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

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.

Research into the gut microbiome has revealed the widespread influence that microbial species have on their host. Host genetics and environmental factors influence the abundance and diversity of the bacterial species living within the gastrointestinal tract. When the normal composition of the gut microbiota is altered, a dysbiotic state incurs. Inflammatory bowel disease (IBD) is a chronic/relapsing inflammatory disorder of the intestinal mucosa, which is characterized by a state of dysbiosis. Despite the large amount of information studying the role dysbiosis has in the pathogenesis of IBD, it is not clear how the altered microbial composition of the gut in IBD patients leads to susceptibility to enteric pathogens such as Clostridium difficile. This study aims to highlight the features of the gastrointestinal tract that are modified as a result of dysbiosis in the IBD population, and how these features facilitate colonization by C. difficile and symptom development. Review of the available literature demonstrated that the depletion of Clostridial cluster XIVa in IBD-associated dysbiosis alters bile acid metabolism and butyrate fermentation in the colon, ultimately promoting germination of C. difficile spores and weakening the gut's immune response against toxin-mediated inflammation. From continued research into the gut microbiota, more will be understood of how these microbial organisms influence human health and disease.

BACKGROUND: Inflammatory Bowel Disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory conditions of the bowel that display a rising prevalence in childhood and adolescence. The diagnosis of a chronic condition, such as IBD, in childhood can be overwhelming and stressful for both the patient and caregiver. Parents and family members can play a critical role in providing emotional support for children with newly diagnosed IBD. We hypothesized that dysfunctional patient and parental coping strategies would correlate with increased anxiety and depression in children, worsening clinical disease activity, and increased healthcare utilization. OBJECTIVE: The primary objective of the IBD Coping Study is to assess the stability of coping strategies and psychological stress over the first year following a new diagnosis of IBD. Secondarily, we aim to assess the impact of child and parental coping strategies on disease activity and emotional well-being over the year. METHODS: This is a prospective, longitudinal cohort study of children with newly diagnosed IBD and their parents at Boston Children's Hospital (BCH). Patients between the ages of 9 and 17 years old that have been diagnosed with CD, UC or Indeterminate colitis (IC) within the last 6 months, are English-speaking, and receive routine care at BCH are approached for participation in our study. Participation includes the completion of previously validated psychological metrics for both child and parent at baseline and then again 12 months later. Our instruments include the Children's Depression Inventory (CDI), the Screen for Child Anxiety Related Disorders (SCARED), the IMPACT-III Questionnaire, the Patient Health Questionnaire (PHQ-9), Healthcare Utilization Survey, Pediatric Inventory for Parents (PIP), and the Hospital Anxiety and Depression Scale (HADS). RESULTS: We screened 187 patients with IBD for participation in our study, and roughly 30% of them were eligible for recruitment. To date, we have enrolled a total of 30 patients. Of these patients, there was an equal distribution of male and female participants. The majority of patients were around 14 years of age at the time of IBD diagnosis with a greater number of patients with CD (17) currently represented. We are approaching patients on average about 1.5 months after their initial diagnosis. The baseline average Pediatric Ulcerative Colitis Activity Index (PUCAI) score was 21.15 ± 20.53, whereas the average Pediatric Crohn's Disease Activity Index (PCDAI) score was 3.75 ± 2.50. On CDI items, teenage girls and boys reported increased raw and standardized scores (Raw: 5.83 and 4.83, respectively; Standardized: 43.67 and 44.67, respectively) than their younger counterparts for depressive behaviors, including negative mood and interpersonal problems. Pediatric patients encountered as inpatients reported an overall lower quality of life on IMPACT-III items (103.29 ± 15.11) than those approached in the ambulatory setting (140.36 ± 7.50). On SCARED items, patients met criteria for the potential presence of one or more anxiety disorders. Inpatients also reported being bothered more frequently with respect to hindrances in their sleep, appetite, and daily routines on the PHQ-9 metric. Parents of children with newly diagnosed IBD rely on increased communication with their child's primary GI provider, and their scores reflected lower emotional functioning during an admission period when compared to scores reported during regular scheduled ambulatory visits. Scores collected from the HADS screen demonstrate that 6% and 33% of parents reported a score great enough to be considered a "borderline case" for depression and anxiety measures, respectively. Primary comparisons between child health assessments and parent healthcare utilization depicted concurrent elevations in the same child-parent pair at baseline. CONCLUSION: Our initial findings suggest a clear disparity between emotional stability in children and their parents in outpatient and inpatient settings following a new IBD diagnosis. Healthcare utilization by parents may be linked to adaptive or maladaptive coping, and continuation of our study will substantiate this prediction. In looking ahead, potential interventions may require approaches stratified by age, gender, and hospital setting. Our study supports the need for further investigations into the impact of targeted interventions that promote an improvement in overall quality of life in children with IBD and their family during the first year of post-diagnosis.

Defensinas são peptídeos antimicrobianos produzidos na mucosa intestinal e fazem parte da imunidade inata, agindo sobre vários microrganismos luminais. Deficiência na expressão de defensinas tem sido relatada em doenças inflamatórias intestinais (DII), no entanto a contribuição de cada tipo de defensina, num cenário de polimorfismo genético, mantém alguma controversa. Βeta-defensinas humanas (HBDs) têm atividade antimicrobiana contra uma ampla variedade de fungos, bactérias e vírus e têm também, um papel na ligação entre a imunidade inata e adaptativa atuando como quimiotáticos. O gene DEFB1 (8p23), codificando a beta-defensina humana 1 (HBD-1), é expresso normalmente por células epiteliais de uma série de tecidos, mas sua expressão pode variar entre indivíduos e pode ser modificada durante processo inflamatório. Produção deficiente de defensinas parece contribuir para a patogênese de DII, e uma diminuição na expressão de HBD-1 tem sido relatada na mucosa de pacientes com doença de Crohn (DC) e retocolite ulcerativa (RCU). Nós avaliamos a possível associação de três polimorfismos do gene DEFB1 com a suscetibilidade a DII, RCU e DC, em 149 pacientes, 79 com DC e 70 com RCU; e 200 controles saudáveis do sul do Brasil. No nosso estudo não se observou diferença estatisticamente significativa entre a distribuição das frequências alélicas para DEFB1 SNPs -52G>A. -44C>G e -20G>A entre o total de pacientes com DII e controles. Porém, quando pacientes com DC foram estratificados de acordo com a localização anatômica, o alelo -20G>A foi mais frequente em pacientes com DC colônica do que em controles (65 % VS 44 %, p=0,048). De forma similar, o genótipo A/A foi mais frequente em pacientes com DC colônica do que em controles (36 % VS 16 %), mas neste caso, a diferença não foi estatisticamente significativa (p=0,07). Embora não se achou uma clara e forte associação entre os SNPs 5’-UTR DEFB1 e suscetibilidade/proteção à doença inflamatória intestinal, nossos resultados sugerem possível envolvimento do gene DEFB1 nestas enfermidades, especialmente com a localização colônica da doença de Crohn. Estudos com amostras maiores e populações diversas serão úteis para avaliar a tendência observada no nosso grupo. / Defensins are antimicrobial peptides produced by the intestinal mucosa and are part of the innate immune system, playing a protective role against various intestinal microorganisms. Deficiency in the expression of defensins has been reported in inflammatory bowel diseases (IBD), however there is some controversy over the contribution of each type of defensine, in a setting of great genetic polymorphism. Beta-defensins (HBDs) have an antimicrobial activity against a great variety of fungi, bacteria and viruses, and also have a role in connecting the innate and the adaptive immunity, acting as a chemostatic agent. Deficient production of defensins appears to contribute to the pathogenesis of IBD, and the lower expression of HBD-1 has been reported on the mucosa of Ulcerative colitis (UC) and Crohn’s disease (CD) patients. We evaluated a possible association of three polymorphisms of gene DEFB1 with susceptibility to develop IBD, UC and CD in 149 patients, 79 with CD and 70 with UC; and 200 healthy controls from the south of Brazil. The gene DEFB1 (8p23), which codifies human beta-defensin 1 (HBD-1), is constitutivelly expressed by epithelial cells of several tissues, but its expression may vary among different individuals and may be modified by inflammation. In our study we did not find a statistically significant difference between the distribution of the allelic frequencies for DEFB1 SNPs -52G>A, -44C.G and -20G>A between the total number of patients and controls. However, when patients were stratified according to the anatomic location, the allele -20G>A was more frequent in patients with colonic CD than in contros (65% VS 44%, p=0,048). Similarly, the genotype A/A was more frequent in patients with colonic CD than in controls (36% vs 16%), however, in this case, the difference wasn’t statistically significant (p=0,07). Although we did not find a clear and strong association between the 5’-UTR DEFB1 SNP and susceptibility to IBD, our results suggest a possible involvement of the DEFB1 gene and these diseases, particularlly colonic CD. Further studies with larger samples and diverse populations will be usefull to evaluate the trend observed by our group.

Detection of evolutionarily conserved pathogen motifs by pattern recognition receptors (PRRs), particularly on dendritic cells (DCs), is crucial for adequate immune responses. Defects in DC function are known to be associated with inflammatory bowel disease (IBD). The endocannabinoid system (ECS) is the system through which exocannabinoids such as Δ⁹-tetrahydrocannabinol and cannabidiol signal. Regarding inflammation, cannabinoids generally exert anti-inflammatory effects, including on experimental colitis. However, most work has been performed in animal models and less is known about the function of this system in human immune cells, particularly DCs. Monoacylglycerol lipase (MGLL) is the key enzyme for hydrolysis of the endocannabinoid 2-arachidonoylglycerol, and a member of the serine hydrolase enzyme superfamily. This thesis defines the activity of serine hydrolase enzymes for the first time in human DCs upon stimulation by NOD2/TLR2 ligands using activity-based protein profiling (ABPP). MGLL is shown to be ubiquitously upregulated upon stimulation of DCs and in monocyte-derived macrophages. Through pharmacological inhibition studies, MGLL is demonstrated to regulate cellular and secreted lipids, not limited to endocannabinoids. However, overall DC function is independent of this enzyme suggesting that the effects of lipid modulation may be on bystander cells. Challenging the current literature, MGLL inhibition with a novel inhibitor worsens murine Citrobacter rodentium colitis. Finally, ABPP demonstrates a rich serine hydrolome in colonic tissue from human IBD with many enzymes previously undefined in this disease. Gene expression of ECS components suggests the enzymes ABHD12 and DAGLα/β may be potential markers of field change in IBD.

There is an unmet need for novel biomarker discovery in Inflammatory Bowel Diseases (IBD) to aid clinical management in several clinical settings including diagnosis and prognosis. With an ever-advancing repertoire of biological therapies on the horizon, it is important to personalise treatments at an early stage. The aim of this thesis is to explore the clinical utility of novel blood-based biomarkers in diagnosis, disease classification and prognosis in 2 cohorts: newly diagnosed IBD and acute severe colitis. Investigating the circulating methylome, 290 probes exhibited Holm significant IBD-associated methylation differences, including VMP1/MIR21 (p=7.5×10-14) and RPS6KA2 (1.1×10-19) and were consistent within the European cohort. 11 Differentially methylated positions (DMPs) predicted treatment escalation after Holm adjustment (top probe p=0.003). A panel of 6 probes identified 2 patient subgroups that have significantly different disease courses (Hazard Ratio (HR) 10.5, 95%CI: 4.3-25.6; logrank p=1.5×10-24). The 6 probe marker outperformed conventional biomarkers in predicting treatment escalation (hsCRP > 4mg/L, HR 3.2(1.7-5.8), logrank p=0.0004 and Alb < 36g/L, HR 2.9(1.5-5.6), p=0.0001). Within the same cohort, a novel proximity extension assay (PEA) was then utilised to identify novel diagnostic and prognostic protein markers. 61 proteins were significantly associated with IBD including MMP12 (Holm-adjusted p=4.1×10-26). A total of 9 proteins predicted disease course in this cohort. Using a panel of 7 randomly selected top prognostic probes, 2 patient groups were identified that had significantly different disease courses: logrank p=2.2×10-10, HR 5.6(2.0-15.6), outperforming conventional biomarkers in predicting treatment escalation (hsCRP > 4mg/L, HR 3.2(1.7- 5.8), logrank p=0.0003 and Alb < 36g/L, HR 2.7(1.4-5.2), p=0.0004). In a subcohort, serum calprotectin (SC) and conventional blood markers were investigated for their utility in diagnosis and prognosis in IBD. SC performed at par with CRP at differentiating IBD from controls with an area under the curve (AUC) of 0.87 (CI 0.81-0.92). For prognostication, both albumin and SC remained significant predictors of treatment escalation in IBD (logrank test p=5.1×10-5). MicroRNAs (miRNA) are small non-coding nucleic acids that have the capacity to modulate gene expression. Using small RNA sequencing in acute severe colitis (ASUC) and healthy controls (HC), 10 serum-based miRNA markers were significantly associated with acute severe colitis, including miR-30a-5p. Validating the findings using qPCR, miR-30a-5p was downregulated in ASUC (p=0.003). Furthermore, miR30a-5p remained a significant predictor of eventual colectomy in acute colitis (logrank test p=0.0014). These data highlight the translational potential for methylation, miRNA and proteomic biomarkers in diagnosing and prognosticating in IBD.

Epigenetic alterations including DNA methylation and microRNAs may provide important insights into gene-environment interaction in complex immune diseases such as inflammatory bowel disease (IBD). An integrative genome-wide approach was used to analyse whole blood genetic, DNA methylation and gene expression data in 240 newly diagnosed IBD patients and 190 controls. Using the Illumina 450k array, differences in whole blood DNA methylation were observed in IBD cases versus controls including 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs). The top DMP (RPS6KA2, discovery Holm adjusted p=1.22×10-16, replication p=1×10-9) and DMRs (VMP1, ITGB2, TXK) were replicated in an independent cohort using pyrosequencing. Paired genetic and epigenetic data allowed the identification of methylation quantitative trait loci (meQTL); two of the five DMRs (VMP1, ITGB2) demonstrated significant association with genetic polymorphisms. Methylation in the VMP1/microRNA-21 region was significantly associated with two single nucleotide polymorphisms (cg18942579 -rs10853015 [meQTL FDR adjusted p=9.4 × 10-5], cg16936953 - rs8078424 [meQTL FDR adjusted p=8.8 × 10-5]), both of which are in linkage disequilibrium with a known IBD susceptibility variant (rs1292053). Separated leukocyte methylation data highlight the cell type of origin of epigenetic signals seen in whole blood. IBD-associated hypermethylation within the TXK gene transcription start-site negatively correlated with gene expression in whole blood and CD8+ T-cells, but not other cell types, highlighting that cell-specificity and gene location-specificity of DNA methylation change is critical when associating methylation and gene expression. These data offer significant translational potential as diagnostic biomarkers. Least absolute shrinkage and selection operator (lasso) modelling identified 30 methylation probes can be used to accurately discriminate IBD cases from controls (Area under receiver operating characteristic curve = 0.898, sensitivity = 90.6%, specificity = 84.7%). MicroRNAs (miRNA) are small non-coding nucleic acids that have the capacity to modulate gene expression. MiRNAs have been increasingly implicated in many of the important IBD pathogenic pathways including autophagy, intestinal epithelial barrier integrity and the Th17 pathway. In common with all epigenetic mechanisms, miRNA expression is dynamic and cell-specific. Small RNA sequencing (RNA-seq) was performed on RNA extracted from CD14+, CD4+ and CD8+ cells isolated from 8 newly diagnosed cases of ileal or ileocolonic CD and 8 age and sex matched controls. There was a median of 2.4 million reads per sample (range 132,800-12.8 million reads per sample). One microRNA was differentially expressed in CD compared with controls (hsa-miR-503-5p log fold change = 0.7, FDR adjusted p = 9.1 × 10-5) in CD4+ lymphocytes, however this finding did not remain significant when alternative normalisation methods were used. The small number of cases used in microRNA analyses raises the possibility of both type I and II error, and limits the ability to draw firm conclusion from this series of experiments. Site-specific differences in DNA methylation in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression. This is the most detailed characterisation of the epigenome carried out in IBD to date. The findings strongly validate this approach in complex disease, are replicable, and provide clear translational opportunities.