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Influence of Ethnicity and Disease in Migrant Populations With UlcerativeColitisJordan Mar, Michelle Nazareth, Uma Mahadevan, Susan Lynch

Though the etiology of Ulcerative colitis (UC) is unknown, it is widely recognized as adisease influenced by environmental, genotypic and microbiological factors. Many establishedUC risk-alleles are involved in host response to microbes, though these genetic risks frequentlydo not span ethnic populations. In addition, both antibiotic use and high fat diet, factorscommon to Western lifestyle, have been shown to independently impact the gastrointestinal(GI) microbiome and increase UC risk. Interestingly, Asian migrants who translocate fromtheir native country to Western nations and adopt a Western lifestyle develop UC at ratessimilar to, or greater than, the background population. These populations afford a uniqueopportunity to study the impact of ethnicity (genotype) and environment (e.g. dietaryexposures) on UC development. Combined with findings regarding the immunomodulatorycapabilities of GI microbiota, these observations have led us to hypothesize that first orsecond generation migrants (Asian or European) to the US, who exhibit distinct UC riskgene profiles, develop a compositionally distinct, dysbiotic microbiome enriched for similarpro-inflammatory metabolites that contribute to disease severity. To test our hypothesis,we employed 16S rRNA phylogenetic microarray analyses to identify differences in stoolmicrobiome composition and used this data, in conjunction with PICRUSt, to predict themetagenomic content of samples obtained from migrant South Asian (SA) and European(EU) UC patients and familial, healthy controls. Consistent with previous reports, a distinctshift in gross stool microbiota composition in patients vs. controls, characterized by significantenrichment of unclassified Streptococcus across all UC patients (p<0.05, q<0.1), wasobserved. Multivariate regression revealed that, in addition to disease status and SimpleClinical Colitis Activity, patient origin was significantly associated with compositional varia-tion in stool microbiome. Indeed, SA UC patients exhibited depletion of unclassified Lach-nospiraceae, while EU patients were depleted in both Odoribacter and Pantoea. Analysis ofthe predicted metagenome of these communities identified a variety of metabolic pathwaysenriched in UC patients compared to controls. This included ubiquitin metabolism, a knownimmunogenic pathway previously linked to UC. Combined, these observations suggest that,though the microbiome of ethnically distinct UC patients is compositionally divergent,enrichment of a common set of immunomodulatory metabolic pathways characterizes thesepatients. Future work involving genetic profiling of UC risk-alleles as well as broad rangefecal metabolite profiling will provide further insight into the mechanisms behind theobserved functional shift in the microbiome and it's relationship to disease severity.

Tu1753

Intestinal Epithelial Vitamin D Receptor Deletion Leads to DefectiveAutophagy and Dysbiosis in ColitisShaoping Wu, Yong-guo Zhang, Rong Lu, Yinglin Xia, Zhongren Zhou, Elaine O. Petrof,Erika C. Claud, Eugene B. Chang, Geert Carmeliet, Jun Sun

Vitamin D and the vitamin D receptor (VDR) appear to be important immunological regulatorsof inflammatory bowel diseases (IBD). VDR expression is significantly decreased in IBDpatients, and, polymorphisms in the VDR gene are associated with susceptibility to IBD.Our previous study demonstrated that VDR negatively regulates bacteria-induced proinflam-matory NF-κB activity in Salmonella-colitis. Bacteria regulate intestinal VDR expressionin both gnotobiotic and bacterial-colitis models. Autophagy is involved in intracellularhomeostasis, as well as in promoting immunity against infection and the removal of intracellu-lar microbes. Defective autophagy has also been implicated in inflammatory bowel diseases(IBD), where interestingly, polymorphisms of genes such as ATG16L1 have been associatedwith increased risk. Although vitamin D, the microbiome, and autophagy are all involvedin pathogenesis of IBD, it remains unclear whether these processes are related or functionindependently. We investigated the effects and mechanisms of intestinal epithelial VDR inhealthy and inflamed states using cell culture models, a conditional VDR knockout mousemodel (VDRΔIEC), colitis models, and human samples. Absence of intestinal epithelial VDRleads to an ecologic change of bacterial profiles characterized by increased E. coli andBacteroides and decreased butyrate-producing bacteria (Butyivibrio). These VDRΔIEC micehave increased susceptibility to dextran sulfate sodium-induced injury. Intestinal epithelialVDR downregulates expressions of ATG16L1 and lysozyme, both components of the autoph-agy pathway that can impair anti-microbial function of Paneth cells. Decreased VDR proteinis also associated with decreased autophagy markers and ATG16L1 expression in vitro andin the IL10-/- experimental colitis model. Gain and loss of function assays showed that VDRlevels regulate ATG6L1 and lysozyme at the transcriptional and translational levels. Moreover,low levels of intestinal epithelial VDR correlated with reduced ATG16L1 and increasedmembership of intestinal Bacteroides in IBD patients. Finally, we explored the possibilityof restoring intestinal VDR in the inflamed gut. Administration of the butyrate (a fermentationproduct of gut microbes) increases intestinal VDR expression and suppresses inflammationin an experimental colitis model. Our study demonstrates key relationships among VDR,autophagy, and gut microbial assemblage that link their actions in maintaining intestinalhomeostasis, but also in contributing to the pathophysiology of IBD. These insights can beleveraged to define therapeutic targets for restoring VDR expression and function, e,g,through microbe-derived small molecules such as short chain fatty acids.

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Fecal Microbial Transplant in Pediatric Crohn's DiseaseDavid Suskind, Ghassan Wahbeh, Heather Vendetoulli, Namita Singh, Samuel Miller

Crohn's disease is characterized by chronic intestinal inflammation in the absence of arecognized etiology. Evidence from human and animal studies support the theory thatpatients with Crohn's disease have a dysregulated immune response to a yet to be determinedtrigger. Given significant fecal microbial dysbiosis in Crohn's disease, this trigger is felt bymany to be the fecal microbiome. The aim of this study was to determine the tolerabilityand potential efficacy of fecal microbial transplantation (FMT) in pediatric patients with

S-834AGA Abstracts

Crohn's disease. This study received an IND (# 14942) from the FDA as well as IRB approvalfrom Seattle Children's Hospital. Ten patients with mild to moderate active Crohn's diseasewere enrolled in the study and received FMT. Patient's continued there maintenance medica-tions for Crohn's disease. Patient's ages were 16.2 ± 2.7 years (range 12-19). 6 were male/4 female. Prior to FMT, stool donors were screened for infectious and noninfectious exclusioncriteria. Patients received bowel cleanout, three days of rifaximin, and one day of omeprazoleprior to FMT. FMT was preformed via nasogastric tube. Patients tolerated FMT well withoutsevere adverse events. After the FMT, 5 patients complained of mild abdominal pain, and5 had mild bloating likely related to FMT. 4 patients had mild diarrhea. Abdominal pain,bloating and diarrhea went back to baseline or improved within 48 hours of FMT. Twoindividuals had vomiting post FMT greater than 4 hours after procedure. No stool was invomitus. One individual went on a long care ride (> 3 hours) after FMT. The other individualhad a large milkshake after FMT. Each individual vomited only once and no fever wasassociated with the vomiting. Four individuals had complaints of a mild stuffy nose/sorethroat/"drippy" nose. 7 out of 10 patients were in clinical remission based upon PCDAIscoring at 2 weeks post FMT, this decreased to 5/10 at 6 weeks post FMT. Three individualshad very little change in clinical symptomatology. Baseline PCDAI was 20.3 ± 7.0, at 2weeks post fecal transplant PCDAI was 8.5 ± 9.1 and at 6 weeks post FMT PCDAI was10.25 ± 6.9. C - reactive protein at baseline 2.6 ± 1.3 mg/dL, 2 weeks post FMT 1.4 ± 0.6mg/dL, and 6 weeks post FMT 1.8 ± 1.1 mg/dL. After FMT, 1 patient required additionalstandard Crohn's medical therapy within a month and another 4 CD patients requiredadditional standard therapy after the 6 week visit. For individuals who did not requireadditional medical therapy, calprotectin went from 614mcg/g ± 621, to 496 mcg/g ± 533at 2 weeks and 430 mcg/g ± 378 at 6 weeks. FMT is well tolerated in pediatric patientswith Crohn's disease. Side effects were mild and self limiting. Both clinical and laboratoryimprovements were seen in the majority of patients. Fecal microbiome analysis is currentlyunderway for this cohort of patients.Pediatric Crohn's Disease Activity Index(PCDAI) in Patients receiving Fecal Microbial Trans-plant

PCDAI score of < 10 denotes remission, 10 -30 mild to moderate active diseases, and > 30severe disease activity. ** denotes individuals who required additional standard Crohn'stherapy after 6 week follow up *** denote individuals who required additional standardCrohn's therapy prior to 6 week follow up

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Reduced B. Pullicaecorum Levels in Mucosa of UC Patients Correlate WithAberrant CLDN1 ExpressionSarah Devriese, Venessa Eeckhaut, Filip Van Immerseel, Richard Ducatelle, Martine DeVos, Debby Laukens

Introduction: Butyrate maintains colonic homeostasis by modulating a wide variety of cellularfunctions including the control of intestinal epithelial integrity. Butyricicoccus pullicaecorumis a butyrate-producing bacterial strain that is found in reduced amounts in stool samplesof patients with ulcerative colitis (UC) and is currently being investigated as a pharmabiotic.Conditioned growth medium of B. pullicaecorum reduces TNF-induced colonic epithelialpermeability in vitro, however its in vivo relevance is unknown. Aim: To investigate therelationship between the presence of B. pullicaecorum in the colonic mucosa and the expressionof tight junction protein 1 (TJP1), occludin (OCLN) and claudin 1 (CLDN1), essentialcomponents of the tight junction complex which are partially regulated by butyrate. Materialsand Methods: The expression of these genes was analyzed by quantitative real-time PCR(qPCR) in a collection of colonic biopsies from healthy controls (N=21) and UC patientswith active disease (N=26). Next, the effect of the conditioned growth medium of B. pullicaec-orum (strain 25-3T) on the expression of these genes was investigated in HT-29 cells in thepresence or absence of TNF. Finally, B. pullicaecorum bacteria were quantified in an extendedcohort of colonic mucosa of UC patients (N=36) and healthy controls (N=31) using a genus-specific qPCR. Results: TJP1 and OCLN were significantly downregulated in colonic biopsiesof UC patients (both P<0.005), whereas CLDN1 expression was increased (P<0.003). Theconditioned growth medium of B. pullicaecorum increased the baseline expression of TJP1and OCLN but did not decrease CLDN1 levels in HT-29 cells. TNF did not affect expressionof TJP1 or OCLN but increased CLDN1 expression which was counteracted by 21% after

co-incubation with the conditioned growth medium. B. pullicaecorum could be detected incolonic biopsies of 71% of healthy controls and in only 42% of UC patients (Fisher exactP=0.026). In addition, in samples where B. pullicaecorum was detected, the absolute amountwas lower in UC samples (P=0.081). Interestingly, the quantity of B. pullicaecorum correlatedwith the deregulated expression of CLDN1 (R= -0.528). Conclusion: Butyricicoccus pullicaec-orum is a mucus-adherent bacterium and is underrepresented in colonic biopsies of UCpatients. Their reduced prevalence correlates with aberrant CLDN1 expression which canbe reversed in vitro by the conditioned growth medium of B. pullicaecorum. Together, thesedata support a role for B. pullicaecorum in the preservation of colonic barrier integrity.

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Significant Shifts of the Microbiome After Bowel Preparation for ColonoscopyAre Transient As Demonstrated by Longitudinal Stool Sampling and High-Throughput SequencingSuBin Kim, Philip Chuang, Emily Walsh, Jose C. Clemente, Jai Ram Rideout, Scott M.Smukalla, Josephine Ni, Ilseung Cho

Background: Polyethylene glycol (PEG) is a widely used bowel preparation for patientsundergoing colonoscopy. However, there are no studies evaluating the effect of PEG bowelpreparation on the gut microbiome. As studies of gastrointestinal diseases involving themicrobiome usually require colonoscopies, it is important to determine if perturbations ofthe gut bacterial communities due to bowel preparation are significant and whether theyare transient or permanent. Aim: To study the effects of polyethylene glycol bowel preparationon the composition of the gut microbiome and determine whether the microbiota returnsto its original state after perturbation with the bowel preparation. Methods: Seven patientsundergoing screening colonoscopies were enrolled at the Manhattan campus of the NewYork Harbor Healthcare system gastroenterology clinic and provided a pre-colonoscopy stoolsample at enrollment. After bowel preparation with polyethylene glycol, samples werecollected both during colonoscopy and at weekly intervals for five weeks after the procedure.Stool DNA was extracted using the MoBio Powersoil kit and total bacterial 16S rRNAquantitative PCR (qPCR) was performed using degenerate primers. The V4 hypervariableregion of the 16S rRNA was sequenced with 2x250bp paired end reads using the IlluminaMiSeq system. Quality filtering, demultiplexing, and downstream bioinformatic analyses,including operational taxonomic unit (out) picking and estimates of alpha and beta diversity,were performed using the QIIME software package (v 1.7.0). Results: Total 16S bacterialcounts were significantly decreased immediately after bowel preparation and returned tobaseline by post-procedure week 2 as measured by qPCR. After quality filtering, we obtained2,510,597 sequences with an average 54,578 reads per sample. Sequence analysis demon-strated significant taxonomic shifts between the pre-bowel prep sample and the intra-colonoscopy sample. In 5 of the 7 patients, the observed differences in taxonomy at theorder level was primarily caused by a bloom of Bacteroidales. In the remaining two patients,the intracolonoscopy shifts were caused by blooms of Burkholderiales or Enterobacteriales.By week 1 after colonoscopy, all 7 patients had returned to their baseline microbiome states.No further significant changes were seen between weeks 1 and 5. Conclusions: Polyethyleneglycol bowel preparation significantly alters the colonic microbiome immediately as deter-mined by intra-colonoscopic sampling, largely due to a bloom in Bacteroidales. One weekafter exposure to bowel preparation, the microbiome had returned to baseline. The resultsof this study demonstrate that shifts in the microbiome due to bowel preparation are largebut transient and that recovery is rapid and complete.

S-835 AGA Abstracts

Sequence analyses of serial stool samples. (A) Principal component analysis plot demonstrat-ing patient-specific clustering of pre-bowel preparation samples with post-colonoscopy sam-ples. Intra-colonoscopy samples deviate significantly from other samples from the samepatient. Samples are denoted as "Patient ID"."collection time". ("81.3" indicates sample frompatient 81 at 3 weeks post-colonoscopy.) (B) Taxonomy abundance plots at the orderlevel demonstrate that intracolonoscopy samples (indicated by the arrows) are significantlydifferent from pre- and post-colonoscopy samples. A bloom of Bacteroidiales is seen in 5of the 7 intracolonoscopy samples and is the primary component of the taxonomic shifts.Post-colonoscopy recovery appears to occur in less than 1 week.

Tu1757

Intestinal Microbiota Is a Heritable Trait in Healthy SubjectsWilliams Turpin, Mark S. Silverberg, Gabriel Moreno-Hagelsieb, David Kevans, Wei Xu,Venus Onay, Ken Croitoru, Andrew D. Paterson

The relative contribution of genetic and environmental factors to the composition of theintestinal microbiota remains poorly understood. While it has been shown previously thatthe microbiota of an identical or fraternal twin is more like their co-twin and mother thanthat of an unrelated individual, the heritability of individual taxa have not been determined.We aimed to determine the genetic contribution to differences in intestinal microbial composi-tion in a cohort of family members of healthy first-degree relatives (FDR) of CD. We examineda cohort of 450 healthy FDRs subjects from 203 families with more than one family memberrecruited to the GEM Project. Each subjects provided stool samples. Bacterial DNA wasextracted from the stool and the V1-V3 hypervariable regions of 16S rRNA genes weresequenced using 454 pyrosequencing. 16s rRNA sequences were processed using QIIMEand USEARCH. The relative abundance of bacterial genera was inverse normally transformed.Polygenic heritability (H2R) of the 16 bacterial genera detected with relative abundance>1% was assessed using SOLAR software. H2R is expressed as a value from 0 - 1 with anaccompanying significance test for the additive genetic heritability (i.e. p value<0.05) afteradjustment for covariates: age, sex, and sequencing plate. The cohort of FDRs (n=450) wasas follows: 17.0 median age [range 6-35] and 49.4% were male. The family structures withinthe cohort were as follows: siblings 58.9%, offspring 36.7%, avuncular 3.1% and monozygotictwins 1.3%. Fecal samples were dominated by three genera, Faecalibacterium, Bacteroidesand Pseudobutyrivibrio with their relative abundance (mean % ± SD %) being 25.7 ±15.9, 19.4 ± 18.9, and 12.7 ± 11.1 respectively. H2R assessment (H2R±SE, p-value) ofFaecalibacterium (0.52±0.17, p<0.002), Alistipes (0.36±0.20, p<0.033), Pseudobutyrivibrio(0.40±0.21, p<0.033), Collinsella (0.51±0.21, p<0.02), and Dialister (0.42±0.19, p<0.02)genera demonstrated heritability that was statistically significant. This is the first report todemonstrate significant heritability of specific constituents of the intestinal microbiota in acohort of healthy humans; however, for the individual heritable genera our estimates are ofa moderate effect further supporting the importance of environmental factors as importantdeterminants of intestinal microbial composition. Submitted on behalf of GEM projectresearch team

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Intestinal Microbiota Makeup Is Not Associated With NOD2 and FUT2Genetic Polymorphisms in a Healthy CohortWilliams Turpin, Mark S. Silverberg, Gabriel Moreno-Hagelsieb, Wei Xu, Andrew D.Paterson, Ken Croitoru

Recent studies in humans have shown that SNPs in the genetic region of the intracellularpattern recognition receptor NOD2 and in the controller of the presence of mucosal fucosy-lated glycan structures FUT2 genes are associated with gut microbiota composition. Thesefindings were discovered in relatively small sample sizes and included subjects with inflamma-tory disease and various other non-inflammatory diseases and require replication. To investi-gate if NOD2 and FUT2 SNPs are associated with fecal microbiota composition in a largepopulation of healthy first degree relatives (FDRs) of Crohn's disease (CD) subjects. BacterialDNA extracted from the stool of 464 healthy FDRs was subjected to 454 pyrosequencingof the V1-V3 hypervariable regions of the 16S rRNA. Sequences were processed using theQIIME and USEARCH. Subjects were genotyped using Sequenom Gold iPlex, TaqManplatforms, or ImmunoChip and a non-parametric test was used to test SNPs for associationwith the sixteen major bacterial genera detected with mean relative abundance >1%. In thiscohort, stool microbiota were dominated by Bacteroides (25.4 ± 23.8), Faecalibacterium(23.5 ± 16.5, mean % ± SD %), and Pseudobutyrivibrio (12.7 ± 11.2 respectively). Ouranalysis demonstrates that there is no significant association between NOD2 rs2066844,rs2066845, rs2066847, or FUT2 rs601338 with microbial genera after correction of the p-value for multiple testing. To further investigate for any possible association of NOD2 orFUT2 SNPs, we fine mapped 155 and 19 polymorphic SNPs located in and around NOD2and FUT2 respectively and assessed for any association with stool microbiota composition.No significant association between these SNPs and the microbiota were founded. Our datasuggests that there is no association between NOD2 or FUT2 SNPs and fecal microbiotacomposition at the genera level in healthy individuals. This is in contrast to previous studies;however, those populations included subjects with IBD or various other non-inflammatorydiseases and were composed of relatively small sample sizes. Discrepancies between thesefindings may be due to the larger sample size or due to the restriction of our study tohealthy individuals. It is further possible that the associations between the host genetics andmicrobiotia makeup differs between healthy subjects and those with IBD, suggesting thatgenetic factors at play in normal homeostasis may differ from that engaged during the processof inflammation. Submitted on behalf of GEM project research team

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