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.

BACKGROUND: Vitamin D deficiency and insufficiency are rising healthcare concerns in the United States (US) and worldwide. The latest data collected by the National Health and Nutrition Examination Surveys (NHANES) between 2002-2006 showed that approximately one third of Americans over one-year-old were vitamin D deficient (serum 25-hydroxy vitamin D (25-OHD) < 12 ng/mL) or insufficient (serum 25-OHD < 20 ng/mL) (Looker et al., 2011). Environmental exposures, acute or chronic disease, and genetics can exacerbate vitamin D deficiency. People with malabsorptive disorders such as Inflammatory Bowel Disease (IBD) are at an even greater risk of becoming vitamin D deficient. Pediatric patients with IBD are particularly vulnerable to the short and long-term effects of vitamin D deficiency, given the prominent role played by this agent on skeletal development. More recent data have demonstrated that vitamin D also plays an important role in maintaining and regulating the immune system. For this reason, investigators have been interested in a better understanding of the relationship between vitamin D and inflammation. Vitamin D may prove to be an important adjunct therapy for people suffering from IBD and other autoinflammatory diseases. OBJECTIVES: Many patients and medical providers understand the importance that vitamin D has in a growing child’s skeletal development. However, compliance with daily supplementation remains low. The design of this study allows patients to receive high-dose vitamin D supplementation during scheduled biologic infusions. The goal is to assess the safety and efficacy of high-dose interval vitamin D therapy. The secondary goal of this study will be to determine if optimal vitamin D levels impact the inflammation observed in the gastrointestinal (GI) tract of patients with IBD. METHODS: 60 patients with IBD, between 5-25 years of age, who received regularly scheduled infliximab or vedolizumab infusions, and had serum 25-OHD levels below 30 ng/mL were recruited for the study. These patients were screened for the exclusion criteria, including underlying liver or kidney disease. Enrolled participants were given eight high-dose oral vitamin D3 supplements during scheduled infliximab or vedolizumab infusions for 8-16 months. Serum 25-OHD levels, urine calcium and creatinine levels, and research blood samples were collected at baseline, midpoint, and final visits. Questionnaires were also dispensed to patients to measure quality of life (QoL). This data was collected and analyzed to assess the safety and efficacy of high-dose interval vitamin D supplementation in pediatric patients with IBD. RESULTS: The data from this study showed statistical significance in the change of serum-25OHD level from baseline to midpoint and final visits. The mean increase from baseline to midpoint was 15.71±10.1 ng/mL for the 30 participants who had completed 3 study doses (2,500 mCg or 5,000 mCg) (mean±95% CI). The mean increase from baseline to final visit was 18.1±11.67 ng/mL for the 19 participants who completed all 7 study doses (2,500 mCg or 5,000 mCg) (mean±95% CI). A single factor ANOVA test confirmed statistical significance with p < 0.0001. Urine calcium and creatinine levels did not have a statistically significant change from baseline to final visit for the 12 participants who had completed both samples. Lastly, IMPACT-III QoL scores were not significantly different from baseline. However, there was an overall increase in the mean scores in all 6 subcategories of the survey. As more participants complete the study, the statistical significance and the validity of results will likely be strengthened. CONCLUSION: High-dose interval vitamin D supplementation was a safe and effective way to achieve serum 25-OHD levels to an optimal range (i.e., 40-60 ng/mL) in pediatric patients and young adult patients with IBD. The data suggests that three doses of high-dose vitamin D may be sufficient to bring levels to an optimal and stable plateau. Patient compliance with supplementation was 100% in this study, because of provider-observed ingestion of vitamin D. Patients also noted that this was their preferred method of supplementation. The safety and efficacy results of this study serve as a framework for developing a more standard approach to vitamin D supplementation for our patients with IBD. Future studies may benefit from expanding this method of delivery to patients who have other inflammatory diseases that require both regular oral vitamin D therapy and in person visits for treatments (i.e., intravenous (IV) medication).

INTRODUCTION: Inflammatory bowel disease (IBD) is a complex, chronic, autoimmune disease of the gastrointestinal tract. Reactive oxygen species (ROS), a product of active leukocytes, have been implicated in the pathogenesis of IBD. The ability to reliably measure ROS in blood, urine, and stool samples could represent a new approach to assessing disease activity and response to therapy in pediatric patients with IBD. OBJECTIVES: To assess the relationship between redox measurements and clinical disease activity in pediatric patients with IBD. METHODS: Biological specimens, including stool, urine, blood plasma, and intestinal aspirates, were collected from patients at Boston Children’s Hospital. Each sample’s oxidation-reduction potential was measured by two oxidation-reduction potential probes (an Arrowdox probe and a Mettler Toledo probe). Probes were directly immersed into the sample, returning a millivolt measurement of oxidation-reduction potential. Linear regression was performed to explore the relationship between patient-reported outcome measures (PROMs) and redox measurements of biological specimens. Patients were also stratified by disease severity, and ANOVA testing was performed to test for differences in oxidation-reduction potential observed in patients with remittent, mild, moderate, and severe disease activity. RESULTS: Redox values in stool, urine, plasma, and intestinal aspirate did not significantly correlate with PROMs or differ significantly among groups categorized by disease severity. CONCLUSIONS: Measurements of oxidation-reduction potential from stool, urine, plasma, and intestinal aspirate do not appear to be useful for assessing disease severity in pediatric patients with inflammatory bowel disease.

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<p>Inflammatory bowel disease (IBD) is chronic, relapsing, intestinal inflammation characterized by periods of remission and exacerbation. Pathogenesis of IBD is a complex process and the exact aetiology is unclear yet. Studies have provided evidences that IBD is a result of a genetic predisposition that leads to a mucosal immune regulatory cell defect, barrier defects, and susceptibility to environmental triggers like luminal bacteria and specific antigens. Innate and adaptive immune responses to commensal bacteria are balanced by the multiple regulatory cells like T regs and B regs. Monoclonal-antibody-based therapies have been identified for the treatment of IBD but, they have side effects and the efficacy is not stable. Hence, great expectations lie towards finding a successful cure for IBD.</p> <p>Recent studies have proved the involvement of several different genes in the pathogenesis of IBD and there expressions were highly reduced in IBD. Homeodomain only protein- Hopx is a nuclear protein required to modulate heart growth and function. Expression of Hopx has been reported in various types of normal tissues but not in malignant tissues. Recent study on Hopx revealed that Hopx is needed for the function of regulatory T cells induced by DCs. However the correlation between Hopx gene and IBD remains unanswered. In this study, we examined the role of Hopx expressing regulatory B cell in IBD.</p> <p>The aim of this study is to investigate whether the expression of Hopx in regulatory B cell play a key role in suppressing colitis in murine model.</p> <p>The expression of Hopx was studied using cellular and molecular techniques including flowcytometry, immunohistochemistry, western blotting and real time RT-PCR.</p> <p>As a first step, we investigated the Hopx expression in colitis and control murine model. After we found the possible involvement of Hopx gene in regulating intestinal inflammation, we further our study to investigate whether Hopx is expressed by regulatory B cell and function together to inhibit intestinal inflammation. After establishing Hopx and B regs association, we used probiotics to modulate Hopx expression in regulatory B cells in order to prevent/reduce intestinal inflammation. Finally we elucidate the importance of Hopx expression by injecting neutralizing anti-Hopx antibody to block Hopx.</p> <p>Together, studies on human intestinal tissue and murine model revealed that Hopx expression is suppressed during inflammation condition. Probiotic administration helps to increase Hopx expressing Breg cell thereby, prevent IBD. Hopx deficient group expressed high frequency of proinflammatory cytokines and reduced immune regulatory cells. This particular study proposes that the down regulation of Hopx contributes to the development of intestinal inflammation.</p> / Master of Health Sciences (MSc)

Serotonin (5-hydroxytryptamine [5-HT]) is a key enteric signaling molecule that is implicated in many gastrointestinal (GI) disorders, including inflammatory bowel disease (IBD). Enterochromaffin (EC) cells are a key subgroup of enteric endocrine cells and produce the majority of 5-HT via tryptophan hydroxylase 1 (Tph1) in the gut. Recently, we have identified a pivotal role of 5-HT in the pathogenesis of experimental colitis, whereby 5-HT plays as a pro-inflammatory molecule. Gut function as well as pathology rely on interactions with gut microbiota. The intestinal epithelial cells produce antimicrobial peptides (AMPs), maintaining the mucosal barrier by shaping gut microbiota composition. Among the AMPs, β-defensins are the most well investigated subtype in the colon. Aberrant β-defensin expression has been reported in association with various GI disease pathogenesis including IBD. As EC cells are dispersed throughout the intestinal epithelium, it seems possible that 5-HT can modify β-defensin production which can regulate gut inflammation by influencing gut microbial composition. Colitis was induced with dextran sulfate sodium (DSS) in Tph1+/+ and Tph1-/- (which have lower amounts of 5-HT in gut). Tph1-/- mice exhibited higher levels of β-defensin in the colon, compared with wild-type littermates post-DSS. In addition, increased expression of β-defensin in Tph1-/- mice was suppressed by 5-hydroxytryptophan (5-HTP; precursor of 5-HT) treatment. 5-HT treatment resulted in decreased human β-defensin (hBD) 1 and hBD-2 expression in HT-29 cells. Peroxisome proliferator-activated receptor gamma (PPAR-γ) is essential for maintaining β-defensin expression in the colon. GW-9662, PPAR-γ antagonist, reduced mouse β-defensin (mBD) 1 and mBD-3 (orthologue of hBD-2). Furthermore, disrupting 5-HT7 receptors, but not 5-HT3 or 5-HT4, led to enhanced expression of PPAR-γ via ERK1/2-dependent mechanism. These observations provide us with novel information on pivotal role of gut-derived 5-HT in innate immune response and highlight the potential benefits of targeting 5-HT signaling in various GI disorders such as IBD. / Thesis / Master of Science (MSc)

Habitual physical activity and fitness are well-established as independent predictors of health in both children and adults. Chronic inflammatory conditions in children have been shown to negatively impact participation and maintenance of physical activity in childhood which can lead to a reduction in fitness. Juvenile idiopathic arthritis (JIA) and inflammatory bowel disease (IBD) are two of the most common childhood chronic conditions, both of which are characterized by inflammation. Alarmingly, even during remission, physical activity and fitness levels are reduced and sickness behaviours persist in these children. As such, it is thought that psychosocial measures, such as self-perceived health and well-being, may have a stronger association with decreased physical activity and fitness levels in this population as compared to healthy controls. The purpose of this project was to examine the relationship between self-perceived health and well-being and physical activity and fitness in children with JIA or IBD. In total, 58 children (32 girls, 26 boys) between the ages of 7 and 17 years with a single diagnosis of either JIA or IBD, and healthy controls were recruited. They completed measures of anthropometry, body composition, physical activity, as well as fitness (aerobic and muscle strength). Questionnaires regarding perceived health and well-being were also completed, and blood samples were obtained to measure specific markers of inflammation. Children with JIA or IBD were found to engage in significantly less moderate-to-vigorous physical activity (MVPA) relative to healthy controls (t = -1.977, p = 0.040). A linear regression established that MVPA, when expressed as an average of minutes per day, could statistically significantly predict self-perceived health, F(1,50) = 6.516, p = 0.014, where MVPA accounted for 11.5% of the explained variability in self-perceived health. This was no longer significant with the controls age, sex, and body fat percentage added into the model. Linear regression models showed that fitness was more predictive of self-perceived well-being, as seen with relative VO2 peak, F(1,38) = 6.683, p = 0.014, where relative VO2 peak accounted for 15% of the variability with self-perceived well-being. Furthermore, composite isometric strength expressed relative to body mass was able to significantly predict a composite blood inflammatory marker score, F(1,49) = 4.447, p = 0.040, where a relative composite isometric strength score accounted for 8.3% of the variability in a composite inflammatory blood marker score. Our findings indicate weak but significant predictive power for physical activity and fitness variables with regards to self-perceived health and well-being. Therefore, it may be important to explore ways to increase self-perceived health and well-being in children with JIA or IBD in order to improve physical activity participation and fitness. / Thesis / Master of Health Sciences (MSc)