Background: Crohn's disease is a chronic inflammatory disease of the intestine in humans, with no known cause. Johne's disease is a chronic intestinal disease of ruminants caused by Mycobacterium avium suhspecies paratuberculosis (MAP), and has some features similar to Crohn's disease. Although MAP has been purported to play an etiologic role in Crohn's disease, this causal link is still under debate. Objective: The overall aim of this project is to analyze MAP strains from different hosts (cattle, sheep and humans) and regions in North Dakota by biochemical and epidemiological methods, in order to better understand the pathogenesis and epidemiology of MAP strains and the relationship between MAP and Crohn's disease. The specific aims of this research are the following: Aim 1. Investigate the epidemiological evidence for MAP as a cause of Crohn's disease. Aim 2. Conduct a comparative causality study to investigate whether MAP or other enteric pathogens cause Crohn's disease. Aim 3. Evaluate the occurrence of MAP infections in cattle in North Dakota, 1995-2005. Aim 4. Analyze MAP strains from symptomatic and asymptomatic cattle. Aim 5. Investigate the biochemical variations of rapid and slow growing MAP strains. Aim 6. Evaluate MAP strains from low shedders and high shedders. Methods: MAP isolates were analyzed by biochemical methods of gas chromatography, high performance liquid chromatography and mass spectrometry. In addition, extensive literature review was performed to (1) determine the epidemiologic causal link between MAP and Crohn' s disease and (2) determine whether MAP or other enteric pathogens cause Crohn's disease. Results: Results from our study indicated the availablity of epidemiologic evidence supporting the causal role of MAP in Crohn's disease. It was also demonstrated that MAP is the most implicated organism in the etiology of Crohn's disease when compared to other infectious agents. Investigation of the occurrence of MAP infection in North Dakota showed an increase in number of MAP cases reported, with seasonal trends. Biochemical typing of MAP strains from symptomatic and asymptomatic cattle indicated that the symptoms status of isolates was significantly associated with mass spectra patterns and shedder status (p < 0.05). However, the association between symptoms status and HPLC and GC patterns was not significant (p > 0.05). Investigation of biochemical variations of rapid and slow growing MAP strains showed associations between the biochemical variability of MAP strains and their growth rate and presence of symptoms in the source cattle. Evaluation of MAP strains of different shedding characteristics by univariate logistic regression revealed that the shedder status of isolates was significantly associated with growth rate of isolates, symptom status, and source regions, but not with mass spectra patterns of isolates. Conclusion: Overall, this study strengthens the theories of strain sharing, intraspecies and interspecies transmission, and supports an association between MAP and Crohn's disease. In addition, the understanding of the biochemical variation among MAP isolates will help in the future design of diagnostics, therapeutics and vaccines for Johne's and Crohn's diseases. / North Dakota State University. Department of Chemistry and Biochemistry

For many years there has been a suggested association between multiple sclerosis (MS) and inflammatory bowel disease (IBD). Aside from their common epidemiological and immunological similarities, there appears to be an association between the incidence of both diseases coexisting. We report a case of a 41-year-old man with chronic diarrhea and weakness, who was found to have concomitant MS and Crohn's Disease. Our report underscores the importance clinicians of maintaining a high degree of suspicion about the potential association of these conditions among these patient populations.

Emerging rise in microbial drug resistance and the slow-growing characteristic of some intracellular pathogens such as MAP (Mycobacterium avium subspecies paratuberculosis) strongly urges the need for an effective approach for unconventional drug susceptibility testing. We designed a molecular-based PCR method for the evaluation of rifabutin (RFB) and rifampicin (RIF) resistance based on probable determinant regions within the rpoB gene of MAP, including the 81 bp variable site located between nucleotides 1363 and 1443. The minimum inhibitory concentration (MIC) for RIF was also determined against 10 MAP isolates in attempt to seek correlation with rpoB sequences. We determined that MAP strain 18 had an MIC ≥ 30 ug/ml and ≥ 5 ug/ml for RIF and RFB respectively, and a significant rpoB mutation C1367T, compared to an MIC of ≤ 1.0 ug/ml for both drugs in the wild type MAP. The 30-fold increase in the MIC was a direct result of the rpoB mutation C1367T, which caused an amino acid change Thr456 to Ile456 in the drug's binding site; the beta subunit of RNA polymerase. Our in vitro induced mutation in MAP strain UCF5 resulted in the generation of a new resistant strain (UCF5-RIF16r) that possessed T1442C rpoB mutation and an MIC ≥ 30 ug/ml and ≥ 10 ug/ml for RIF and RFB respectively. The T1442C mutation resulted in a Leu481 to Pro481 amino acid change, consequently altering the beta subunit sequence. Sequencing the entire 3.5 kb rpoB in strains 18 and UCF5-RIF16r revealed no additional expressed nucleotide mutation. Of the 10 MAP strains analyzed, an additional one strain (UCF4) exhibited a slight increase in the MIC against RIF and RFB compared to the wild-type. Nucleotide sequencing of the rpoB gene revealed an A2284C mutation in strain UCF4 that occurred further downstream of the expected probable rpoB region and resulted in an amino acid alteration Asn762 to His762. The location of this mutation outside the binding site and its correlation with the minor increase in MIC suggests a possible secondary interaction between the drug and the beta subunit. We have provided three dimensional images through the utilization of PyMol Molecular-based Graphics to display a clear comparison of the mutations observed in the beta subunit for MAP strains 18, UCF5-RIF16r, and UCF4. We propose that these alterations may have caused a less stable interaction between RIF and the beta subunit, resulting in the observed increased in MIC. Furthermore, the change in amino acid sequence did not affect the viability for our RIF resistant strains. The data clearly illustrates that clinical and in vitro-induced MAP mutants with rpoB mutations result in resistance to RIF and RFB. Consequently, unconventional drug susceptibility testing such as our molecular approach will be beneficial for evaluation of antibiotic effectiveness. This molecular approach may also serve as a model for other drugs used for treatment of MAP infections.

Diet is a key factor in the development and progression of Inflammatory Bowel Disease (IBD). A variety of diets have been studied with IBD patients. This cross-sectional survey identified current healthcare practitioner views on different diets and their efficacy with IBD patients. Diets were rated on awareness, compliance, and contributors to success by participants (n = 181). Frequencies were conducted, and ANOVA with Duncan pairwise comparison or chi-square analysis were used to determine significant differences. Most participants (96%) and 98% of registered dietitians (RD) considered using diet to help treat IBD patients. RDs perceived the low fiber or low residue diet easiest for patient compliance (4.2 ± 1.0, P < .05), and the specific carbohydrate diet hardest for patient compliance (2.4 ± 1.4). Initial and follow up consultations with a RD significantly contributed to patient success across all diets, and greater involvement from the RD may solve issues with compliance.

<p>Currently, there is no effective cure for Inflammatory Bowel Disease (IBD), medications are aimed solely at alleviating symptoms and not curative. Great scientific efforts have been aimed at elucidating the mechanisms underlying the pathogenesis of IBD. Macrophages—antigen presenting cells—play a chief role in the pathophysiology of IBD. It has been proposed that CD36 receptor present on the surface of macrophages, may play a role in the inflamed intestine. CD36-expressing macrophages have been implicated in a variety of human diseases; however the role of CD36+ macrophages in the intestine has been limited. The aim of this study was to decipher whether or not CD36+ F4/80+ macrophages are inflammatory in the colonic intestine. Our study discovered the proportion of CD36+ F4/80+ macrophages were markedly upregulated in active IBD patients and TNBS-induced colitis mice. CD36+ macrophages isolated from the LPMC of the small and large intestine of Balb/c treatment groups(a) 12TNBS, (b) MHS+12TNBS, and (c) MHS+PA mice confirmed these macrophages expressed some level of proinflammatory cytokine TNF-α. Two macrophage ligands, LCFA: Palmitic Acid and PGN, in conjunction or separately, appeared to be two culprits which induced MHS macrophages to produce TNF-α both in vitro and in vivo. It is possible these two ligands may work in concert, however the mouse model has yet to be examined. The precise role(s) of these CD36+ F4/80+ macrophages requires further scientific inquiry and elucidation in the context of intestinal inflammation. It is quite possible understanding the mechanisms and roles of these macrophages will greatly advance our knowledge in the pathophysiology of IBD and potential therapeutic treatments or targets.</p> / Master of Science (MSc)

There are over 700,000 individuals living with Crohn's disease in the United States. Crohn's disease is a chronic inflammatory bowel illness that can have debilitating effects on individuals and their partners. There is ample literature on the various medical effects of Crohn's disease on the diagnosed individual but a dearth of literature on how Crohn's disease affects young adult individuals and their partners. This dissertation endeavors to fill this gap in the literature. Through Interpretative Phenomenological Analysis, this study explores how an individual's diagnosis of Crohn's disease is perceived to affect the couple relationship and young adult life-cycle transitions. From this study, healthcare providers can better understand how the numerous physical symptoms of Crohn's disease cause psychological and social implications for the diagnosed individual and partner. This understanding will allow healthcare providers to complete couple level clinical assessment and increase systemic interventions, promoting greater resilience among young adult couples. / Ph. D.

The number of patients who are receiving home total parenteral nutrition (TPN) is increasing. This phenomenon is a result of rising hospital costs and legislation which encourages shorter hospital stays. Previous research has not demonstrated thoroughly change in nutritional status associated with TPN given over a long period of time to patients with singular disease entities such as Crohn's disease. Therefore, this study was undertaken. Two groups of patients were obtained: a sample of five hospitalized patients from a veterans' hospital, and a sample of fifteen home patients followed by a hospital-based nutritional support team. Both groups had Crohn's disease and were receiving TPN. The following parameters were measured: serum albumin, percentage of ideal body weight, and total iron binding capacity. Mean levels and standard deviations of each parameter per time period measured were obtained. These means were plotted across time periods. For each parameter, slopes for each time period were compared using a t-test. For the hospital group, time periods consisted of 20 day periods. For the home patients, time periods were 12 months in length. Mean levels of each parameter remained within normal limits within nearly all time periods for both groups. However, significant changes in each parameter in the home group occurred at the following time periods: 60- 72 months and 72-84 months for serum albumin; and 0-48 months and 48-120 months for total iron binding capacity. (No periods of significance occurred for changes in percentage of ideal body weight.) These changes might indicate periods of significant response to TPN. Other factors which also might have influenced these results include level of compliance by patient, other major illness, iron status, hydration status, effects of sample size, and protein-losing enteropathy. / M.S.

Vitamin D plays a vital role in modulating both innate and adaptive immune systems. Therefore, vitamin D deficiency has been associated with higher levels of autoimmune response and increased susceptibility to infections. CYP27B1 encodes a member of the cytochrome P450 superfamily of enzymes. It is instrumental in the conversion of circulating vitamin D (calcifediol) to active vitamin D (calcitriol). This is a crucial step for macrophages to express Cathelicidin Anti-microbial Peptide (CAMP), an anti-bacterial factor released during the immune response. Our recent study indicated that Crohn's disease (CD)-associated pathogen known as Mycobacterium avium paratuberculosis (MAP) decreases vitamin D activation in macrophages, thereby impeding cathelicidin production and MAP infection clearance. The mechanism by which MAP infection exerts these effects on the vitamin D metabolic axis remains elusive. In this study, we found that MAP infection interferes with vitamin D activation inside THP-1 macrophages by reducing levels of CYP27B1 and vitamin D receptor (VDR) gene expression via interaction with the TLR2-dependent p38/MAPK pathway. MAP infection exerts its effects in a time-dependent manner, with the maximal inhibition observed at 24 hours post-infection. We also demonstrated the necessity to have toll-like receptor 2 (TLR2) for MAP infection to influence CYP27B1 and CAMP expression, as TLR2 gene knockdown resulted in an average increase of 7.78±0.88 and 13.90±3.5 folds in their expression, respectively. MAP infection also clearly decreased the levels of p38 phosphorylation and showed dependency on the p38/MAPK pathway to influence the expression of CYP27B1, VDR, and CAMP which was evident by the average fold increase of 1.93±0.28, 1.86±0.27, and 6.34±0.51 in their expression, respectively, following p38 antagonism. Finally, we showed that calcitriol treatment, and p38/MAPK blockade reduce cellular oxidative stress and inflammatory markers in Caco-2 monolayers following macrophage-mediated MAP infection. In conclusion, this study characterized the primary mechanism by which MAP infection leads to diminished levels of active vitamin D and cathelicidin in CD patients, which may explain the exacerbated vitamin D deficiency state in these cases.

Crohn's disease is a chronic inflammatory bowel disease which may affect any part of the gastrointestinal tract with a preference for the terminal ileum and ileocaecal region. The disease was first described in 1932 and has increased during the last decades. The clinical manifestations could be referred to as inflammation, malabsorption and obstruction. The annual incidence of Crohn's disease in the county of Västerbotten, North Sweden, was found to be 4.9/105 inhabitants. In a study of 87 patients in a medical gastrointestinal unit, 23% of non-operated patients and 66% of resected patients had increased fecal fat excretion. D-xylose test and lactose tolerance test were abnormal 1n 19% and 24% respectively of the non-operated patients. No clear relation could be found between the outcome of these malabsorption tests and localization, extension or activity of the disease. This suggests the cause of malabsorption 1n Crohn's disease to be complex and multi- factorial . The morphology of jejunal biopsies from 18 patients with Crohn's disease elsewhere 1n the gastrointestinal tract demonstrated an abnormal picture 1n 13 patients when assessed by light microscopy and scanning electron microscopy. A high proportion of these patients had abnormal Intestinal absorptive tests. Skeletal muscle biopsies were performed 1n 13 patients showing a depletion of muscle potassium content and more Infrequently low skeletal muscle magnesium content. This depletion 1s not reflected by subnormal plasma concentration. In the Initial clinical assessment of a new gamma labelled synthetic bile ac1d-SeHCAT, 45 patients, 19 of whom had Crohn's disease, were studied. The outcome of the test correlated well with the excretion of fecal bile acids. It was possible to discriminate patients with terminal Ileal disease from other patient groups. In a follow-up study, the SeHCAT test was modified as to make it simpler and to shorten the test period. Nine patients with Crohn's disease were tested, showing a suffi cent accuracy of the outcome of the test within 48 hours, using simple equipment available in many hospitals. The elimination of radioactivity was calculated as WBR50*» the time for 50% of the administered dose to be excreted. This gives information as to the rate of excretion, reflecting the degree of terminal ileal malfunction. / <p>S. 1-41: sammanfattning, s. 43-115: 5 uppsatser</p> / digitalisering@umu

<p>Malignant tumours of the small intestine are rare. Age-standardised incidence in Europe is between 0.5-1.5 per 100 000. As the small intestine represents more than 90 % of the gastrointestinal mucosal surface, it is surprising that it gives rise to less than 2 % of gastrointestinal malignancies. The dominating histological subtypes are carcinoids and adenocarcinomas. </p><p>We used three population-based registries in Sweden to study survival, second malignant tumours, causes of death, and Crohn’s disease as a risk factor for small intestinal adenocarcinoma and carcinoid.</p><p>We evaluated tumour site, sex, age, and year of diagnosis as prognostic factors. For adenocarcinomas there was no difference in survival between duodenal and jejunal/ileal tumours. Women with jejunal/ileal adenocarcinomas showed higher probabilities of survival than men, while no such relation was found for duodenal tumours. Old age correlated with poor survival for duodenal tumours, and prognosis has improved in later years. For carcinoids, duodenal tumours had a more favourable prognosis than jejunal/ileal tumours. There was no difference in survival between sexes. Old age correlated with poor survival, and survival has improved in recent years.</p><p>Female patients with adenocarcinoma had increased risk of acquiring cancer in the genital organs and breasts, and both sexes had increased risks of second tumours in the gastrointestinal tract and skin. Men with carcinoid tumours had increased risk of prostate cancer. Both sexes had increased risk of malignant melanoma and malignancies of endocrine organs.</p><p>Patients with adenocarcinoma had increased risk of dying from malignant diseases other than the primary small intestinal cancer and from gastrointestinal disease. The cohort with carcinoid had higher than expected risk of dying from malignant disease, gastrointestinal disease, and cardiovascular disease.</p><p>Patients with Crohn’s disease had increased risk of small intestinal adenocarcinoma and carcinoid, and the risk has increased for patients diagnosed in recent years.</p>