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Immune responses following SARS-CoV-2 vaccination in patients with inflammatory bowel disease (IBD) are not well characterized. The aims of this study were to explore the serological response associated with IBD, and immunosuppressive medications including serum concentrations of biologics and thiopurine metabolites. This prospective, observational study included adult patients with ulcerative colitis (UC) and Crohn’s disease (CD), and healthy controls. Antibodies to the receptor-binding domain of SARS-CoV-2 spike proteins, and serum concentrations of ongoing biologic and immunomodulatory medications were assessed prior to, and 2-5 weeks after the second vaccine dose. Serologic response was defined as anti-Spike antibodies ≥70 AU/ml. In 958 IBD patients (380 UC, 578 CD) and 323 healthy controls, the median (Q1; Q3) anti-Spike antibody level (AU/ml) was lower in patients (618 (192; 4370)) compared to controls (3355 (896; 7849)) (p < 0.001). The antibody levels were lower in CD (439 (174; 3304)) compared to UC (1088 (251; 5975)) (p < 0.001). No associations were demonstrated between antibody levels and serum drug concentrations for TNF inhibitor (TNFi), vedolizumab and ustekinumab. Patients receiving TNFi + thiopurines with a subtherapeutic 6-thioguanine nucleotide (6-TGN) level had higher response rate (93%) compared to patients with 6-TGN within the therapeutic range (53%) (p = 0.003). A diagnosis of UC, mRNA-1273 vaccine, and other treatments than TNFi + thiopurines were associated with humoral response. Patients with CD had an attenuated humoral response to SARS-COV-2 vaccination as compared to patients with UC. The lack of association between serum levels of biologics and serologic response indicates vaccination regardless of proximity to drug administration.

Purpose This unselected, population-based cohort study aimed to determine the level of health-related quality of life (HRQoL) in patients with Crohn’s disease (CD) and ulcerative colitis (UC) at the time of diagnosis compared with a reference population and identify the demographic factors, psychosocial measures, and disease activity markers associated with HRQoL. Methods Adult patients newly diagnosed with CD or UC were prospectively enrolled. HRQoL was measured using the Short Form 36 (SF-36) and Norwegian Inflammatory Bowel Disease Questionnaires. Clinical significance was assessed using Cohen’s d effect size and further compared with a Norwegian reference population. Associations between HRQoL and symptom scores, demographic factors, psychosocial measures, and disease activity markers were analyzed. Results Compared with the Norwegian reference population, patients with CD and UC reported significantly lower scores in all SF-36 dimensions, except for physical functioning. Cohen’s d effect sizes for men and women in all SF-36 dimensions were at least moderate, except for bodily pain and emotional role for men with UC and physical functioning for both sexes and diagnoses. In the multivariate regression analysis, depression subscale scores ≥ 8 on the Hospital Anxiety and Depression Scale, substantial fatigue, and high symptom scores were associated with reduced HRQoL. Conclusion Patients newly diagnosed with CD and UC reported statistically and clinically significantly lower scores in seven of the eight SF-36 dimensions than the reference population. Symptoms of depression, fatigue, and elevated symptom scores were associated with poorer HRQoL.

Background Epigenetic alterations may provide valuable insights into gene–environment interactions in the pathogenesis of inflammatory bowel disease [IBD]. Methods Genome-wide methylation was measured from peripheral blood using the Illumina 450k platform in a case-control study in an inception cohort (295 controls, 154 Crohn’s disease [CD], 161 ulcerative colitis [UC], 28 IBD unclassified [IBD-U)] with covariates of age, sex and cell counts, deconvoluted by the Houseman method. Genotyping was performed using Illumina HumanOmniExpressExome-8 BeadChips and gene expression using the Ion AmpliSeq Human Gene Expression Core Panel. Treatment escalation was characterized by the need for biological agents or surgery after initial disease remission. Results A total of 137 differentially methylated positions [DMPs] were identified in IBD, including VMP1/MIR21 [p = 9.11 × 10−15] and RPS6KA2 [6.43 × 10−13], with consistency seen across Scandinavia and the UK. Dysregulated loci demonstrate strong genetic influence, notably VMP1 [p = 1.53 × 10−15]. Age acceleration is seen in IBD [coefficient 0.94, p Conclusion These data demonstrate consistent epigenetic alterations at diagnosis in European patients with IBD, providing insights into the pathogenetic importance and translational potential of epigenetic mapping in complex disease.

Objectives Peptic ulcers and erosions are the most common causes of upper gastrointestinal bleeding. The aim of this study was to investigate the management and outcomes of these patients. Materials and methods A total of 543 patients with endoscopically confirmed bleeding from peptic ulcers and erosions were included from March 2015 to December 2017. The patient characteristics, endoscopic findings, Forrest classification and endoscopic treatment were recorded. Moreover, the rebleeding rates, repeated endoscopies and transcatheter angiographic embolization and surgery incidences were registered. A follow-up endoscopy after discharge from the hospital was scheduled. Results Among the patients, high-risk stigmata ulcers were present in 36% (198/543) and low-risk stigmata ulcers and erosions in 60% (327/543) at first endoscopy. Endoscopic therapy was performed in 30% (165/543) of the patients, and hemostasis was achieved in 94% (155/165). The incidence of rebleeding was 9% (49/543) for the whole cohort and 14.8% (23/155) for those patients who had received successful endoscopic treatment. Moreover, rebleeding was significantly more frequent in duodenal ulcers than in gastric ulcers (11.9% vs 4.0%, p = .004). In a multivariable analysis, rebleeding was significantly related to comorbidity and Forrest classification. Transcatheter angiographic embolization and surgery were required in 6% (34/543) and 0.07% (4/543) of patients, respectively. Complete peptic ulcer healing was found at follow-up in 73.3% (270/368) of patients. Conclusions Endoscopic hemostasis was achieved in the majority of patients with high-risk ulceration, although the occurrence of rebleeding is a significant challenge, especially in patients with duodenal ulcers. Clinical trial registration: Bleeding Ulcer and Erosions Study (BLUE Study), ClinicalTrials.gov identifier: NCT03367897.

Aims i] To evaluate the evolution of pregnancies and offspring after inflammatory bowel disease [IBD] surgery during pregnancy; and ii] to describe the indications, the surgical techniques, and the frequency of caesarean section concomitant with surgery. Methods Patients operated on due to IBD during pregnancy after 1998 were included. Participating clinicians were asked to review their databases to identify cases. Data on patients’ demographics, IBD characteristics, medical treatments, IBD activity, pregnancy outcomes, surgery, delivery, and foetal and maternal outcomes, were recorded. Results In all, 44 IBD patients were included, of whom 75% had Crohn’s disease; 18% of the surgeries were performed in the first trimester, 55% in the second, and 27% in the third trimester. One patient had complications during surgery, and 27% had postsurgical complications. No patient died. Of deliveries, 70% were carried out by caesarean section. There were 40 newborns alive. There were four miscarriages/stillbirths [one in the first, two in the second, and one in the third trimester]; two occurred during surgery, and another two occurred 2 weeks after surgery; 14% of the surgeries during the second trimester and 64% of those in the third trimester ended up with a simultaneous caesarean section or vaginal delivery. Of the 40 newborns, 61% were premature and 47% had low birth weight; 42% of newborns needed hospitalisation [25% in the intensive care unit]. Conclusions IBD surgery during pregnancy remains an extremely serious situation. Therefore, surgical management should be performed in a multidisciplinary team, involving gastroenterologists, colorectal surgeons, obstetricians, and neonatal specialists.

Objectives Acute upper gastrointestinal bleeding is a well-recognized complication of peptic ulcers and erosions. The aim of this study was to assess the incidence rate and identify risk factors for this complication in southeastern Norway. Materials and methods Between March 2015 and December 2017, a prospective observational study was conducted at two Norwegian hospitals with a total catchment area of approximately 800,000 inhabitants. Information regarding patient characteristics, comorbidities, drug use, H. pylori status and 30-day mortality was recorded. Results A total of 543 adult patients were included. The incidence was 30/100,000 inhabitants per year. Altogether, 434 (80%) of the study patients used risk medication. Only 46 patients (8.5%) used proton pump inhibitors (PPIs) for more than 2 weeks before the bleeding episode. H. pylori testing was performed in 527 (97%) patients, of whom 195 (37%) were H. pylori-positive. The main comorbidity was cardiovascular disease. Gastric and duodenal ulcers were found in 183 (34%) and 275 (51%) patients, respectively. Simultaneous ulcerations at both locations were present in 58 (10%) patients, and 27 (5%) had only erosions. Overall, the 30-day mortality rate was 7.6%. Conclusions The incidence of upper gastrointestinal bleeding due to peptic ulcers and erosions was found to be lower than previously demonstrated in comparable studies, but the overall mortality rate was unchanged. The consumption of risk medication was high, and only a few patients had used prophylactic PPIs. Concurrent H. pylori infection was present in only one-third of the patients.

Background MicroRNAs [miRNAs] are cell-specific small non-coding RNAs that can regulate gene expression and have been implicated in inflammatory bowel disease [IBD] pathogenesis. Here we define the cell-specific miRNA profiles and investigate its biomarker potential in IBD. Methods In a two-stage prospective multi-centre case control study, next generation sequencing was performed on a discovery cohort of immunomagnetically separated leukocytes from 32 patients (nine Crohn’s disease [CD], 14 ulcerative colitis [UC], eight healthy controls) and differentially expressed signals were validated in whole blood in 294 patients [97 UC, 98 CD, 98 non-IBD, 1 IBDU] using quantitative PCR. Correlations were analysed with phenotype, including need for early treatment escalation as a marker of progressive disease using Cox proportional hazards. Results In stage 1, each leukocyte subset [CD4+ and CD8+ T-cells and CD14+ monocytes] was analysed in IBD and controls. Three specific miRNAs differentiated IBD from controls in CD4+ T-cells, including miR-1307-3p [p = 0.01], miR-3615 [p = 0.02] and miR-4792 [p = 0.01]. In the extension cohort, in stage 2, miR-1307-3p was able to predict disease progression in IBD (hazard ratio [HR] 1.98, interquartile range [IQR]: 1.20–3.27; logrank p = 1.80 × 10–3), in particular CD [HR 2.81; IQR: 1.11–3.53, p = 6.50 × 10–4]. Using blood-based multimarker miRNA models, the estimated chance of escalation in CD was 83% if two or more criteria were met and 90% for UC if three or more criteria are met. Interpretation We have identified and validated unique CD4+ T-cell miRNAs that are differentially regulated in IBD. These miRNAs may be able to predict treatment escalation and have the potential for clinical translation; further prospective evaluation is now indicated.

AIM: To assess the pathobiological and translational importance of whole blood transcriptomic analysis in inflammatory bowel disease (IBD). METHODS: We analyzed whole blood expression profiles from paired-end sequencing in a discovery cohort of 590 Europeans recruited across six countries in the IBD Character initiative (newly diagnosed patients with Crohn's disease [CD, n = 156], ulcerative colitis [UC, n = 167], and controls [n = 267]), exploring differential expression (DESeq2), co-expression networks (WGCNA), and transcription factor involvement (EPEE, ChEA, DoRothEA). Findings were validated by analysis of an independent replication cohort (99 CD, 100 UC, and 95 controls). In the discovery cohort, we also defined baseline expression correlates of future treatment escalation using cross-validated elastic-net and random forest modelling, along with a pragmatic ratio detection procedure. RESULTS: Disease-specific transcriptomes were defined in IBD (8697 transcripts), CD (7152), and UC (8521), with the most highly significant changes in single genes, including CD177 (log2-fold change [LFC] = 4.63, p = 4.05 × 10 -118), MCEMP1 (LFC = 2.45, p = 7.37 × 10 -109), and S100A12 (LFC = 2.31, p = 2.15 × 10 -93). Significantly over-represented pathways included IL-1 (p = 1.58 × 10 -11), IL-4, and IL-13 (p = 8.96 × 10 -9). Highly concordant results were obtained using multiple regulatory activity inference tools applied to the discovery and replication cohorts. These analyses demonstrated central roles in IBD for the transcription factors NFE2, SPI1 (PU.1), CEBPB, and IRF2, all regulators of cytokine signaling, based on a consistent signal across cohorts and transcription factor ranking methods. A number of simple transcriptome-based models were associated with the need for treatment escalation, including the binary CLEC5A/CDH2 expression ratio in UC (hazard ratio = 23.4, 95% CI 5.3-102.0). CONCLUSION: Transcriptomic analysis has allowed for a detailed characterization of IBD pathobiology, with important potential translational implications., Funding agency:Polish National Science Centre 2017/25/B/NZ5/02783

Simen Svendsen Vatn,1,2,* Jonas Christoffer Lindstrøm,3,4,* Aina EF Moen,1,4,5 Stephan Brackmann,1,2 Tone M Tannæs,1,5 Christine Olbjørn,1,6 Daniel Bergemalm,7 Åsa V Keita,8 Fernando Gomollon,9 Trond Espen Detlie,1,2 Torben Lüders,5 Rahul Kalla,10 Alex Adams,10,11 Jack Satsangi,10,11 Jørgen Jahnsen,1,2 Morten H Vatn,1 Jonas Halfvarson,7 Petr Ricanek,2 Hilde Nilsen1,5 On behalf of IBD-CHARACTER consortium1Department of Clinical Molecular Biology, Institute of Clinical Medicine, University of Oslo, Oslo, Norway; 2Department of Gastroenterology, Division of Medicine, Akershus University Hospital, Lørenskog, Norway; 3Health Services Research Unit (HØKH), Akershus University Hospital, Lørenskog, Norway; 4Department of Methods Development and Analytics, Division of Infectious Disease Control and Environmental Health, Norwegian Institute of Public Health, Oslo, Norway; 5Section for Clinical Molecular Biology (EpiGen), Akershus University Hospital, Lørenskog, Norway; 6Department of Pediatric and Adolescent Medicine, Akershus University Hospital, Lørenskog, Norway; 7Department of Gastroenterology, Faculty of Medicine and Health, Örebro University, Örebro, Sweden; 8Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden; 9Digestive Diseases Unit, IIS Aragón, Zaragoza, Spain; 10Gastrointestinal Unit, Centre for Genomics and Molecular Medicine, Division of Medical and Radiological Sciences, University of Edinburgh, Edinburgh, UK; 11Translational Gastroenterology Unit, Medical Sciences/ Experimental medicine Division, University of Oxford, Oxford, UK*These authors contributed equally to this workCorrespondence: Simen Svendsen Vatn, Akershus University Hospital, Postbox 1000, Lørenskog, 1478, Norway, Tel +47 94277594, Email bikkjas@hotmail.comBackground: Studies of the mucosal transcriptomic landscape have given new insight into the pathogenesis of inflammatory bowel disease (IBD). Recently, the predictive biomarker potential of gene expression signatures has been explored. To further investigate the mucosal gene expression in IBD, we recruited a cohort of treatment naïve patients and compared them to both symptomatic and healthy controls.Methods: Altogether, 323 subjects were included: Crohn’s disease (N = 75), ulcerative colitis (N = 87) and IBD unclassified (N = 3). Additionally, there were two control groups: symptomatic controls (N = 131) and healthy controls (N = 27). Mucosal biopsies were collected during ileocolonoscopy and gene expression in inflamed and non-inflamed mucosa was explored. Gene expression profiling was performed using Agilent G3 Human Gene Expression 860K v3 One-Color microarray. We recorded information about treatment escalation to anti-TNF agents or surgery, and anti-TNF response, to explore predictive opportunities of the mucosal transcriptome.Results: Gene expression profiles in symptomatic controls in whom IBD had been excluded resembled that of IBD patients and diverged from that of healthy controls. In non-inflamed Crohn’s disease and ulcerative colitis, gene set enrichment analysis revealed dysregulation of pathways involved in basic cellular biological processes. Mitochondria-associated pathways were dysregulated both in non-inflamed and inflamed Crohn’s disease and ulcerative colitis (> 2.6 normalized enrichment scores

Objective Immunogenicity and safety following receipt of the standard SARS–CoV-2 vaccination regimen in patients with immune-mediated inflammatory diseases (IMIDs) are poorly characterized, and data after receipt of the third vaccine dose are lacking. The aim of the study was to evaluate serologic responses and adverse events following the standard 2-dose regimen and a third dose of SARS–CoV-2 vaccine in IMID patients receiving immunosuppressive therapy. Methods Adult patients receiving immunosuppressive therapy for rheumatoid arthritis, spondyloarthritis, psoriatic arthritis, Crohn's disease, or ulcerative colitis, as well as healthy adult controls, who received the standard 2-dose SARS–CoV-2 vaccination regimen were included in this prospective observational study. Analyses of antibodies to the receptor-binding domain (RBD) of the SARS–CoV-2 spike protein were performed prior to and 2–4 weeks after vaccination. Patients with a weak serologic response, defined as an IgG antibody titer of ≤100 arbitrary units per milliliter (AU/ml) against the receptor-binding domain of the full-length SARS–Cov-2 spike protein, were allotted a third vaccine dose. Results A total of 1,505 patients (91%) and 1,096 healthy controls (98%) had a serologic response to the standard regimen (P

Background and aim: Modern treatment strategies for inflammatory bowel disease (IBD) are postulated to change the natural disease course. Inception cohort studies are the gold standard for investigating such changes. We have initiated a new population-based inception cohort study; Inflammatory bowel disease in South Eastern Norway III (IBSEN III). In this article, we describe the study protocol and baseline characteristics of the cohort. Methods: IBSEN III is an ongoing, population-based observational inception cohort study with prospective follow-up. Adult and pediatric patients with suspected IBD in the South-Eastern Health Region of Norway (catchment area of 2.95 million inhabitants in 2017), during the 3-year period from 2017 to 2019, were eligible for inclusion. Comprehensive clinical, biochemical, endoscopic, demographic, and patient-reported data were collected at the time of diagnosis and throughout standardized follow-up. For a portion of the patients, extensive biological material was biobanked. Results: The study included 2168 patients, of whom 1779 were diagnosed with IBD (Crohn's disease: 626, ulcerative colitis: 1082, IBD unclassified: 71). In 124 patients, there were subtle findings indicative of, but not diagnostic for, IBD. The remaining 265 patients were classified as symptomatic non-IBD controls. Conclusion: We have included patients in a comprehensive population-based IBD cohort from a catchment population of 2.95 million, and a unique biobank with materials from newly diagnosed and treatment-naïve IBD patients and symptomatic non-IBD controls. We believe this cohort will add important knowledge about IBD in the years to come.

Background Success in personalized medicine in complex disease is critically dependent on biomarker discovery. We profiled serum proteins using a novel proximity extension assay [PEA] to identify diagnostic and prognostic biomarkers in inflammatory bowel disease [IBD]. Methods We conducted a prospective case-control study in an inception cohort of 552 patients [328 IBD, 224 non-IBD], profiling proteins recruited across six centres. Treatment escalation was characterized by the need for biological agents or surgery after initial disease remission. Nested leave-one-out cross-validation was used to examine the performance of diagnostic and prognostic proteins. Results A total of 66 serum proteins differentiated IBD from symptomatic non-IBD controls, including matrix metallopeptidase-12 [MMP-12; Holm-adjusted p = 4.1 × 10–23] and oncostatin-M [OSM; p = 3.7 × 10–16]. Nine of these proteins are associated with cis-germline variation [59 independent single nucleotide polymorphisms]. Fifteen proteins, all members of tumour necrosis factor-independent pathways including interleukin-1 (IL-1) and OSM, predicted escalation, over a median follow-up of 518 [interquartile range 224–756] days. Nested cross-validation of the entire data set allowed characterization of five-protein models [96% comprising five core proteins ITGAV, EpCAM, IL18, SLAMF7 and IL8], which define a high-risk subgroup in IBD [hazard ratio 3.90, confidence interval: 2.43–6.26], or allowed distinct two- and three-protein models for ulcerative colitis and Crohn’s disease respectively. Conclusion We have characterized a simple oligo-protein panel that has the potential to identify IBD from symptomatic controls and to predict future disease course. Further prospective work is required to validate our findings.

SummaryBackgroundSuccess in personalised medicine in complex disease is critically dependent on biomarker discovery. We profiled serum proteins using a novel proximity extension assay (PEA) to identify diagnostic and prognostic biomarkers in inflammatory bowel disease (IBD).MethodsWe conducted a prospective case-control study in an inception cohort of 552 patients (328 IBD, 224 non-IBD), profiling proteins recruited across 6 centres. Treatment escalation was characterised by the need for biological agents or surgery after initial disease remission. Nested leave-one-out cross validation was used to examine the performance of diagnostic and prognostic proteins.ResultsA total of 66 serum proteins differentiated IBD from symptomatic non-IBD controls including Matrix Metalloproteinase-12 (Holm adjusted p=4.1×10−23) and Oncostatin-M (OSM, p=3.7×10−16). Nine of these proteins associate with cis- germline variation (59 independent SNPs). Fifteen proteins, all members of TNF independent pathways including interleukin-1 and OSM predicted escalation, over a median follow-up of 518 (IQR 224-756) days. Nested cross-validation of the entire data set allows characterisation of 5-protein-models (96% comprising five core proteins ITGAV, EpCAM, IL18, SLAMF7, and IL8) which define a high-risk subgroup in IBD (HR 3.90, 95% CI: 2.43-6.26), or allows distinct 2, and 3 protein models for UC and CD respectively.ConclusionWe have characterised a simple oligo-protein panel that has the potential to identify IBD from symptomatic controls and predicts the evolution of disease over time. The technology could be suitable as a point of care testing in defining risk. Further prospective work is required to characterise the utility of the approach.

Method We examined faecal samples, using the GA-map™ Dysbiosis Test, to associate gut microbiota composition with Crohn’s disease (CD) and ulcerative colitis (UC) and to identify markers for future biomarker identification. We conducted a prospective case-control study (EU-ref. no. 305676) in an inception cohort of 324 individuals (64 CD, 84 UC, 116 symptomatic non-IBD controls and 44 healthy controls) across five European centres and examined 54 predetermined bacterial markers. We categorized patients according to the Montreal Classification and calculated the dysbiosis index (DI). Non-parametric tests were used to compare groups and the Bonferroni correction to adjust for multiple comparisons. Results The fluorescent signals (FSSs) for Firmicutes and Eubacterium hallii were lower in inflammatory bowel disease (IBD) vs. symptomatic controls (p

Variability in functional outcome after ileal-pouch anal anastomosis (IPAA) is to a large extent unexplained. The aim of this study was to perform multiple physiological and biochemical tests including an endoscopic examination with histology on IPAA patients with well and poorly functioning pouches to determine factors, or combinations thereof, contributing to functional outcome.All patients with ulcerative colitis undergoing restorative proctocolectomy between 2000 and 2013 (N = 108) were interviewed using a pouch functioning score. The best and worst functioning quartiles were invited to undergo examination with a barostat measuring pouch volume at preset variable distension pressures, and a pouch endoscopy.Forty five of 58 eligible patients agreed to participate. The most significant physiological parameter differing between the well and poorly functioning pouches was pouch volume at first sensation, urge and discomfort (p value.001). Urge volumes were 213 (CI 171-256) ml for poorly and 352 (CI 305-401) ml for well functioning pouches. Pouchitis episodes were negatively correlated to function. The poorly functioning patients had a higher prevalence of histological signs of inflammation and hand-sewn anastomosis, and a longer remaining rectal cuff, however, nonsignificant. The pouch pressure at sensation thresholds did not differ between the groups.Pouch volume is the most dominant predictor of pouch function in this study. The present comprehensive study of a multitude of different factors that possibly could be contributing to functional outcome, failed to shed much further light on the functional variability among pouch patients. The pouch physiology remains to a large extent unexplained.

Christine Olbjørn,1,2 Milada Cvancarova Småstuen,3 Espen Thiis-Evensen,4 Britt Nakstad,1,2 Morten Harald Vatn,5 Jørgen Jahnsen,2,6 Petr Ricanek,2,6 Simen Vatn,2,6 Aina EF Moen,5 Tone M Tannæs,5 Jonas C Lindstrøm,7 Johan D Söderholm,8 Jonas Halfvarson,9 Fernando Gomollón,10 Christina Casén,11 Magdalena K Karlsson,11 Rahul Kalla,12 Alex T Adams,12,13 Jack Satsangi,12,13 Gøri Perminow14 1Department of Pediatric and Adolescent Medicine, Akershus University Hospital, Lørenskog, Norway; 2Institute of Clinical Medicine, Campus Ahus, University of Oslo, Oslo, Norway; 3Faculty of Health Sciences, Oslo Metropolitan University, Oslo, Norway; 4Department of Gastroenterology, Oslo University Hospital, Rikshospitalet, Oslo, Norway; 5Department of Clinical Molecular Biology (EpiGen), Division of Medicine, Akershus University Hospital, Lørenskog, and University of Oslo, Oslo, Norway; 6Department of Gastroenterology, Akerhus University Hospital, Lørenskog, Norway; 7Institute of Clinical Medicine, University of Oslo, Health Services Research Unit, Akershus University Hospital, Lørenskog, Norway; 8Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden; 9Department of Clinical and Experimental Medicine, Faculty of Medicine and Health Sciences, Linköping University, Linköping, Sweden; 10Digestive Diseases Unit, IIS Aragón, Zaragoza, Spain; 11Genetic-Analysis AS, Oslo, Norway; 12Gastrointestinal Unit, Centre for Genomics and Molecular Medicine, University of Edinburgh, Edinburgh, UK; 13Translational Gastroenterology Unit, Experimental Medicine Division, University of Oxford, Oxford, UK; 14Department of Pediatrics, Oslo University Hospital, Ullevål, Oslo, Norway Purpose: Imbalance in the microbiota, dysbiosis, has been identified in inflammatory bowel disease (IBD). We explored the fecal microbiota in pediatric patients with treatment-naïve IBD, non-IBD patients with gastrointestinal symptoms and healthy children, its relation to IBD subgroups, and treatment outcomes.Patients and methods: Fecal samples were collected from 235 children below 18 years of age. Eighty children had Crohn’s disease (CD), 27 ulcerative colitis (UC), 3 IBD unclassified, 50 were non-IBD symptomatic patients, and 75 were healthy. The bacterial abundance of 54 predefined DNA markers was measured with a 16S rRNA DNA-based test using GA-Map™ technology at diagnosis and after therapy in IBD patients.Results: Bacterial abundance was similarly reduced in IBD and non-IBD patients in 51 of 54 markers compared to healthy patients (P

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., Epigenetic perturbations may be an important factor in diseases where both genes and environment play a role. Here, Ventham and colleagues show that DNA methylation changes in inflammatory bowel disease are related to the underlying genotype, and are associated with cell-specific changes to gene expression.

Background Nucleic acid purification methods are of importance when performing microbiota studies and especially when analysing the intestinal microbiota as we here find a wide range of different microbes. Various considerations must be taken to lyse the microbial cell wall of each microbe. In the present article, we compare several tissue lysis steps and commercial purification kits, to achieve a joint RNA and DNA purification protocol for the purpose of investigating the microbiota and the microbiota-host interactions in a single colonic mucosal tissue sample. Results A further optimised tissue homogenisation and lysis protocol comprising mechanical bead beating, lysis buffer replacement and enzymatic treatment, in combination with the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) resulted in efficient and simultaneous purification of microbial and human RNA and DNA from a single mucosal colonic tissue sample. Conclusions The present work provides a unique possibility to study RNA and DNA from the same mucosal biopsy sample, making a direct comparison between metabolically active microbes and total microbial DNA. The protocol also offers an opportunity to investigate other members of a microbiota such as viruses, fungi and micro-eukaryotes, and moreover the possibility to extract data on microbiota and host interactions from one single mucosal biopsy. Electronic supplementary material The online version of this article (doi:10.1186/s13104-016-2110-7) contains supplementary material, which is available to authorized users.

Introduction Proximity extension assays (PEA) allows multiprotein profiling and utilises the specificity of antibody proximity and the sensitivity of polymerase chain reaction to detect proteins of interests. As part of IBD Character, we performed high-throughput prospective case-control serum profiling to identify proteins that can predict Inflammatory Bowel Disease (IBD) and its disease course. Method Serum profiling was performed in newly diagnosed IBD and Non-IBD cases using PEA panels (Olink Proteomics) from 6 centres in Europe. Phenotypic data were obtained for all patients and follow up outcome data were captured for the Edinburgh and Oslo IBD cohorts. Treatment escalation was defined as the need for surgery and/or biologic therapies after initial induction of remission. Statistical analysis was performed using R. Results Protein profiles were available in 635 patients (152 CD, 159 UC, 26 IBD-U, 298 non-IBD). 61 protein markers were significantly associated with IBD including MMP12 (Holm-adjusted p=4.1×10–26). Mapping top markers to cell-specific FANTOM 5 [1], several differentially expressed proteins originate from innate and adaptive immune cells such as dendritic cells. 5 proteins differentiate UC from CD including MMP-12 (p=4.6×10–4) Follow up data were available for 206 patients of which 49 patients required treatment escalation. The data were randomly split into a testing (n=130) and a validation cohort (n=76). Using multivariable analyses with age, sex and follow up time as covariates, 9 proteins survived Holm adjustment and 8 of these proteins remained signficant in the validation cohort. 1000 iterations of unsupervised linear discriminant consensus clustering were performed using 7 randomly selected top proteins and identified 2 patients groups that had significantly different disease courses(Image):logrank p=2.2×10–10, HR 5.6 (2.0–15.6); outperforming conventional biomarkers in predicting treatment escalation (hsCRP >4 mg/L, HR 3.2 (1.7–5.8), p=0.0003 and Alb Conclusion We have identified immune cell-specific PEA-based serum proteins that can diagnose IBD and predict disease course. These data demonstrate the translational potential of a PEA based technology in IBD Reference . FANTOM Consortium(2014), A promoter level mammalian expression atlas, Nature2014, 507:462–70 Disclosure of Interest R. Kalla: None Declared, A Adams Conflict with: EC FP7, S Vatn: None Declared, D Bergemalm: None Declared, P Ricanek: None Declared, J Lindstrom: None Declared, A Ocklind Conflict with: Olink is an SME within IBD Character, N Nordberg Conflict with: Olink is an SME within IBD Character, N Kennedy: None Declared, N Ventham: None Declared, M Vatn: None Declared, J Soderholm: None Declared, M Pierik: None Declared, L Torkvist: None Declared, F Gomollon: None Declared, J Jahnsen: None Declared, J Halfvarson: None Declared, J Satsangi: None Declared

Microbiota related disease activity and distribution in subgroups of inflammatory bowel disease

The present population based adult cohort was part of a new prospective study of patients with inflammatory bowel disease (IBD) in South-Eastern Norway, the Inflammatory Bowel South-Eastern Norway II study, investigating disease characteristics in an attempt to improve our knowledge regarding factors related to early clinical phenotype and disease activity.Patients suspected to have IBD on the basis of predefined symptoms, including abdominal pain, diarrhea, and/or blood in stool for more than 10 days were examined at the local hospital. Colonoscopy with biopsies was performed and blood and stool samples were taken.In ulcerative colitis (UC) patients, the median Simple Clinical Colitis Activity Index (SCCAI) was 4 (range 0-10) in mild and 6 (range 0-14) in patients with moderate or severe endoscopic activity of inflammation (p = 0.002). The calprotectin concentration in feces was significantly related to the SCCAI (p = 0.034) and the Mayo endoscopic subscore (p = 0.031). There was a significant association between the C-reactive protein (CRP) value, leucocytes and thrombocytes and the SCCAI, but only leucocytes were significantly associated with the Mayo endoscopic subscore. In Crohn's disease (CD) patients, there was no statistical significant association between the Harvey-Bradshaw Index (HBI) and the endoscopic grade of mucosal inflammation (p = 0.8). The calprotectin concentration in feces was significantly related to the endoscopic activity score (p = 0.004), but not to the HBI (p = 0.5). HBI was significantly related to the CRP value (p = 0.047) and thrombocytes (p = 0.03).In UC, both biochemical and fecal markers are related to disease activity and extent of disease, whereas in CD, the fecal calprotectin concentration is a reliable marker of mucosal affection, but not for systemic disease activity.

The presence of Mycobacterium avium subspecies paratuberculosis (MAP) has previously been inferred in the genesis of Crohn's disease (CD), and a higher incidence of MAP PCR positivity has been demonstrated in the gut and peripheral blood of CD patients than in healthy individuals. The objective of this prospective study was to assess the potential etiological role of MAP in the pathogenesis of CD.The presence of mycobacteria was assessed in bowel biopsies from newly diagnosed, treatment naïve Norwegian patients with IBD, including CD and ulcerative colitis (UC), as compared to a hospital-based cohort of CD and UC patients. Biopsies were collected from the small and large bowel in 354 individuals with suspected IBD. Detection of mycobacteria was performed by long-term cultivation in combination with direct detection by MAP IS900-specific PCR.Among the specimens included from the patients with early IBD, samples from only two of the patients with CD (2.7%) and two of the non-IBD controls (1.5%) exhibited a positive growth signal. None of the CD patients and only one of the non-IBD controls was MAP PCR positive. Only the single PCR positive non-IBD control was also mycobacterial culture positive with Mycobacterium avium subsp. hominissuis. In the referral patients with long-term IBD, the prevalence of growth signal and MAP PCR positivity was higher (52 and 9%, respectively).These findings demonstrate the paucity of MAP in the gut of treatment naïve CD patients. This study does not provide evidence for a role of MAP in early IBD.

Faecal microbiota in newly diagnosed Crohn's disease and its relation to treatment escalation

Faecal microbiota in treatment-naive ulcerative colitis and its relation to treatment escalation

Objective: To assess the efficacy, tolerability, and safety of CT-P13 (Remsima®) in patients with Crohn’s disease (CD) or ulcerative colitis (UC). Methods: This was a prospective observational study performed in a single center in Norway. Patients with CD (n = 46) or UC (n = 32) received CT-P13 (5 mg/kg) by intravenous infusion at weeks 0, 2, and 6. Efficacy end points included remission at week 14, measured by a Harvey-Bradshaw Index score of ≤4 or partial Mayo score of ≤2. Levels of the inflammatory markers C-reactive protein and calprotectin were measured. Adverse events up to week 14 were also recorded. Results: Seventy-nine percent of CD and 56% of UC patients achieved remission at week 14. Significant reductions in C-reactive protein and calprotectin occurred between baseline and week 14. There were no unexpected adverse events reported during the study. Conclusion: CT-P13 is efficacious and well tolerated in patients with CD or UC.

Background Exploring DNA methylation in inflammatory bowel disease might provide an insight into the complex gene–environment interactions in disease pathogenesis. Our study aimed to characterise disease-associated methylation changes in newly diagnosed inflammatory bowel disease and explore its association with germline variation and gene expression. Methods Samples were obtained from new onset inflammatory bowel disease in six European centres (IBD-Character project). Genome-wide methylation was measured (450k platform, Illumina, San Diego, CA, USA) in 641 whole blood DNA samples (298 controls, 150 Crohn's disease, 167 ulcerative colitis, 26 inflammatory bowel disease unclassified). Genotyping and gene expression were performed using HumanOmniExpressExome-8 BeadChips (Illumina) and Ion AmpliSeq (ThermoFisher, Waltham, MA, USA) platforms, respectively. Correlation and pathway analyses were performed between the top differentially methylated regions and gene expression. Findings M195 probes exhibited Holm-significant inflammatory bowel disease-associated methylation differences, including MIR21 (p=3·7 × 10 − 20 ) and RPS6KA2 (p=1·1 × 10 −19 ) with only one probe differentiating Crohn's disease from ulcerative colitis ( NAV2 , p=6·82 × 10 −8 ). Paired genetic and methylation data showed 1037 significant methylation quantitative trait loci indicating a genetic influence on several key loci: RPS6KA2 (p=8·6 × 10 −34 ) and MIR21 (rs8078424, p=4·4 × 10 − 25 ; rs10853015, p=7·4 × 10 −21 ). 1543 differentially methylated regions (DMRs) were identified and included MIR21 , RPS6KA2 , and TNF . These DMRs mapped to 8214 mRNA profiles of which 1916 showed Holm-significant correlation with methylation such as IL32 (seven probes: r =–0·50 to −0·66, p=6·0 × 10 −76 ). GO term analyses of these highly correlated genes revealed pathways that regulate cell–cell adhesion and immune cell differentiation. Interpretation Our data provide a comprehensive multicentre genome-wide profile of the circulating methylome and give an insight into the complex interplay between differential methylation, germline variation, and gene expression in immune mediated disease. Funding RK and AA are funded by IBD-Character (a European Union 7th Framework Programme project).

Proximity Extension Assay technology identifies novel serum biomarkers for predicting Inflammatory Bowel Disease : IBD Character Consortium

The knowledge about correlation patterns between the fecal microbiota and inflammatory bowel diseases (IBD)—comprising the two subforms Crohn's disease (CD) and ulcerative colitis (UC)—for newly diagnosed untreated children is limited. To address this knowledge gap, a selection of faecal specimens (CD,n=27and UC,n=16) and non-IBD controls (n=30) children (age < 18 years) was analysed utilising bacterial small subunit (SSU) rRNA. We found, surprising age dependence for the fecal microbiota correlating to IBD. The most pronounced patterns were thatE. coliwas positively (R2=0.16,P=0.05) and Bacteroidetes, negatively (R2=0.15,P=0.05) correlated to age for CD patients. For UC, we found an apparent opposite age-related disease correlation for bothBacteroidesandEscherichia. In addition, there was an overrepresentation ofHaemophilusfor the UC children. From our, results we propose a model where the aetiology of IBD is related to an on-going immunological development in children requiring different age-dependent bacterial stimuli. The impact of our findings could be a better age stratification for understanding and treating IBD in children.

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

Tap-water has recently been identified as an important epidemiological parameter in inflammatory bowel disease (IBD). Whether bacteria present in tap-water are involved in IBD still remains unknown. To address this question, the dominant microbiota in tap-water samples from households of patients with IBD, and from those of non-IBD control patients, were screened. Tap-water from both pediatric patients (

Introduction Advances in protein profiling using proximity extension assays (PEA) have transformed our ability to compare concentrations of multiple proteins across biological samples. PEA utilises the specificity of antibody proximity and the sensitivity of polymerase chain reaction (qPCR) to detect proteins of interest. As part of the EC funded IBD Character initiative to discover biomarkers for clinical use using multiomic technologies, we performed high-throughput prospective case-control serum profiling to identify protein biomarkers that can predict Inflammatory Bowel Disease (IBD). Method Utilising Proseek Multiplex panels (Olink Bioscience, Sweden), serum profiling was performed in patients with a new diagnosis of IBD. Our control group consisted of symptomatic individuals. Phenotypic data was captured including age, sex, diagnosis and IBD medications. 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 186 proteins was available for analysis. Results A total of 245 patient serum samples (n = 153) newly diagnosed IBD, n = 92 symptomatic controls) from 4 IBD centres from Norway, United Kingdom and Sweden were included in the study from December 2012 to June 2014. 70 had Crohn’s disease (CD), 70 ulcerative colitis (UC) and 13 Inflammatory Bowel Disease Unclassified (IBDU). The mean age of the entire cohort was 33 years (range 0–79 years) and 54% were female. Multivariable analysis identified a set of 48 protein markers that were significantly associated with IBD. The 5 most significant protein markers were MMP-12 (Holm-adjusted p = 3.3 × 10 –13 ), OSM (p = 2.4 × 10 –12 ), CXCL9 (p = 1.7 × 10 –9 ), MMP10 (p = 1.7 × 10 –9 ) and EGFR (p = 1.8 × 10 –9 ). Of these five, all except EGFR were upregulated in IBD. The top 2 markers, MMP-12 and OSM were able to discriminate IBD from controls with an area under the receiver operator characteristics curve of 0.81 and 0.75 respectively. Using linear discriminant analysis, a combined biomarker consisting of MMP-12 and OSM was able to discriminate IBD from controls with a sensitivity and specificity of 80% and 72% respectively. Conclusion We have identified serum biomarkers that can predict IBD. These data demonstrate a potential for a PEA multiplex technology in IBD diagnostics and its ability to identify novel proteins that may be relevant in disease pathogenesis. Disclosure of interest R. Kalla Grant/Research Support from: IBD Character, N. Kennedy Grant/Research Support from: Wellcome Trust, Conflict with: Abbvie, MSD, Warner Chilcott, Ferring speaker fees, F. Hjelm: None Declared, E. Modig: None Declared, M. Sundell: None Declared, J. Soderholm: None Declared, B. Andreassen: None Declared, D. Bergemalm: None Declared, N. Ventham Grant/Research Support from: IBD BIOM, H. Hjortswang: None Declared, R. Petr: None Declared, M. Vatn: None Declared, J. Halfvarson: None Declared, M. Gullberg: None Declared, J. Satsangi Grant/Research Support from: EC Grants, Wellcome, CSO, MRC, Consultant for: Takeda, Conflict with: MSD speaker fees.

Petr Ricanek,1,2 Lisa K Lunde,3 Stephan A Frye,1 Mari Støen,1 Ståle Nygård,4 Jens P Morth,5,6 Andreas Rydning,2 Morten H Vatn,7,8 Mahmood Amiry-Moghaddam,3 Tone Tønjum,1,9 1Department of Microbiology, Oslo University Hospital, Rikshospitalet, Oslo, 2Department of Gastroenterology, Akershus University Hospital, Lørenskog and Campus Ahus, Institute of Clinical Medicine, University of Oslo, Lørenskog, 3Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, 4Bioinformatics Core Facility, Institute for Medical Informatics, Oslo University Hospital and University of Oslo, 5Centre for Molecular Medicine, Nordic EMBL Partnership, University of Oslo, 6Institute for Experimental Research, Oslo University Hospital (Ullevaal), Oslo, 7EpiGen Institute, Campus Ahus, Institute of Clinical Medicine, University of Oslo, Lørenskog, 8Section of Gastroenterology, Oslo University Hospital, Rikshospitalet, Oslo, 9Department of Microbiology, University of Oslo, Oslo, Norway Objectives: The aim of this study was to investigate the relationship between aquaporin (AQP) water channel expression and the pathological features of early untreated inflammatory bowel disease (IBD) in humans. Methods: Patients suspected to have IBD on the basis of predefined symptoms, including abdominal pain, diarrhea, and/or blood in stool for more than 10 days, were examined at the local hospital. Colonoscopy with biopsies was performed and blood samples were taken. Patients who did not meet the diagnostic criteria for IBD and who displayed no evidence of infection or other pathology in the gut were included as symptomatic non-IBD controls. AQP1, 3, 4, 5, 7, 8, and 9 messenger RNA (mRNA) levels were quantified in biopsies from the distal ileum and colon by quantitative real-time polymerase chain reaction. Protein expression of selected AQPs was assessed by confocal microscopy. Through multiple alignments of the deduced amino acid sequences, the putative three-dimensional structures of AQP1, 3, 7, and 8 were modeled. Results: AQP1, 3, 7, and 8 mRNAs were detected in all parts of the intestinal mucosa. Notably, AQP1 and AQP3 mRNA levels were reduced in the ileum of patients with Crohn's disease, and AQP7 and AQP8 mRNA levels were reduced in the ileum and the colon of patients with ulcerative colitis. Immunofluorescence confocal microscopy showed localization of AQP3, 7, and 8 at the mucosal epithelium, whereas the expression of AQP1 was mainly confined to the endothelial cells and erythrocytes. The reduction in the level of AQP3, 7, and 8 mRNA was confirmed by immunofluorescence, which also indicated a reduction of apical immunolabeling for AQP8 in the colonic surface epithelium and crypts of the IBD samples. This could indicate loss of epithelial polarity in IBD, leading to disrupted barrier function. Conclusion: AQPs 1 and 8 and the aquaglyceroporins AQPs 3 and 7 are the AQPs predominantly expressed in the lower intestinal tract of humans. Their expression is significantly reduced in patients with IBD, and they are differentially expressed in specific bowel segments in patients with Crohn's disease and ulcerative colitis. The data present a link between gut inflammation and water/solute homeostasis, suggesting that AQPs may play a significant role in IBD pathophysiology. Keywords: inflammatory bowel disease, Crohn's disease, ulcerative colitis, aquaporins, aquaglyceroporins

to a labeled end of an amplified gene. T-RFLP is an established technique for analysing bacterial species in faecal samples. Methods: This was a quantitative analysis with a major focus on evaluating a biomarker based on intestinal microbiota. The subjects were 69 UC patients together with an 80 relatives as controls. Twenty-three patients had active UC (group I) and 46 had quiescent UC (group II). The latter included 17 with mild inflammation in the large intestine (group IIa), and 29 without inflammation (group IIb). The patients’ relatives were consanguineous (group III, n = 47), and non-consanguineous (group IV, n = 33). The Discriminant analysis of operationaltaxonomic-unit (OTU) on T-RFLP fingerprints was done. The Canonical Discriminant Function Coefficient (Df) for each OTU was calculated by using group I and group IV, in whom the difference of intestinal flora was thought to be greatest. The individual OTUs were multiplied by the Df value, and the sum was termed the Discriminant Score (Ds). The analysis was by SPSS (IBM Statistics 20.0). Results: The Ds decreased thus: group I > group IIa > group IIb > group III > group IV. As certain strains of bacteria are associated with the development of UC, while others not, this should show up in the OTU values in T-RFLP analysis. The analysis was set to factor this assumption, and the OUTs with 0 value 95% of all cases together with the OTUs of structure matrix value

Petr Ricanek,1,2 Sheba M Lothe,1 Stephan A Frye,1 Andreas Rydning,2 Morten H Vatn,3,4 Tone Tønjum1,51Centre for Molecular Biology and Neuroscience and Department of Microbiology, Oslo University Hospital, Rikshospitalet, Oslo, 2Department of Gastroenterology, Akershus University Hospital, Lørenskog and Faculty Division Akershus University Hospital, University of Oslo, Lørenskog, 3EpiGen Institute, Faculty Division Akershus University Hospital, University of Oslo, Lørenskog, 4Department of Medicine, Oslo University Hospital, Rikshospitalet, Oslo, 5Centre for Molecular Biology and Neuroscience and Department of Microbiology, University of Oslo, Oslo, NorwayObjectives: The aim of this study was to define the composition of the gut bacterial flora in Norwegian patients with early stage Crohn's disease (CD). Methods: By using a nonselective metagenomics approach, the general bacterial composition in mucosal biopsies from the ileum and the colon of five subjects, four patients with different phenotypes of CD, and one noninflammatory bowel disease control, was characterized. After partial 16S ribosomal RNA (rRNA) gene sequencing, BLAST homology searches for species identification and phylogenetic analysis were performed.Results: An overall biodiversity of 106 different bacterial operational taxonomic units (OTUs) was detected in the cloned libraries. Nearly all OTUs belonged to the phylae Bacteroidetes (42% in CD, 71% in the control) or Firmicutes (42% in CD, 28% in the control), except for some OTUs that belonged to the phylum Proteobacteria (15% in CD, 0% in the control) and a few OTUs that could not be assigned to a phylum (2% in CD, 1% in the control).Conclusion: Based on the high incidence of inflammatory bowel disease (IBD) in Norway, this pilot study represents a relevant determination of the gut microbiota in Norwegian patients compared to previous findings in other countries. The bacterial profile of Norwegian CD patients was found to be similar to that of CD patients in other countries. The findings do not support a particular bacterial composition as a predominant causative factor for the high incidence of IBD that exists in some countries.Keywords: Crohn's disease, gut microbiota composition, inflammatory bowel disease, IBD, metagenomics, 16S rRNA gene sequences

Greenstein et al. have commented on the report “Paucity of mycobacteria in mucosal bowel biopsies from adults and children with early inflammatory bowel disease”,1 which negates the role of Mycobacterium avium subspecies paratuberculosis (MAP) in the early pathogenesis of Crohn's disease (CD), and suggest that this scenario opens up for an alternative mycobacterial species in the genesis of CD. We thank the Authors and the Editor for initiating such an illustrious discussion and would like to address two points, anti-inflammatory drugs in IBD and the potential role of mycobacteria other than MAP in the …