Background: Reliable biomarkers are needed to evade the risk of injury, invasiveness and discomfort of endoscopies, which are required for inflammatory bowel disease (IBD) diagnosis and extent of disease assessment in ulcerative colitis (UC) patients. The need for biomarkers is accentuated in children, wherein the most frequently used IBD biomarker yields low specificity. Proteomics of clinical samples or their enriched components is a means to evaluate and identify alterations in proteins reflective of disease, with the potential for use as biomarkers and for providing insight on disease pathogenesis. Methods: Proteins were isolated from the intestinal mucosal-luminal interface (MLI), collected from the ascending and descending colon of pediatric treatment-naive patients. The intestinal MLI proteomes of 42 IBD and 18 control patients were analyzed by high resolution mass spectrometry (HRMS). Multivariate analysis and receiver operating characteristics curves were performed to develop protein biomarker panels to discriminate IBD from control, and for UC extent of disease. ELISAs were used to assess a subset of biomarker candidates in stool samples from an independent pediatric cohort (n=24). Extracellular vesicles (EVs) were isolated by ultracentrifugation from the intestinal MLI of 11 IBD and seven control patients, and analyzed by electron microscopy, nanoparticle tracking analysis and HRMS. Results: A biomarker panel of four proteins classified patients as either controls or active IBD with 97.5% accuracy. A second biomarker panel correctly classified 100% of UC patients as presenting with pancolitis or non-pancolitis. The differential protein expression of two biomarker candidates (catalase and leukotriene A-4 hydrolase) identified from the intestinal MLI was comparable in stool samples. Comparison of EV proteomes isolated from IBD patients and controls identified differential expression of processes related to host defense and immunity. Conclusions: Proteomic analysis of clinical samples identified differentially expressed proteins that can classify IBD patients from non-IBD controls and distinguish UC patients with pancolitis from those without pancolitis; proteins identified in intestinal aspirates displayed consistent differential expression in stool. Furthermore enrichment of EVs from the intestinal MLI indicates that these may contribute to the dysregulated host response against the intestinal microbiota which is observed in IBD.

The inflammatory bowel diseases (IBD) are chronic conditions of the intestinal tract, divided into two main subtypes Crohn’s disease (CD) and ulcerative colitis (UC). The exact pathogenesis is unclear but the current paradigm is thought to be an aberrant immune response in a genetically susceptible individual. The incidence and prevalence of IBD has traditionally been higher in North America, Europe, Australia and Israel compared to other regions of the world including China, Japan, India and Korea. More recently there is evidence of an increase in immigrant populations. Studies have also suggested that the clinical characteristics differ across ethnic groups. This has been mirrored by genetic studies that suggest different genetic susceptibilities between groups. A systematic review was performed to define the relevance of gene variants to IBD in a South Asian population. This found that few studies (n=6) have genotyped susceptibility variants in the South Asian population. The majority of these studies examined three common polymorphisms (R702W, G908R, 1007fs) in NOD2/CARD15 in Caucasians and have determined that these are absent in South Asians. The first hypothesis of this study was that clinical characteristics and mucosal distribution differed in South Asians compared with White British in the North of England. A total of 1318 individuals (314 South Asians) with a diagnosis of IBD were recruited. In the South Asian cohort 59% had a diagnosis of UC, 41% CD. In contrast the Caucasian cohort 56% had CD and 44% had UC. South Asians had twice the rate of extensive colitis compared to White British cohort (46% SA vs. 24% White British) and a younger age of diagnosis (30 years vs. 40 years). In the CD cohort South Asians were twice as likely to have colonic disease than White British (54% vs. 20%). Also they had a younger age of onset and were less likely to need surgery for CD.The second hypothesis was that common variants in the same genes described in Caucasian IBD were relevant in South Asians. 13 known SNPs from GWA Studies robustly associated with IBD in Caucasian cohorts were sequenced in South Asians IBD cohort (n=255) and unrelated ethnically matched controls (n=275) to determine if they were relevant to IBD in South Asians. These were genotyped by Sequenom MassArray and no significant associations were discovered. The final hypothesis was that rare highly penetrant variants underlie a group of IBD in consanguineous families in South Asian IBD. A consanguineous family in which the proband had inflammatory colitis diagnosed at 18 months of age was recruited. No disease causing mutations were present in IL10RA, IL10RB and ADAM17. DNA from other family members was used to perform autozygosity mapping of the proband and family. Exome sequence analysis identified 6099 variants in autozygous regions. Further analysis focused on three novel variants. One variant (PPP1R3G) was considered a likely candidate and Sanger sequencing was performed which confirmed it was homozygous in the proband, but it did not segregate in the family and so unlikely to underlie IBD in this individual. In summary this thesis has shown that few genetic studies have been done in South Asian IBD. Also there are significant differences in the clinical characteristics and mucosal distribution between groups and that 13 SNPs associated with IBD in Caucasians were not replicated in the South Asian IBD cohort. Finally autozygosity mapping and exome sequencing has not been successful in identifying a rare novel variant responsible for IBD in the consanguineous family but work is continuing.

Inflammatory bowel disease (IBD) affects millions of people in the United States, with the number of diagnoses steadily rising. It has been associated with poor quality of life and a host of comorbidities. Most notably, IBD patients are at an increased risk of developing colorectal cancer (CRC). The American Gastroenterological Association (AGA) recommends that IBD patients with involvement of 1/3 or more of the colon undergo colonoscopy regularly to screen for CRC starting 8 years after initial IBD diagnosis. Colonoscopy techniques for IBD-related CRC screening are highly variable and differ widely between clinical practices. Currently, high-definition white-light colonoscopy (HD-WLC) and dye spraying chromoendoscopy (DCE) are both standard of care. The use of these technologies requires a high level of skill that is typically attained during clinicians’ 3-year gastroenterology (GI) fellowship. This study intends to compare outcomes of screening colonoscopies performed by GI fellows and attending physicians in patients with longstanding IBD (>8 years) and to assess the impact of GI fellow involvement on these procedures. Additionally, the current research intends to draw distinctions between HD-WLC and DCE procedures. The research was performed in the Division of Gastroenterology at Beth Israel Deaconess Medical Center (BIDMC) as part of a large randomized controlled trial (RCT) that aims to evaluate the comparative efficacy of HD-WLC and DCE. Patients were screened for study eligibility using relevant criteria and then randomized to undergo colonoscopy using HD-WLC technique or DCE technique. Data from 128 procedures were included in the study. Of these procedures, 59 (46.1%) were attending-performed procedures while 69 (53.9%) were fellow-performed, attending-supervised procedures. Of the attending-performed procedures, 30 (50.8%) were performed using the DCE technique and 29 (49.2%) were performed using the HD-WLC technique. Of the fellow-performed, attending-supervised procedures, 32 (46.4%) were performed using the DCE technique while 37 (53.6%) were performed using the HD-WLC technique. Fellow-performed, attending-supervised procedures were associated with longer total procedure time (TPT) and increased intra-procedure administration of sedation medications without superior lesion detection. Thus, fellows appear to be on par with attendings in terms of lesion detection but this level of proficiency comes at the cost of increased TPT. Assessing the short-term and long-term impacts of this could be a valuable area of future investigation. Also, DCE procedures took longer for all clinicians to perform, especially fellows, and are not associated with enhanced lesion detection. Further research is needed to understand the usefulness of DCE.

Inflammatory Bowel Disease (IBD), divided into Crohn’s disease (CD), ulcerative colitis (UC), and indeterminate colitis (IC), is a chronic, crippling autoimmune condition characterized by gastrointestinal (GI) inflammation. The methods used to diagnose IBD and assess its activity can be invasive and costly and typically include a combination of histologic, endoscopic, radiologic, clinical, and biochemical measures. Currently, there is an increasing need for the development of noninvasive assessment measures to detect an interval response to prescribed therapy. Previous studies have found serial and fecal biomarkers to be reliable, but non-specific indicators of GI tract inflammation. At present, they cannot be used to distinguish between inflammation resulting from infection and that caused by chronic inflammation in patients with IBD. The aim of this study is to measure changes in serum and fecal biomarkers over time in individual children and adolescents with CD, UC, and IC initiating infliximab therapy while investigating any parallels between fluctuations in biomarker levels and endoscopic, clinical, and biochemical outcomes. The inflammatory biomarkers evaluated in this study include fecal and serum anti-Saccharomyces-Cerevisiae Antibody (ASCA), fecal and serum lactoferrin, fecal hemoglobin, fecal calprotectin, fecal IL1-α, fecal IL1-β, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR). The data for this study was collected from a multicenter longitudinal prospective cohort study following pediatric patients over the course of six consecutive infliximab infusion appointments. Study sites include Boston Children’s Hospital and Riley Children’s Hospital in Indianapolis. Participants were recruited from a pool of CD, UC, and IC patients who were either new to infliximab, had been receiving infliximab for less than six months, or had been receiving infliximab for more than one year. Patients brought in stool samples at each of their scheduled infliximab infusions, biochemical labs (ESR and CRP) were obtained, and patients completed a health-related quality of life survey (IMPACT-III Questionnaire). Forty-three patients (26 with CD, 16 with UC, and one with IC) completed this study. There was no significant difference in mean serum or fecal ASCA levels between participants with CD and those with UC. However, average serum and fecal ASCA were higher in patients with CD than those with UC at almost every infusion. The baseline mean CRP level in patients with CD was significantly higher than that observed in patients with UC (p<0.05). In patients with CD, the mean IMPACT-III score was significantly higher (improved quality of life) at Infusion 5 than at baseline. The data collected in this study suggest serial biomarker measurements may be useful in monitoring a patient’s response to infliximab therapy. This study is not yet complete and requires further data analysis to more definitively conclude if a single or a composite metric including several fecal and/or serum inflammatory biomarkers would provide a more robust assessment of disease activity in children and young adults with IBD.

Inflammatory Bowel Disease (IBD) is a chronic, idiopathic autoimmune disease characterized by the inflammation of the GI tract. The main subcategories of IBD include Crohn Disease (CD), ulcerative colitis (UC), and Indeterminate Colitis (IC). Currently, IBD is diagnosed and evaluated using clinical, endoscopic, biochemical, and histologic measures - which can be invasive and costly. Previous studies have shown that measurement of serum and fecal inflammatory biomarkers might be effective both for the assessment of intestinal infectious or inflammatory processes, as well as for gauging IBD disease activity. Inflammatory biomarkers investigated in this study include serum and fecal Anti-Saccharomyces-Cerevisiae Antibody (ASCA), serum and fecal lactoferrin, fecal calprotectin, fecal hemoglobin, IL1-𝛼, IL1-β, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP). IMPACT-III questionnaires are utilized to measure the quality of life in enrolled subjects. Data collected in this study investigated the relationship between changes in serum and fecal biomarker levels, clinical disease activity, and the mental wellbeing of patients. The primary objective was to measure changes in inflammatory biomarker levels in patients with CD, UC, and IC after being treated with the anti-TNF therapy Infliximab (Remicade). The second objective is to study the relationship between changes in these biomarker levels and the clinical, biochemical, and endoscopic outcome parameters of IBD in patients. This is a multicenter, longitudinal, cohort study following 50 pediatric patients with IBD over the course of six Remicade infusion appointments. The project was conducted in partnership with Riley’s Children Hospital in Indianapolis. Patients with CD, UC, and IC were recruited for the study and stratified with respect to the temporal phase of their Remicade infusions, including: • Induction • Past induction, but less than 6 months • Those who have been receiving Remicade for over one year. A total of 58 eligible IBD patients are currently enrolled. 13 patients have withdrawn from the study, leaving 45 active patients: 27 CD, 17 UC, and 1 IC remaining in the prospective cohort. Patients provided serum and fecal samples and completed IMPACT-III questionnaires at the time of each Remicade infusion. Baseline serum ASCA levels were 0.014 ±0.029 OD from (n=15) patients with CD, 0.0083 ±0.022 OD in (n=12) patients with UC, and 0.002 OD in one patient with IC. Baseline fecal ASCA levels were 0.068 ±0.186 OD in (n=10) patients with CD, 0.0018 ±0.0013 OD in (n=9) patients with UC, and 0.001 OD in one patient with IC. At both baseline and at the time of the first infusion, CRP levels were significantly higher in patients with CD (p<0.05). At the time of the first infusion, the ESR in patients with CD was significantly higher than that in patients with UC (p<0.10). Serum lactoferrin was significantly higher in patients with UC at infusion 2 (p<0.05). ESR was significantly higher in patients with UC (p<0.10) at their fourth infusion. Our data support the hypothesis that serum and fecal biomarkers are useful in evaluating the response of intestinal inflammation to Remicade therapy. This study is ongoing, and further sample collection and data analysis are needed to more conclusively determine the accuracy of inflammatory biomarkers as a diagnostic tool for use in the diagnosis and interval assessment of patients with IBD.

Over the last couple of decades changes to pediatric inflammatory bowel disease (IBD) care may have altered health services use among these children. I used a retrospective matched cohort design and population-based health administrative data to first quantify trends in IBD health services and surgical outcomes in Ontario IBD children diagnosed between 1994-2012. I then used these results to validate the distributed network analysis method – a method being increasingly used in Canadian multi-province studies where privacy regulations prevent sharing of individual-level data across provincial borders - using Ontario’s Local Health Integration Networks. I found (1) decreasing hospitalizations and surgical outcomes but increasing outpatient visit rates, suggesting changing patterns of health care use in Ontario children with IBD, and, (2) distributed network analyses is a satisfactory privacy-preserving alternative to individual-level analysis under the conditions tested in my study, providing a tested analysis method for researchers using multi-jurisdictional data.

Hydrogen sulfide (H2S) has emerged as an important mediator of host function. In the gastrointestinal tract H2S is enzymatically produced and plays a vital role in cytoprotection, inflammation, and tissue repair. During a bout of colitis, the ability of the colon to produce H2S is markedly increased and drives the resolution of colitis. However, little is known about how the production of H2S is regulated in the colon and how dysregulated production can affect the course of colitis in vivo. Additionally, the mechanisms through which H2S can promote the resolution of colitis remain to be fully investigated. In Chapter 3 of this dissertation, the regulation of H2S production in the colon was explored by examining the contributions of three enzymatic pathways to colonic H2S synthesis. The largest source of the H2S synthesis was from a pathway previously unrecognized in the GI tract involving the enzyme 3-mercaptopyruvate sulfurtransferase (3MST). Additionally we found that the upregulation of H2S production during colitis occurred specifically at sites of mucosal ulceration. At the same time H2S inactivation via the enzyme sulfide quinone reductase (SQR) was significantly reduced at these sites. We propose that the site-specific alterations in H2S production and inactivation during colitis promote the resolution of inflammation and injury. Chapter 4 examined whether the ability of hyperhomocysteinemia (Hhcy) to exacerbate colonic inflammation occurred through impaired H2S synthesis. Hyperhomocysteinemia is often reported in patients with inflammatory bowel disease and is a consequence of decreased vitamin B intake. In all three models tested, diet-induced Hhcy significantly exacerbated colitis. Being dependent on vitamin B6 as a co-factor, the increased H2S production normally observed during colitis was abolished during Hhcy. Administration of an H2S donor to Hhcy rats significantly decreased the severity of colitis. These results also uncovered a novel role for IL-10 in promoting H2S production and homocysteine metabolism, which may have therapeutic value in conditions characterized by Hhcy. Finally, in Chapter 5 we looked for a mechanism through which H2S can promote resolution of colitis. Using CSE-deficient mice we found that H2S production was required to maintain HIF-1α signaling in the colon. Additionally, proper HIF-1α signaling was required for H2S-donating molecules to promote the resolution of colitis. These results suggest that HIF-1α signaling is a critical event through which H2S promotes resolution of colitis. Collectively, these chapters further highlight the importance of H2S production in colon during inflammation and injury and offer insight into new therapeutic targets mediated through H2S. / Dissertation / Doctor of Philosophy (PhD)

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