The overall aims of this thesis were to investigate the incidence of inflammatory bowel disease in the Uppsala Region of Sweden, to study the clinical course and the impact of the disease with regards to complications. Patients in Uppsala County were included in the study from the 1st of January 2005 and patients in Falun, Eskilstuna and Åland counties from the 1st of January 2007. The study was closed for all centres on the 31st of December 2009. Mean population in the study region was 305,381 in 2005–2006 and 642,117 in 2007–2009. The mean incidence for ulcerative colitis (UC) during the time period 2005-2009 was 20.0 /100,000/year (95% CI: 16.1-23.9) and for Crohn’s disease (CD) it was 9.9/100,000/year (95% CI: 7.1-12.6). The combined incidence for UC or CD in the area was thus 29.9/100,000/year (95% CI: 25.1-34.7). Half of the UC patients relapsed during the first year. Risk factors for relapse were female gender and young age. Colectomy during the first year was uncommon (2.5%). CD patients with complicated disease had longer symptom duration before diagnosis and less often diarrhoea and blood in stools compared to patients with non-complicated disease. The risk for surgery during the first year was 12%. The prevalence of anaemia at the time of diagnosis was 30% and after one year 18%. Anaemia was more common among newly diagnosed patients with CD compared with UC. 13% of the UC patients developed an acute severe episode. During the first 90 days 22% of these patients were subjected to colectomy. There was a significant difference between University and County hospitals in colectomy frequency (7.5% vs. 41%). The cumulative prevalence of treatment complications was 12% at the hospital with low colectomy rate versus 41% at the hospitals with high colectomy rate. In conclusion, the incidence of UC and CD in Sweden was high compared to international studies. Colectomy frequency for UC during the first year was low. Patients with complicated CD at the time of diagnosis had longer symptom duration and less alarming symptoms compared to uncomplicated disease. Anaemia was a common trait among patients with newly diagnosed IBD and more effort is needed to treat anaemia in these patients. Severe UC can be treated safely with prolonged medical therapy instead of colectomy.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder that occurs as a consequence of a genetic mutation that results in an overly aggressive immune response to normal bacteria. Metabolomics is a new born cousin to genomics and proteomics and involves a high throughput identification, characterization and quantification of small molecule metabolites generated by the organism. This study will show that metabolomics can be an effective tool in studying the differences between wild type and IL 10 KO mice as they age in axenic and conventional environments, and the onset of disease in a conventional environment. I show specific changes upon colonizing axenic mice with fecal bacteria that are similar to changes occurring over 16 weeks of conventional growth. Several bacterial metabolites have been identified that may play a role in the pathogenesis or provide clues to the interactions of the gut microbiota with the intestinal immune system. / Experimental Medicine

Abstract Nutritional status affects every aspect of a child?s health. Thorough nutritional assessment is hampered by the lack of a single comprehensive tool, which can cover all aspects of nutritional assessment. In three distinct studies, this thesis investigated the nutritional status of hospitalised children, children with coeliac disease and children with inflammatory bowel disease. Study 1 The objectives of this study were to assess prevalence of malnutrition and nutritional risk, and define demographic and anthropometric factors associated with nutritional risk among hospitalized children. In this cross sectional study, 157 hospitalised children were assessed for nutritional status using nutritional risk score (NRS) and anthropometric measurements. We found that 4.5%, 8.9%, 15.1% and 10.4% children were wasted, stunted, overweight and obese respectively. However, with the NRS, 47.8% of the children were at high nutritional risk. These children at high risk had lower weight for age (p=0.02), lower BMI percentiles for age (p=0.001) and longer hospitalization (p=0.001) than children at no risk. Study 2 The objectives of this study were to determine nutritional parameters in children with coeliac disease. Twenty-five children with coeliac disease and an equal number of age and gender matched controls were enrolled and anthropometric measurements, BIA and leptin levels were analysed in all. No significant differences were found between the children with coeliac disease and controls in these parameters. BMI percentile correlated with leptin levels in children with coeliac disease. Study 3 The objectives of this study were to determine anthropometric parameters and leptin levels in children with IBD and ascertain if BMI correlates with leptin levels in these children. Thirty children with IBD and 60 age and gender matched controls were enrolled. Anthropometric measurements and leptin levels were analysed and compared with controls. IBD children had significantly low weight for age (p=0.002), BMI percentiles (p=0.001) and leptin levels (p=0.009) compared to controls. There was a correlation between BMI and leptin levels in IBD children. In conclusion, this thesis has shown that one quarter of hospitalized children were overweight or obese, and further, that half of the hospitalised children were at high risk of nutritional deterioration and these children had longer hospital stay than children at no risk. Children with coeliac disease had similar anthropometric measurements, body compartments and leptin levels to controls. However, children with IBD had lower anthropometric measurements and leptin levels, indicating under-nutrition. Nutritional assessment should be a mandatory part of clinical management with nutritional status assessed by various tools including NRS, anthropometry, BIA and leptin levels.

Abstract Nutritional status affects every aspect of a child?s health. Thorough nutritional assessment is hampered by the lack of a single comprehensive tool, which can cover all aspects of nutritional assessment. In three distinct studies, this thesis investigated the nutritional status of hospitalised children, children with coeliac disease and children with inflammatory bowel disease. Study 1 The objectives of this study were to assess prevalence of malnutrition and nutritional risk, and define demographic and anthropometric factors associated with nutritional risk among hospitalized children. In this cross sectional study, 157 hospitalised children were assessed for nutritional status using nutritional risk score (NRS) and anthropometric measurements. We found that 4.5%, 8.9%, 15.1% and 10.4% children were wasted, stunted, overweight and obese respectively. However, with the NRS, 47.8% of the children were at high nutritional risk. These children at high risk had lower weight for age (p=0.02), lower BMI percentiles for age (p=0.001) and longer hospitalization (p=0.001) than children at no risk. Study 2 The objectives of this study were to determine nutritional parameters in children with coeliac disease. Twenty-five children with coeliac disease and an equal number of age and gender matched controls were enrolled and anthropometric measurements, BIA and leptin levels were analysed in all. No significant differences were found between the children with coeliac disease and controls in these parameters. BMI percentile correlated with leptin levels in children with coeliac disease. Study 3 The objectives of this study were to determine anthropometric parameters and leptin levels in children with IBD and ascertain if BMI correlates with leptin levels in these children. Thirty children with IBD and 60 age and gender matched controls were enrolled. Anthropometric measurements and leptin levels were analysed and compared with controls. IBD children had significantly low weight for age (p=0.002), BMI percentiles (p=0.001) and leptin levels (p=0.009) compared to controls. There was a correlation between BMI and leptin levels in IBD children. In conclusion, this thesis has shown that one quarter of hospitalized children were overweight or obese, and further, that half of the hospitalised children were at high risk of nutritional deterioration and these children had longer hospital stay than children at no risk. Children with coeliac disease had similar anthropometric measurements, body compartments and leptin levels to controls. However, children with IBD had lower anthropometric measurements and leptin levels, indicating under-nutrition. Nutritional assessment should be a mandatory part of clinical management with nutritional status assessed by various tools including NRS, anthropometry, BIA and leptin levels.

Abstract Nutritional status affects every aspect of a child?s health. Thorough nutritional assessment is hampered by the lack of a single comprehensive tool, which can cover all aspects of nutritional assessment. In three distinct studies, this thesis investigated the nutritional status of hospitalised children, children with coeliac disease and children with inflammatory bowel disease. Study 1 The objectives of this study were to assess prevalence of malnutrition and nutritional risk, and define demographic and anthropometric factors associated with nutritional risk among hospitalized children. In this cross sectional study, 157 hospitalised children were assessed for nutritional status using nutritional risk score (NRS) and anthropometric measurements. We found that 4.5%, 8.9%, 15.1% and 10.4% children were wasted, stunted, overweight and obese respectively. However, with the NRS, 47.8% of the children were at high nutritional risk. These children at high risk had lower weight for age (p=0.02), lower BMI percentiles for age (p=0.001) and longer hospitalization (p=0.001) than children at no risk. Study 2 The objectives of this study were to determine nutritional parameters in children with coeliac disease. Twenty-five children with coeliac disease and an equal number of age and gender matched controls were enrolled and anthropometric measurements, BIA and leptin levels were analysed in all. No significant differences were found between the children with coeliac disease and controls in these parameters. BMI percentile correlated with leptin levels in children with coeliac disease. Study 3 The objectives of this study were to determine anthropometric parameters and leptin levels in children with IBD and ascertain if BMI correlates with leptin levels in these children. Thirty children with IBD and 60 age and gender matched controls were enrolled. Anthropometric measurements and leptin levels were analysed and compared with controls. IBD children had significantly low weight for age (p=0.002), BMI percentiles (p=0.001) and leptin levels (p=0.009) compared to controls. There was a correlation between BMI and leptin levels in IBD children. In conclusion, this thesis has shown that one quarter of hospitalized children were overweight or obese, and further, that half of the hospitalised children were at high risk of nutritional deterioration and these children had longer hospital stay than children at no risk. Children with coeliac disease had similar anthropometric measurements, body compartments and leptin levels to controls. However, children with IBD had lower anthropometric measurements and leptin levels, indicating under-nutrition. Nutritional assessment should be a mandatory part of clinical management with nutritional status assessed by various tools including NRS, anthropometry, BIA and leptin levels.

Chronic inflammatory conditions such as Crohn's disease (CD) and Ulcerative colitis (UC) are risk factors for colon cancer. TGFβ has been shown to be dysregulated in colon cancer. Bacteria-induced inflammation is necessary for the induction of colon cancer in TGFβ mouse models. However, the mechanism by which TGFβ regulates the inflammatory response in these models is not well elucidated. It was our thought that we needed to be able to distinguish what was TGFβ dependent and what was inflammation dependent. To do this we created 2 colonies of Smad3 mice. One colony was housed with normal colonic bacteria (Smad3-uninfected animals) and the other colony (Smad3-infected animals) had chronic H. hepaticus infection. As previously seen the Smad3⁻/⁻- infected animals developed colitis and carcinoma (~40%). In the absence of H. hepaticus infection SMAD3 was found to negatively regulate TLR4 expression. This was then exacerbated with the addition of H. hepaticus resulting extreme up-regulation of TLR4 and the downstream effectors IRAK4 and NF-κB in Smad3⁻/⁻-infected colonic tissues. Examination of adaptive immune regulation in this model demonstrated that SMAD3 was necessary for FOXP3 expression in H. hepaticus-infected splenocytes. Loss of SMAD3 resulted in up-regulation of IL17 and reduced iTreg populations. These data demonstrate the important role SMAD3 has in maintaining tolerance to microbial populations through both the innate and adaptive immune systems.

IL-33 is a pleiotropic cytokine that orchestrates both innate and adaptive immunity. It is commonly associated with type 2 immune responses but recently expression of the IL-33 receptor, ST2, was reported on Treg cells found preferentially in non-lymphoid tissues, such as the visceral adipose tissue, muscle or colon. A crucial role of Tregs in maintaining intestinal homeostasis has been well described. However, little is known about the functional relevance of the ST2-expressing Treg population in the colon. Phenotypic and functional characterisation of Tregs in the gut revealed the presence of two distinct populations: ST2⁺/Gata3⁺ and Rorγt⁺ Tregs. Thymic-derived ST2⁺/Gata3⁺ Tregs showed a more activated phenotype and produced IL-10 under homeostatic conditions. Upon microbial challenge and colitis, ST2+/Gata3+ Tregs were decreased, while Rorγt⁺ Tregs expanded. Furthermore, in vitro experiments demonstrated that IL-33 directly induced activation of the Gata3 pathway in Tregs, which enhanced expression of Foxp3 and ST2. Additionally, amphiregulin was also induced in Tregs upon stimulation with IL-33. However, in vivo, IL-33 was dispensable for both the maintenance of Treg cells under homeostatic conditions and Treg function in Helicobacter hepaticus-driven colitis. Investigation of the negative regulators of IL-33 showed that IL-23 inhibited IL-33-mediated effects on Tregs. We also observed increased production of soluble ST2 by stromal cells during intestinal inflammation, which likely contributed to the reduction of IL-33 bioavailability. Finally, a systematic analysis of the cellular source of IL-33 revealed that PDGRFα⁺ stromal cells located in the T cell zone of secondary and tertiary lymphoid tissues were a major IL-33-producing cell population in the gut. Collectively, our findings suggest that signals received by the stromal compartment upon cell injury may trigger a specific phenotype of Tregs with a repair capacity, and thus, promote intestinal homeostasis. These findings improve our understanding of tissue-resident Tregs and open an exciting avenue to explore heterocellular signalling between stromal cells and Tregs.

Inflammatory Bowel Disease (IBD) is a debilitating, life- long disease that affects about 1.4 millions Americans. Little is known about the pathogenesis of IBD and an effective cure still remains to be discovered. While there are numerous T cell targeting therapies for IBD, more research is still needed. Bispecific T Cell Engagers, BiTES, is a modified protein capable of engaging two antigens simultaneously; it is capable of activating T cells by circumventing the MHC protein molecule. This provides an alternative to the current molecular therapies for IBD. In addition to monoclonal therapy research, there has been a plethora of research on immunomodulatory molecules, such as β- glucan. The benefit of β-glucan has been shown with supplements and food sources alike in animal models. In this study, we used BiTES, CMPD-1, with an anti-CD3/ Dectin-1 epitopes capable of engaging T Cells and β-glucan in beads and fungi cell wall. CMPD-1 is capable of engaging Splenic and Lamina Propia T Cells from a C57BL/6 mice. Likewise, CMPD-1 engaged T cells to hyphae of C. albicans and A. fumigatus, which have a higher concentration of β-glucan than in the candida form. The data show a delayed in hyphae growth in yeast with CMPD-1 and a decrease in yeast growth for the first four hours when compared to non- BiTES molecules. Additionally, qualitative analysis of CMPD-1 shows a decrease A. fumigatus growth after a 72-hour incubation period. Splenic T cells from mice lacking Dectin-1 and Wild-type (WT) mouse strains where incubated with BiTES compound and yeast for 23 hours followed by a PrestoBlue killing assay to assess yeast cell viability. The PrestoBlue assay showed that CMPD-1 killed more A. fumigatus in both T cell subsets; although, the difference lacked statistical significance. The applications of this molecule as a therapeutic agent for IBD are promising, although, still in its infancy. An alternative use for this molecule is to train the immune system with the BiTES molecule in conjunction with β-glucan supplements to build immunity against opportunistic pathogens such as A. fumigatus and C. albicans that often cause havoc in IBD patients as a result of the changes in microbiota, and compromised integrity of the GI tract. / 2017-06-16T00:00:00Z

INTRODUCTION: The management of pediatric patients with ulcerative colitis (UC) is dependent upon the ability to detect meaningful changes in disease status. This is currently done using validated patient-reported clinical disease activity indices, including the Pediatric Ulcerative Colitis Activity Index (PUCAI). While useful for global assessments completed during ambulatory office visits, the sensitivity of this metric may be insufficient to reflect more subtle changes in disease activity or response to medical therapy in hospitalized patients. Intravenous steroids are typically employed in the management of patients admitted for acute exacerbations of UC symptoms. These are typically manifest by worsening bloody diarrhea, abdominal pain, and worsening anemia. There is presently no way of predicting whether a patient admitted for UC will respond to steroid therapy. Current paradigms dictate a five-day trial before considering a transition to more potent medical or definitive surgical approaches to the management of refractory colitis. The development of more sensitive and reliable biomarkers or disease activity metrics could enable clinicians to more expediently identify steroid non-responders. This would minimize patient morbidity, decrease risk of complication, and lower overall cost of care. Previous studies have demonstrated that changes in fecal lactoferrin (FLA) correlate with disease activity in patients with UC. OBJECTIVES: To analyze the predictive value of FLA in the response to steroid treatment of patients admitted for management of UC. METHODS: We recruited pediatric inpatients with UC in the Division of Gastroenterology, Hepatology and Nutrition at Boston Children’s Hospital who were hospitalized for treatment of a flare of their UC symptoms. After obtaining patient consent, we collected a stool sample on days 1 and 3 of their hospital stay. We sent samples to TECHLAB® Inc. (Blacksburg, VA) to be analyzed for levels of FLA. We compared Day 1, Day 3 and ∆FLA (Day 1 – Day 3) in two patient groups: those that responded to conventional steroid therapy and those that required rescue medical or surgical therapy. We reported statistical significance with the Wilcoxon signed-rank test. RESULTS: Of 67 patients consented for the study, 30 provided stool samples on both days 1 and 3 of their inpatient hospitalization. Of the 30 patients, 63.3% responded to steroids while 36.7% required rescue therapy with immunomodulators. ∆FLA for responders, 43.6μg/mL(-239.0, 331.6) (median(interquartile range)), did not differ significantly from non-responders, -74.1μg/mL(-296.7, 221.7), P = 0.3. CONCLUSIONS: Our findings do not demonstrate that measurement of changes in quantitative FLA over three days can be used to assess acute responses to steroid therapy. Increasing the sample size may allow us to better delineate subtle differences between responders and non-responders to steroid therapy.

Introduction Inflammatory bowel disease (IBD) is a chronic gastrointestinal disorder that encompasses two major subtypes; Crohn’s Disease (CD) and Ulcerative Colitis (UC). Our knowledge regarding disease pathogesis is rapidly increasing. However, these disease entities provide challenges in diagnosis, monitoring of disease activity and assessing individual response to treatment, because there is a lack of validated clinical biomarkers. Metabolomics involves the study of numerous analytes that have very diverse physical and chemical properties and occur in a wide concentration range. Early evidence suggests there is potential for metabolomic profiling to be used in the differentiation of CD and UC. However, knowledge is limited regarding the metabolic changes seen in relation to disease activity or to medical or surgical treatments. Aims A metabolomics approach was taken to determine whether metabolomic profiles could distinguish between patients with CD or UC and healthy controls. We also aimed to define the relationship between metabolomic profile and disease activity, and to determine the effect of medical (anti-TNFa agents) and surgical treatment on the metabolome. Methods A metabolomics approach was undertaken. Serum and urine sample sets were collected from a total of 41 patients with ulcerative colitis, 43 patients with Crohn’s disease, and 62 healthy controls (HC). In order to allow a comparison of metablomic profile and disease activity, 4 sample sets were taken from the same patient at 3 monthly intervals over the period of one year. Those patients undergoing either surgical or biological treatment had sample sets taken pre and post intervention. Metabolomic analysis using gas chromatography time of flight mass spectrometry (GC-ToF-MS) and ultra-high performance liquid chromatography Fourier Transform mass spectrometry (UHPLC-FTMS) was carried out on both serum and urine. Results Serum and urine GC-ToF-MS and UHPLC-FTMS metabolomic analyses show differentiation between UC, CD and healthy controls, most significantly in urine analyses. No significant differentiation was seen in pre- and post-surgical patients, or pre- and post-biological therapy patients. It was possible to differentiate surgical patients from healthy controls, especially in the urine analyses. Metabolite identification revealed consistently more dietary variation in the healthy controls than in the IBD patients. Significant differences (p < 0.05) were seen between healthy controls and IBD patients in classes of metabolites relating to the citric acid cycle and the uronic acid pathway, as well as amino acids, fatty acids and cholesterols. The behaviour or location of disease, or the disease activity score did not appear to influence the metabolome in either serum or urine analyses using GC-ToF-MS and UHPLC-FTMS. Conclusion Metabolomic profiling of urine and serum in IBD may provide a novel methodology aiding both clinical diagnosis through biomarker development, and advancing knowledge of disease pathogenesis.