- Email: [email protected]
Cecal Microbiota Directly Affect Colitis Development in a Mouse Model of Induced Colitis
lymph nodes, liver, spleen, ileum, colon, and stomach were analyzed through RT-qPCR, immunohistochemistry, ELISA, and flow cytometry for Th1/Th2/Th17 characterization. Intestinal villous morphology was evaluated using 3D stereomicroscopy, with isolation of normal and abnormal specimens for myeloperoxidase (MPO) analyses. Fixed tissues were processed for PAS/AB staining. Histological assessments were performed blinded using validated techniques. 16s rRNA gene sequencing was used to identify bacterial populations. Results: Within 14 days, the CD, UC and non-IBD FMT-treated group had a significant increase in mean baseline diarrhea scores [0.08(0.20) vs 2.17(0.41), p=0.002; 0.17(0.41) vs 0.5(0.55), p=0.008; 0.08(0.20) vs 0.5(0.83); p=0.002, respectively), with severe dermatitis observed in 3 hGB. Pairwise Friedman's tests of differences among repeated measures revealed a significant increase in baseline endoscopic scores by week 4 in the CD [x2(3)= 9.58, p= 0.001] and non-IBD [x2(3)= 9.33, p=0.005] groups, while histological analysis showed decreased ileitis severity in the UC and non-IBD groups, compared to PBS/DMSO controls [0.33 ± 0.81 vs 3.4 ± 1.14, p=0.025; 0.00 ± 0.00 vs 3.4 ± 1.14, p=0.0136, respectively]. Quantification of MPO activity showed no significant difference between groups. 16s rRNAbased sequencing time-series analyses across groups are in progress. Conclusion: Our results suggest differences in phenotypic parameters relative to bacterial colonization adaptations over time in humanized and murine gnotobiotic mice. Therefore, hGB mice via FMT may represent an excellent model to study functional and metabolic effects of human microbiota.
Tu1848 CO-HOUSING DSS TREATED MICE WITH HEALTHY MICE RESULTS IN FASTER NORMALIZATION OF THE INTESTINAL MICROBIOTA AND PROMOTES RECOVERY Marianne Spalinger, Claudia Gottier, Larissa Hering, Silvia Lang, Gerhard Rogler, Michael M. Scharl Background: The intestine is populated with myriads of bacteria, which form a complex ecosystem and have tremendous impact on our health. In inflammatory bowel disease (IBD), shifts in microbiota composition and a reduction in overall diversity have been described. There are attempts to therapeutically transfer the microbiota from healthy subjects to persons suffering from intestinal disease. While in case of Clostridium difficile infections, this approach proves to be very efficient; the therapeutic value of fecal microbial transfer (FMT) in IBD is still unclear. In mouse models of intestinal inflammation, the effect of FMT has been studied poorly and if so, germ-free or antibiotic-treated animals have been used - models that poorly reflect the situation in human IBD patients. Here, we addressed how transmission of microbiota from healthy to diseased mice affects recovery from acute colitis. Methods: Acute colitis was induced in 12-14 week old C57B6 mice by administration of 2% DSS in the drinking water for 7 days. Mice with colitis were co-housed with healthy mice after removal of DSS. Due to coprophagy, this results in fast transfer of the microbiota between co-housed mice. To analyse changes in the composition of microbiota over time, stool samples were taken every second day and sequenced for the V4 hyper-variable region in the bacterial 16S DNA. Results: As expected, DSS treatment resulted in severe weight loss, and even 7 days after withdraw of DSS (day 15), histology confirmed severe colitis. Intestinal inflammation was accompanied by an overall reduction of microbial diversity (decreased Shannon index, p<0.01), and a marked shift in the composition of the microbiota (increased abundance of Verrucomicrobia, Cyanobacteria and some families of Firmicutes [mainly Clostridiacea], although overall abundance of Firmicutes was decreased [p<0.01 for all]). However, on day 15, these changes were less pronounced, indicating a normalization of the microbiota composition upon recovery. DSS-treated mice which were co-housed with healthy littermates after colitis induction, showed faster recovery (earlier weight gain, reduced histological scores, reduced levels of the infiltration marker myeloperoxidase (MPO), less pronounced shortening of the colon, p<0.01 for all) and an earlier normalization of the microbiota composition. Conclusions: Our results indicate that co-housing of DSS-treated mice with healthy mice results in transfer of healthy microbiota to diseased mice, and promotes recovery from colitis. This indicates that introduction of a "healthy" microbiota might have beneficial effects during intestinal inflammation and opens the possibility to systematically study the effect of genetic alterations in donor and/or recipient on the efficacy of FMT.
Tu1846 GENOTYPE-SEROTYPE INTERACTIONS SHED LIGHT ON OF THE PATHOPHYSIOLOGY INFLAMMATORY BOWEL DISEASES Shay Ben-Shachar, Yael Finezilber, Hofit Elad, Henit A. Yanai, Iris Dotan Introduction: Multiple genetic variants are associated with inflammatory bowel diseases (IBD) but their role in IBD pathophysiology is mostly unclear. Variants in NOD2 and ATG16L1 genes were related to defects in microorganism sensing in IBD. These variants are more prevalent Crohn's disease (CD), but not ulcerative colitis (UC), compared with controls. Serologic responses may reflect loss of tolerance towards luminal microorganisms. Aims: To evaluate the effect of having variants in NOD2 and ATG16L1 genes on serologic responses in IBD patients. Methods: IBD patients and healthy controls were recruited in a tertiary IBD Center. NOD2 variants (1007fs, G908R, R702W) and the ATG16L1 A300T variant were analyzed in leukocytes DNA using TaqMan chemistry. Anti-glycan-antibodies: AntiS. cerevisiae antibodies (ASCA), anti-mannobioside carbohydrate antibodies (AMCA), antichitobioside carbohydrate antibodies (ACCA) and anti-laminaribioside carbohydrate antibodies (ALCA), were analyzed using Elisa. Results: Patients [144 CD, 195 UC of whom 126 underwent pouch surgery (pouch group)], and healthy controls (90) were recruited. NOD2 G908 allele was detected in 15% of CD patients compared with up to 4% in the other groups. The ATG16L1 A300T variant was detected in 61% of controls compared with 7985% in the IBD groups (p<0.05). ASCA levels were elevated in CD compared to all other groups (p<0.01). CD patients with the NOD2 100fs variant had increased ASCA levels compared with CD patients negative for the variant (64% vs. 36% positive ASCA, p<0.01, and 78.0±31.1IU vs. 52.9±50.5IU absolute levels, p<0.05). No increased ASCA levels were detected in CD patients with the missense NOD2 variants compared to those without the variants. Similarly, pouch patients having the NOD2 100fs variant had elevated ASCA levels with 33% of them showing positive ASCA levels compared to 8% in these without this variant (P<0.05). The 100fs variant, detected in two UC patients and six controls, did not affect serologic responses. ACCA levels were highest in CD, and significantly elevated in UC compared to normal controls (p<0.05). Positive ACCA was detected in 26% of CD patients having an ATG16L1 A300T variant (either heterozygote or homozygote state) compared with zero among those without the ATG16L1 A300T variant (p<0.01). Concordantly, ACCA levels were doubled in CD patients with compared to those without the ATG16L1 A300T variant (p<0.05). Conclusions: Genetic variants impact serologic responses in CD and pouch patients, but not in UC and healthy controls. The difference in genotype- serotype interactions may imply diverse response towards microorganisms in IBD patients with different genetic backgrounds, such as the NOD2 and ATG16L1 functional variants.
Tu1849 CECAL MICROBIOTA DIRECTLY AFFECT COLITIS DEVELOPMENT IN A MOUSE MODEL OF INDUCED COLITIS Ramya Movva, Amy S. Purdon, Saleh Y. Alabbas, Paraic O Cuiv, Timothy H. Florin, Iulia Oancea A dysbiotic microbiome is associated with inflammatory bowel diseases (IBD). This has raised the possibility that a dysbiosis is causal rather than only associated with inflammation but despite this, most IBD treatments are directed at suppressing inflammation. The early results of fecal microbiota transplantation (FMT) have been variable. Positive trials may not always have been satisfactorily controlled. If the dysbiosis is causal then it should also be able to transmit colitis. Winnie mice have a Muc2 SNP mutation resulting in increased ER stress, a barrier defect and a spontaneous Th17-predominant colonic inflammation resembling ulcerative colitis. Winnie has proved an excellent model for preclinical testing of treatments for ulcerative colitis (Das 2012, Oancea 2016). We have also been able to ameliorate Winnie colitis with an antibiotic cocktail, but could not distinguish if the improvement was due to antibiotic-altered microbiome structure versus a direct effect of the antibiotics on mucosal inflammation. To investigate this, we studied the effect of FMT donated from untreated or antibiotic-treated Winnie, or WT mice, in a low dose DSS-induced colitis model recipient. Donor mice were bred in-house in a pathogen-free animal facility. Recipient mice were outsourced, but acclimatised for a week in the same animal facility. Cecal contents were collected from the donor mice and processed under anaerobic conditions using Ringer solution to produce supernatants. These or vehicle control were gavaged daily for 7 days into 1%-DSS treated WT mice. Disease activity (a composite of diarrhea, rectal bleeding and body weight) was scored daily. At sacrifice, colon weight/length ratio as a measure of colitis severity was recorded. Histology scoring was blinded. We have analysed data for the donor WT and donor untreated Winnie cecal contents into the WT DSS treated mice. Gavage
Tu1847 FUNCTIONAL CHARACTERIZATION OF A HUMANIZED FECAL MICROBIOTA TRANSPLANTATION (FMT) MODEL IN GNOTOBIOTIC SAMP1/YITFC MICE: A VALIDATION STUDY Abigail R. Basson, Adrian S. Gomez-Nguyen, Paola Menghini, Luca Di Martino, Alexander Rodriguez-Palacios, Fabio Cominelli Background: Validated, humanized gnotobiotic (hGB), inflammatory bowel disease (IBD) mice models, present an exceptional opportunity to conduct proof-of-principal studies that test the effect of microbiota-mediated effects on host immune and metabolic responses. However, the persistence of human flora in mice remains unclear, and in the SAMP1/YitFc (SAMP) model of CD-ileitis, no such literature exists. The aim of this study was thus to characterize the functional recovery of fecal microbiota isolated from IBD (Crohn's, CD; ulcerative colitis, UC) and healthy non-IBD patients, and of specific pathogen-free (SPF) mice donors after FMT in SAMP germ-free (GF) mice recipients, and to obtain preliminary data on mechanistic and phenotypic parameters over time. Methods: Five groups (6/group) of young (7 week), sex-matched, GF SAMP mice were administered 200µL anaerobically prepared FMT inoculum, or sham (PBS/7%DMSO), weekly, via oral gavage, for 8 weeks. In a second experiment, groups received a single inoculation (same donors) and were sacrificed one week later. Fecal samples, body weight and diarrhea scores were measured weekly. Endoscopic assessment was performed at baseline, week 4 and sacrifice. Mesenteric
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adherence or mucin fucosylation. This alters bacterial and mucous protection against pathogens, causing immune disequilibrium and mucosal inflammation. FUT2 products also interact with the IL 12/23 inflammatory pathways. Supporting genome wide association studies strongly associate homozygous FUT2 SNPs in strong linkage disequilibrium (LD) (rs602662 (A), rs676388 (C), rs492602 (G), rs504963 (A), rs601338 (A), rs485186 (G)), non-secretion, and CD. rs601338 (W143X) is the common null allele in Caucasians associated with the ABO non-secretory phenotype. Methods: We conducted a cross-sectional observational study of consecutive adult CD outpatients at the McGill University Health Center (20132015). Clinical and biochemical data were prospectively collected at a single routine office visit. We analyzed associations between CD and FUT2 mutation status. Results: Sixty-two CD patients were recruited. FUT2M homozygotes (rs602662, rs601338 or any mutation in LD) were detected in 27% of CD (17/62). Compared to Wild type (n=18), CD FUT2M homozygotes (n=17) had less penetrating CD (18% vs. 56%, p=0.02) and higher clinical remission without biologic or immunomodulator therapy (47% vs 6%, p=0.006). The latter was not observed in heterozygotes (22%, n=27, p=0.15). CD FUT2M homozygotes had similar disease location in the ileum (L2 p=0.31) and colon (L1, L3, p=0.56). Similarly, patients with homozygous rs601338 (n=15) had increased clinical remission rates without biologic or immunomodulator therapy (53% vs 5%, p=0.0016) compared to patients with wild type status (n=19). This was not observed in heterozygotes (24%, n=25, p=0.09). Both rs601338 homozygotes (20%) and heterozygotes (24%) had less penetrating disease than Wild type (53%, p=0.0495, 0.0478). rs601338 homozygotes (67%) but not heterozygotes (56%) had more luminal phenotype compared to non-mutants (32%, p=0.042). Conclusion: FUT2 homozygous mutations in CD was associated with a milder disease course: lower rates of penetrating disease and higher rates of remission without need for biologic or immunomodulator therapy. FUT2 may play a role in the natural history of CD through mofification of expression of adherence molecules and gut dysbiosis. Further studies are needed to confirm these findings.
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of donor untreated Winnie into WT DSS recipient dramatically exacerbated colitis with 3.5fold increased disease activity from day 4 of DSS (p<0.0001) and almost doubled colon weight/length ratio. There was no change in disease activity in donor WT to WT DSS recipients. Blinded histology will be presented for all groups. Conclusion The data strongly supports a direct role for the cecal microbiome in determining colitic development. We predict that the results will also show that the microbiome is critical in restoring normal colonic physiology in this model.
rate, following one session of treatment with FMT, was 78% (confidence interval [CI]: 71.2%-83.6%; Cochran's Q, P: 0.67; I2: 0%) (figure 1A). The pooled estimate of overall cure rate was 87.7% (CI: 81.6%-92%; Cochran's Q , P: 0.67; I2: 14.1%) (figure 1B). The pooled estimate of overall cure rate of CDI in IBD was not inferior to non-IBD patients (risk ratio: 0.97, CI: 0.85-1.11, P: 0.7) (figure 2). Although this was with moderate risk of heterogeneity (Cochran's Q , P: 0.02; I2: 65.6%). Conclusion: Treatment of CDI with FMT in IBD patients is associated with a high overall cure rate. If initial treatment fails, multiple administration of FMT may increase the cure rate. The efficacy of FMT for CDI in IBD patients is similar to that in non-IBD patients.
Figure 1. (A) Pooled estimate of cure rate; (B) pooled estimate of overall cure rate Tu1850 ALTERATION IN MICROBIOTA COMPOSITION CONTRIBUTES TO CHRONIC INFLAMMATORY RESPONSE TRIGGERED BY CROHN'S DISEASE-ASSOCIATED AIEC INFECTION IN GCN2 DEFICIENT MICE Alexis Bretin, Guillaume Dalmasso, Nicolas Barnich, Richard Bonnet, Hang T. Nguyen Background and Aims: A high prevalence of the adherent-invasive E. coli (AIEC) in the intestinal mucosa of Crohn's disease (CD) patients has been shown. We previously showed that AIEC persist in the intestine and induce inflammation in transgenic CEABAC10 mice expressing human CEACAM6, the receptor for AIEC. We also showed that upon infection, autophagy is induced in host cells to restrain AIEC intracellular replication and that this requires activation of the GCN2/eIF2alpha/ATF4 pathway. In mice deficient for Gcn2 (gcn2-/mice), autophagy response upon AIEC infection was inhibited, leading to increased AIEC colonization and AIEC-induced inflammation. Here, we investigated whether transient AIEC colonization of gcn2-/- mice induces changes in the gut microbiota, which might contribute to the development of chronic inflammation. Methods: Wild type (WT) and gcn2-/- mice were infected with the AIEC reference strain LF82 or the non-pathogenic E. coli MG1655 strain by gavage. Feces were collected at different time points and used to determine AIEC persistence, to quantify the amounts of the inflammatory marker lipocalin-2 and to analyse microbiota composition by 454 pyrosequencing. The microbiota composition data were analysed using Quantitative Insights Into Microbial Ecology (QIIME) software package. Results: In WT and gcn2-/- mice, AIEC LF82 colonized better and persisted longer compared to the non-pathogenic MG1655 strain. However, LF82 was undetectable in the feces of mice at day 4 post-infection, indicating that in this model of infection, in which neither a cocktail of antibiotics nor DSS was used, the gut colonization by AIEC was transient. The microbiota composition analyzed by UniFrac metrics and principal coordinates analysis (PCoA) did not show any pattern of clustering or significant change at the family level in WT mice upon LF82 infection. However, for the LF82-infected gcn2-/- mice, PC analysis showed a clear pattern of clustering at day 14 and 21 post-infection. This was associated with significant changes at the family level with an increase in Bacteroidetes and Proteobacteria and a decrease in Firmicutes. These changes are consistent with those observed in CD patients. Furthermore, lipocalin-2 levels were increased in LF82-infected gcn2-/- mice at day 21 post-infection compared to other groups of mice. These data showed that chronic intestinal inflammation was developed in gcn2-/- mice after AIEC clearance and more importantly after the modification in the microbiota composition. Conclusion: Herein, we show that, in genetically predisposed hosts, a transient colonization of a pathobiont AIEC strain could lead to a change in the gut microbiota composition that might contribute to the development of chronic inflammation as observed in CD.
Figure 2. Pooled estimate of overall cure rate in IBD versus non-IBD patients
Tu1852 FECAL MICROBIOTA TRANSPLANTATION IS EFFICACIOUS TO ACHIEVE CLINICAL RESPONSE AND REMISSION IN ADULT PATIENTS WITH ULCERATIVE COLITIS; A META-ANALYSIS Amir Kashani, David Q. Shih Background: The efficacy of fecal microbiota transplantation (FMT) for the treatment of ulcerative colitis (UC) is conflicting. Systematic reviews of FMT for UC treatment have included children or Crohn's disease (CD) patient. We aimed to evaluate the efficacy of FMT in adult patients with UC using meta-analysis. Method: A systematic literature search was performed through October 2016. Only prospective studies on treatment of UC with FMT which included adult patients were selected. If a study included children or CD patients, then the study was selected only if we were able to identify and exclude those subjects (patients ≥ 17 years included). Pooled estimate of clinical response and remission were calculated. Subgroup analysis was performed based on UC disease activity scores using the Mayo score (mild to moderate: Mayo score<10; moderate to severe Mayo score>6), and number of FMT sessions (1 session vs. >1 session). Results: Twelve studies (cohort studies: 8; randomized clinical trials: 3; case control: 1) with a total of 218 patients met eligibility criteria. The pooled estimate of clinical response was 0.46 (confidence interval [CI]: 0.36, 0.57; Cochran's Q, P<0.04; I2: 46.81) (Figure 1A). The pooled estimate of clinical remission was 0.28 (CI: 0.18, 0.41; Cochran's Q, P<0.01; I2: 56.12) (Figure 1B). On meta-regression, those studies which administered more than one session of FMT showed a better pooled estimate of clinical remission (0.34 vs. 0.17; P= 0.07). Subgroup analyses between studies is shown in table 1. Conclusion: FMT is efficacious in 28% of patients with UC. About 46% of patients may experience a clinical response. The efficacy of FMT appears to be unrelated to baseline UC disease activity. Randomized placebo-controlled trials are needed to clarify the efficacy of FMT for the treatment of UC. Subgroup analyses between studies
Tu1851 FECAL MICROBIOTA TRANSPLANTATION IS HIGHLY EFFECTIVE FOR TREATMENT OF CLOSTRIDIUM DIFFICILE INFECTION IN PATIANTS WITH INFLAMMATORY BOWEL DISEASE; A META-ANALYSIS Amir Kashani, David Q. Shih Background: The efficacy of fecal microbiota transplantation (FMT) in patients with Clostridium difficile infection (CDI) has been demonstrated in randomized clinical trials. However, data on the efficacy of FMT in the subgroup of patients with concurrent CDI and inflammatory bowel disease (IBD) is limited to subsets of larger patient samples, and to small single-center case series. We aimed to perform meta-analysis to evaluate the efficacy of FMT for CDI in patients with IBD. Methods: A systematic literature search was performed through October 2016. Studies which recruited IBD patients who were treated with FMT for CDI infection were included in the analysis. The efficacy of the FMT for CDI was assessed by pooled estimate of cure rate after one FMT administration. Some patients underwent more than one FMT administration due to initial failure. Thus the overall efficacy was defined by the pooled estimate of overall cure rate in patients who received ≥ 1 session of FMT. In addition, the pooled estimate of overall cure rate was compared in IBD versus non-IBD patients among the studies which recruited both groups of patients. Results: A total of 7 studies met eligibility criteria (5 retrospective, and 2 prospective case series) which included 242 patients with IBD and 563 patients without IBD. Two studies were conducted on the same cohort of patients at different time; one of these was included in analysis. The pooled estimate of cure
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(confidence interval)
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Tu1848 CO-HOUSING DSS TREATED MICE WITH HEALTHY MICE RESULTS IN FASTER NORMALIZATION OF THE INTESTINAL MICROBIOTA AND PROMOTES RECOVERY Marianne Spalinger, Claudia Gottier, Larissa Hering, Silvia Lang, Gerhard Rogler, Michael M. Scharl Background: The intestine is populated with myriads of bacteria, which form a complex ecosystem and have tremendous impact on our health. In inflammatory bowel disease (IBD), shifts in microbiota composition and a reduction in overall diversity have been described. There are attempts to therapeutically transfer the microbiota from healthy subjects to persons suffering from intestinal disease. While in case of Clostridium difficile infections, this approach proves to be very efficient; the therapeutic value of fecal microbial transfer (FMT) in IBD is still unclear. In mouse models of intestinal inflammation, the effect of FMT has been studied poorly and if so, germ-free or antibiotic-treated animals have been used - models that poorly reflect the situation in human IBD patients. Here, we addressed how transmission of microbiota from healthy to diseased mice affects recovery from acute colitis. Methods: Acute colitis was induced in 12-14 week old C57B6 mice by administration of 2% DSS in the drinking water for 7 days. Mice with colitis were co-housed with healthy mice after removal of DSS. Due to coprophagy, this results in fast transfer of the microbiota between co-housed mice. To analyse changes in the composition of microbiota over time, stool samples were taken every second day and sequenced for the V4 hyper-variable region in the bacterial 16S DNA. Results: As expected, DSS treatment resulted in severe weight loss, and even 7 days after withdraw of DSS (day 15), histology confirmed severe colitis. Intestinal inflammation was accompanied by an overall reduction of microbial diversity (decreased Shannon index, p<0.01), and a marked shift in the composition of the microbiota (increased abundance of Verrucomicrobia, Cyanobacteria and some families of Firmicutes [mainly Clostridiacea], although overall abundance of Firmicutes was decreased [p<0.01 for all]). However, on day 15, these changes were less pronounced, indicating a normalization of the microbiota composition upon recovery. DSS-treated mice which were co-housed with healthy littermates after colitis induction, showed faster recovery (earlier weight gain, reduced histological scores, reduced levels of the infiltration marker myeloperoxidase (MPO), less pronounced shortening of the colon, p<0.01 for all) and an earlier normalization of the microbiota composition. Conclusions: Our results indicate that co-housing of DSS-treated mice with healthy mice results in transfer of healthy microbiota to diseased mice, and promotes recovery from colitis. This indicates that introduction of a "healthy" microbiota might have beneficial effects during intestinal inflammation and opens the possibility to systematically study the effect of genetic alterations in donor and/or recipient on the efficacy of FMT.
Tu1846 GENOTYPE-SEROTYPE INTERACTIONS SHED LIGHT ON OF THE PATHOPHYSIOLOGY INFLAMMATORY BOWEL DISEASES Shay Ben-Shachar, Yael Finezilber, Hofit Elad, Henit A. Yanai, Iris Dotan Introduction: Multiple genetic variants are associated with inflammatory bowel diseases (IBD) but their role in IBD pathophysiology is mostly unclear. Variants in NOD2 and ATG16L1 genes were related to defects in microorganism sensing in IBD. These variants are more prevalent Crohn's disease (CD), but not ulcerative colitis (UC), compared with controls. Serologic responses may reflect loss of tolerance towards luminal microorganisms. Aims: To evaluate the effect of having variants in NOD2 and ATG16L1 genes on serologic responses in IBD patients. Methods: IBD patients and healthy controls were recruited in a tertiary IBD Center. NOD2 variants (1007fs, G908R, R702W) and the ATG16L1 A300T variant were analyzed in leukocytes DNA using TaqMan chemistry. Anti-glycan-antibodies: AntiS. cerevisiae antibodies (ASCA), anti-mannobioside carbohydrate antibodies (AMCA), antichitobioside carbohydrate antibodies (ACCA) and anti-laminaribioside carbohydrate antibodies (ALCA), were analyzed using Elisa. Results: Patients [144 CD, 195 UC of whom 126 underwent pouch surgery (pouch group)], and healthy controls (90) were recruited. NOD2 G908 allele was detected in 15% of CD patients compared with up to 4% in the other groups. The ATG16L1 A300T variant was detected in 61% of controls compared with 7985% in the IBD groups (p<0.05). ASCA levels were elevated in CD compared to all other groups (p<0.01). CD patients with the NOD2 100fs variant had increased ASCA levels compared with CD patients negative for the variant (64% vs. 36% positive ASCA, p<0.01, and 78.0±31.1IU vs. 52.9±50.5IU absolute levels, p<0.05). No increased ASCA levels were detected in CD patients with the missense NOD2 variants compared to those without the variants. Similarly, pouch patients having the NOD2 100fs variant had elevated ASCA levels with 33% of them showing positive ASCA levels compared to 8% in these without this variant (P<0.05). The 100fs variant, detected in two UC patients and six controls, did not affect serologic responses. ACCA levels were highest in CD, and significantly elevated in UC compared to normal controls (p<0.05). Positive ACCA was detected in 26% of CD patients having an ATG16L1 A300T variant (either heterozygote or homozygote state) compared with zero among those without the ATG16L1 A300T variant (p<0.01). Concordantly, ACCA levels were doubled in CD patients with compared to those without the ATG16L1 A300T variant (p<0.05). Conclusions: Genetic variants impact serologic responses in CD and pouch patients, but not in UC and healthy controls. The difference in genotype- serotype interactions may imply diverse response towards microorganisms in IBD patients with different genetic backgrounds, such as the NOD2 and ATG16L1 functional variants.
Tu1849 CECAL MICROBIOTA DIRECTLY AFFECT COLITIS DEVELOPMENT IN A MOUSE MODEL OF INDUCED COLITIS Ramya Movva, Amy S. Purdon, Saleh Y. Alabbas, Paraic O Cuiv, Timothy H. Florin, Iulia Oancea A dysbiotic microbiome is associated with inflammatory bowel diseases (IBD). This has raised the possibility that a dysbiosis is causal rather than only associated with inflammation but despite this, most IBD treatments are directed at suppressing inflammation. The early results of fecal microbiota transplantation (FMT) have been variable. Positive trials may not always have been satisfactorily controlled. If the dysbiosis is causal then it should also be able to transmit colitis. Winnie mice have a Muc2 SNP mutation resulting in increased ER stress, a barrier defect and a spontaneous Th17-predominant colonic inflammation resembling ulcerative colitis. Winnie has proved an excellent model for preclinical testing of treatments for ulcerative colitis (Das 2012, Oancea 2016). We have also been able to ameliorate Winnie colitis with an antibiotic cocktail, but could not distinguish if the improvement was due to antibiotic-altered microbiome structure versus a direct effect of the antibiotics on mucosal inflammation. To investigate this, we studied the effect of FMT donated from untreated or antibiotic-treated Winnie, or WT mice, in a low dose DSS-induced colitis model recipient. Donor mice were bred in-house in a pathogen-free animal facility. Recipient mice were outsourced, but acclimatised for a week in the same animal facility. Cecal contents were collected from the donor mice and processed under anaerobic conditions using Ringer solution to produce supernatants. These or vehicle control were gavaged daily for 7 days into 1%-DSS treated WT mice. Disease activity (a composite of diarrhea, rectal bleeding and body weight) was scored daily. At sacrifice, colon weight/length ratio as a measure of colitis severity was recorded. Histology scoring was blinded. We have analysed data for the donor WT and donor untreated Winnie cecal contents into the WT DSS treated mice. Gavage
Tu1847 FUNCTIONAL CHARACTERIZATION OF A HUMANIZED FECAL MICROBIOTA TRANSPLANTATION (FMT) MODEL IN GNOTOBIOTIC SAMP1/YITFC MICE: A VALIDATION STUDY Abigail R. Basson, Adrian S. Gomez-Nguyen, Paola Menghini, Luca Di Martino, Alexander Rodriguez-Palacios, Fabio Cominelli Background: Validated, humanized gnotobiotic (hGB), inflammatory bowel disease (IBD) mice models, present an exceptional opportunity to conduct proof-of-principal studies that test the effect of microbiota-mediated effects on host immune and metabolic responses. However, the persistence of human flora in mice remains unclear, and in the SAMP1/YitFc (SAMP) model of CD-ileitis, no such literature exists. The aim of this study was thus to characterize the functional recovery of fecal microbiota isolated from IBD (Crohn's, CD; ulcerative colitis, UC) and healthy non-IBD patients, and of specific pathogen-free (SPF) mice donors after FMT in SAMP germ-free (GF) mice recipients, and to obtain preliminary data on mechanistic and phenotypic parameters over time. Methods: Five groups (6/group) of young (7 week), sex-matched, GF SAMP mice were administered 200µL anaerobically prepared FMT inoculum, or sham (PBS/7%DMSO), weekly, via oral gavage, for 8 weeks. In a second experiment, groups received a single inoculation (same donors) and were sacrificed one week later. Fecal samples, body weight and diarrhea scores were measured weekly. Endoscopic assessment was performed at baseline, week 4 and sacrifice. Mesenteric
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adherence or mucin fucosylation. This alters bacterial and mucous protection against pathogens, causing immune disequilibrium and mucosal inflammation. FUT2 products also interact with the IL 12/23 inflammatory pathways. Supporting genome wide association studies strongly associate homozygous FUT2 SNPs in strong linkage disequilibrium (LD) (rs602662 (A), rs676388 (C), rs492602 (G), rs504963 (A), rs601338 (A), rs485186 (G)), non-secretion, and CD. rs601338 (W143X) is the common null allele in Caucasians associated with the ABO non-secretory phenotype. Methods: We conducted a cross-sectional observational study of consecutive adult CD outpatients at the McGill University Health Center (20132015). Clinical and biochemical data were prospectively collected at a single routine office visit. We analyzed associations between CD and FUT2 mutation status. Results: Sixty-two CD patients were recruited. FUT2M homozygotes (rs602662, rs601338 or any mutation in LD) were detected in 27% of CD (17/62). Compared to Wild type (n=18), CD FUT2M homozygotes (n=17) had less penetrating CD (18% vs. 56%, p=0.02) and higher clinical remission without biologic or immunomodulator therapy (47% vs 6%, p=0.006). The latter was not observed in heterozygotes (22%, n=27, p=0.15). CD FUT2M homozygotes had similar disease location in the ileum (L2 p=0.31) and colon (L1, L3, p=0.56). Similarly, patients with homozygous rs601338 (n=15) had increased clinical remission rates without biologic or immunomodulator therapy (53% vs 5%, p=0.0016) compared to patients with wild type status (n=19). This was not observed in heterozygotes (24%, n=25, p=0.09). Both rs601338 homozygotes (20%) and heterozygotes (24%) had less penetrating disease than Wild type (53%, p=0.0495, 0.0478). rs601338 homozygotes (67%) but not heterozygotes (56%) had more luminal phenotype compared to non-mutants (32%, p=0.042). Conclusion: FUT2 homozygous mutations in CD was associated with a milder disease course: lower rates of penetrating disease and higher rates of remission without need for biologic or immunomodulator therapy. FUT2 may play a role in the natural history of CD through mofification of expression of adherence molecules and gut dysbiosis. Further studies are needed to confirm these findings.
AGA Abstracts
of donor untreated Winnie into WT DSS recipient dramatically exacerbated colitis with 3.5fold increased disease activity from day 4 of DSS (p<0.0001) and almost doubled colon weight/length ratio. There was no change in disease activity in donor WT to WT DSS recipients. Blinded histology will be presented for all groups. Conclusion The data strongly supports a direct role for the cecal microbiome in determining colitic development. We predict that the results will also show that the microbiome is critical in restoring normal colonic physiology in this model.
rate, following one session of treatment with FMT, was 78% (confidence interval [CI]: 71.2%-83.6%; Cochran's Q, P: 0.67; I2: 0%) (figure 1A). The pooled estimate of overall cure rate was 87.7% (CI: 81.6%-92%; Cochran's Q , P: 0.67; I2: 14.1%) (figure 1B). The pooled estimate of overall cure rate of CDI in IBD was not inferior to non-IBD patients (risk ratio: 0.97, CI: 0.85-1.11, P: 0.7) (figure 2). Although this was with moderate risk of heterogeneity (Cochran's Q , P: 0.02; I2: 65.6%). Conclusion: Treatment of CDI with FMT in IBD patients is associated with a high overall cure rate. If initial treatment fails, multiple administration of FMT may increase the cure rate. The efficacy of FMT for CDI in IBD patients is similar to that in non-IBD patients.
Figure 1. (A) Pooled estimate of cure rate; (B) pooled estimate of overall cure rate Tu1850 ALTERATION IN MICROBIOTA COMPOSITION CONTRIBUTES TO CHRONIC INFLAMMATORY RESPONSE TRIGGERED BY CROHN'S DISEASE-ASSOCIATED AIEC INFECTION IN GCN2 DEFICIENT MICE Alexis Bretin, Guillaume Dalmasso, Nicolas Barnich, Richard Bonnet, Hang T. Nguyen Background and Aims: A high prevalence of the adherent-invasive E. coli (AIEC) in the intestinal mucosa of Crohn's disease (CD) patients has been shown. We previously showed that AIEC persist in the intestine and induce inflammation in transgenic CEABAC10 mice expressing human CEACAM6, the receptor for AIEC. We also showed that upon infection, autophagy is induced in host cells to restrain AIEC intracellular replication and that this requires activation of the GCN2/eIF2alpha/ATF4 pathway. In mice deficient for Gcn2 (gcn2-/mice), autophagy response upon AIEC infection was inhibited, leading to increased AIEC colonization and AIEC-induced inflammation. Here, we investigated whether transient AIEC colonization of gcn2-/- mice induces changes in the gut microbiota, which might contribute to the development of chronic inflammation. Methods: Wild type (WT) and gcn2-/- mice were infected with the AIEC reference strain LF82 or the non-pathogenic E. coli MG1655 strain by gavage. Feces were collected at different time points and used to determine AIEC persistence, to quantify the amounts of the inflammatory marker lipocalin-2 and to analyse microbiota composition by 454 pyrosequencing. The microbiota composition data were analysed using Quantitative Insights Into Microbial Ecology (QIIME) software package. Results: In WT and gcn2-/- mice, AIEC LF82 colonized better and persisted longer compared to the non-pathogenic MG1655 strain. However, LF82 was undetectable in the feces of mice at day 4 post-infection, indicating that in this model of infection, in which neither a cocktail of antibiotics nor DSS was used, the gut colonization by AIEC was transient. The microbiota composition analyzed by UniFrac metrics and principal coordinates analysis (PCoA) did not show any pattern of clustering or significant change at the family level in WT mice upon LF82 infection. However, for the LF82-infected gcn2-/- mice, PC analysis showed a clear pattern of clustering at day 14 and 21 post-infection. This was associated with significant changes at the family level with an increase in Bacteroidetes and Proteobacteria and a decrease in Firmicutes. These changes are consistent with those observed in CD patients. Furthermore, lipocalin-2 levels were increased in LF82-infected gcn2-/- mice at day 21 post-infection compared to other groups of mice. These data showed that chronic intestinal inflammation was developed in gcn2-/- mice after AIEC clearance and more importantly after the modification in the microbiota composition. Conclusion: Herein, we show that, in genetically predisposed hosts, a transient colonization of a pathobiont AIEC strain could lead to a change in the gut microbiota composition that might contribute to the development of chronic inflammation as observed in CD.
Figure 2. Pooled estimate of overall cure rate in IBD versus non-IBD patients
Tu1852 FECAL MICROBIOTA TRANSPLANTATION IS EFFICACIOUS TO ACHIEVE CLINICAL RESPONSE AND REMISSION IN ADULT PATIENTS WITH ULCERATIVE COLITIS; A META-ANALYSIS Amir Kashani, David Q. Shih Background: The efficacy of fecal microbiota transplantation (FMT) for the treatment of ulcerative colitis (UC) is conflicting. Systematic reviews of FMT for UC treatment have included children or Crohn's disease (CD) patient. We aimed to evaluate the efficacy of FMT in adult patients with UC using meta-analysis. Method: A systematic literature search was performed through October 2016. Only prospective studies on treatment of UC with FMT which included adult patients were selected. If a study included children or CD patients, then the study was selected only if we were able to identify and exclude those subjects (patients ≥ 17 years included). Pooled estimate of clinical response and remission were calculated. Subgroup analysis was performed based on UC disease activity scores using the Mayo score (mild to moderate: Mayo score<10; moderate to severe Mayo score>6), and number of FMT sessions (1 session vs. >1 session). Results: Twelve studies (cohort studies: 8; randomized clinical trials: 3; case control: 1) with a total of 218 patients met eligibility criteria. The pooled estimate of clinical response was 0.46 (confidence interval [CI]: 0.36, 0.57; Cochran's Q, P<0.04; I2: 46.81) (Figure 1A). The pooled estimate of clinical remission was 0.28 (CI: 0.18, 0.41; Cochran's Q, P<0.01; I2: 56.12) (Figure 1B). On meta-regression, those studies which administered more than one session of FMT showed a better pooled estimate of clinical remission (0.34 vs. 0.17; P= 0.07). Subgroup analyses between studies is shown in table 1. Conclusion: FMT is efficacious in 28% of patients with UC. About 46% of patients may experience a clinical response. The efficacy of FMT appears to be unrelated to baseline UC disease activity. Randomized placebo-controlled trials are needed to clarify the efficacy of FMT for the treatment of UC. Subgroup analyses between studies
Tu1851 FECAL MICROBIOTA TRANSPLANTATION IS HIGHLY EFFECTIVE FOR TREATMENT OF CLOSTRIDIUM DIFFICILE INFECTION IN PATIANTS WITH INFLAMMATORY BOWEL DISEASE; A META-ANALYSIS Amir Kashani, David Q. Shih Background: The efficacy of fecal microbiota transplantation (FMT) in patients with Clostridium difficile infection (CDI) has been demonstrated in randomized clinical trials. However, data on the efficacy of FMT in the subgroup of patients with concurrent CDI and inflammatory bowel disease (IBD) is limited to subsets of larger patient samples, and to small single-center case series. We aimed to perform meta-analysis to evaluate the efficacy of FMT for CDI in patients with IBD. Methods: A systematic literature search was performed through October 2016. Studies which recruited IBD patients who were treated with FMT for CDI infection were included in the analysis. The efficacy of the FMT for CDI was assessed by pooled estimate of cure rate after one FMT administration. Some patients underwent more than one FMT administration due to initial failure. Thus the overall efficacy was defined by the pooled estimate of overall cure rate in patients who received ≥ 1 session of FMT. In addition, the pooled estimate of overall cure rate was compared in IBD versus non-IBD patients among the studies which recruited both groups of patients. Results: A total of 7 studies met eligibility criteria (5 retrospective, and 2 prospective case series) which included 242 patients with IBD and 563 patients without IBD. Two studies were conducted on the same cohort of patients at different time; one of these was included in analysis. The pooled estimate of cure
AGA Abstracts
(confidence interval)
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