Philosophiae Doctor - PhD / Background: A subtype of inflammatory bowel disease, Crohn' s disease is thought to represent a complex interaction between environmental factors, a defective immune system, the gastrointestinal microbiome and genetic' susceptibility; however; the-prevalence of different susceptibility mutations appears to vary between population groups, implying distinctions in disease pathogenesis or risk. Vitamin D, signaling through the vitamin D receptor, appears to have numerous effects on the immune system, and deficiency has been shown to playa role in both the pathogenesis and severity of experimental inflammatory bowel disease. However, the literature surrounding the association between vitamin D concentrations and disease severity in Crohn's disease is limited, and no such literature exists in South Africa. Furthermore, a paucity of data exists on the racial variability of Crohn' s disease phenotype in the Western Cape population of South Africa, as well as environmental factors in childhood associated with future Crohn's disease development. Aims: The three primary aims of the study were to investigate: 1) the racial variability of, Crohn's disease phenotype, defined by the Montreal classification scheme, as well as Crohn's disease behavior, using predefined definitions, stratified as 'complicated' or 'uncomplicated', based on a cross-sectional study design; 2) the association between childhood environmental exposures and the subsequent development of Crohn's disease, with specific emphasis on the timing of exposure, based on a case-control study design; and 3) the association between serum 25(OH)D concentration with Crohn's disease activity, measured by the Harvey Bradshaw Index, based on a cross-sectional study design; in this process, various vitamin D thresholds for predicting a high disease activity score were investigated, and the serum 25(OH)D concentrations were compared with those of the healthy controls to evaluate the prevalence of vitamin D deficiency. Design: This was a case control study, as well as two cross-sectional evaluations of the case control study data, of all consecutive Crohn's disease patients (ages 18-70 years) seen between September 2011 and January 2013 during their normally scheduled appointments at Schuur Hospital and Tygerberg Hospital. Control subjects for the study were identified from the same populations giving rise to the Crohn's disease cases. An investigator-administrated questionnaire was used to identify numerous demographic and lifestyle variables, as well as childhood environmental exposures during three age intervals; 0-5, 6-10 and 11-18 years. Clinical variables at diagnosis and time of study enrolment were determined via a review of medical and pharmacy records, as well as clinical examination by the consulting gastroenterologist. Serum 25(OH)D was measured using the SIEMENS ADIVA Centaur® XP Vitamin D Immunoassay [Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA]. Vitamin D status was classified as either 'deficient' or 'sufficient', and was analyzed in 2 ways: ~20 ng/mL versus ~21 ng/mL; and ~29 ng/mL versus ~30 ng/mL, respectively. One year after study completion, a total of 40 (10%) randomly selected participants from the cohort completed the interviewer-administered questionnaire for a second time. A kappa statistic was used in order to measure the agreement between repeated data for the questionnaire. Only data pertaining to the three age intervals (0-5, 6-10 and 11-18 years) was extracted in this process. Results: One hundred and ninety four Crohn's disease patients and 213 controls meeting our inclusion criteria were identified; 35 (18%) and 19 (9%) were White, 152 (78%) and 177 (83%) were Coloured, and 7(4%) and 17 (8%) were South African Black, respectively. No subjects reported being of Asian or Indian ethnicity. Overall, 125 (31%) of the cohort were male. On multiple logistic regression analysis, Coloured Crohn's disease patients were significantly more likely to develop 'complicated' Crohn's disease (60% versus 9%, P = 0.023) during the disease course when compared to White Crohn's disease patients. In addition, significantly more White subjects had successfully discontinued cigarette smoking at study enrolment (31% versus 7% reduction, P = 0.02). No additional interracial differences were found. A low proportion inflammatory bowel diseases family history was observed among the Coloured and Black subjects. When evaluating childhood environmental exposures, multiple logistic regression analysis showed that during the age interval 6-10 years, never having consumed unpasteurized milk [(OR = 6.43; 95% Cl, 3.02-14.81), (K =0.79; 95% Cl, 0.39-1.00)] and never having a donkey, horse, sheep or cow on the property [(OR = 3.10; 95% Cl, 1.42-7.21), (K = 0.84; 95% Cl, 0.12-1.00)], significantly increased the risk of developing future Crohn's disease. During the age interval 11-18 years, an independent risk-association was identified for; never having consumed unpasteurized milk (OR = 2.60; 95% Cl, 1.17-6.10) and second-hand cigarette smoke exposure (OR = 1.93; 95% Cl, 1.13-3.35). For the vitamin Danalysis, 186 Crohn's disease patients and 199 control subjects met the study inclusion criteria. Overall, 113 (29%) of the cohort were male. Forty four percent of the cohort had a deficient vitamin D concentration (::;20 ng/ml.), no participants had severely deficient vitamin D concentrations, and 26% of the cohort had sufficient vitamin D concentrations (:::30 ng/mL). Fifty-three percent of the controls and 34% of the cases had vitamin D concentrations ::;20 ng/mL (P < 0.001). On multiple logistic regression analysis, higher Harvey Bradshaw Index scores and not having taken vitamin D supplementation in the six months prior to enrolment were identified as independent predictors of vitamin D deficiency in Crohn's disease patients; defined either as ::;20 ng/mL, or as ::;29 ng/mL (P < 0.001). Compared to patients with Harvey Bradshaw Index <5, those with Harvey Bradshaw Index 2:8 were 2.5-times more likely to have vitamin D concentrations ::;21 ng/mL (PR = 2.5; 95% Cl, 1.30-6.30). The risk was similar, though not as high, if deficiency was defined as ::;29ng/ml. (PR = 2.0; 95% Cl, 1.20-3.50). Conclusions: Coloured Crohn's disease patients were significantly more likely to develop 'complicated' Crohn's disease over time when compared to White Crohn's disease patients. Limited microbial exposures and exposure to second-hand cigarette smoke during childhood is associated with future development Crohn's diseases. However the inconsistencies between each age interval with regards to the identified risk factors may imply that the effect of different viruses or bacteria on the development of immune structures varies according to the timing of exposure. The finding that lower serum 25(OH)D was associated with moderate to severe Crohn's disease activity suggests that this patient population may benefit from vitamin D supplementation in order to achieve, or maintain a serum 25(OH)D concentration of at least 30 ng/mL.

Due to the intrauterine environment required to maintain pregnancy it may be that neonatal animals are born type-2 immune response (IR) biased, which consequently may increase susceptibility to certain infectious and immune mediated diseases, such as allergy. Recently, the prevalence of both allergic and autoimmune diseases has increased, leading to the development of the hygiene hypothesis. The hypothesis states that lack of early environmental stimulus leading to inappropriate development and bias in IR, may contribute to this increase. The objectives of this thesis, therefore, were to: (a) Determine the IR bias of neonatal pigs. (b) Investigate the effect of heat-killed Escherichia coli, lipopolysaccharide (LPS) and muramyl dipeptide (MDP) on the IR phenotype and the frequency of allergy in pigs sensitized to the egg white allergen ovomucoid (Ovm). (c) Establish IR phenotypes of pigs allergic or clinically tolerant to Ovm. Immune response bias was determined using an established phenotyping protocol and compared between two groups of pigs, (A) and (B). A difference in IR bias was observed. Bias in IR was not consistently towards type-2. Increase in indicators of type-1 IR, were greater in A and the frequency of type-2 IR correlates were greater in B. It’s likely that unidentified environmental variables may have induced this change, although etiology was not pursued. Treatment with heat-killed Escherichia coli, LPS and MDP had an effect on IR bias and frequency of allergy. Muramyl dipeptide-treatments promoted type-2 bias and were associated with increased frequency of allergy. Pre-treatment with E. coli did not affect allergic frequency, but did elicit the production of a relatively balanced allergen-specific IR phenotype. Lipopolysaccharide-pre-treatment was associated with decreased frequency of allergy. Correlates of an allergic IR phenotype in pigs were also established. The measurement of allergen-specific IgG, IgG1 and/or IgE activity and evaluation of late-phase intradermal skin tests were proposed to be useful in identifying allergic IR phenotypes. This thesis emphasizes the importance of considering the potential for variation in IR in terms of pig health and experimental reproducibility. Further, given the physiological similarities of pigs and humans, these findings may be extended to studies of food allergy in humans. / NSERC, OMAFRA, Ontario Pork, AllerGen NCE