Rosica Valcheva

Mucosal Barrier Depletion And Loss Of Bacterial Diversity Are Primary Abnormalities In Paediatric Ulcerative Colitis

BACKGROUND AND AIMS Ulcerative colitis [UC] is associated with colonic mucosa barrier defects and bacterial dysbiosis, but these features may simply be the result of inflammation. Therefore, we sought to assess whether these features are inherently abrogated in the terminal ileum [TI] of UC patients, where inflammation is absent. METHODS TI biopsies from paediatric inflammatory bowel disease [IBD] subsets [Crohns disease [CD; n = 13] and UC [n = 10]], and non-IBD disease controls [n = 12] were histologically graded, and alcian blue/periodic acid-Schiff stained biopsies were quantified. The mucosal barrier was assessed for mucin [MUC2], immunoglobulin [Ig]A, IgG, and total bacteria (fluorescence in-situ hybridisation [FISH probe EUB338]) by immunofluorescence. The regulation of mucin secretion was investigated by NLRP6 gene expression and immunofluorescence. The composition of the active mucosa-associated microbiota was explored by sequencing the 16S rRNA amplicon generated from total RNA. RESULTS Despite the absence of ileitis, UC patients displayed ileal barrier depletion illustrated by reductions in mucin-containing goblet cells and mucin production and altered epithelial NLRP6 expression. In both CD patients with ileitis and UC patients with normal histology, bacteria coated with IgA and IgG penetrated the TI mucin layer. Biopsy 16S rRNA sequencing revealed a reduction in α-diversity by three methods [Shannon, Simpson, and Equitability indices] between UC and non-IBD paediatric patients. CONCLUSIONS These findings suggest an underlying defect in the UC-afflicted intestinal tract even in the absence of inflammation, implicating barrier and microbial changes as primary abnormalities in UC that may play a causative role in disease development.

Prebiotics: Definition and protective mechanisms.

The increase in chronic metabolic and immunologic disorders in the modern society is linked to major changes in the dietary patterns. These chronic conditions are associated with intestinal microbiota dysbiosis where important groups of carbohydrate fermenting, short-chain fatty acids-producing bacteria are reduced. Dietary prebiotics are defined as a selectively fermented ingredients that result in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health. Application of prebiotics may then restore the gut microbiota diversity and activity. Unlike the previously accepted prebiotics definition, where a limited number of bacterial species are involved in the prebiotic activity, new data from community-wide microbiome analysis demonstrated a broader affect of the prebiotics on the intestinal microbiota. These new findings require a revision of the current definition. In addition, prebiotics may exert immunomodulatory effects through microbiota-independent mechanisms that will require future investigations involving germ-free animal disease models.

The Role of Intestinal Microbiota in Development of Irinotecan Toxicity and in Toxicity Reduction through Dietary Fibres in Rats

CPT-11 is a drug used as chemotherapy for colorectal cancer. CPT-11 causes toxic side-effects in patients. CPT-11 toxicity has been attributed to the activity of intestinal microbiota, however, intestinal microbiota may also have protective effects in CP!-11 chemotherapy. This study aimed to elucidate mechanisms through which microbiota and dietary fibres could modify host health. Rats bearing a Ward colon carcinoma were treated with a two-cycle CPT-11/5-fluorouracil therapy recapitulating clinical therapy of colorectal cancer. Animals were fed with a semi-purified diet or a semi-purified diet was supplemented with non-digestible carbohydrates (isomalto-oligosaccharides, resistant starch, fructo-oligosaccharides, or inulin) in 3 independent experiments. Changes in intestinal microbiota, bacteria translocating to mesenteric lymphnodes, cecal GUD activity, and cecal SCFA production, and the intestinal concentration of CPT-11 and its metabolites were analysed. Non-digestible carbohydrates significantly influenced feed intake, body weight and other indicators of animal health. The identification of translocating bacteria and their quantification in cecal microbiota indicated that overgrowth of the intestine by opportunistic pathogens was not a major contributor to CPT-11 toxicity. Remarkably, fecal GUD activity positively correlated to body weight and feed intake but negatively correlated to cecal SN-38 concentrations and IL1-β. The reduction in CPT-11 toxicity by non-digestible carbohydrates did not correlate to stimulation of specific bacterial taxa. However, cecal butyrate concentrations and feed intake were highly correlated. The protective role of intestinal butyrate production was substantiated by a positive correlation of the host expression of MCT1 (monocarboxylate transporter 1) with body weight as well as a positive correlation of the abundance of bacterial butyryl-CoA gene with cecal butyrate concentrations. These correlations support the interpretation that the influence of dietary fibre on CPT-11 toxicity is partially mediated by an increased cecal production of butyrate.

Microbial ecology of sorghum sourdoughs: Effect of substrate supply and phenolic compounds on composition of fermentation microbiota

The choice of the cereal substrate determines sourdough microbiota, however, the substrate-associated ecological factors for this phenomenon have not been elucidated. This study investigated the competitiveness of Lactobacillus sanfranciscensis LTH 2590, a wheat sourdough isolate, and four isolates from sorghum sourdoughs (ting), Lactobacillus casei FUA3166, Lactobacillus harbinensis FUA3199, Lactobacillus parabuchneri FUA3169, and Lactobacillus coryniformis FUA3307, in sorghum sourdoughs, sorghum sourdoughs supplemented with maltose, or wheat sourdoughs. Fermentations were characterised by determination of cell counts, pH, and quantification of metabolites. Maltose was the main carbon source in wheat sourdoughs whereas glucose was the major carbon source in sorghum. L. coryniformis and L. parabuchneri produced 1,3- and 1,2-propanediol from glycerol and lactate, respectively, metabolites that were previously not observed in sourdough. To determine the competitiveness of strains, wheat and sorghum slurries were inoculated with equal cells counts of L. sanfranciscensis, L. parabuchneri, and L. casei fermented at 28°C or 34°C and propagated by back-slopping every 24h. Lactobacilli in sourdough were quantified by plating and species-specific quantitative PCR (qPCR). Generally, sorghum and wheat sourdoughs inoculated with isolates from ting gave no appreciable differences in the metabolites produced during the fermentation process. L. sanfranciscensis grew in wheat but not in sorghum sourdoughs, or sorghum sourdoughs supplemented with 2% maltose, 1% tryptone, 0.1% l-cysteine and 2% sucrose. Furthermore, L.sanfranciscensis decreased progressively during propagation of sorghum sourdoughs but ting isolates were overgrown by L. sanfranciscensis after three propagations in wheat sourdoughs independent of the incubation temperature. The anti-microbial activity of four different types of sorghum extracts was tested against L. sanfranciscensis, L. parabuchneri, and L. casei to correlate the resistance to phenolic compounds to growth in wheat or sorghum sourdoughs. L. sanfranciscensis was inhibited by phenolic extracts from sorghum flours whereas ting isolates were resistant. In conclusion, microbiota of sorghum sourdough differ from wheat and rye because sorghum contains active concentrations of antimicrobial phenolic compounds, and offers glucose as major carbon source.

Chemically Defined Diet Alters the Protective Properties of Fructo-Oligosaccharides and Isomalto-Oligosaccharides in HLA-B27 Transgenic Rats

Non-digestible oligosaccharides (NDO) were shown to reduce inflammation in experimental colitis, but it remains unclear whether microbiota changes mediate their colitis-modulating effects. This study assessed intestinal microbiota and intestinal inflammation after feeding chemically defined AIN-76A or rat chow diets, with or without supplementation with 8 g/kg body weight of fructo-oligosaccharides (FOS) or isomalto-oligosaccharides (IMO). The study used HLA-B27 transgenic rats, a validated model of inflammatory bowel disease (IBD), in a factorial design with 6 treatment groups. Intestinal inflammation and intestinal microbiota were analysed after 12 weeks of treatment. FOS and IMO reduced colitis in animals fed rat chow, but exhibited no anti-inflammatory effect when added to AIN-76A diets. Both NDO induced specific but divergent microbiota changes. Bifidobacteria and Enterobacteriaceae were stimulated by FOS, whereas copy numbers of Clostridium cluster IV were decreased. In addition, higher concentrations of total short-chain fatty acids (SCFA) were observed in cecal contents of rats on rat chow compared to the chemically defined diet. AIN-76A increased the relative proportions of propionate, iso-butyrate, valerate and iso-valerate irrespective of the oligosaccharide treatment. The SCFA composition, particularly the relative concentration of iso-butyrate, valerate and iso-valerate, was associated (P≤0.004 and r≥0.4) with increased colitis and IL-1 β concentration of the cecal mucosa. This study demonstrated that the protective effects of fibres on colitis development depend on the diet. Although diets modified specific cecal microbiota, our study indicates that these changes were not associated with colitis reduction. Intestinal inflammation was positively correlated to protein fermentation and negatively correlated with carbohydrate fermentation in the large intestine.

Dietary and metabolomic determinants of relapse in ulcerative colitis patients: A pilot prospective cohort study

AIM To identify demographic, clinical, metabolomic, and lifestyle related predictors of relapse in adult ulcerative colitis (UC) patients. METHODS In this prospective pilot study, UC patients in clinical remission were recruited and followed-up at 12 mo to assess a clinical relapse, or not. At baseline information on demographic and clinical parameters was collected. Serum and urine samples were collected for analysis of metabolomic assays using a combined direct infusion/liquid chromatography tandem mass spectrometry and nuclear magnetic resolution spectroscopy. Stool samples were also collected to measure fecal calprotectin (FCP). Dietary assessment was performed using a validated self-administered food frequency questionnaire. RESULTS Twenty patients were included (mean age: 42.7 ± 14.8 years, females: 55%). Seven patients (35%) experienced a clinical relapse during the follow-up period. While 6 patients (66.7%) with normal body weight developed a clinical relapse, 1 UC patient (9.1%) who was overweight/obese relapsed during the follow-up (P = 0.02). At baseline, poultry intake was significantly higher in patients who were still in remission during follow-up (0.9 oz vs 0.2 oz, P = 0.002). Five patients (71.4%) with FCP > 150 μg/g and 2 patients (15.4%) with normal FCP (≤ 150 μg/g) at baseline relapsed during the follow-up (P = 0.02). Interestingly, baseline urinary and serum metabolomic profiling of UC patients with or without clinical relapse within 12 mo showed a significant difference. The most important metabolites that were responsible for this discrimination were trans-aconitate, cystine and acetamide in urine, and 3-hydroxybutyrate, acetoacetate and acetone in serum. CONCLUSION A combination of baseline dietary intake, fecal calprotectin, and metabolomic factors are associated with risk of UC clinical relapse within 12 mo.

Increased Intestinal Permeability in Relatives of Patients With Crohn’s Disease Is Not Associated With Small Bowel Ulcerations

BACKGROUND & AIMS: Many first‐degree relatives of patients with Crohn’s disease (CD) have increased intestinal permeability. Video capsule endoscopy (VCE) is the most sensitive imaging test to identify small bowel mucosal lesions that could indicate subclinical CD. We aimed to estimate the association of increased intestinal permeability with small bowel ulcerations detectable by VCE in healthy first‐degree relatives of patients with CD. METHODS: We conducted a cross‐sectional study of 223 healthy, asymptomatic first‐degree relatives of patients with CD (parents, siblings, and children; 9–45 years old) enrolled at the University of Alberta between 2009 and 2012. Patients were given the lactulose and mannitol test to measure small bowel permeability; we used high‐performance liquid chromatography to measure concentrations of lactulose and mannitol in urine samples (increased permeability defined as a ratio of lactulose/mannitol 0.025 or greater). Patients with increased permeability (n = 39) and randomly selected subjects with normal permeability (n = 59) were then examined by VCE for signs of small bowel inflammation and subclinical CD. The prevalence of small bowel lesions was compared among groups. We performed logistic regression analyses to estimate odds ratios for the association of small bowel ulcerations with intestinal permeability. RESULTS: Among 223 first‐degree relatives of patients with CD, 30% were found to have increased intestinal permeability; VCE examination found 24% of subjects to have 3 or more small bowel ulcers. Three or more small bowel ulcers were detected in 28% of patients with increased intestinal permeability and 20% of patients with normal intestinal permeability (P = .37). The adjusted odds ratio for the association of 3 or more small bowel ulcers with increased intestinal permeability was 1.5 (95% confidence interval, 0.6–3.8; P = .46). CONCLUSIONS: Thirty percent of healthy, asymptomatic first‐degree relatives of patients with CD have increased intestinal permeability. However, a strong association of small bowel ulceration seen on VCE with increased intestinal permeability was not observed.

P-323 Immunoglobulin G Selectively Identifies Pathosymbionts in Paediatric Inflammatory Bowel Diseases

Background: Inflammatory bowel diseases (IBD) are a group of complex and multifactorial disorders with unknown etiology. Both environmental and genetic factors contribute to disease pathogenesis. This complexity is further exacerbated with an uncontrolled immune response to the gut microbiota. Given that immunoglobulin (Ig) G is formed in response to invasive microbes, we anticipated that bacteria bound by IgG are more likely to be virulent; their isolation could help identify such invasive strains and define mechanisms of immune activation. We hypothesized that host IgG immune response can be used to identify bacteria that may be involved in the pathogenesis of IBD. Methods: Aspirate washes from the terminal ileum of pediatric IBD (including Crohn disease [CD] and ulcerative colitis [UC]) and non-IBD controls were collected during endsocopy, stringently washed, and then fixed in paraformaldehyde. An aliquot was stored (presort) and the remaining washes were labeled with propidium iodide and anti-IgG fluorescent antibody to allow for identification of bacteria bound by IgG and those that are not. Using fluorescence-activated cell sorting (FACS), the samples were then fractioned for IgG bound (IgG+) and non-IgG bound (IgG−) bacteria. Some samples were also imaged by image cytometry to validate the FACS procedure. After sorting, DNA was extracted using beads and purified by phenol/chloroform. DNA was then analyzed by 16S sequencing using the Ilumina MiSeq platform. Results: A total of 36 washes from children without IBD (n = 10), and with CD (n = 17), and UC (n = 9), were suitable for analysis with DNA meeting quality control criteria. The ileal mucosa-associated microbiome composition of non-IBD and IBD groups did not differ significantly, with predominance of Firmicutes and Bacteroidetes. When sorted for IgG coated bacteria, the ratio of IgG+/IgG−bacteria (indicating taxa over-represented by IgG binding) were increased in CD and UC patients by 2- and 1.5-fold, respectively. Notably, the IgG+/IgG−ratio of Bacteroidetes and Proteobacteria taxa were increased in CD, Actinobacteria was increased in UC patients, and there was no changes in Firmicutes, the major phyla of the ileal mucosal microbiome. Although there was considerable inter-individual variation, at the family level, IgG binding favored Porphyromonadaceae (Bacteroidetes) and Enterobacteriaceae (Proteobacteria) in children with CD; Bifidobacteriaceae (Actinobacteria) and Barnesiellaceae (Bacteroidetes) in UC; and Clostridiaceae and Veillonellaceae (both Firmicutes) in non-IBD. Conclusions: IgG coating of mucosa associated bacteria in the ileum of IBD patients is elevated and is selective for unique phyla compared to non-IBD controls. Taxa identified by IgG include bacteria that have been associated with disease and virulence. Identification of individual microbes using this method could facilitate development of targeted diagnostic and therapeutic approaches for IBD.

1030 Distinctive Urinary Metabolite Profiles Correlate With Microbial Composition and Are Linked With Endoscopic Post-Operative Disease Recurrence in Crohn's Disease Patients

Endoscopic disease recurrence after ileocolonic resection and re-anastomosis in Crohns disease (CD) patients occurs in a majority of patients. The aim of this study was to examine environmental exposures, the gut microbiome and urinary metabolites in CD patients following ileocolonic resection to determine if specific gut microbes, their metabolites or toxins or environmental factors could be linked with either relapse or remission. Methods: Biopsies were obtained from the neo-terminal ileum after ileocecal resection from CD patients in endoscopic remission (n=20) or in relapse (n=18). Microbial DNA was extracted from biopsies using the QIAamp DNA stool mini kit. Multitag pyrosequencing of the 16S gene was used to characterize the mucosa-associated microbiota, and the RDP 10 classifier used to assign taxonomic groups. Genes encoding for colibactin, C. difficile toxin B, C. perfringens alpha toxin, shiga-like toxin 1 were quantified by qPCR. Urine samples were collected and metabolites measured using 1H NMR. Peak identification was done using Chenomx NMRSuite v7.5 and multivariate analysis performed using MetaboAnalyst 2.0. An environmental questionnaire was obtained that examined early environmental exposures, family history, diet, smoking behaviours, animal exposure and current medication. Principle component analysis (PCA) and support vector machines were used to determine differences between patients with endoscopic relapse and those that remained in remission. Results: Support vector machines applied to total microbial abundance at the genus level and PCA did not show a clear separation between CD patients in relapse or remission. In addition, there were no differences in any toxin encoding genes. In contrast, patients with endoscopic disease recurrence could be separated from patients without ileal inflammation through partial least squares discriminant analysis of urinary metabolites. In addition, correlation analysis showed a positive correlation between urinary metabolites involved in essential processes such as energy production and amino acid metabolism and Bacteroidaceae and negative correlations between amino acid metabolism and Clostridia in the inflamed group. In contrast, the non-inflamed group showed positive correlations between metabolites involved in energy production and Enterobacteriaceae. The most important environmental features associated with disease relapse included smoking cigarettes and/or pipes. Age, gender, years since last surgery, domicile location, or use of immunosuppressants did not differ between CD patients suffering relapse or remaining in endoscopic remission. Conclusions: Recurrence of Crohns disease after surgery was associated with a unique urinary metabolomics signature that could be linked with specific gut microbes. The major risk factor associated with recurrence of disease was smoking.

Fructo-oligosaccharides as therapeutics for active Crohn's disease: Adequate power and no effect

Benjamin JL, Hedin CRH, Koutsoumpas A, et al. Randomised, double-blind, placebo-controlled trial of fructo-oligosaccharides in active Crohns disease. (Inflamm Bowel Dis 2011)