Persistent Diarrhea in Patients With Crohn's Disease After Mucosal Healing Is Associated With Lower Diversity of the Intestinal Microbiome and Increased Dysbiosis.

BACKGROUND & AIMS: In patients with inflammatory bowel diseases (IBDs), symptoms do not always associate with the severity of endoscopic inflammation and can persist after mucosal healing. We investigated whether symptoms in patients with successfully treated IBD are related to the composition of the intestinal microbiome. METHODS: We analyzed 590 tissue biopsy specimens from 215 patients with IBD and 48 healthy individuals (controls). We obtained mucosal biopsy specimens from 2 colon sites (ascending and rectosigmoid) and from the terminal ileum along with clinical data. Bacterial DNA was extracted from the biopsy specimens and the V4 region of 16s ribosomal RNA sequenced by Miseq and processed using the QIIME v1.9 pipeline. RESULTS: Mucosal biopsy specimens from patients with Crohn's disease (CD) who achieved mucosal healing (Mayo scores of 0-1 or segmental endoscopic severity CD scores of 0-5) had lower Chao1 diversity than biopsy specimens from patients with ulcerative colitis (UC) or unclassified IBD (IBD-U), or controls. After endoscopic evidence of improvement in patients with UC or IBD-U, diversity of the tissue-associated microbiota did not differ significantly from that of controls. Colon biopsy specimens from patients with CD had lower microbial diversity, before and after healing (segmental endoscopic severity CD scores, 0-2), than colon biopsy specimens from controls (P < .002). In patients with CD who achieved mucosal healing, residual clinical activity (CD activity index scores >150; P = .03) and persistent diarrhea were associated with reduced microbial diversity (P = .01). Continued diarrhea was associated with a trend toward dysbiosis, based on the microbial dysbiosis index (P = .059). In patients with UC or IBD-U with moderate to severe inflammation, increasing severity of diarrhea was associated with reduced microbial diversity (P = .03). CONCLUSIONS: In an analysis of biopsy specimens from patients with IBD and controls, we found that despite endoscopic evidence of improvement or remission, α-diversity of the tissue-associated intestinal microbiome remained lower in patients with CD than in controls. This observation, along with the reduced Chao1 diversity and greater dysbiosis in intestinal microbiota of patients with residual symptoms of IBD, indicates that microbiome composition could be associated with persistent diarrhea.

Combined Histo-endoscopic Remission but not Endoscopic Healing Alone in Ulcerative Colitis is Associated with a Mucosal Transcriptional Profile Resembling Healthy Mucosa.

BACKGROUND AND AIMS: A composite endpoint of histological and endoscopic remission is proposed to be the most complete measure of mucosal healing in ulcerative colitis [UC]. We aim to establish the prognosis, and transcriptional and microbial features of histo-endoscopic remission and activity. METHODS: A cross-sectional endoscopic rectosigmoid colon sample collection from UC patients and healthy controls [HC] was performed for histopathology and host genome-wide RNA-sequencing. Histo-endoscopic remission and histo-endoscopic activity were defined as Mayo endoscopic subscore [MES] 0-1 with and without histological activity, respectively. UC relapse, defined as symptomatic and endoscopic worsening, was retrospectively recorded for survival analysis. Unsupervised and differential gene expression analyses were performed, and the interaction between transcriptomics and mucosal gut microbiota was analysed based on the 16S rRNA gene sequencing profile. RESULTS: UC patients with histo-endoscopic remission showed a significantly lower risk of relapse compared to histo-endoscopic activity. Unsupervised analysis of the transcriptomic profile showed that histo-endoscopic remission and histo-endoscopic activity samples clustered with HC and MES 2-3 samples, respectively. A total of 452 host genes enriched for humoral immune response, antimicrobial defence, chemokine and TH17 signalling pathway were upregulated in histo-endoscopic activity compared to histo-endoscopic remission. A set of host genes with antimicrobial properties showed significant associations with mucosal microbiota. CONCLUSIONS: The rectosigmoid mucosa transcriptional profile of UC patients in histo-endoscopic remission resembles that of HC mucosa and confers a lower risk of relapse. These data support that the combination of histo-endoscopic remission could be the most appropriate definition of mucosal healing in UC.

Ustekinumab induction concentrations are associated with clinical and biochemical outcomes at week 12 of treatment in Crohn's disease.

BACKGROUND: We investigated relationships between induction ustekinumab levels and clinical and biochemical outcomes in Crohn's disease. METHODS: Following standard IV induction, ustekinumab levels were measured at week 2 (wk2) and week 6 (wk6). Ustekinumab levels were compared in patients receiving 260, 390 and 520 mg at induction. Crohn's disease activity index (CDAI), serum albumin, C-reactive protein (CRP) and fecal calprotectin (FCP) were measured at baseline and week 12 (wk12). Associations between ustekinumab levels and these parameters were assessed. Ustekinumab levels were compared between patients requiring dose intensification within one year of induction and those remaining on standard dosing. RESULTS: Of 23 wk2 ustekinumab levels, 22(95.7%) were above the upper limit of quantification of the assay (25 µg/mL). Median wk6 ustekinumab level (n = 25) was 14.2 µg/mL [interquartile range (IQR), 9.6-20.1]. Median wk6 ustekinumab levels in patients receiving 260, 390 and 520 mg were 8.6, 16.3 and 25.0 µg/mL, respectively, P = 0.01. There were significant correlations between baseline albumin and wk6 ustekinumab levels; r = 0.644 [95% confidence interval (CI), 0.304-0.839], P < 0.001, and between baseline FCP and wk6 ustekinumab levels; r = -0.678 (95% CI, -0.873 to -0.296), P < 00.01. Median wk12 CDAI (n = 18), CRP (n = 22) and FCP (n = 13) were 78 (IQR, 52.5-152), 1.75 mg/L (IQR, 0.93-7.03) and 746 µg/g (IQR, 259-2100), respectively. There were significant correlations between wk6 ustekinumab levels and wk12 CDAI; r = -0.513 (95% CI, -0.796 to -0.046), P = 0.03; and between wk6 ustekinumab levels and wk12 CRP; r = -0.578 (95% CI, -0.808 to -0.194), P < 0.01. Wk6 ustekinumab levels were lower in patients undergoing subsequent dose intensification; 12.5 vs. 19.6 µg/mL, P = 0.04. CONCLUSION: Wk6 ustekinumab levels are significantly associated with baseline Crohn's disease biomarkers and subsequent clinical and biochemical outcomes.

Integrative Analysis of Colonic Biopsies from Inflammatory Bowel Disease Patients Identifies an Interaction Between Microbial Bile Acid-inducible Gene Abundance and Human Angiopoietin-like 4 Gene Expression.

BACKGROUND AND AIMS: Microbial-derived bile acids can modulate host gene expression, and their faecal abundance is decreased in active inflammatory bowel disease [IBD]. We analysed the impact of endoscopic inflammation on microbial genes involved in bile acid biotransformation, and their interaction with host transcriptome in the intestinal mucosa of IBD patients. METHODS: Endoscopic mucosal biopsies were collected from non-inflamed and inflamed terminal ileum, ascending and sigmoid colon of IBD patients. Prediction of imputed metagenome functional content from 16S rRNA profile and real-time quantitative polymerase chain reaction [qPCR] were utsed to assess microbial bile acid biotransformation gene abundance, and RNA-seq was used for host transcriptome analysis. Linear regression and partial Spearman correlation accounting for age, sex, and IBD type were used to assess the association between microbial genes, inflammation, and host transcriptomics in each biopsy location. A Bayesian network [BN] analysis was fitted to infer the direction of interactions between IBD traits and microbial and host genes. RESULTS: The inferred microbial gene pathway involved in secondary bile acid biosynthesis [ko00121 pathway] was depleted in inflamed terminal ileum of IBD patients compared with non-inflamed tissue. In non-inflamed sigmoid colon, the relative abundance of bile acid-inducible [baiCD] microbial genes was positively correlated with the host Angiopoietin-like 4 [Angptl4] gene expression. The BN analysis suggests that the microbial baiCD gene abundance could affect Angptl4 expression, and this interaction appears to be lost in the presence of inflammation. CONCLUSIONS: Endoscopic inflammation affects the abundance of crucial microbial bile acid-metabolising genes and their interaction with Angptl4 in intestinal mucosa of IBD patients.

Serum Ustekinumab Concentrations Are Associated With Remission in Crohn's Disease Defined by a Serum-Based Endoscopic Healing Index.

Background: Optimal ustekinumab levels (UST) in Crohn disease (CD) treatment have not been defined. We set out to define the optimal UST to differentiate between remission and active CD, as defined using the serum-based endoscopic healing index (EHI). Methods: Paired serum UST and EHI tests were analyzed. Remission was defined as EHI <20. Active disease was defined as EHI ≥50. The proportion of patients in remission was compared across UST quartiles. UST in subjects with EHI <20 and EHI ≥50 were compared. An area under receiver operating characteristic curve was generated to identify an optimal UST to differentiate between active disease and remission. Results: A total of 337 unique patients were identified; median UST and EHI were 5.0 µg/mL [interquartile range (IQR) 2.7-9.1] and 37 (IQR 26-53), respectively. EHI <20 (remission) was found in 57 (16.9%) patients. EHI ≥50 (active disease) was found in 97 (28.8%) patients. Higher proportions of subjects were in remission for increasing UST quartiles, P = 0.01. Median UST in patients with EHI <20 and EHI ≥50 were 7.5 µg/mL (IQR 4.6-10.9) and 3.1 µg/mL (IQR 1.8-6.6), respectively, P < 0.001. An UST threshold of 3.75 µg/mL optimally differentiated between active disease and remission (area under the curve 0.725). UST levels >3.75 µg/mL were associated with a lower proportion of subjects with active disease (EHI ≥50; 18.9%) compared with UST levels ≤3.75 µg/mL (45.6%, P < 0.001). Conclusions: Using the EHI, we identified a threshold UST level of 3.75 µg/mL to optimally differentiate between active and quiescent CD. These data suggest that UST serum concentrations of >3.75 µg/mL are optimally associated with endoscopic remission in CD.

Mucosa-Associated Microbiota in Ileoanal Pouches May Contribute to Clinical Symptoms, Particularly Stool Frequency, Independent of Endoscopic Disease Activity.

INTRODUCTION: Pouchitis is a common complication after ileal pouch-anal anastomosis (IPAA). However, there is a poor correlation between symptoms and endoscopic appearance of the pouch, and many patients can have debilitating symptoms in the absence of overt inflammation. It is unknown whether these clinical symptoms are independently associated with the microbiota. The objective of this work was to examine whether the individual clinical components of the pouch activity scoring systems are associated with specific microbiota. METHODS: Pouch biopsies from 233 patients (50% male, 100% IPAA/ulcerative colitis) post-IPAA were included. Clinical phenotyping was performed, and patients were classified using both clinical and endoscopic components of the Pouch Activity Scale. Scoring for symptoms examined 24-hour stool frequency, urgency, incontinence, and rectal bleeding as described by the Pouchitis Disease Activity Index Score. RESULTS: In the absence of inflammation, an increase in stool frequency reported over 24 hours was associated with a decrease in Bacteroidetes relative abundance, and this was the strongest association found. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis in inflamed groups showed that an increase in 24-hour stool frequency was associated with an increase in biofilm formation. DISCUSSION: These findings indicate that in patients with IPAA, the composition of mucosa-associated microbiota of the pouch may contribute to clinical symptoms, particularly stool frequency, independent of endoscopic disease activity.

The Unsolved Link of Genetic Markers and Crohn's Disease Progression: A North American Cohort Experience.

BACKGROUND: While progress has been made in the identification of Crohn's disease (CD) susceptibility loci, efforts to identify a genetic basis for disease progression have been less fruitful. The specific aim of this study was to build upon the major genetic advances made in IBD by applying genome-wide technologies toward predicting disease progression in CD. METHODS: Crohn's disease cases (n = 1495) from 3 IBD centers were reviewed by experienced physicians. Clinical and demographic details were collected, focusing on the time to first disease progression. Genome-wide association (GWA) analysis was carried out on 3 clinical outcomes: 1) time to disease progression; 2) time to first abdominal surgery; and 3) a binary analysis of indolent vs progressive disease. Cox-proportional hazard and logistic regression models were used. RESULTS: A GWA analysis was carried out to determine any genetic variation associated with the time to disease progression; 662 cases were included after quality control (QC) and exclusion of any cases with B2/B3 behavior at baseline (n = 450). There were 1360 cases included after QC in the time to abdominal surgery analysis. No variant reached genome-wide significance in any of the 3 analyses performed. Eight known IBD susceptibility single nucleotide polymorphism (SNPs) were found to be associated with time-to-abdominal surgery SMAD3 (rs17293632), CCR6 (rs1819333), CNTF (rs11229555), TSPAN14 (rs7097656), CARD9 (rs10781499), IPMK (rs2790216), IL10 (rs3024505), and SMURF1 (rs9297145) (P < 0.05). CONCLUSION: Our GWA study failed to show any SNP-phenotype association reaching genome-wide significance. It is likely that multiple variables affect disease progression, with genetic factors potentially having only a small effect size.

IBD Genetic Risk Profile in Healthy First-Degree Relatives of Crohn's Disease Patients.

BACKGROUND: Family history provides important information on risk of developing inflammatory bowel disease [IBD], and genetic profiling of first-degree relatives [FDR] of Crohn's disease [CD]- affected individuals might provide additional information. We aimed to delineate the genetic contribution to the increased IBD susceptibility observed in FDR. METHODS: N = 976 Caucasian, healthy, non-related FDR; n = 4997 independent CD; and n = 5000 healthy controls [HC]; were studied. Genotyping for 158 IBD-associated single nucleotide polymorphisms [SNPs] was performed using the Illumina Immunochip. Risk allele frequency [RAF] differences between FDR and HC cohorts were correlated with those between CD and HC cohorts. CD and IBD genetic risk scores [GRS] were calculated and compared between HC, FDR, and CD cohorts. RESULTS: IBD-associated SNP RAF differences in FDR and HC cohorts were strongly correlated with those in CD and HC cohorts, correlation coefficient 0.63 (95% confidence interval [CI] 0.53 - 0.72), p = 9.90 x 10(-19). There was a significant increase in CD-GRS [mean] comparing HC, FDR, and CD cohorts: 0.0244, 0.0250, and 0.0257 respectively [p < 1.00 x 10(-7) for each comparison]. There was no significant difference in the IBD-GRS between HC and FDR cohorts [p = 0.81]; however, IBD-GRS was significantly higher in CD compared with FDR and HC cohorts [p < 1.00 x 10(-10) for each comparison]. CONCLUSION: FDR of CD-affected individuals are enriched with IBD risk alleles compared with HC. Cumulative CD-specific genetic risk is increased in FDR compared with HC. Prospective studies are required to determine if genotyping would facilitate better risk stratification of FDR.

Visual comparison of multiple gene expression datasets in a genomic context.

The need for novel methods of visualizing microarray data is growing. New perspectives are beneficial to finding patterns in expression data. The Bluejay genome browser provides an integrative way of visualizing gene expression datasets in a genomic context. We have now developed the functionality to display multiple microarray datasets simultaneously in Bluejay, in order to provide researchers with a comprehensive view of their datasets linked to a graphical representation of gene function. This will enable biologists to obtain valuable insights on expression patterns, by allowing them to analyze the expression values in relation to the gene locations as well as to compare expression profiles of related genomes or of different experiments for the same genome.