs and the efficacy of lactoferrin in differentiating ISO from ISS and healthy controls. CD was best differentiated from those with IBS by presence or absence of urine VOCs when compared to UC. Active UC versus IBS comparison, using the presence or absence of urine VOCs, yielded a higher sensitivity and specificity than when inactive UC was compared with ISS (Inactive UC vs ISS: 54.2%, 30.4% and Active uC vs IBS: 86%, 77% respectively). Active and inactive UC comparisons with IBS groups, based on peak area, resulted in significant urine VOCs with high sensitivity but moderate specificity (Inactive UC vs ISS: 83%, 52% and Active UC vs ISS: 100%, 78% respectively). Moderate sensitivity and specificity were recorded for faecal VOCs with comparisons based on peak area measures for inactive CD versus IBS (67%; 77%) and inactive UC versus ISS (100%; 59%). Faecal vacs in the active CD and ISS comparison recorded the be.st sensitivity and specificity (92%; 74%). Faecal vac profiles were better than urine for the group separation of active disease. Correct classification was much improved with peak area measure than the presence or absence of volatiles: Lactoferrin levels were considerably higher in 180 patients than those with IBS, while in IBS and healthy controls were similar. Lactoferrin levels were lower in inactive UC when compared with active UC. No such statistical difference was noted when active and inactive CD were compared. Lactoferrin levels in inactive IBD groups were higher compared to the healthy controls. Faecal and urinary biomarkers have potential to enhance management of patients with IBS and IBD.