Contrast enhanced endoscopic ultrasound detects early therapy response following anti-TNF-therapy in patients with ulcerative colitis.

BACKGROUND AND AIM: Though colonoscopy plays a crucial role in assessing active ulcerative colitis (aUC), its scope is limited to the mucosal surface. Endoscopic ultrasound (EUS) coupled with contrast-enhancement (dCEUS) can precisely quantify bowel wall thickness and microvascular circulation, potentially enabling the quantitative evaluation of inflammation.We conducted a prospective, longitudinal study to assess therapy response using dCEUS in aUC patients undergoing treatment with adalimumab (ADA) or infliximab (IFX). METHODS: 30 ADA- and 15 IFX-treated aUC patients were examined at baseline and at 2, 6, 14 weeks of therapy and 48 weeks of follow-up. Bowel wall thickness (BWT) was measured by EUS in the rectum. Vascularity was quantified by dCEUS using Rise Time (RT) and Time To Peak (TTP). Therapy response was defined after 14 weeks using the Mayo Score. RESULTS: Patients with aUC displayed a mean BWT of 3.9±0.9 mm. In case of response to ADA/IFX a significant reduction in BWT was observed after 2 weeks (p=0.04), whereas non-responders displayed no significant changes. The TTP was notably accelerated at baseline and significantly normalised by week 2 in responders (p=0.001), while non-responders exhibited no significant alterations (p=0.9). At week 2, the endoscopic Mayo score did not exhibit any changes, thus failing to predict treatment responses. CONCLUSION: dCEUS enables the early detection of therapy response in patients with aUC, which serves as a predictive marker for long term clinical success. Therefore, dCEUS serves as a diagnostic tool for assessing the probability of future therapy success.

Selection of cross-reactive T cells by commensal and food-derived yeasts drives cytotoxic TH1 cell responses in Crohn's disease.

Aberrant CD4+ T cell reactivity against intestinal microorganisms is considered to drive mucosal inflammation in inflammatory bowel diseases. The disease-relevant microbial species and the corresponding microorganism-specific, pathogenic T cell phenotypes remain largely unknown. In the present study, we identified common gut commensal and food-derived yeasts, as direct activators of altered CD4+ T cell reactions in patients with Crohn's disease (CD). Yeast-responsive CD4+ T cells in CD display a cytotoxic T helper cell (TH1 cell) phenotype and show selective expansion of T cell clones that are highly cross-reactive to several commensal, as well as food-derived, fungal species. This indicates cross-reactive T cell selection by repeated encounter with conserved fungal antigens in the context of chronic intestinal disease. Our results highlighted a role of yeasts as drivers of aberrant CD4+ T cell reactivity in patients with CD and suggest that both gut-resident fungal commensals and daily dietary intake of yeasts might contribute to chronic activation of inflammatory CD4+ T cell responses in patients with CD.

Interobserver Reliability of the Nancy Index for Ulcerative Colitis: An Assessment of the Practicability and Ease of Use in a Single-Centre Real-World Setting.

BACKGROUND: Histological disease severity assessment in ulcerative colitis [UC] has become a mainstay in the definition of clinical endpoints ['histological remission'] in clinical trials of UC. Several scores have been established in the microscopic assessment of disease activity, but the Nancy index [NI] stands out as being the histological index with the fewest scoring items. To what extent histological assessment using the NI is affected by interobserver reliability in a real-word setting is poorly understood. We therefore performed a single-centre retrospective analysis of NI assessment in patients with UC. METHODS: We retrospectively evaluated the NI in two independent cohorts [total: 1085 biopsies, 547 UC patients] of clinically diagnosed UC patients, who underwent colonoscopy between 2007 and 2020. Cohort #1 consisted of 637 biopsies from 312 patients, while Cohort #2 consisted of 448 biopsies from 235 patients. Two blinded pathologists with different levels of expertise scored all biopsies from each cohort. A consensus conference was held for cases with discrepant scoring results. Finally, an overall consensus scoring was obtained from both cohorts. RESULTS: The interobserver agreement of the NI was substantial after the assessment of 1085 biopsy samples (κ = 0.796 [95% confidence interval, CI: 0.771-0.820]). An improvement of the interobserver agreement was found with increasing numbers of samples evaluated by both observers (Cohort #1: κ = 0.772 [95% CI: 0.739-0.805]; Cohort #2: κ = 0.829 [95% CI: 0.793-0.864]). Interobserver discordance was highest in NI grade 1 [observer 1: n = 128; observer 2: n = 236]. Interobserver discordance was lowest in NI grades 0 [observer 1: n = 504; observer 2: n = 479] and 3 [observer 1: n = 71; observer 2: n = 66]. CONCLUSION: The NI is an easy-to-use index with high interobserver reliability for assessment of the histological disease activity of UC patients in a real-world setting. While NI grades 0 and 3 had a high level of agreement between observers, NI grade 1 had a poorer level of agreement. This highlights the clinical need to specify histological characteristics leading to NI grade 1.

DNA methyltransferase 3A controls intestinal epithelial barrier function and regeneration in the colon.

Genetic variants in the DNA methyltransferase 3 A (DNMT3A) locus have been associated with inflammatory bowel disease (IBD). DNMT3A is part of the epigenetic machinery physiologically involved in DNA methylation. We show that DNMT3A plays a critical role in maintaining intestinal homeostasis and gut barrier function. DNMT3A expression is downregulated in intestinal epithelial cells from IBD patients and upon tumor necrosis factor treatment in murine intestinal organoids. Ablation of DNMT3A in Caco-2 cells results in global DNA hypomethylation, which is linked to impaired regenerative capacity, transepithelial resistance and intercellular junction formation. Genetic deletion of Dnmt3a in intestinal epithelial cells (Dnmt3aΔIEC) in mice confirms the phenotype of an altered epithelial ultrastructure with shortened apical-junctional complexes, reduced Goblet cell numbers and increased intestinal permeability in the colon in vivo. Dnmt3aΔIEC mice suffer from increased susceptibility to experimental colitis, characterized by reduced epithelial regeneration. These data demonstrate a critical role for DNMT3A in orchestrating intestinal epithelial homeostasis and response to tissue damage and suggest an involvement of impaired epithelial DNMT3A function in the etiology of IBD.

Longitudinal multi-omics analysis identifies early blood-based predictors of anti-TNF therapy response in inflammatory bowel disease.

BACKGROUND AND AIMS: Treatment with tumor necrosis factor α (TNFα) antagonists in IBD patients suffers from primary non-response rates of up to 40%. Biomarkers for early prediction of therapy success are missing. We investigated the dynamics of gene expression and DNA methylation in blood samples of IBD patients treated with the TNF antagonist infliximab and analyzed the predictive potential regarding therapy outcome. METHODS: We performed a longitudinal, blood-based multi-omics study in two prospective IBD patient cohorts receiving first-time infliximab therapy (discovery: 14 patients, replication: 23 patients). Samples were collected at up to 7 time points (from baseline to 14 weeks after therapy induction). RNA-sequencing and genome-wide DNA methylation data were analyzed and correlated with clinical remission at week 14 as a primary endpoint. RESULTS: We found no consistent ex ante predictive signature across the two cohorts. Longitudinally upregulated transcripts in the non-remitter group comprised TH2- and eosinophil-related genes including ALOX15, FCER1A, and OLIG2. Network construction identified transcript modules that were coherently expressed at baseline and in non-remitting patients but were disrupted at early time points in remitting patients. These modules reflected processes such as interferon signaling, erythropoiesis, and platelet aggregation. DNA methylation analysis identified remission-specific temporal changes, which partially overlapped with transcriptomic signals. Machine learning approaches identified features from differentially expressed genes cis-linked to DNA methylation changes at week 2 as a robust predictor of therapy outcome at week 14, which was validated in a publicly available dataset of 20 infliximab-treated CD patients. CONCLUSIONS: Integrative multi-omics analysis reveals early shifts of gene expression and DNA methylation as predictors for efficient response to anti-TNF treatment. Lack of such signatures might be used to identify patients with IBD unlikely to benefit from TNF antagonists at an early time point.

Detailed Transcriptional Landscape of Peripheral Blood Points to Increased Neutrophil Activation in Treatment-Naïve Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Inflammatory bowel disease [IBD] is a chronic relapsing disorder of the gastrointestinal tract, which generally manifests as Crohn's disease [CD] or ulcerative colitis [UC]. These subtypes are heterogeneous in terms of disease location and histological features, while sharing common clinical presentation, genetic associations and, thus, common immune regulatory pathways. METHODS: Using miRNA and mRNA coupled transcriptome profiling and systems biology approaches, we report a comprehensive analysis of blood transcriptomes from treatment-naïve [n = 110] and treatment-exposed [n = 177] IBD patients as well as symptomatic [n = 65] and healthy controls [n = 95]. RESULTS: Broadly, the peripheral blood transcriptomes of CD and UC patients were similar. However, there was an extensive gene deregulation in the blood of IBD patients, while only a slight deregulation in symptomatic controls, when compared with healthy controls. The deregulated mRNAs and miRNAs are mainly involved in the innate immunity and are especially enriched in neutrophil activation-related pathways. Oxidative phosphorylation and neutrophil activation-related modules were found to be differentially co-expressed among treatment-naïve IBD as compared to healthy controls. In the deregulated neutrophil activation-related co-expression module, IL1B was identified as the central gene. Levels of co-expression among IL1B and chemosensing receptor [CXCR1/2 and FPR1/2] genes were reduced in the blood of IBD patients when compared with healthy controls. CONCLUSIONS: Immune dysregulation seen in peripheral blood transcriptomes of treatment-naïve IBD patients is mainly driven by neutrophil activation.

Humoral protection to SARS-CoV2 declines faster in patients on TNF alpha blocking therapies.

BACKGROUND: The persistence of the SARS-CoV2 pandemic, partly due to the appearance of highly infectious variants, has made booster vaccinations necessary for vulnerable groups. Questions remain as to which cohorts require SARS-CoV2 boosters. However, there is a critical lack of data on the dynamics of vaccine responses in patients with chronic inflammatory diseases (CID) undergoing immunosuppressive/disease modifying anti-rheumatic (DMARD) treatment. Here, we present the first data regarding the decline of the vaccine-induced humoral immune responses in patients with CID. METHODS: 23 patients with CID were monitored clinically and for anti-spike IgG and IgA levels, neutralization efficacy and antigen-specific CD4+ T cell responses over the first 6 months after SARS-CoV2 vaccination. 24 healthy individuals were included as controls. RESULTS: While anti-spike IgG-levels declined in CID patients and healthy controls, patients receiving anti-TNF treatment showed significantly greater declines at 6 months post second vaccination in IgG and especially neutralizing antibodies. IgA levels were generally lower in CID patients, particularly during anti-TNF therapy. No differences in SARS-CoV2 spike-specific CD4+ T-cell frequencies were detected. CONCLUSION: Although the long-term efficacy of SARS-CoV2 vaccination in CID patients undergoing disease-modifying therapy is still not known, the pronounced declines in humoral responses towards SARS-CoV2 6 months after mRNA vaccination in the context of TNF blockade should be considered when formulating booster regimens. These patients should be considered for early booster vaccinations.

Therapeutic Interleukin-6 Trans-signaling Inhibition by Olamkicept (sgp130Fc) in Patients With Active Inflammatory Bowel Disease.

BACKGROUND & AIMS: A large unmet therapeutic need exists in inflammatory bowel disease (IBD). Inhibition of interleukin (IL)-6 appears to be effective, but the therapeutic benefit of a complete IL6/IL6 receptor (IL6R) blockade is limited by profound immunosuppression. Evidence has emerged that chronic proinflammatory activity of IL6 is mainly mediated by trans-signaling via a complex of IL6 bound to soluble IL6R engaging the gp130 co-receptor without the need for membrane-bound IL6R. We have developed a decoy protein, sgp130Fc, that exclusively blocks IL6 proinflammatory trans-signaling and has shown efficacy in preclinical models of IBD, without signs of immunosuppression. METHODS: We present a 12-week, open-label, prospective phase 2a trial (FUTURE) in 16 patients with active IBD treated with the trans-signaling inhibitor olamkicept (sgp130Fc) to assess the molecular mechanisms, safety, and effectiveness of IL6 trans-signaling blockade in vivo. We performed in-depth molecular profiling at various timepoints before and after therapy induction to identify the mechanism of action of olamkicept. RESULTS: Olamkicept was well tolerated and induced clinical response in 44% and clinical remission in 19% of patients. Clinical effectiveness coincided with target inhibition (reduction of phosphorylated STAT3) and marked transcriptional changes in the inflamed mucosa. An olamkicept-specific transcriptional signature, distinguishable from remission signatures of anti-tumor necrosis factor (infliximab) or anti-integrin (vedolizumab) therapies was identified. CONCLUSIONS: Our data suggest that blockade of IL6 trans-signaling holds great promise for the therapy of IBD and should undergo full clinical development as a new immunoregulatory therapy for IBD. (EudraCT no., Nu 2016-000205-36).

Metabolic Functions of Gut Microbes Associate With Efficacy of Tumor Necrosis Factor Antagonists in Patients With Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Altered interactions between the mucosal immune system and intestinal microbiota contribute to pathogenesis of inflammatory bowel diseases (IBD). It is not clear how inhibitors of cytokines, such as antagonists of tumor necrosis factor (anti-TNF), affect the intestinal microbiome. We investigated the effects of anti-TNF agents on gut microbe community structure and function in a longitudinal 2-step study of patients with IBD. We correlated our findings with outcomes of treatment and investigated patterns of metabolites in fecal samples before and after anti-TNF therapy. METHODS: We performed a prospective study of 2 cohorts of patients in Germany; the discovery cohort comprised 12 patients with IBD, 17 patients with rheumatic disease, and 19 healthy individuals (controls); fecal samples were collected at baseline and 2, 6, and 30 weeks after induction of anti-TNF therapy. The validation cohort comprised 23 patients with IBD treated with anti-TNF or vedolizumab (anti-α4ß7 integrin) and 99 healthy controls; fecal samples were collected at baseline and at weeks 2, 6, and 14. Fecal microbiota were analyzed by V3-V4 16S ribosomal RNA gene amplicon sequencing. Clinical response and remission were determined by clinical disease activity scores. Metabolic network reconstruction and associated fecal metabolite level inference was performed in silico using the AGORA (Assembly of Gut Organisms through Reconstruction and Analysis) resource. Metabolomic analyses of fecal samples from a subset of patients were performed to validate metabolites associated with treatment outcomes. RESULTS: Anti-TNF therapy shifted the diversity of fecal microbiota in patients with IBD, but not with rheumatic disease, toward that of controls. Across timepoints, diversity indices did not vary significantly between patients with IBD who did or did not achieve clinical remission after therapy. In contrast, in silico modeling of metabolic interactions between gut microbes found metabolite exchange to be significantly reduced at baseline in fecal samples from patients with IBD and to be associated with later clinical remission. Predicted levels of butyrate and substrates involved in butyrate synthesis (ethanol or acetaldehyde) were significantly associated with clinical remission following anti-TNF therapy, verified by fecal metabolomic analyses. CONCLUSIONS: Metabolic network reconstruction and assessment of metabolic profiles of fecal samples might be used to identify patients with IBD likely to achieve clinical remission following anti-TNF therapy and increase our understanding of the heterogeneity of IBD.

Vedolizumab is associated with changes in innate rather than adaptive immunity in patients with inflammatory bowel disease.

OBJECTIVE: Vedolizumab, a monoclonal antibody directed against the integrin heterodimer α4ß7, is approved for the treatment of Crohn's disease and ulcerative colitis. The efficacy of vedolizumab has been suggested to result from inhibition of intestinal T cell trafficking although human data to support this conclusion are scarce. We therefore performed a comprehensive analysis of vedolizumab-induced alterations in mucosal and systemic immunity in patients with inflammatory bowel disease (IBD), using anti-inflammatory therapy with the TNFα antibody infliximab as control. DESIGN: Immunophenotyping, immunohistochemistry, T cell receptor profiling and RNA sequencing were performed using blood and colonic biopsies from patients with IBD before and during treatment with vedolizumab (n=18) or, as control, the anti-TNFα antibody infliximab (n=20). Leucocyte trafficking in vivo was assessed using single photon emission computed tomography and endomicroscopy. RESULTS: Vedolizumab was not associated with alterations in the abundance or phenotype of lamina propria T cells and did not affect the mucosal T cell repertoire or leucocyte trafficking in vivo. Surprisingly, however, α4ß7 antibody treatment was associated with substantial effects on innate immunity including changes in macrophage populations and pronounced alterations in the expression of molecules involved in microbial sensing, chemoattraction and regulation of the innate effector response. These effects were specific to vedolizumab, not observed in response to the TNFα antibody infliximab, and associated with inhibition of intestinal inflammation. CONCLUSION: Our findings suggest that modulation of innate immunity contributes to the therapeutic efficacy of vedolizumab in IBD. TRIAL REGISTRATION NUMBER: NCT02694588.

Evaluation of interleukin-6 and its soluble receptor components sIL-6R and sgp130 as markers of inflammation in inflammatory bowel diseases.

PURPOSE: Interleukin-6 (IL-6) production and signalling are increased in the inflamed mucosa in inflammatory bowel diseases (IBD). As published serum levels of IL-6 and its soluble receptors sIL-6R and sgp130 in IBD are from small cohorts and partly contradictory, we systematically evaluated IL-6, sIL-6R and sgp130 levels as markers of disease activity in Crohn's disease (CD) and ulcerative colitis (UC). METHODS: Consecutive adult outpatients with confirmed CD or UC were included, and their disease activity and medication were monitored. Serum from 212 CD patients (815 measurements) and 166 UC patients (514 measurements) was analysed, and 100 age-matched healthy blood donors were used as controls. RESULTS: IL-6 serum levels were significantly elevated in active versus inactive CD and UC, also compared with healthy controls. However, only a fraction of IBD patients showed increased serum IL-6. IL-6 levels ranged up to 32.7 ng/mL in active CD (> 5000-fold higher than in controls), but also up to 6.9 ng/mL in inactive CD. Increases in active UC (up to 195 pg/mL) and inactive UC (up to 27 pg/mL) were less pronounced. Associations between IL-6 serum levels and C-reactive protein concentrations as well as leukocyte and thrombocyte counts were observed. Median sIL-6R and sgp130 levels were only increased by up to 15%, which was considered of no diagnostic significance. CONCLUSIONS: Only a minority of IBD patients shows elevated IL-6 serum levels. However, in these patients, IL-6 is strongly associated with disease activity. Its soluble receptors sIL-6R and sgp130 do not appear useful as biomarkers in IBD.

Mucus Detachment by Host Metalloprotease Meprin ß Requires Shedding of Its Inactive Pro-form, which Is Abrogated by the Pathogenic Protease RgpB.

The host metalloprotease meprin ß is required for mucin 2 (MUC2) cleavage, which drives intestinal mucus detachment and prevents bacterial overgrowth. To gain access to the cleavage site in MUC2, meprin ß must be proteolytically shed from epithelial cells. Hence, regulation of meprin ß shedding and activation is important for physiological and pathophysiological conditions. Here, we demonstrate that meprin ß activation and shedding are mutually exclusive events. Employing ex vivo small intestinal organoid and cell culture experiments, we found that ADAM-mediated shedding is restricted to the inactive pro-form of meprin ß and is completely inhibited upon its conversion to the active form at the cell surface. This strict regulation of meprin ß activity can be overridden by pathogens, as demonstrated for the bacterial protease Arg-gingipain (RgpB). This secreted cysteine protease potently converts membrane-bound meprin ß into its active form, impairing meprin ß shedding and its function as a mucus-detaching protease.

Increased Tryptophan Metabolism Is Associated With Activity of Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Administration of tryptophan and some of its metabolites reduces the severity of colitis in mice, whereas removing tryptophan from the diet increases susceptibility to colitis. Transfer of the intestinal microbiome transfers the colitogenic phenotype from tryptophan starved animals to normally nourished mice. We aimed to systematically evaluate serum levels of tryptophan and its metabolites in patients with inflammatory bowel diseases (IBD), and study their association with clinical and serologic features. METHODS: We studied 535 consecutive patients with IBD (211 with ulcerative colitis [UC], 234 with Crohn's disease [CD]; 236 male), enrolled in Germany from August 2013 through April 2014 and followed until July 2016. Serum samples were collected from patients and 291 matched individuals without IBD (controls); levels of tryptophan were measured using high-performance liquid chromatography. Metabolites of tryptophan were measured in serum from 148 patients and 100 controls by mass spectrometry. We measured levels of interleukin 22 in serum from 28 patients by enzyme-linked immunosorbent assay. Paired stool and serum samples were collected from a subset of patients with active UC (n = 10) or CD (n = 8) to investigate associations between serum levels of tryptophan and composition of the fecal microbiota, analyzed by 16S ribosomal DNA amplicon sequencing. We used real-time polymerase chain reaction to measure levels of messenger RNAs in colonic biopsies from 60 patients with UC, 50 with CD, and 30 controls. We collected information on patients' disease activity scores, medications, laboratory assessments, and clinical examinations during recruitment and follow-up visits. RESULTS: Serum levels of tryptophan were significantly lower in patients with IBD than in controls (P = 5.3 × 10-6) with a stronger reduction in patients with CD (vs control; P = 1.1 × 10-10) than UC (vs control; P = 2.8 × 10-3). We found a negative correlation between serum levels of tryptophan and disease activity or levels of C-reactive protein. Levels of messenger RNAs encoding tryptophan 2,3-dioxygenase-2 and solute carrier family 6 member 19 (also called B0AT1) were significantly decreased in colonic biopsies from patients with IBD compared with controls, whereas level of messenger RNA encoding indoleamine 2,3-dioxygenase-1 was significantly increased. The composition of the fecal microbiota associated with serum levels of tryptophan. Analysis of tryptophan metabolites revealed activation of the kynurenine pathway, based on high levels of quinolinic acid, in patients with IBD compared with controls. Serum concentration of interleukin 22 associated with disease activity in patients with IBD; there was an inverse association between levels of interleukin 22 and serum levels of tryptophan. CONCLUSIONS: In an analysis of serum samples from more than 500 patients with IBD, we observed a negative correlation between serum levels of tryptophan and disease activity. Increased levels of tryptophan metabolites-especially of quinolinic acid-indicated a high activity of tryptophan degradation in patients with active IBD. Tryptophan deficiency could contribute to development of IBD or aggravate disease activity. Interventional clinical studies are needed to determine whether modification of intestinal tryptophan pathways affects the severity of IBD.

Genome-wide association analysis for chronic venous disease identifies EFEMP1 and KCNH8 as susceptibility loci.

Chronic venous disease (CVD) is a multifactorial condition representing one of the most common disorders among populations of Western countries. The heritability of about 17% suggests genetic risk factors in CVD etiology. However, so far the genetic causes are unknown. We undertook the hitherto first genome-wide association study (GWAS) for CVD, analyzing more than 1.93 M SNPs in 4,942 German individuals, followed by replication in two independent German data sets. The combined analysis of discovery and replication stages (2,269 cases and 7,765 controls) yielded robust associations within the two genes EFEMP1 and KCNH8 (rs17278665, rs727139 with P < 5 × 10-8), and suggestive association within gene SKAP2 (rs2030136 with P < 5 × 10-7). Association signals of rs17278665 and rs727139 reside in regions of low linkage disequilibrium containing no other genes. Data from the ENCODE and Roadmap Epigenomics projects show that tissue specific marks overlap with the variants. SNPs rs17278665 and rs2030136 are known eQTLs. Our study demonstrates that GWAS are a valuable tool to study the genetic component of CVD. With our approach, we identified two novel genome-wide significant susceptibility loci for this common disease. Particularly, the extracellular matrix glycoprotein EFEMP1 is promising for future functional studies due to its antagonistic role in vessel development and angiogenesis.

Uncoupling of mucosal gene regulation, mRNA splicing and adherent microbiota signatures in inflammatory bowel disease.

OBJECTIVE: An inadequate host response to the intestinal microbiota likely contributes to the manifestation and progression of human inflammatory bowel disease (IBD). However, molecular approaches to unravelling the nature of the defective crosstalk and its consequences for intestinal metabolic and immunological networks are lacking. We assessed the mucosal transcript levels, splicing architecture and mucosa-attached microbial communities of patients with IBD to obtain a comprehensive view of the underlying, hitherto poorly characterised interactions, and how these are altered in IBD. DESIGN: Mucosal biopsies from Crohn's disease and patients with UC, disease controls and healthy individuals (n=63) were subjected to microbiome, transcriptome and splicing analysis, employing next-generation sequencing. The three data levels were integrated by different bioinformatic approaches, including systems biology-inspired network and pathway analysis. RESULTS: Microbiota, host transcript levels and host splicing patterns were influenced most strongly by tissue differences, followed by the effect of inflammation. Both factors point towards a substantial disease-related alteration of metabolic processes. We also observed a strong enrichment of splicing events in inflamed tissues, accompanied by an alteration of the mucosa-attached bacterial taxa. Finally, we noted a striking uncoupling of the three molecular entities when moving from healthy individuals via disease controls to patients with IBD. CONCLUSIONS: Our results provide strong evidence that the interplay between microbiome and host transcriptome, which normally characterises a state of intestinal homeostasis, is drastically perturbed in Crohn's disease and UC. Consequently, integrating multiple OMICs levels appears to be a promising approach to further disentangle the complexity of IBD.

Diagnosing fatty liver disease: a comparative evaluation of metabolic markers, phenotypes, genotypes and established biomarkers.

BACKGROUND: To date, liver biopsy is the only means of reliable diagnosis for fatty liver disease (FLD). Owing to the inevitable biopsy-associated health risks, however, the development of valid noninvasive diagnostic tools for FLD is well warranted. AIM: We evaluated a particular metabolic profile with regard to its ability to diagnose FLD and compared its performance to that of established phenotypes, conventional biomarkers and disease-associated genotypes. METHODS: The study population comprised 115 patients with ultrasound-diagnosed FLD and 115 sex- and age-matched controls for whom the serum concentration was measured of 138 different metabolites, including acylcarnitines, amino acids, biogenic amines, hexose, phosphatidylcholines (PCs), lyso-PCs and sphingomyelins. Established phenotypes, biomarkers, disease-associated genotypes and metabolite data were included in diagnostic models for FLD using logistic regression and partial least-squares discriminant analysis. The discriminative power of the ensuing models was compared with respect to area under curve (AUC), integrated discrimination improvement (IDI) and by way of cross-validation (CV). RESULTS: Use of metabolic markers for predicting FLD showed the best performance among all considered types of markers, yielding an AUC of 0.8993. Additional information on phenotypes, conventional biomarkers or genotypes did not significantly improve this performance. Phospholipids and branched-chain amino acids were most informative for predicting FLD. CONCLUSION: We show that the inclusion of metabolite data may substantially increase the power to diagnose FLD over that of models based solely upon phenotypes and conventional biomarkers.

Association between variants of PRDM1 and NDP52 and Crohn's disease, based on exome sequencing and functional studies.

BACKGROUND & AIMS: Genome-wide association studies (GWAS) have identified 140 Crohn's disease (CD) susceptibility loci. For most loci, the variants that cause disease are not known and the genes affected by these variants have not been identified. We aimed to identify variants that cause CD through detailed sequencing, genetic association, expression, and functional studies. METHODS: We sequenced whole exomes of 42 unrelated subjects with CD and 5 healthy subjects (controls) and then filtered single nucleotide variants by incorporating association results from meta-analyses of CD GWAS and in silico mutation effect prediction algorithms. We then genotyped 9348 subjects with CD, 2868 subjects with ulcerative colitis, and 14,567 control subjects and associated variants analyzed in functional studies using materials from subjects and controls and in vitro model systems. RESULTS: We identified rare missense mutations in PR domain-containing 1 (PRDM1) and associated these with CD. These mutations increased proliferation of T cells and secretion of cytokines on activation and increased expression of the adhesion molecule L-selectin. A common CD risk allele, identified in GWAS, correlated with reduced expression of PRDM1 in ileal biopsy specimens and peripheral blood mononuclear cells (combined P = 1.6 × 10(-8)). We identified an association between CD and a common missense variant, Val248Ala, in nuclear domain 10 protein 52 (NDP52) (P = 4.83 × 10(-9)). We found that this variant impairs the regulatory functions of NDP52 to inhibit nuclear factor κB activation of genes that regulate inflammation and affect the stability of proteins in Toll-like receptor pathways. CONCLUSIONS: We have extended the results of GWAS and provide evidence that variants in PRDM1 and NDP52 determine susceptibility to CD. PRDM1 maps adjacent to a CD interval identified in GWAS and encodes a transcription factor expressed by T and B cells. NDP52 is an adaptor protein that functions in selective autophagy of intracellular bacteria and signaling molecules, supporting the role of autophagy in the pathogenesis of CD.

mTNF reverse signalling induced by TNFα antagonists involves a GDF-1 dependent pathway: implications for Crohn's disease.

OBJECTIVE: Mechanisms of action (MoA) of anti-tumour necrosis factor α (TNFα) therapies in Crohn's disease (CD) may critically involve induction of immune cell apoptosis via membrane-bound TNFα (mTNFα) binding. Certolizumab pegol (CZP), which is effective in induction and maintenance of remission in CD lacks the ability to induce apoptosis. The aim of this study was to analyse transcriptomal responses of reverse signalling induced by the TNFα binding agents infliximab (IFX) and CZP in myelomonocytic cells. DESIGN: Induction of transcriptional patterns upon anti-TNFα stimulation was assessed using oligonucleotide microarrays. mRNA expression of GDF-1/ LASS1, which was identified as a shared target, was studied in inflammatory bowel disease by real-time PCR, while signalling pathways induced by growth and differentiation factor 1 (GDF-1) were investigated using western blots and ELISA. RESULTS: IFX and CZP induced a common signature of 20 transcripts that could be categorised into control of cell cycle, transcription activation and pre-mRNA processing. We selected GDF-1/LASS1 for functional follow-up, which was found to be upregulated in inflamed CD tissues. We show that downregulation of GDF-1/LASS1 depends on autocrine release of transforming growth factor ß after mTNFα ligation. We demonstrate that GDF-1 itself acts as a novel proinflammatory factor via induction of interleukin 6 and signal transducer and activator of transcription 3 and is downregulated after IFX treatment. CONCLUSION: Commonalities in the MoA of IFX and CZP comprise modulation of non-apoptotic pathways through downregulation of proinflammatory GDF-1. Further characterisation of the molecular role of GDF-1 in complex inflammatory processes in vivo is warranted to decide whether this proinflammatory molecule is a promising therapeutic target in patients with CD.

Combined analysis of genome-wide association studies for Crohn disease and psoriasis identifies seven shared susceptibility loci.

Psoriasis (PS) and Crohn disease (CD) have been shown to be epidemiologically, pathologically, and therapeutically connected, but little is known about their shared genetic causes. We performed meta-analyses of five published genome-wide association studies on PS (2,529 cases and 4,955 controls) and CD (2,142 cases and 5,505 controls), followed up 20 loci that showed strongest evidence for shared disease association and, furthermore, tested cross-disease associations for previously reported PS and CD risk alleles in additional 6,115 PS cases, 4,073 CD cases, and 10,100 controls. We identified seven susceptibility loci outside the human leukocyte antigen region (9p24 near JAK2, 10q22 at ZMIZ1, 11q13 near PRDX5, 16p13 near SOCS1, 17q21 at STAT3, 19p13 near FUT2, and 22q11 at YDJC) shared between PS and CD with genome-wide significance (p < 5 × 10(-8)) and confirmed four already established PS and CD risk loci (IL23R, IL12B, REL, and TYK2). Three of the shared loci are also genome-wide significantly associated with PS alone (10q22 at ZMIZ1, p(rs1250544) = 3.53 × 10(-8), 11q13 near PRDX5, p(rs694739) = 3.71 × 10(-09), 22q11 at YDJC, p(rs181359) = 8.02 × 10(-10)). In addition, we identified one susceptibility locus for CD (16p13 near SOCS1, p(rs4780355) = 4.99 × 10(-8)). Refinement of association signals identified shared genome-wide significant associations for exonic SNPs at 10q22 (ZMIZ1) and in silico expression quantitative trait locus analyses revealed that the associations at ZMIZ1 and near SOCS1 have a potential functional effect on gene expression. Our results show the usefulness of joint analyses of clinically distinct immune-mediated diseases and enlarge the map of shared genetic risk loci.

Anti-TNF-α antibodies improve intestinal barrier function in Crohn's disease.

BACKGROUND AND AIMS: Intestinal barrier function in Crohn's disease patients and their first degree healthy relatives is impaired. The increased intestinal permeability may result in an enhanced mucosal immune response and thereby aggravate intestinal inflammation. Humanised anti-TNF-α antibodies have been shown to be effective in the treatment of active Crohn's disease and in the treatment of entero-cutaneous fistula. The aim of the present study was to investigate the influence of anti-TNF-α antibody (infliximab) treatment on the intestinal barrier function of patients with active Crohn's disease. METHODS: The differential intestinal uptake of lactulose and mannitol was measured to quantify intestinal permeability in patients with long standing active Crohn's disease (n=17) directly before and seven days after treatment with infliximab (5 mg/kg bodyweight). In parallel, intestinal permeability was studied in a healthy control group (n=20). Serum samples were analysed with pulsed amperometric detection after separation on an anion exchange column. RESULTS: Intestinal permeability was significantly increased in all patients with Crohn's disease (L/M ratio 0.24±0.17) prior to infliximab treatment compared to the control group (L/M ratio 0.01±0.02; p-value <1×10(-7)). Treatment of patients with infliximab resulted in a marked decrease of intestinal permeability as measured by L/M ratio from 0.24±0.17 before to 0.02±0.02 (p-value <1×10(-7)) seven days after infliximab application. CONCLUSIONS: Treatment with anti-TNF-α antibodies improved impaired intestinal barrier function in patients with Crohn's disease. This effect may correlate to the well documented anti-inflammatory effect of TNF-α blockade in this intestinal disease.