OBJECTIVES:There is an unmet need for novel blood-based biomarkers that offer timely and accurate diagnostic and prognostic testing in inflammatory bowel diseases (IBD). We aimed to investigate the diagnostic and prognostic utility of serum calprotectin (SC) in IBD. METHODS:A total of 171 patients (n=96 IBD, n=75 non-IBD) were prospectively recruited. A multi-biomarker model was derived using multivariable logistic regression analysis. Cox proportional hazards model was derived to assess the contribution of each variable to disease outcomes. RESULTS:SC correlated strongly with current biomarkers, including fecal calprotectin (FC) (n=50, ρ=0.50, P=1.6 × 10-4). SC was the strongest individual predictor of IBD diagnosis (odds ratio (OR): 9.37 (95% confidence interval (CI): 2.82-34.68), P=4.00 × 10-4) compared with other markers (C-reactive protein (CRP): OR 8.52 (95% CI: 2.75-28.63), P=2.80 × 10-4); albumin: OR 6.12 (95% CI: 1.82-22.16), P=0.004). In a subset of 50 patients with paired SC and FC, the area under receiver operating characteristic discriminating IBD from controls was better for FC than for SC (0.99, (95% CI 0.87-1.00) and 0.87 (95% CI:0.78-0.97), respectively; P=0.01). At follow-up (median 342 days; interquartile range: 88-563), SC predicted treatment escalation and/or surgery in IBD (hazard ratio (HR) 2.7, 95% CI: 1.1-4.9), in particular Crohn's disease (CD) (HR 4.2, 95% CI 1.2-15.3). A model incorporating SC and either CRP or albumin has a positive likelihood ratio of 24.14 for IBD. At 1 year, our prognostic model can predict treatment escalation in IBD in 65% of cases (95% CI: 43-79%) and 80% (95% CI: 31-94%) in CD if ≥2 blood marker criteria are met. CONCLUSIONS:A diagnostic and prognostic model that combines SC and other blood-based biomarkers accurately predicts the inflammatory burden in IBD and has the potential to predict disease and its outcomes. Our data warrant further detailed exploration and validation in large multicenter cohorts.

Background: Proximity extension assays (PEA) can compare concentrations of multiple proteins across biological samples and utilises the specificity of antibody proximity and the sensitivity of polymerase chain reaction to detect proteins of interests (1).As part of the IBD Character biomarker discovery initiative (www.ibdcharacter.eu),we performed highthroughput prospective case-control serum profiling to identify protein biomarkers that can predict Inflammatory Bowel Disease (IBD).Methods: Utilising currently available and custom designed IBD specific multiplex panels (Olink Bioscience), serum profiling was performed in patients with a new diagnosis of IBD.Our control group consisted of symptomatic individuals.Phenotypic data was captured including demographics and disease classification.Statistical analysis was performed using R. Data were normalised and then batch corrected using ComBAT.Linear models were created for each protein including age and sex as covariates.After quality control, data from 280 proteins was available for analysis.Results: 625 patient serum samples (n=324 newly diagnosed IBD, n=301 symptomatic controls) from 6 IBD centres across Europe were included in the study from May 2012 to Sept 2015.The mean age was 33 years (range 2-79 years) and 51% were female.145 had Crohn's disease (CD), 157 ulcerative colitis (UC) and 22 Inflammatory Bowel Disease Unclassified (IBDU).Multivariable analysis identified 59 protein markers that were significantly associated with IBD.The 5 most significant proteins were upregulated in IBD including MMP12 (Holmadjusted p=9.9×10 -28) and CXCL9 (p=3.4×10 -2).These 2 markers were able to discriminate IBD from controls with a similar area under the receiver operator characteristics curve (AUC) of 0.78.Using linear discriminant analysis, a combined biomarker consisting of the top 2 proteins improved the AUC to 0.81.This 2 marker test outperformed conventional blood markers (n=263, AUC CRP 0.50 p=1.6x10 -9; albumin 0.69 p=0.004) while a combined panel of the top 5 markers performed similar to faecal calprotectin (n=296, AUC 0.80 and 0.86 respectively, p=0.12) in diagnosing IBD.Five protein markers differentiated UC from CD.In UC, MMP12 levels were significantly higher in extensive disease (Paris classification E3 vs E1 and 2, p=1.8x10 -7) while in CD, MMP12 and CXCL9 levels were higher in those with colonic involvement (Montreal Classification L2 and L3 vs. L1 and L4; p=0.007 and 0.002 respectively).Conclusion: We have identified PEA-based serum biomarkers that can diagnose and classify IBD.These data demonstrate the translational potential of a PEA based technology in IBD diagnostics.

proliferation that was mediated by AHR as siRNA knockdown of AHR abrogated ITEinduced suppressive function.To investigate the mechanisms of ITE-mediated suppression we performed transcriptional profiling of ITE treated T cells and found upregulation of FOXP3, IL10, GZMB, and ENTPD1, molecules associated with Tregs.We then tested whether the suppressive mechanism of ITE was mediated by CD39 and granzyme B (GZMB) using blocking antibodies and pharmacological inhibitors.Both CD39 and GZMB were involved in the suppressive effects of ITE.We then wanted to evaluate the translational potential of ITE in an in vivo setting.We utilized a well-known model of colitis using 2,4,6-trinitrobenzenesulfonic acid (TNBS) in immunodeficient NOD.Prkdc scid .Il2rg -/- (NSG) mice lacking MHCII and instead expressing human leukocyte antigen-DR1 (NSGAb o DR1) that were reconstituted with human CD4 + T cells isolated from HLA-DR1 + donors.We first tested whether the human CD4 + T cells would cause colitis in NSGAb o DR1 mice reconstituted with human CD4 + T cells following TNBS rectal challenge.TNBS induced colitis only in mice reconstituted with human CD4 + T cells that infiltrated the colonic lamina propria and produced elevated levels of human cytokines including TNF and IFNg.We then tested the ability of ITE to ameliorate TNBS-induced colitis by injecting daily ITE or vehicle control prior to the colitis induction with TNBS.AHR activation by ITE led to a significant amelioration of colitis that was associated with increased transcripts of IL10, FOXP3 GZMB, and ENTPD1, similar to our in vitro data, and down-modulation of inflammatory pathways including TNF and NFkB.Collectively, these results describe the development of a new humanized murine experimental model to investigate the immune response in IBD, and identify the non-toxic AHR agonist ITE as a potential therapy for expanding human Tregs and promoting immune tolerance in the intestine.

Proximity extension assay immunoassay technology identifies novel serum biomarkers that can diagnose and classify inflammatory bowel diseases : IBD Character Consortium

Epigenetic alterations may provide important insights into gene-environment interaction in inflammatory bowel disease (IBD). Here we observe epigenome-wide DNA methylation differences in 240 newly-diagnosed IBD cases and 190 controls. These include 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs), which we study in detail using whole genome bisulphite sequencing. We replicate the top DMP (RPS6KA2) and DMRs (VMP1, ITGB2 and TXK) in an independent cohort. Using paired genetic and epigenetic data, we delineate methylation quantitative trait loci; VMP1/microRNA-21 methylation associates with two polymorphisms in linkage disequilibrium with a known IBD susceptibility variant. Separated cell data shows that IBD-associated hypermethylation within the TXK promoter region negatively correlates with gene expression in whole-blood and CD8+ T cells, but not other cell types. Thus, site-specific DNA methylation changes in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression.

BACKGROUND:Glycobiology is an underexplored research area in inflammatory bowel disease (IBD), and glycans are relevant to many etiological mechanisms described in IBD. Alterations in N-glycans attached to the immunoglobulin G (IgG) Fc fragment can affect molecular structure and immunological function. Recent genome-wide association studies reveal pleiotropy between IBD and IgG glycosylation. This study aims to explore IgG glycan changes in ulcerative colitis (UC) and Crohn's disease (CD). METHODS:IgG glycome composition in patients with UC (n = 507), CD (n = 287), and controls (n = 320) was analyzed by ultra performance liquid chromatography. RESULTS:Statistically significant differences in IgG glycome composition between patients with UC or CD, compared with controls, were observed. Both UC and CD were associated with significantly decreased IgG galactosylation (digalactosylation, UC: odds ratio [OR] = 0.71; 95% confidence interval [CI], 0.5-0.9; P = 0.01; CD: OR = 0.41; CI, 0.3-0.6; P = 1.4 × 10) and significant decrease in the proportion of sialylated structures in CD (OR = 0.46, CI, 0.3-0.6, P = 8.4 × 10). Logistic regression models incorporating measured IgG glycan traits were able to distinguish UC and CD from controls (UC: P = 2.13 × 10 and CD: P = 2.20 × 10), with receiver-operator characteristic curves demonstrating better performance of the CD model (area under curve [AUC] = 0.77) over the UC model (AUC = 0.72) (P = 0.026). The ratio of the presence to absence of bisecting GlcNAc in monogalactosylated structures was increased in patients with UC undergoing colectomy compared with no colectomy (FDR-adjusted, P = 0.05). CONCLUSIONS:The observed differences indicate significantly increased inflammatory potential of IgG in IBD. Changes in IgG glycosylation may contribute to IBD pathogenesis and could alter monoclonal antibody therapeutic efficacy. IgG glycan profiles have translational potential as IBD biomarkers.

Background: Although endoscopy is the gold standard for the assessment of activity in Crohn's disease (CD), it should be integrated with clinical, laboratory, and radiological data.In particular, it is still unclear when MRI should be performed.Aim: To evaluate the advantages and limitations of endoscopy and MRI to assess disease activity and severity in a series of CD patients.Material and methods: 50 consecutive patients with endoscopically proven CD underwent MRI enterography for the staging at diagnosis or follow-up.Endoscopic activity was measured by SES-CD (range 0-40) with active mild,moderate ad severe disease defined as a scores 4-10,11-19 and >20 respectively.MRI activity was measured by a validated quantitative score which integrates both mural and extramural involvement (Magnetic Resonance Enterography global score,MEGS,range 0-296), with active disease present for a ≥ 1 score.CDAI, CRP and fecal calprotectin (FC) were measured in all participants (positivity cut-off respectively >0,50 mg/dl and >150 μg/gr).Results: We enrolled 20 males and 30 females (diagnosis in 62%, follow-up in 38%), mean age 38±15 years,mean disease duration 5,4±5 years.SES-CD and MEGS were well correlated (r=0,42, p=0.001); both SESCD and MEGS show correlation with clinical (CDAI r=0,51,r=0,59, p<0.001) and biological activity (CRP r=0,37,r=0,43 p<0.005,FC r=0.27, p=0.02 respectively).According to SES-CD,90% of patient had active disease (64% mild,20% moderate and 6% severe); at MEGS,86% of patients had active disease (sensibility 89%,specificity 40%,VPP 93%,VPN 29% vs. endoscopy).MEGS scoring did not show ability to distinguish severity of disease as determined at endoscopy (p=0.14),but revealed trasmural/extramural signs of inflammation, indipendently from CD activity (60% of patients in remission,84% mild,100% with moderate and severe disease), mostly with CRP positivity.Increasing severity at endoscopy was significantly correlated to the risk of trasmural/extramural involvement, only in CRP positive patients (p=0.007).CRP positivity was associated with the presence of extraintestinal involvement (p=0.006),not of mural involment (p=0.4).Conclusions: MRI does not identify luminal CD severity; however,transmural inflammation,which is more frequent in severe disease, may still be present regardless of endoscopical activity.Positive CRP suggests the need of MRI for the staging of patients with CD independently from endoscopic severity.

We read with great interest the article by Shanahan et al 1 describing the roles of environmental conditions, notably co-housing with wild type (WT) littermates, and mouse genetic background in nucleotide-binding oligomerisation domain-containing protein 2 (NOD2)-dependent production of anti-microbial peptides in the mouse intestine. These authors demonstrate that expression, translation and anti-microbial activity of α-defensins are independent of NOD2.1 Robertson et al 2 recently confirmed that housing conditions rather than NOD2 status influenced intestinal microbiota composition. Shanahan et al address the question whether an increase in the number of Paneth cells could compensate for a NOD2-dependent reduction in the level of defensin production in Paneth cells. Using a combination of hematoxylin and eosin staining (to assess crypt numbers), immunohistochemistry (using anti-lyzozyme staining) and flow cytometry (sorting for expression of lyzozyme and lack of expression of CD45—a haematopoietic cell marker) of the entire ilea of WT and NOD2-deficient mice showed NOD2 status did not influence Paneth cell numbers.1 CD24, a heavily glycosylated protein marker of intestinal crypt stem cells and Paneth cells, is upregulated in inflammatory bowel disease (IBD).3 ,4 Sato et al 3 demonstrated that the combination of CD24hi and …

Aims Autoimmune diseases (AIDs) affect up to 10% of individuals living in Europe, so are a significant cause of chronic morbidity. High rates of immune-mediated comorbidity and familial clustering suggest that genetic predisposition underlies AI disease susceptibility, yet few clinical studies have defined the prevalence rates of co-morbid AIDs in specific paediatric populations. This study aims to document the occurrence of Juvenile Idiopathic Arthritis (JIA) and other AIDs in a Scotland-wide cohort of paediatric inflammatory bowel disease (PIBD; diagnosed <17 years of age) patients. Methods The Paediatric-onset IBD Cohort and Treatment Study (PICTS) is a nationwide Scottish study of incident and prevalent PIBD patients, collecting a wide range of data, including rigorous phenotyping, with continuous long-term follow-up. The PICTS database was interrogated to identify patients enrolled up to 30/06/12 (follow-up to 30/06/14) with a diagnosis of at least one associated AID by last follow-up. Cases believed to be related to use of anti-TNFα treatment were excluded. Results Of 809 patients in the PICTS cohort, 43 had one or more associated AID, an overall co-morbid immune disease rate of 5.3%; 49% (21/43) male. There were 44 AIDs in 43 patients; one patient had dual AIDs (psoriasis [PSOR] and spondyloarthropathy [SPA]) co-existing with IBD. Otherwise, there were 7 cases of JIA, 3 cases of SPA and 9 cases of PSOR. Additionally there were 4 cases of coeliac disease, 2 of thyroiditis and 2 cases of type 1 diabetes. No cases of Systemic Lupus Erythematosus (SLE) were identified. There were 15 cases of autoimmune liver disease (Primary Sclerosing Cholangitis [PSC], Auto-Immune Hepatitis [AIH] and Autoimmune Sclerosing Cholangitis [ASC]) in this cohort, accounting for 35% of all PIBD-associated AID. Conclusion Over 5% of PIBD patients in this large cohort study have associated AIDs. Autoimmune liver disease is the commonest AID in this cohort of PIBD patients, followed by PSOR accounting for 23%; JIA accounted for 16% of PIBD-associated AID.

Background: As a result of technological and analytical advances, genome-wide characterization of key epigenetic alterations is now feasible in complex diseases. We hypothesized that this may provide important insights into gene-environmental interactions in Crohn's disease (CD) and is especially pertinent to early onset disease.Methods: The Illumina 450K platform was applied to assess epigenome-wide methylation profiles in circulating leukocyte DNA in discovery and replication pediatric CD cohorts and controls. Data were corrected for differential leukocyte proportions. Targeted replication was performed in adults using pyrosequencing. Methylation changes were correlated with gene expression in blood and intestinal mucosa.Results: We identified 65 individual CpG sites with methylation alterations achieving epigenome-wide significance after Bonferroni correction (P < 1.1 x 10(-7)), and 19 differently methylated regions displaying unidirectional methylation change. There was a highly significant enrichment of methylation changes around GWAS single nucleotide polymorphisms (P = 3.7 x 10(-7)), notably the HLA region and MIR21. Two-locus discriminant analysis in the discovery cohort predicted disease in the pediatric replication cohort with high accuracy (area under the curve, 0.98). The findings strongly implicate the transcriptional start site of MIR21 as a region of extended epigenetic alteration, containing the most significant individual probes (P = 1.97 x 10(-15)) within a GWAS risk locus. In extension studies, we confirmed hypomethylation of MIR21 in adults (P = 6.6 x 10(-5), n = 172) and show increased mRNA expression in leukocytes (P < 0.005, n = 66) and in the inflamed intestine (P = 1.4 x 10(-6), n = 99).Conclusions: We demonstrate highly significant and replicable differences in DNA methylation in CD, defining the disease-associated epigenome. The data strongly implicate known GWAS loci, with compelling evidence implicating MIR21 and the HLA region.

in the CD4+aEb7 compartment (p < 0.001). IL-10 was not differentially expressed between CD4+aEb7+ and CD4+aEb7 lymphocytes in controls or UC, and a low frequency of aEb7+FOXP3+ cells was observed by IHC. qPCR array confirmed higher mRNA levels of IFNg (p < 0.001), TNFa (p < 0.01) and IL-17A (p < 0.01), and lower transcription of FOXP3 (p < 0.0001) in CD4+aEb7+ cells compared to CD4+aEb7 cells. Conclusions: aEb7 expression was associated with an enrichment of pro-inflammatory Th17, Th1 and Th17/Th1 T lymphocytes, and not associated with a regulatory phenotype. These data suggest therapeutic interventions targeting aE expressing T cells and the aEb7 integrin itself may be viable approaches for reducing aberrant inflammatory responses in UC.

Introduction Historical cohort studies have clearly demonstrated that over time the majority of patients with Crohn’s disease (CD) will progress from inflammatory (B1) to stricturing (B2) or fistulating (B3) disease. Emerging data suggest that more intensive treatment targeted towards mucosal healing will help to prevent disease progression. Faecal calprotectin (FC) is an established surrogate biomarker for endoscopic mucosal healing. It has yet to be established whether tailoring therapy to FC levels prevents disease progression. In the present study we aimed to determine whether FC levels in patients with established CD were predictive of disease progression. Methods The Edinburgh Faecal Calprotectin Registry (EFCR) comprises data on 22,130 FC assays in 16,278 patients from 2005–2012. Detailed phenotypic information was obtained on patients with CD by retrospective casenote review. Data collected included demographics, disease location, disease behaviour over time, CD-related surgery, investigations, hospitalisations and drug therapy. Patients were included in the main analysis if they had at least 12 months’ follow-up since first FC. The a priori primary endpoint was a composite of progression in Montreal luminal behaviour, hospitalisation for flare and resectional surgery. Results There were 881 CD patients identified with at least one FC, of which 723 had at least one year’s follow-up, with median follow-up time 40 months (IQR 25–60). The median age was 28y (IQR 20–42) at diagnosis and 40y (28–53) at time of first FC. 239 patients (33%) reached the primary endpoint, of whom 68 had had progression of their Montreal behaviour from B1 to B2 or B3, or from B2 to B3. The median of the earliest FC was significantly higher in the group that reached the primary endpoint at 586 µg/g (IQR 210–1235) vs. 289 (75–1001) in those that did not (p < 0.0001). Survival analysis (Fig 1) revealed significant differences in time to progression, hospitalisation or surgery with calprotectin ≥ 200 (p < 0.0001). Conclusion This large single-centre study presents compelling evidence that measurement of FC can be used to predict disease course, which creates the opportunity for physicians to intervene earlier and perhaps alter the disease course. Disclosure of Interest None Declared

Background Acute severe ulcerative colitis (ASUC) remains a common reason for urgent colectomy, yet there are relatively few large cohort studies exploring prognosis and outcome. This study aims to examine presentation and management of ASUC in the Western General Hospital, a tertiary referral centre in Edinburgh, UK and to identify prognostic factors. Methods Patients were identified from a large database of participants in genetic studies in Edinburgh, as well as from two previous small cohorts of ASUC studied in Edinburgh. More recent cases were found from minutes of a weekly IBD meeting. Cases were included if they met the standard clinical, radiological and pathological criteria for ulcerative colitis and required an admission for 3 days or more requiring intravenous corticosteroids and/or colectomy. Two cohorts were analysed, one with full clinical detail (97 admissions in 86 patients) and one with more basic detail (444 admissions in 323 patients). Results Overall colectomy rate was 31.8%. Haemoglobin, C-reactive protein and albumin at days 0 and 3 were significant predictors of colectomy in both cohorts (p<0.05 in each case), while in the detailed cohort day 3 but not day 0 stool frequency was predictive (p<0.001 and 0.81 respectively). A simple score was derived to predict colectomy at admission based on disease extent, albumin and CRP. Scores of 0, 1, 2 and 3 corresponded to risks for colectomy of 10%, 31%, 61% and 75%. For day 3 parameters, both the Edinburgh acute colitis (Ho) score (figure 1) and Travis criteria performed well. Conclusions ASUC remains an important cause of colectomy. This study confirms the prognostic value of the Ho score and Travis criteria at day 3, but also indicates that day 0 CRP and albumin are strong predictors of outcome.

Background: Germline variation in the 71 Crohn's disease (CD) loci implicated by genome‐wide association studies (GWAS) only accounts for approximately 25% of estimated heritability. The contribution of epigenetic alterations to disease pathogenesis is emerging as a research priority. Materials and Methods: The methylation status of 27,578 CpG sites across the genome was analyzed using the Illumina Human Methylation27 assay in DNA extracted from whole blood samples from 40 adult females (21 ileal CD, 19 healthy controls) and 16 girls with childhood‐onset CD, all nonsmokers. Our primary analysis compared methylation profiles in adult cases and controls. Results: Our data define a global methylation profile characteristic of ileal CD. In all, 1117 sites were differentially methylated (corrected P < 0.01); 50 showed significantly altered methylation in cases compared with controls (uncorrected P < 10−6, corrected P < 0.0006), including genes altering immune activation: MAPK13, FASLG, PRF1, S100A13, RIPK3, and IL‐21R. Gene ontology analyses implicated immunity‐related pathways as targets of epigenetic modification (immune system processes [P = 1.3 × 10−22], immune response [P = 8.1 × 10−16], defense responses to bacteria [P = 1.8 × 10−15]). Ingenuity canonical pathway analyses implicated dendritic cell activity (P = 2.4 × 10−8) and differential regulation of cytokines by interleukin (IL)‐17A and IL‐17F (P = 5.8 × 10−7). We identified a significant enrichment of methylation changes within 50 kb of CD GWAS loci (8.6‐fold [P = 0.021] in adults; 2.4‐fold [P = 0.009] in adults and children combined), including IL‐27, IL‐19, TNF, MST1, and NOD2. Methylation status was predictive of disease status (sensitivity 0.71, specificity 0.83). Disease activity, drug therapy, NOD2 and DNMT3A genotypes were not associated with methylation changes. Conclusions: These data provide an important insight into the impact of epigenetic mechanisms in the pathogenesis of CD. (Inflamm Bowel Dis 2011;)

BACKGROUND:An accurate indication of the changing incidence of pediatric inflammatory bowel disease (PIBD) within a population is useful in understanding concurrent etiological factors. We aimed to compare the current incidence and other demographic attributes of PIBD in the Scottish population to previous data. METHODS:A national cohort of prospectively and retrospectively acquired incident cases of PIBD diagnosed less than 16 years old in pediatric services in Scotland was captured for the period 2003-2008; historical Scottish data were used for comparison (1990-1995). Age/sex-adjusted incidences were calculated and statistical comparisons made using Poisson regression. RESULTS:During the 2003-2008 study period 436 patients were diagnosed with PIBD in Scotland, giving an adjusted incidence of 7.82/100,000/year. The incidence of Crohn's disease (CD) was 4.75/100,000/year, ulcerative colitis (UC) 2.06/100,000/year, and inflammatory bowel disease-unclassified (IBDU) 1.01/100,000/year. Compared with data from 1990-1995 when 260 IBD patients were diagnosed, significant rises in the incidence of IBD (from 4.45/100,000/year, P < 0.0001), CD (from 2.86/100,000/year, P < 0.0001), and UC (from 1.59/100,000/year, P = 0.023) were seen. There was also a significant reduction in the median age at IBD diagnosis from 12.7 years to 11.9 years between the periods (P = 0.003), with a continued male preponderance. CONCLUSIONS:The number of Scottish children diagnosed with IBD continues to rise, with a statistically significant 76% increase since the mid-1990 s. Furthermore, PIBD is now being diagnosed at a younger age. The reason for this continued rise is not yet clear; however, new hypotheses regarding disease pathogenesis and other population trends may provide further insights in future years.