After surgically induced remission, ileal and colonic mucosa-associated microbiota predicts Crohn's disease recurrence.

BACKGROUND AND AIMS: Investigating the tissue-associated microbiota after surgically induced remission may help to understand the mechanisms initiating intestinal inflammation in Crohn's disease. METHODS: Crohn's disease patients undergoing ileocolic resection were prospectively recruited in six academic centers. Biopsy samples from the neoterminal ileum, colon and rectosigmoid were obtained from colonoscopies performed after surgery. Microbial DNA was extracted for 16S rRNA gene sequencing. Microbial diversity and taxonomic differential relative abundance were analyzed. A random forest model was applied to analyze the performance of clinical and microbial features to predict recurrence. A Rutgeerts score ≥i2 was deemed as endoscopic recurrence. RESULTS: A total of 349 postoperative colonoscopies and 944 biopsy samples from 262 Crohn's disease patients were analyzed. Ileal inflammation accounted for most of the explained variance of the ileal and colonic mucosa-associated microbiota. Samples obtained from 97 patients who were in surgically induced remission at first postoperative colonoscopy who went on to develop endoscopic recurrence at second colonoscopy showed lower diversity and microbial deviations when compared to patients who remained in endoscopic remission. Depletion of genus Anaerostipes and increase of several genera from class Gammaproteobacteria at the three biopsy sites increase the risk of further recurrence. Gut microbiome was able to predict future recurrence better than clinical features. CONCLUSION: Ileal and colonic mucosa-associated microbiome deviations precede development of new onset ileal inflammation after surgically induced remission and show good predictive performance for future recurrence. These findings suggest that targeted microbial modulation is a plausible modality to prevent postoperative Crohn's disease recurrence.

Inflammatory bowel disease associated with primary sclerosing cholangitis is associated with an altered gut microbiome and bile acid profile.

BACKGROUND: Primary sclerosing cholangitis associated with inflammatory bowel disease (IBD-PSC) carries significant morbidity compared to IBD without PSC. Alterations in microbial composition and bile acid (BA) profiles have been shown to modulate chronic inflammation in IBD, but data in IBD-PSC is scarce. We aimed to assess the differences in gut microbiome composition as well as in the BA profile and BA-related microbial functions between IBD-PSC and IBD-only. METHODS: 54 IBD-PSC and 62 IBD-only subjects were enrolled from 2012 to 2021. Baseline samples were collected for fecal DNA shotgun metagenomic sequencing, fecal and serum BA quantitation using mass spectrometry and fecal calprotectin. Liver fibrosis measured by transient elastography (TE) was assessed in the IBD-PSC group. Data was analyzed using general linear regression models and Spearman rank correlation tests. RESULTS: Patients with IBD-PSC had reduced microbial gene richness (p=0.004) and significant compositional shifts (PERMANOVA: R2=0.01, p=0.03) compared to IBD-only. IBD-PSC was associated with altered microbial composition and function, including decreased abundance of Blautia obeum, increased abundance of Veillonella atypica, Veillonella dispar and Clostridium scindens (q<0.05 for all), and increased abundance of microbial genes involved in secondary BA metabolism. Decreased serum sulfated and increased serum conjugated secondary BA were associated with IBD-PSC and increased liver fibrosis. CONCLUSION: We identified differences in microbial species, functional capacity and serum BA profiles in IBD-PSC compared with IBD-only. Our findings provide insight into the pathophysiology of IBD associated with PSC and suggest possible targets for modulating the risk and course of IBD in subjects with PSC.

Faecal microbiota of schoolchildren is associated with nutritional status and markers of inflammation: a double-blinded cluster-randomized controlled trial using multi-micronutrient fortified rice.

Faecal microbiota plays a critical role in human health, but its relationship with nutritional status among schoolchildren remains under-explored. Here, in a double-blinded cluster-randomized controlled trial on 380 Cambodian schoolchildren, we characterize the impact of six months consumption of two types of rice fortified with different levels of vitamins and minerals on pre-specified outcomes. We investigate the association between the faecal microbiota (16SrRNA sequencing) and age, sex, nutritional status (underweight, stunting), micronutrient status (iron, zinc and vitamin A deficiencies, anaemia, iron deficient anaemia, hemoglobinopathy), inflammation (systemic, gut), and parasitic infection. We show that the faecal microbiota is characterised by a surprisingly high proportion of Lactobacillaceae. We discover that deficiencies in specific micronutrients, such as iron and vitamin A, correlate with particular microbiota profiles, whereas zinc deficiency shows no such association. The nutritional intervention with the two rice treatments impacts both the composition and functions predicted from compositional analysis in different ways. (ClinicalTrials.gov (Identifier: NCT01706419)).

Environmental Factors Associated with Risk of Crohn's Disease Development in the Crohn's and Colitis Canada - Genetic, Environmental, Microbial Project.

BACKGROUND & AIMS: To date, it is unclear how environmental factors influence Crohn's disease (CD) risk and how they interact with biological processes. This study investigates the association between environmental exposures and CD risk and evaluates their association with pre-disease biomarkers. METHODS: We studied 4289 healthy first-degree relatives (FDRs) of patients with CD from the Crohn's and Colitis Canada - Genetic, Environmental, Microbial (CCC-GEM) project. Regression models identified environmental factors associated with future CD onset and their association with pre-disease biological factors, including altered intestinal permeability measured by urinary fractional excretion of lactulose to mannitol ratio (LMR); gut inflammation via fecal calprotectin (FCP) levels; and fecal microbiome composition through 16S rRNA sequencing. RESULTS: Over a 5.62-year median follow-up, 86 FDRs developed CD. Living with a dog between ages 5 and 15 (hazard ratio [HR], 0.62; 95% confidence interval [CI], 0.40-0.96; P = .034), and living with a large family size in the first year of life (HR, 0.43; 95% CI, 0.21-0.85; P = .016) were associated with decreased CD risk, whereas having a bird at the time of recruitment (HR, 2.78; 95% CI, 1.36-5.68; P = .005) was associated with an increased CD risk. Furthermore, living with a dog was associated with reduced LMR, altered relative abundance of multiple bacterial genera, and increased Chao1 diversity, whereas bird owners had higher FCP levels. Large family during participants' first year of life was associated with altered microbiota composition without affecting FCP or LMR. CONCLUSION: This study identifies environmental variables associated with CD risk. These variables were also associated with altered barrier function, subclinical inflammation, and gut microbiome composition shifts, suggesting potential roles in CD pathogenesis.

The chromatin and single-cell transcriptional landscapes of CD4 T cells in inflammatory bowel disease link risk loci with a proinflammatory Th17 cell population.

Introduction: The imbalance between Th17 and regulatory T cells in inflammatory bowel diseases (IBD) promotes intestinal epithelial cell damage. In this scenario, T helper cell lineage commitment is accompanied by dynamic changes to the chromatin that facilitate or repress gene expression. Methods: Here, we characterized the chromatin landscape and heterogeneity of intestinal and peripheral CD4 T cellsfrom IBD patients using in house ATAC-Seq and single cell RNA-Seq libraries. Results: We show that chromatin accessibility profiles of CD4 T cells from inflamed intestinal biopsies relate to genes associated with a network of inflammatory processes. After integrating the chromatin profiles of tissue-derived CD4 T cells and in-vitro polarized CD4 T cell subpopulations, we found that the chromatin accessibility changes of CD4 T cells were associated with a higher predominance of pathogenic Th17 cells (pTh17 cells) in inflamed biopsies. In addition, IBD risk loci in CD4 T cells were colocalized with accessible chromatin changes near pTh17-related genes, as shown in intronic STAT3 and IL23R regions enriched in areas of active intestinal inflammation. Moreover, single cell RNA-Seq analysis revealed a population of pTh17 cells that co-expresses Th1 and cytotoxic transcriptional programs associated with IBD severity. Discussion: Altogether, we show that cytotoxic pTh17 cells were specifically associated with IBD genetic variants and linked to intestinal inflammation of IBD patients.

Gut Microbiome Composition Is Associated With Future Onset of Crohn's Disease in Healthy First-Degree Relatives.

BACKGROUND & AIMS: The cause of Crohn's disease (CD) is unknown, but the current hypothesis is that microbial or environmental factors induce gut inflammation in genetically susceptible individuals, leading to chronic intestinal inflammation. Case-control studies of patients with CD have cataloged alterations in the gut microbiome composition; however, these studies fail to distinguish whether the altered gut microbiome composition is associated with initiation of CD or is the result of inflammation or drug treatment. METHODS: In this prospective cohort study, 3483 healthy first-degree relatives (FDRs) of patients with CD were recruited to identify the gut microbiome composition that precedes the onset of CD and to what extent this composition predicts the risk of developing CD. We applied a machine learning approach to the analysis of the gut microbiome composition (based on 16S ribosomal RNA sequencing) to define a microbial signature that associates with future development of CD. The performance of the model was assessed in an independent validation cohort. RESULTS: In the validation cohort, the microbiome risk score (MRS) model yielded a hazard ratio of 2.24 (95% confidence interval, 1.03-4.84; P = .04), using the median of the MRS from the discovery cohort as the threshold. The MRS demonstrated a temporal validity by capturing individuals that developed CD up to 5 years before disease onset (area under the curve > 0.65). The 5 most important taxa contributing to the MRS included Ruminococcus torques, Blautia, Colidextribacter, an uncultured genus-level group from Oscillospiraceae, and Roseburia. CONCLUSION: This study is the first to demonstrate that gut microbiome composition is associated with future onset of CD and suggests that gut microbiome is a contributor in the pathogenesis of CD.

Immune response and barrier dysfunction-related proteomic signatures in preclinical phase of Crohn's disease highlight earliest events of pathogenesis.

OBJECTIVE: The measure of serum proteome in the preclinical state of Crohn's disease (CD) may provide insight into biological pathways involved in CD pathogenesis. We aimed to assess associations of serum proteins with future CD onset and with other biomarkers predicting CD risk in a healthy at-risk cohort. DESIGN: In a nested case-control study within the Crohn's and Colitis Canada Genetics Environment Microbial Project (CCC-GEM) cohort, which prospectively follows healthy first-degree relatives (FDRs), subjects who developed CD (n=71) were matched with four FDRs remaining healthy (n=284). Using samples at recruitment, serum protein profiles using the Olink Proximity Extension Assay platform was assessed for association with future development of CD and with other baseline biomarkers as follows: serum antimicrobial antibodies (AS: positive antibody sum) (Prometheus); faecal calprotectin (FCP); gut barrier function using the fractional excretion of lactulose-to-mannitol ratio (LMR) assay. RESULTS: We identified 25 of 446 serum proteins significantly associated with future development of CD. C-X-C motif chemokine 9 (CXCL9) had the highest OR with future risk of CD (OR=2.07 per SD, 95% CI 1.58 to 2.73, q=7.9e-5), whereas matrix extracellular phosphoglycoprotein had the lowest OR (OR 0.44, 95% CI 0.29 to 0.66, q=0.02). Notably, CXCL9 was the only analyte significantly associated with all other CD-risk biomarkers with consistent direction of effect (FCP: OR=2.21; LMR: OR=1.67; AS: OR=1.59) (q<0.05 for all). CONCLUSION: We identified serum proteomic signatures associated with future CD development, reflecting potential early biological processes of immune and barrier dysfunction.

Anti-Integrin αvß6 Autoantibodies Are a Novel Biomarker That Antedate Ulcerative Colitis.

BACKGROUND & AIMS: Better biomarkers for prediction of ulcerative colitis (UC) development and prognostication are needed. Anti-integrin αvß6 (anti-αvß6) autoantibodies have been described in patients with UC. We tested for the presence of anti-αvß6 antibodies in the preclinical phase of UC and studied their association with disease-related outcomes after diagnosis. METHODS: Anti-αvß6 autoantibodies were measured in 4 longitudinal serum samples collected from 82 subjects who later developed UC and 82 matched controls from a Department of Defense preclinical cohort (PREDICTS [Proteomic Evaluation and Discovery in an IBD Cohort of Tri-service Subjects]). In a distinct, external validation cohort (Crohn's and Colitis Canada Genetic Environmental Microbial project cohort), we tested 12 pre-UC subjects and 49 matched controls. Furthermore, anti-αvß6 autoantibodies were measured in 2 incident UC cohorts (COMPASS [Comprehensive Care for the Recently Diagnosed IBD Patients], n = 55 and OSCCAR [Ocean State Crohn's and Colitis Area Registry], n = 104) and associations between anti-αvß6 autoantibodies and UC-related outcomes were defined using Cox proportional hazards model. RESULTS: Anti-αvß6 autoantibodies were significantly higher among individuals who developed UC compared with controls up to 10 years before diagnosis in PREDICTS. The anti-αvß6 autoantibody seropositivity was 12.2% 10 years before diagnosis and increased to 52.4% at the time of diagnosis in subjects who developed UC compared with 2.7% in controls across the 4 time points. Anti-αvß6 autoantibodies predicted UC development with an area under the curve of at least 0.8 up to 10 years before diagnosis. The presence of anti-αvß6 autoantibodies in preclinical UC samples was validated in the GEM cohort. Finally, high anti-αvß6 autoantibodies was associated with a composite of adverse UC outcomes, including hospitalization, disease extension, colectomy, systemic steroid use, and/or escalation to biologic therapy in recently diagnosed UC. CONCLUSIONS: Anti-integrin αvß6 autoantibodies precede the clinical diagnosis of UC by up to 10 years and are associated with adverse UC-related outcomes.

Long Term Weight Cycling Affects Fecal Microbiota of Mice.

SCOPE: Fighting obesity and associated comorbidities through dieting is not always sustained and results in a subsequent weight gain, a phenomenon referred to as weight cycling. Diet is among the most important factors in modifying the composition of gut microbiota. The objective of this work is to determine whether weight cycling affects the composition and the predicted function of mouse fecal bacteria on a long-term basis. METHODS AND RESULTS: Mice fed for 40 weeks with either high fat (HF), low fat (LF), or cycled diets (starting and ending by one of the two diets, and the reverse) exhibit a bacterial profile specific to each of the four groups. A higher proportion of Firmicutes and Bacteroidota phyla are observed in mice on Hf and LF diet, respectively. The proportion of functions dedicated to amino acid metabolism is higher in mice on HF or LF/HF diets, whereas the mice on LF or HF/LF diets have a higher proportion of functions involve in carbohydrate metabolism and vitamin B biosynthesis. CONCLUSION: Compared to continuous HF or LF diets, cyclic diet specifically alters the composition and function of the mouse fecal microbiota, suggesting that fight against weight gain should be considered on a long-term basis.

Altered Gut Microbiome Composition and Function Are Associated With Gut Barrier Dysfunction in Healthy Relatives of Patients With Crohn's Disease.

BACKGROUND & AIMS: The gut microbiome has been suggested to play a role in gut barrier hemostasis, but data are scarce and limited to animal studies. We therefore aimed to assess whether alterations in gut microbial composition and functional pathways are associated with gut barrier function in a cohort of healthy first-degree relatives of patients with Crohn's disease. METHODS: We used the Crohn's and Colitis Canada Genetic Environmental Microbial (CCC-GEM) cohort of healthy first-degree relatives of patients with Crohn's disease. Gut barrier function was assessed using the urinary fractional excretion of lactulose-to-mannitol ratio (LMR). Microbiome composition was assessed by sequencing fecal 16S ribosomal RNA. The cohort was divided into a discovery cohort (n = 2472) and a validation cohort (n = 655). A regression model was used to assess microbial associations with the LMR. A random forest classifier algorithm was performed to assess microbial community contribution to barrier function. RESULTS: Individuals with impaired barrier function (LMR >0.025) had reduced alpha-diversity (Chao1 index, P = 4.0e-4) and altered beta-diversity (Bray-Curtis dissimilarity index, R2 = 0.001, P = 1.0e-3) compared with individuals with an LMR ≤0.025. When taxa were assessed individually, we identified 8 genera and 52 microbial pathways associated with an LMR >0.025 (q < 0.05). Four genera (decreased prevalence of Adlercreutzia, Clostridia UCG 014, and Clostridium sensu stricto 1 and increased abundance of Colidextribacter) and 8 pathways (including decreased biosynthesis of glutamate, tryptophan, and threonine) were replicated in the validation cohort. The random forest approach revealed that the bacterial community is associated with gut barrier function (area under the curve, 0.63; P = 1.4e-6). CONCLUSIONS: The gut microbiome community and pathways are associated with changes in gut barrier function. These findings may identify potential microbial targets to modulate gut barrier.

Mediterranean-Like Dietary Pattern Associations With Gut Microbiome Composition and Subclinical Gastrointestinal Inflammation.

BACKGROUND & AIMS: Case-control studies have shown that patients with Crohn's disease (CD) have a microbial composition different from healthy individuals. Although the causes of CD are unknown, epidemiologic studies suggest that diet is an important contributor to CD risk, potentially via modulation of bacterial composition and gut inflammation. We hypothesized that long-term dietary clusters (DCs) are associated with gut microbiome compositions and gut inflammation. Our objectives were to identify dietary patterns and assess whether they are associated with alterations in specific gut microbial compositions and subclinical levels of gut inflammation in a cohort of healthy first-degree relatives (FDRs) of patients with CD. METHODS: As part of the Genetic, Environmental, Microbial (GEM) Project, we recruited a cohort of 2289 healthy FDRs of patients with CD. Individuals provided stool samples and answered a validated food frequency questionnaire reflecting their habitual diet during the year before sample collection. Unsupervised analysis identified 3 dietary and 3 microbial composition clusters. RESULTS: DC3, resembling the Mediterranean diet, was strongly associated with a defined microbial composition, with an increased abundance of fiber-degrading bacteria, such as Ruminococcus, as well as taxa such as Faecalibacterium. The DC3 diet was also significantly associated with lower levels of subclinical gut inflammation, defined by fecal calprotectin, compared with other dietary patterns. No significant associations were found between individual food items and fecal calprotectin, suggesting that long-term dietary patterns rather than individual food items contribute to subclinical gut inflammation. Additionally, mediation analysis demonstrated that DC3 had a direct effect on subclinical inflammation that was partially mediated by the microbiota. CONCLUSIONS: Overall, these results indicated that Mediterranean-like dietary patterns are associated with microbiome and lower intestinal inflammation. This study will help guide future dietary strategies that affect microbial composition and host gut inflammation to prevent diseases.

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.

Anti-Microbial Antibody Response is Associated With Future Onset of Crohn's Disease Independent of Biomarkers of Altered Gut Barrier Function, Subclinical Inflammation, and Genetic Risk.

BACKGROUND AND AIMS: Altered host immune reactivity to microbial antigens is hypothesized to trigger the onset of Crohn's disease (CD). We aimed to assess whether increased serum anti-microbial antibody response in asymptomatic first-degree relatives (FDRs) of CD patients is an independent risk factor for future CD development. METHODS: We measured host serum antibody response to 6 microbial antigens at enrollment (Prometheus enzyme-linked immunosorbent assay test: anti-Saccharomyces cerevisiae antibodies immunoglobulin A/immunoglobulin G, anti-OmpC, anti-A4-Fla2, anti-FlaX, anti-CBir1) and derived the sum of positive antibodies (AS). We used samples at enrollment of prospectively followed healthy FDRs from a nested case-control cohort of the Crohn's and Colitis Canada Genetics Environment Microbial Project. Those who later developed CD (n = 77) were matched 1:4 by age, sex, follow-up duration, and geographic location with control FDRs remaining healthy (n = 307). To address our research aims, we fitted a multivariable conditional logistic regression model and performed causal mediation analysis. RESULTS: High baseline AS (≥2) (43% of cases, 11% of controls) was associated with higher risk of developing CD (adjusted odds ratio, 6.5; 95% confidence interval, 3.4-12.7; P < .001). Importantly, this association remained significant when adjusted for markers of gut barrier function, fecal calprotectin, C-reactive protein, and CD-polygenic risk score, and in subjects recruited more than 3 years before diagnosis. Causal mediation analysis showed that the effect of high AS on future CD development is partially mediated (42%) via preclinical gut inflammation. CONCLUSIONS: Our results suggest that increased anti-microbial antibody responses are associated with risk of future development of CD, independent of biomarkers of abnormal gut barrier function, subclinical inflammation, and CD-related genetic risks. This suggests that anti-microbial antibody responses are an early predisease event in the development of CD.

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 Zonulin Measured by Commercial Kit Fails to Correlate With Physiologic Measures of Altered Gut Permeability in First Degree Relatives of Crohn's Disease Patients.

Intestinal epithelial cell tight junctions (TJs) contribute to the integrity of the intestinal barrier allowing for control of the physical barrier between external antigens or bacterial products and the internal environment. Zonula occludens-1 (ZO-1) is a protein that modulates intestinal TJs, and serum levels of ZO-1 has been suggested as a biomarker of disrupted barrier function in humans. Previous studies suggested that increased intestinal permeability was associated with evidence of TJ abnormalities. However, there is limited information on the serological measurement of ZO-1 and its relation to other tests of barrier function in healthy subjects. We investigated the correlation of serum ZO-1, with physiologic measures of intestinal permeability (as the ratio of the fractional excretion of lactulose-mannitol or LMR) in a cohort of 39 healthy FDRs of Crohn's disease (CD) patients. No significant correlation was found between LMR and ZO-1 levels (r2 = 0.004, P < 0.71), or intestinal fatty acid binding proteins (I-FABP) (r2 = 0.004, P < 0.71). In conclusion, our data show that ZO-1 and I-FABP are not a marker of gut permeability as defined by LMR.