Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 77-90).

Thèse (M.Med.Exp.) -- Université du Québec à Chicoutimi, extensionné de l'Université Laval, 2004. / Bibliogr.: f. [99]-108. Document électronique également accessible en format PDF. CaQCU

Thesis (Ph. D.)--University of Louisville, 2009. / Department of Educational and Counseling Psychology. Vita. "August 2009." Includes bibliographical references (leaves 129-167).

Includes bibliographical references and index.

Includes bibliographical references and index.

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.

L’homéostasie des muqueuses intestinale et cutanée dépend des interactions complexes entre le microbiote, l’épithélium et le système immunitaire de l’hôte. Des mécanismes régulateurs divers coopèrent afin de maintenir l’équilibre physiologique, et un défaut dans ces mécanismes entrainent des situations pathologiques. Le glucagon like peptide 2 (GLP-2) est un neuropeptide caractérisé par des propriétés prolifératives et anti-inflammatoires. Le potentiel thérapeutique des analogues de GLP-2 est actuellement évalué dans des essais cliniques pour des maladies digestives. Les effets du GLP-2 dans l’intestin sont médiés par son récepteur GLP-2 receptor (GLP-2R). Malgré la présence des nombreuses études évaluant l’expression cellulaire et la distribution tissulaire du GLP-2R, celles-ci restent controversées, que ca soit chez l’homme ou les rongeurs. Il est admis que l’expression du GLP-2R était confinée au tube digestif, principalement à l’intestin proximal, malgré des études évoquant une expression extra-intestinale. Une meilleure compréhension de l’expression et de la distribution de GLP-2R est nécessaire pour élucider les fonctions biologiques de GLP-2. Nous avons réalisé une cartographie de l’expression de GLP-2R dans différents organes murins ainsi que dans des lignées immortalisées humaines et murines. Nous avons également évalué l’expression intestinale du GLP-2R dans 2 modèles de colites induites chez la souris et dans des biopsies intestinales des patients atteints de maladies inflammatoires chroniques de l’intestin (MICI). Nous avons démontré que GLP-2R était exprimé en dehors du tube digestif notamment dans la vessie, le système nerveux central, le mésentère, les ganglions mésentériques, la rate et le foie. Nous avons également montré que la plus forte expression de GLP-2R dans le tube digestif était détectée dans le colon proximal et le rectum. Dans les modèles murins de colites et chez les patients atteints de MICI, l’expression du GLP-2R était diminuée, notamment dans les zones inflammatoires. Ceci pose la question le rôle physiopathologique des analogues de GLP-2 dans les maladies inflammatoires digestives. En conclusion, les hypothèses précédentes considérant une expression du GLP-2R majoritaire dans le tube proximal doivent être reconsidérées. Les fonctions physiologiques extra-intestinales du GLP-2 doivent être explorés afin d’anticiper des effets indésirables extra-digestifs des analogues de GLP2. / Intestinal and skin physiologies are quite similar as far as the homeostasis in both depends on complex interactions between the microbiota, the epithelium and the host immune system. Diverse regulatory mechanisms cooperate to maintain the equilibrium, and a breakdown in these pathways may precipitate pathological conditions. Glucagon like peptide 2 (GLP-2) represents one of the hot topics in research in intestinal physiology. Its dual function as an anti-inflammatory agent and growth factor has led to its consideration in therapeutic strategies and GLP-2 analogs are currently in clinical trials for several digestive diseases. The integrative responses to GLP-2 are mediated via the GLP-2 receptor (GLP-2R). Despite extensive research, precise tissue distribution of GLP-2R expression remains controversial both in rodents and humans. It is widely believed that GLP-2R expression is restricted to the gastrointestinal tract, mainly to the proximal bowel, despite the presence of few studies reporting extra-intestinal expression. Thus, to enhance our knowledge concerning the potential functions of GLP-2 analogs, a better understanding for GLP-2R expression is considered necessary. We therefore realized a panel of GLP-2R expression in mice tissues and in several human, murine and rat cells lines. Given the therapeutic beneficial effects of GLP-2 analogs in intestinal disorders, we investigated the intestinal expression of GLP-2R in mice models of chemically-induced colitis and in inflammatory bowel disease (IBD) patients. We demonstrated that GLP-2R is more widely expressed than expected with significant expression in several mice tissues including bladder, central nervous system, mesenteric adipose tissue, mesenteric lymph nodes, spleen, and liver. We also showed that the expression of GLP-2R in the gastrointestinal tract follows an increasing gradient toward the distal gut with highest expression in the colon and rectum. Interestingly, the intestinal expression of GLP-2R is significantly decreased in experimental mice models of colitis and in IBD patients which raised the point of the physiological role of GLP-2 analogs in digestive disease patients. Overall, previous hypotheses limiting GLP-2R expression and function to proximal bowel need to be revisited, and further studies should address the extra-intestinal biological function of GLP-2.

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 diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory immune-mediated diseases of the gastrointestinal tract. Two-thirds of IBD patients will develop severe disease, with complications that will require frequent surgeries and hospital admissions, and will seriously impair their quality of life. The ultimate clinical challenge of precision medicine in IBD is to find predictive markers to anticipate the development of severe disease and to monitor treatment in these patients.In the first part of my PhD thesis, we have carried out several studies monitoring the biologics used in IBD patients with severe disease. We have evaluated the pharmacokinetics of the following biologics used in IBD patients: infliximab, vedolizumab, and ustekinumab. We have focused on measuring trough levels (TLs) (defined as the serum drug level measured just before the next drug administration) early on after initiating biologic treatment to predict patient outcomes, including long- term responses in patients treated with infliximab and vedolizumab. In addition, we are currently conducting a prospective multicentric study that aims to design a pharmacokinetic model of infliximab at induction in IBD patients (EudraCT: CT 2015- 004618-10) (End of study expected by December 2019 but interim analysis available in the present work). Moreover, we have reported on the efficacy of ustekinumab in a large national cohort of highly refractory CD patients and have also examined the benefit of early measurement of ustekinumab TLs in these patients. Finally, we have reported novel findings on the impact of different wash-out periods (defined as the time frame between the discontinuation of one biologic and the initiation of a second biologic on the pharmacokinetics of the second-line biologic). Altogether, over the past 3 years, our data suggest the importance of measuring TLs early on during induction to predict long-term response to biologics during maintenance therapyIn the second part of my PhD thesis, we have analysed the inter-variability of the immune response in healthy subjects. Inflammation is the obvious key driver and underlying mechanism of disease severity in IBD. Therefore, the magnitude of inflammation must help define the phenotype of mild to severe disease. Delineating the inter-variability of the immune response in a healthy cohort constitutes a fundamental step to uncovering the genetic factors underlying this variability. We have performed whole blood cell cultures in a highly selected population of more than 400 healthy subjects stimulated with several Toll-like receptor (TLR) agonists and a T-cell receptor (TCR) antagonist. We found that the magnitude of the immune response (the high- or low-cytokine producer phenotype) was independent of the cytokine measured and the TLR agonists used. Thus, a donor exhibits a specific immune (cytokine) response or “immunotype” across cytokines released and TLR stimulation. Importantly, the high- or low-cytokine producer phenotype was different and did not overlap between the TLR and TCR stimulation conditions. In other words, a donor who is ahigh-cytokine producer following TLR stimulation will not be a high-cytokine producer following TCR stimulation (or the inverse). Therefore, we have defined TLR- or TCR- related Immunotypes (IT) as “a grading classification of the magnitude of the cytokine immune response” with IT1, IT2, and IT3 as low, intermediate, and high immunotypes. This suggests that two independent TLR and TCR ITs (TLR IT1 and TCR IT3) can co-exist in the same subject. We are now currently evaluating the genetic markers underlying these ITs before validating them in large cohort of IBD patients with mild-to-moderate and severe disease.This PhD thesis provides some data suggesting that the assessment of the pharmacokinetics of biologics early on at the initiation of treatment could help predict how the patient will respond in the long run. In parallel, this PhD thesis provides some advances in the understanding of the inter-variability of the immune response, a fundamental step before the identification of potential genetic markers underlying the inter-variability of inflammation and, hence, the severity of disease in IBD. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished