Crohn's Disease (CD) is a debilitating condition characterised by chronic intermittent intestinal inflammation. More than 90 genetic polymorphisms are associated with CD susceptibility, including several in genes of the innate immune system. Here I present a series of experiments designed to enhance our knowledge of the roles of CD-associated polymorphisms in pathogenesis. Many therapeutic regimens are employed in CD treatment, but patients' responses to treatment and disease progression vary widely. There is great interest in studying whether analysis of patients' genotype at CD-associated polymorphisms can be used to predict their disease course, and guide clinical decision-making. To answer these questions, it is essential to be able routinely and cost- effectively to genotype patients at the full range of known CD-associated polymorphisms. The first project presented here describes the design and initial successful testing of a CD-specific genotyping microarray for use in genotype-phenotype studies. The polymorphism most strongly associated with CD susceptibility is in the pattern recognition receptor NOD2; the remaining experiments presented here study the function of NOD2 in primary human monocyte-derived Dendritic Cells (DCs). First, a microarray study is presented which characterises global transcriptional responses to NOD2 stimulation in DCs. NOD2 stimulation is shown to enhance transcriptional changes induced by Toll-Like Receptor 2 stimulation, and NOD2-mediated transcriptional regulation is shown to be lost in DCs expressing CD-associated NOD2 variants. Second, experiments are presented which describe development of a new protocol for proteomic analysis of post-translational protein modifications, and which identify a number of novel candidate targets of NOD2 signalling in DCs. Finally, a project is presented which demonstrates for the first time that NOD2 stimulation induces autophagy in DCs, in an NF-kB and RIPK2-dependent pathway. CD-associated polymorphisms in NOD2 and ATG 16Ll abolish NOD2-mediated autophagy induction, resulting in impaired bacterial handling and antigen presentation.

Irritable bowel syndrome (IBS) är en sjukdom som leder till en funktionell motorisk störning i mag-tarmkanalen och symtom som förstoppning, diarré, gasbesvär, buksmärta och uppspänd buk är vanligt förekommande. Symtomen leder ofta till stress, depression, ångest vilket kan medföra ett begränsat vardagsliv. Individens livskvalitet påverkas av vad symtomen orsakar för individen i vardagslivet vilket kan leda till lidande. Syftet med litteraturstudien var att granska och sammanställa vårdvetenskaplig forskning som belyser patienters upplevelser av att leva med IBS. Metoden är en litteraturstudie utifrån Fribergs modell. Sex kvalitativa artiklar och tre kvantitativa artiklar har analyserats. Resultatet visade att kroppen var opålitlig då sjukdomen orsakade fysiska symtom och gav oönskade symtom som gasbesvär, smärta i buken, diarré, förstoppning och uppspändhet. Patienter upplevde begränsningar i vardagen i form av symtom som ökade gasbesvär, uppspändhet, diarré. Patienter hade ständigt uppsikt efter toaletter då de gick hemifrån vilket ofta skapade en ökad oro. Symtom som gasbildningar och diarré innebar att patienter ofta behövde gå på toaletten vilket var besvärligt i samband med möte, arbete, skola. Detta upplevde patienter vara skamligt. Sjukdomen bidrog i längden till ökad stress, ångest, depression och isolering hos patienterna. Detta har orsakat minskat förtroende till vården, patienterna upplevde sig missförstådda och välbefinnandet minskade. Strategier och planering bidrog till bekräftelse, medvetenhet, kunskap och förståelse. Patienter upplevde sig ha bättre koll på symtomen och kunde agera tills värken släppte. Sjuksköterskan har en viktig roll där hen kan hjälpa patienten att känna välbefinnande, hopp, livslust och ökad självkänsla genom bekräftelse av att bli sedd och hörd samt ge nyttig kunskap för att patienter ska kunna hantera sin sjukdom med bra planering och strategier.

INTRODUCTION: The techniques currently in practice to diagnose and assess interval disease activity in patients with inflammatory bowel disease (IBD) are costly and invasive. Physicians typically rely on information derived from a combination of endoscopic, radiologic, and histologic studies to diagnose and determine the extent and severity of the two most common forms of IBD, Crohn disease (CD) and ulcerative colitis (UC). The development of noninvasive methods of assessing response to therapy is of increasing importance to pediatric healthcare providers. Previous studies have demonstrated that serum and fecal biomarkers are reliable measures of inflammation in the gastrointestinal tract. However, existing biomarkers are non-specific and their levels can be elevated in the context of either acute and chronic inflammation (IBD) or infection. As such, further studies are required to develop newer and novel biomarkers that have greater specificity for use in the diagnosis and interval assessment in children and adults with IBD. OBJECTIVES: The goal of this study is to further assess the relationship between biomarkers in the stool and serum of patients with IBD that are being treated with the anti-TNF therapy, infliximab (Remicade). To accomplish this, we will assess the changes in serum and fecal biomarker levels over the course of treatment and correlate the changes in fecal and serum biomarker levels with clinical, biochemical, and endoscopic outcome variables. METHODS: We conducted a prospective longitudinal cohort study in pediatric patients with IBD receiving long-term immunosuppressive therapy with Remicade. Pediatric patients diagnosed with either CD or UC who receive Remicade at Boston Children’s Hospital were recruited. Patients were drawn from subsets of patients that were either naïve to Remicade, had received Remicade for less than 6 months, or had received Remicade for more than one year at the time of enrollment. We collected longitudinal data over the course of their first 6 consecutive infusions following enrollment, including blood and stool samples, disease activity indexes, as well as a patient-reported outcome measure (IMPACT-III Questionnaire) at each infusion session. RESULTS: A total of 33 patients with IBD who fit our eligibility criteria and provided informed consent were enrolled to date. Of these, 20 had a CD diagnosis and 13 had a UC diagnosis. We collected baseline serum, fecal, and IMPACT-III score data and followed enrolled patients over the course of subsequent infusions. Mean baseline fecal ASCA levels from 8 CD and 6 UC patients were 0.08±0.021 OD and 0.02±0.0015 OD, respectively. At baseline, serum lactoferrin (p<0.10), calprotectin (p<0.10), ESR (p<0.05), and CRP (p<0.10) were significantly higher among CD patients. CONCLUSION: Our data demonstrate the potential for serum and fecal biomarkers to evaluate therapeutic response to Remicade. Completion of study enrollment and data collection will be necessary to determine if individual or combinations of fecal and serum biomarkers yield the most robust measures for use in the diagnosis and interval assessment of children and adults with IBD.

Iron deficiency anemia (IDA) is a common extra-intestinal manifestation of inflammatory bowel disease (IBD). It is particularly common in the pediatric population, with 40-60% of pediatric patients with IBD meeting criteria for anemia (Aljomah et al, 2018). A number of studies have examined the use of both IV and oral iron treatments to treat anemia in IBD, but few have examined the safety and efficacy of these treatments in children and how they impact a patient’s health-related quality of life. We conducted a prospective cohort study of 79 pediatric patients admitted to Boston Children’s Hospital for management of their IBD. 48 of these patients received IV iron, 13 received oral iron, and 12 were not treated with either. Treatment with IV iron resulted in a statistically significant increase in hemoglobin of 1.75g/dL+/-1.4g/dL (mean +/- SD) from admission to the time of their first post-discharge visit (p=0.0001). Prescription of oral iron at the time of discharge did not result in a significant increase in hemoglobin over the same interval (p=0.481). Though there was a positive trend, IV iron treatment did not result in a significant change in health-related quality of life (HRQOL) measurements as measured by the IMPACT-III questionnaire (p=0.06). Our study suggests that IDA is common in patients admitted for management of their IBD, IV iron is more efficacious in raising hemoglobin, and that larger studies will be needed to more fully demonstrate the impact of effective iron repletion on overall quality of life in these patients.

INTRODUCTION: Physiologic and pathophysiologic inflammation is mediated, at least in part, by the generation and release of reactive oxygen species into the local tissue milieu. The chronic inflammation observed in patients with inflammatory bowel disease (IBD) is thought to begin in the lining of the intestine and may progress to involve the entire bowel wall.In an effort to assess disease activity, clinicians rely on costly and technically invasive procedures such as colonoscopies. As such, there is currently a need for the development of less invasive and more cost-effective methods for use in the diagnosis and interval assessment of children and adults with these chronic intestinal inflammatory disorders. OBJECTIVES: The objective of this study was to first determine if ambient redox status can be reliably measured in the stool of patients with IBD. A second aim of the study was to determine if ambient stool redox status was related to underlying diagnosis, clinical disease activity, or response to therapy in patients with IBD . METHODS: We first our ability to measure redox redox standards using three different commercially available devices. Once demonstrated, we then the process of performing sample analysis under various conditions (room tempererture, refrigerated, frozen, or spun/unspun) to determine the conditions under which we were able to achieve the most stable redox assessments. Finally, we conducted a small pilot cohort study in hospitalized pediatric patients with IBD to assess if stool redox status informed about disease activityWe collected stool samples from seven patients admitted to the inpatient gastrointestinal service at Boston Children’s Hospital during a period extending from November of 2018 to March of 2019. RESULTS: Preliminary studies confirmed our ability to accurately measure relative redox status (RRS) using three different apparatuses. Furthermore, we were able to generate dilution curves using juices known to include oxidants, with linear regression r2 values of 0.99. In our patient population, we confirmed our ability to generate a reliable readings and consistent RRS measurements over. Frozen samples displayed less stable and higher RRS than those either refrigerated or kept at room temperature for up to 8-hours. This suggests that freeze-thaw cycles may impact adversely on the stability of oxidants and antioxidants in our samples. The RRS measurements from stool samples collected from patients who were exhibiting active symptoms of their IBD measured about -400 mV, while samples collected from hospitalized patients without IBD manifest RRS readings of about 100 mV. CONCLUSION: This preliminary study demonstrates our ability to measure RRS in the stool of patients with and without IBD. The stability we observed in samples that were either stored at room temperature or refrigerated demonstrated that these represented optimal storage options. Additionally, measurements from homogenized stool samples appeared to be more variable when compared to the relatively smaller range from centrifuged samples. Initial studies indicated a strong difference in RRS measurements between patients with inflammatory and non-inflammatory GI disease or inactive IBD. This difference suggests that measurements of RRS could provide a quantitative real-time assessments of disease activity and response to therapy in patients with IBD.

As Doenças Inflamatórias Intestinais (DII), como a colite ulcerativa e a doença de Crohn, são resultados de uma resposta imune desregulada no intestino de indivíduos geneticamente suscetíveis apresentando disbiose intestinal. Essas doenças geralmente são tratadas com anticorpos monoclonais, como o anti-TNF, Infliximab (IFX). No entanto, pouco se sabe como o bloqueio do TNF afeta a resposta do hospedeiro quando ocorre uma nova quebra da homeostase intestinal, durante a recidiva da doença. Neste estudo, avaliamos os efeitos tardios do tratamento com IFX na colite experimental com um novo desafio de disbiose. Camundongos tratados com IFX tiveram remissão da colite. No entanto, o tratamento não protegeu contra a recidiva da doença, o que resultou no aumento de células mononucleares circulantes e diminuição de neutrófilos, em contraste com a redução da atividade de macrófagos e aumento da infiltrado de neutrófilos no cólon após o desafio. Esses animais também apresentaram diminuição da barreira linfocitária epitelial e aumento de linfócitos na lâmina própria do cólon, que também continha elevado número de células dendríticas inflamatórias CD11b+CD11c+CD103-. O tratamento da colite com IFX seguido de um desafio de microbiota levou à redução de linfócitos TCD4, TCD8 e ?? intraepiteliais, com acúmulo de células T ativadas, memória residentes e efetoras na lâmina própria, em comparação com o número reduzido desses linfócitos na ausência de tratamento com IFX. Além disso, o bloqueio do TNF reduziu a expressão de IL-1? e IL-6 e aumentou a expressão de CYP11B1, uma enzima esteroidogênica responsável pela produção de cortisol anti-inflamatório. Porém o desafio antigênico elevou significativamente a expressão de IL-13, mas reduziu a expressão das enzimas esteroidogênicas, CYP11A1 e CYP11B1O. O mais interessante é que a permeabilidade intestinal foi aumentada, assim como a atividade de proliferação das células nos linfonodos mesentéricos. Esses dados sugerem que, embora o IFX possa controlar a inflamação na colite aguda, seus efeitos tardios comprometem a capacidade do hospedeiro de lidar com um novo desafio, como uma disbiose intestinal que ocorre durante a recaída da doença / Inflammatory Bowel Diseases (IBD), such as ulcerative colitis and Crohn\'s disease, are the result of a dysregulated immune response in the intestine of genetically susceptible individuals presenting with intestinal dysbiosis. These diseases are usually treated with monoclonal antibodies, such as anti-TNF, Infliximab (IFX). However, little is known about how TNF blockade affects host response when a new break in intestinal homeostasis occurs during relapse of the disease. In this study, we evaluated the late effects of IFX treatment in experimental colitis with a new challenge of dysbiosis. Mice treated with IFX had remission of colitis. However, the treatment did not protect against disease recurrence, which resulted in increased circulating mononuclear cells and decreased neutrophils, in contrast to reduced macrophage activity and increased neutrophil infiltrate in the colon after challenge. These animals also had a decrease in the lymphocyte epithelial barrier and increased lymphocytes in the lamina propria of the colon, which also contained a high number of inflammatory dendritic cells CD11b+CD11c+CD103-. Treatment of IFX colitis followed by a microbiota challenge led to reduction of intraepithelial TCD4, TCD8 and ?? lymphocytes with accumulation of activated T cells, resident and effector memory in the lamina propria, compared to the reduced number of these lymphocytes in the absence of treatment with IFX. In addition, TNF blockade reduced IL-1? and IL-6 expression and increased expression of CYP11B1, a steroidogenic enzyme responsible for the production of anti-inflammatory cortisol. However, antigen challenge significantly elevated IL-13 expression, but reduced expression of steroidogenic enzymes, CYP11A1 and CYP11B1. Interestingly, the intestinal permeability was increased, as was the proliferation activity of the cells in the mesenteric lymph nodes. These data suggest that although IFX can control inflammation in acute colitis, its late effects compromise the host\'s ability to cope with a new challenge, such as intestinal dysbiosis that occurs during relapse of the disease

Introdução: A Síndrome do Intestino Curto (SIC) é definida como um conjunto de sinais e sintomas conseqüentes de alterações nutricionais e metabólicas secundária a insuficiência intestinal funcional e/ou orgânica, como nos casos de grandes ressecções do intestino delgado. Ressecções intestinais eliminam sítios de colonização, alteram a área de absorção e, o uso freqüente de antibióticos devido a infecções recorrentes, presença de alimentos não digeridos no cólon, trânsito acelerado e diarréia, nestes mesmos pacientes, podem contribuir para a alteração da microbiota intestinal. Objetivo: Caracterizar a microbiota intestinal de pacientes portadores da Síndrome do Intestino Curto atendidos na Unidade Metabólica e do HCFMRP/USP. Casuística e Métodos: Foram recrutados os pacientes portadores de síndrome do intestino curto atendidos na Unidade Metabólica do HCFMRP/USP (uso de terapia nutricional parenteral). Para o grupo controle foram recrutados indivíduos sadios e eutróficos da comunidade e pareados segundo sexo e idade. Foram avaliadas amostras de fezes e exames bioquímicos, estes últimos foram somente dos pacientes. A avaliação do consumo alimentar foi feita por meio do inquérito Diário Alimenta e a avaliação do estado nutricional a partir de medidas antropométricas. Para detecção de cepas patogênicas foram realizados cultivos e testes bioquímicos específicos em meio aeróbio para determinação de espécies da família Enterobacteriaceae. Em cada cepa de E. coli isolada foram aplicados anti-soros para determinação de possível patogenicidade. Metodologia molecular também foi utilizada para determinação do perfil da microbiota intestinal bacteriana: sequenciamento de bibliotecas de DNAr 16S e PCR para detecção de genes característicos de cepas patogênicas de E. coli. Resultados: Verificou-se a presença de subnutrição protéico-calórica no grupo Paciente mesmo com terapia nutricional para recuperação deste estado nutricional. Quanto a microbiota, observou-se maior diversidade de Gram negativas, porém menor quantidade por grama de fezes da família Enterobacteriaceae em pacientes com SIC. Porém a análise molecular mostrou a manutenção na proporção de espécies bacterianas, equivalente a microbiota intestinal saudável. Conclusão: Apesar da subnutrição, retirada maciça do intestino delgado, uso freqüente de antibióticos, depressão do sistema imune, presença de alimentos não digeridos no trato gastrintestinal e transito intestinal acelerado, a relação e proporção entre as espécies bacterianas intestinais permanecem similares à normalidade. / Introduction The short bowel syndrome (SBS) is defined as a set of signs and symptoms resulting from nutritional and metabolic changes after major small bowel resections. Intestinal resections eliminate colonization sites and alter the absorption area. Besides, the frequent use of antibiotics due to recurrent infections, the presence of undigested food remaining in the intestinal tract, rapid transit and diarrhea in these same patients may contribute to the change of intestinal microbiota. Objective: To characterize the intestinal microbiota of patients with short bowel syndrome treated at the Metabolic Unit and HCFMRP / USP. Materials and Methods: We recruited all the patients with short bowel syndrome treated at the Metabolic Unit of HCFMRP / USP (use of parenteral nutrition therapy). The control group was composed by healthy individuals matched by age and sex. Samples of feces and biochemical tests from the patient group were evaluated. The control group had no biochemical tests, only feces samples to be analysed. The nutritional status was evaluated through anthropometric measurements and the assessment of food intake through food diary survey. The pathogenic strains from Enterobacteriaceae were detected by cultivation and specific biochemical tests in aerobic environment. In each strain of isolated E. coli antisera were applied for determination of pathogenicity. PCR analysis was also used to determine the profile of intestinal bacterial microbiota: sequencing libraries of 16S rDNA and PCR for detection of genes characteristic of pathogenic strains of E. coli. Results: Although receiving periodic parenteral nutrition the Patient group had protein-calorie malnutrition. In regards to the microbiota there was greater diversity, but lower concentration of the family Enterobacteriaceae in patients with SBS. However, the molecular analysis showed the a proportion of bacterial species equivalent to the intestinal flora from the control group. Conclusion: Although the protein-calorie malnutrition, massive resection of the small intestine, frequent use of antibiotics, immune system depression, presence of undigested food in the gastrointestinal tract and rapid intestinal transit rate, the ratio and the proportion of the intestinal bacterial species remain similar to normal.

Escherichia coli tem um predomínio anormal em pacientes com doença inflamatória intestinal (DII), mas a razão deste aumento numérico é desconhecida. Amostras de E. coli (131) foram isoladas de biópsias retais de 27 pacientes com retocolite ulcerativa (RCU), 8 pacientes com doença de Chron (DC) e 19 controles. E. coli enteroagregativa (EAEC) foram detectadas no ensaio de adesão de 3 horas a células HEp-2 em 71,4% dos pacientes com DII e em 26,3% dos controles (P<0,02). As cepas de pacientes com DC foram negativas para outros marcadores. Duas cepas invasivas foram detectadas entre pacientes com RCU, três deles tinham cepas com plasmídio de adesão agregativa (pAA) e fímbria de adesão agreagativa (aggR) e outras quatro possuíam o gene attaching and effacing (eae). Nenhuma cepa abrigava locus associado à invasão (ial) e antígeno plasmidial de invasão (ipaH). De acordo com esses resultados, EAEC é o grupo dominante encontrado na mucosa colônica de pacientes com DII, mas somente aquelas encontradas em pacientes com RCU abrigam marcadores de virulência tradicionais. / Escherichia coli are increased in inflammatory bowel disease (IBD) patients, but the reason for this elevation is unknown. Amongst their properties is the interaction with cultured epithelial cells. Rectal biopsies from 27 ulcerative colitis (UC), 8 Crohns disease (CD) and 19 control patients were cultured for E. coli, given 131 isolates. Enteroagregative E. coli (EAEC) strains, as detected in 3h adherence assays to HEp-2 cells, were found in 71.4% of 35 IBD patients and in 26.3% of controls (P<0.02). Two highly invasive strains were detected among UC patients, three of whom had also strains with both plasmid of aggregative adhesion (pAA) and aggregative adherence fimbriae R (aggR) and another four E. coli attaching and effacing (eae). No strains had invasion-associated locus (ial) and invasion plasmid antigen H (ipaH), and those from CD were also negative for other markers. According to these results, EAEC are the dominant E. coli group found in the colonic mucosa of IBD patients, but only those found in UC patients seem to harbor traditional virulence markers.

For over two decades, the study of genetics has been making significant progress towards understanding the causes of common disease. Across a wide range of complex disorders there have been hundreds of associated loci identified, largely driven by common genetic variation. Now, with the advent of next-generation sequencing technology, we are able to interrogate rare and low frequency variation in a high throughput manner for the first time. This provides an exciting opportunity to investigate the role of rarer variation in complex disease risk on a genome-wide scale, potentially o↵ering novel insights into the biological mechanisms underlying disease pathogenesis. In this thesis I will assess the potential of this technology to further our understanding of the genetics of complex disease, using inflammatory bowel disease (IBD) as an example. After first reviewing the history of genetic studies into IBD, I will describe the analytical challenges that can occur when using sequencing to perform case-control association testing at scale, and the methods that can be used to overcome these. I then test for novel IBD associations in a low coverage whole genome sequencing dataset, and uncover a significant burden of rare, damaging missense variation in the gene NOD2, as well as a more general burden of such variation amongst known inflammatory bowel disease risk genes. Through imputation into both new and existing genotyped cohorts, I also describe the discovery of 26 novel IBD-associated loci, including a low frequency missense variant in ADCY7 that approximately doubles the risk of ulcerative colitis. I resolve biological associations underlying several of these novel associations, including a number of signals associated with monocyte-specific changes in integrin gene expression following immune stimulation. These results reveal important insights into the genetic architecture of inflammatory bowel disease, and suggest that a combination of continued array-based genome- wide association studies, imputed using substantial new reference panels, and large scale deep sequencing projects will be required in order to fully understand the genetic basis of complex diseases like IBD.

There is increasing evidence that psychological stress and associated mood disorders are linked with, and can adversely affect the course of inflammatory bowel disease (IBD). Stress is perceived to be relieved by smokers, and this, like a lack of knowledge about its adverse effects, and nicotine dependence, could contribute to continued smoking by patients with Crohn’s disease (CD). Stress has previously been shown to influence disease course in patients with inactive ulcerative colitis (UC) but its influence in acute severe UC is not known. Emerging trial evidence supports the suggestion that psychologically-orientated therapy may ameliorate IBD-associated mood disorders, but there is no strong data yet to indicate that stress management has a beneficial effect on the activity or course of IBD. In addition gut-focussed hypnotherapy has been successfully used in the setting of functional bowel disorders. The 4 main hypotheses tested in thesis are: 1. In patients with IBD: (1) poor knowledge of the effects of smoking on their disease and/or (2) high nicotine dependence explain the higher prevalence of smoking in CD than UC 2. Anxiety, depression and stress are more common and worsen outcome in patients with acute severe UC. 3. Stress management in the form of psychotherapy given by a counsellor has a beneficial effect on the activity and course of IBD. 4. Gut-focussed hypnotherapy reduces the relapse rate in patients with UC. The major findings are as follows: 1. Despite more patients with CD being smokers, they were better informed about the effects of smoking on their own disease than UC patients. Nicotine dependence was no higher in patients with CD than UC. In IBD patients as a whole, nicotine dependence was lower than in smokers’ clinic clients and comparable to that of the general population, suggesting that most IBD patients could be weaned off smoking successfully in the IBD clinic.