Zachary Marsh

Development and Validation of a Biomarker for Diarrhea-Predominant Irritable Bowel Syndrome in Human Subjects

Diarrhea-predominant irritable bowel syndrome (IBS) is diagnosed through clinical criteria after excluding “organic” conditions, and can be precipitated by acute gastroenteritis. Cytolethal distending toxin B (CdtB) is produced by bacteria that cause acute gastroenteritis, and a post-infectious animal model demonstrates that host antibodies to CdtB cross-react with vinculin in the host gut, producing an IBS-like phenotype. Therefore, we assessed circulating anti-CdtB and anti-vinculin antibodies as biomarkers for D-IBS in human subjects. Subjects with D-IBS based on Rome criteria (n=2375) were recruited from a large-scale multicenter clinical trial for D-IBS (TARGET 3). Subjects with inflammatory bowel disease (IBD) (n=142), subjects with celiac disease (n=121), and healthy controls (n=43) were obtained for comparison. Subjects with IBD and celiac disease were recruited based on the presence of intestinal complaints and histologic confirmation of chronic inflammatory changes in the colon or small intestine. Subjects with celiac disease were also required to have an elevated tTG and biopsy. All subjects were aged between 18 and 65 years. Plasma levels of anti-CdtB and anti-vinculin antibodies were determined by ELISA, and compared between groups. Anti-CdtB titers were significantly higher in D-IBS subjects compared to IBD, healthy controls and celiac disease (P<0.001). Anti-vinculin titers were also significantly higher in IBS (P<0.001) compared to the other groups. The area-under-the-receiver operating curves (AUCs) were 0.81 and 0.62 for diagnosis of D-IBS against IBD for anti-CdtB and anti-vinculin, respectively. Both tests were less specific in differentiating IBS from celiac disease. Optimization demonstrated that for anti-CdtB (optical density≥2.80) the specificity, sensitivity and likelihood ratio were 91.6%, 43.7 and 5.2, respectively, and for anti-vinculin (OD≥1.68) were 83.8%, 32.6 and 2.0, respectively. These results confirm that anti-CdtB and anti-vinculin antibodies are elevated in D-IBS compared to non-IBS subjects. These biomarkers may be especially helpful in distinguishing D-IBS from IBD in the workup of chronic diarrhea.

Role of Cytolethal Distending Toxin in Altered Stool Form and Bowel Phenotypes in a Rat Model of Post-infectious Irritable Bowel Syndrome

Background/Aims Campylobacter jejuni infection is a leading cause of acute gastroenteritis, which is a trigger for post-infectious irritable bowel syndrome (PI-IBS). Cytolethal distending toxin (CDT) is expressed by enteric pathogens that cause PI-IBS. We used a rat model of PI-IBS to investigate the role of CDT in long-term altered stool form and bowel phenotypes. Methods Adult Sprague-Dawley rats were gavaged with wildtype C. jejuni (C+), a C. jejuni cdtB knockout (CDT-) or saline vehicle (controls). Four months after gavage, stool from 3 consecutive days was assessed for stool form and percent wet weight. Rectal tissue was analyzed for intraepithelial lymphocytes, and small intestinal tissue was stained with anti-c-kit for deep muscular plexus interstitial cells of Cajal (DMP-ICC). Results All 3 groups showed similar colonization and clearance parameters. Average 3-day stool dry weights were similar in all 3 groups, but day-to-day variability in stool form and stool dry weight were significantly different in the C+ group vs both controls (P < 0.01) and the CDT- roup (P < 0.01), but were not different in the CDT- vs controls. Similarly, rectal lymphocytes were significantly higher after C. jejuni (C+) infection vs both controls (P < 0.01) and CDT-exposed rats (P < 0.05). The counts in the latter 2 groups were not significantly different. Finally, c-kit staining revealed that DMP-ICC were reduced only in rats exposed to wildtype C. jejuni. Conclusions In this rat model of PI-IBS, CDT appears to play a role in the development of chronic altered bowel patterns, mild chronic rectal inflammation and reduction in DMP-ICC.

Effect of repeated Campylobacter jejuni infection on gut flora and mucosal defense in a rat model of post infectious functional and microbial bowel changes

Campylobacter jejuni infection is a leading cause of gastroenteritis and post infectious irritable bowel syndrome (PI‐IBS). Unanswered questions include the role of cytokines, effects on gut flora, and why IBS is not more prevalent in countries with higher gastroenteritis rates. Therefore, we determined the effects of early and repeat C. jejuni infections on post infectious phenotypes, gut flora, and cytokine levels in a rat model of functional bowel and microbial changes.

Metabolic effects of eradicating breath methane using antibiotics in prediabetic subjects with obesity

Methanogens colonizing the human gut produce methane and influence host metabolism. This study examined metabolic parameters in methane‐producing subjects before and after antibiotic treatment.

Mo2051 Lovastatin Improves Stool Form in Methanobrevibacter Smithii Colonized Rats With Constipation

30 adult, male Sprague-Dawley rats were placed on a high-fat diet (60.3% kcal from fat, Teklad high-fat diet TD.06414, Harlan Laboratories Inc, Madison, WI) for 7 weeks. The rats were assessed for increased M. smithii by qPCR before and after the diet, and then divided into 3 groups. Group 1 was given lovastatin in its lactone form, Group 2 was given lovastatin hydroxy acid (each 1.5 mg/rat), and Group 3 was gavaged with a placebo. Each group was gavaged daily for 10 days. Three day stool collections were performed to assess average stool wet weight and daily variability prior to commencing the highfat diet, after 7 weeks of high-fat diet, and the final days of the lovastatin gavage (still on high-fat diet). On day 10 of the gavage, rats were euthanized and DNA was extracted from contents of ligated bowel segments (duodenum, jejunum, ileum, cecum and left colon). qPCR was performed using primers for total luminal bacteria and M. smithii. RESULTS

311 Large Scale Validation of a Biomarker for Diarrhea-Predominant Irritable Bowel Syndrome

of these patients had fasting FGF19 measured. Alanine transaminase (ALT) and appearance of fatty liver on imaging (ultrasound, CT or MR) were retrospectively added to the database. Where multiple investigations had been performed, the test nearest to the date of the SeHCAT test was recorded. Patients with known chronic liver disease or alcohol abuse were excluded from the final analysis. Results: Of 578 SeHCAT values on the database, 303 (52%) were positive with a value 31IU/L (36% v 21%, p 31IU/L (21% v 7%, p 31IU/L (43% v 22%, p 31IU/L (23% v 7%, p 40 IU/L (40% vs 12%, p<0.05), OR 5.13 (95%CI 1.28-20.61, p<0.05). Conclusions: Primary bile acid diarrhea is associated with NAFLD and may share a common pathology in low FGF19. Both conditions may be presentations of the metabolic syndrome associated with low FGF19.

Su2020 Autoimmunity to Vinculin in Humans May Be Important in the Pathophysiology of IBS

G A A b st ra ct s tissue myeloperoxidase (MPO) activity and qPCR quantification of TLR4 gene expression, as markers for immune cell infiltration and host-gut flora interactions, respectively (6 samples/ mouse). Results: Our studies showed a strong, yet conditional, correlation between stereomicroscopy and histology, and treatment effect. For ileitis, there was a strong exponential correlation (y=6.7216*e0.1889x; R2=0.8919), which was explained by a plateau effect of histological scores between (10-12/18) as the severity of 3D-steromicroscopic abnormalities in SAMP increased. Such correlation differed from that of other studies with ileitis-free mice (R2<0.3), indicating that histological scores need to be validated or adjusted for a diverse categories of intestinal pathologies. For colitis, the correlation between stereomicroscopy and histology was strong as it correlated with the high histological scores observed in DSScolitis, and intermediate or low after dexamethasone or placebo treatments (y=8.03x+13.369; R2=0.908). The variability observed between the two methods, was explained by nonrandom segmental distribution of 3-D abnormal pathologies, not captured by the integer histological scoring system. Of immunological and statistical relevance, the ability for MPO and TLR4 to differentiate the groups was significantly enhanced when the analysis was stratified by the degree of 3-Dmucosal abnormalities (p<0.01). Conclusion: Stereomicroscopy is a very cost effective visualization tool that can be used as a complementary research and diagnostic tool in studies of therapeutic and intestinal pathology.

Su2119 Introduction of Enteric Methanogens Reduces Intestinal Transit and Alters Oral Glucose Tolerance

min functional MRI scan, followed by 10 min acid infusion, and followed by a 6 min functional MRI scan. This protocol was repeated after bilateral vagotomy. For functional imaging we acquired gradient ECHO images on a 9.4T Bruker MRI scanner with the following EPI parameters-matrix 96 x 96,FOV 35mm,TR=2s,TE=18.76 ms, 1 mm slice. All analysis was performed using FSL/FLIRT for image registration and AFNI(NIH) for connectivity analysis. We used seed-based connectivity analysis with placement of seeds within the cingulate cortex (anterior, middle, and retrosplenial). Group comparisons were made using ANOVA and Monte-Carlo simulations for multiple comparison correction. Correlation maps were generated and plotted with a corrected p-value. Results: We found that across all correlation coefficient thresholds, esophageal acid infusion was associated with significantly fewer correlated cortical voxels to the cingulate cortex (Figure 1). We found that when placing seeds within the anterior cingulate cortex, there was statistically significant diminished connectivity with the primary and secondary somatosensory, and primary and secondary motor areas after acid infusion. These alternations were not observed following bilateral cervical vagotomy (Figure 2). The findings of diminished connectivity were also demonstrated when placing seeds in the middle cingulate cortex and retrosplenial cortex. (p-value threshold of 0.02, cluster size of 6 voxels) Conclusions: Esophageal acid stimulation alters functional connectivity of the acid responsive regions of the brain. Esophageal acid induced brain functional connectivity changes are vagally mediated. These findings may play a role in further understanding of acid-related esophageal disorders.

Tu1990 Expression of Mucosal Defense Mediators and Cytokines are Dependent on the Number of Exposures to C. Jejuni in a Rat Model of Post-Infectious IBS

Background and Aims: The question was raised, whether the herbal medicine STW 5 acts on contractions elicited after electrical field stimulation (EFS) and on intestinal slow wave activity in the small intestine of mice, in a new type of an In Vitro model of trinitrobenzene sulfonic acid (TNBS) triggered inflammation. Methods: In the organ bath, segments of distal ileum of Balb/C mice were used for registration of spontaneous and EFS stimulated contractions. In the electrophysiological studies, segments of distal ileum (with minimum 50% increase in EFS induced contraction after 90 min of intraluminal TNBS application) were used for intracellular recordings, after removal of mucosa and submucosa. STW 5 was tested in a dilution of 1:100 in an organ bath and compared to vehicle (31% ethanol solution diluted 1:100), following intraluminal application of TNBS (0,01M solution, 3 cm H2O pressure) or Krebs solution for 90 min. Results: TNBS induced a significant time dependent enhancement of contractility (90 min: 155.1 ± 6.5% vs. control, n=15). STW 5 applied into the organ bath reduced basal tone (-22.1±2.1% TNBS vs. -17.7±1.5% no TNBS n=15), EFS induced contractility (-61.3±3.5% TNBS vs. -67.6±3.7% no TNBS n=10) and significantly prevented TNBS induced increase of EFS induced contractions (97.4 ± 4.1% STW5+TNBS vs. TNBS/no STW 5 155.1 ± 6.5% n=10). Electrophysiological parameters were not significantly influenced in TNBS pretreated or control preparations. No significant effects on intracellular recordings of resting membrane potential, slow wave amplitude and frequency were observed making neuronal and muscular effects unlikely. Summary and Conclusions: Stimulation with TNBS results in inflammation induced changes of motility, which are reversed or prevented by STW 5. The underlying mechanisms may be of relevance in the treatment of functional gastrointestinal and subclinical inflammatory gastrointestinal diseases.

Expression of B-Defensin-2 and Other PRO-Inflammatory Mediators Notable in IBS Are Increased in Rat Mucosa During Acute Campylobacter Jejuni Infection

G A A b st ra ct s and jejunum after Rikkunshito administration was significantly higher than that in the control and increased in a dose-dependent manner. In the postprandial state, Rikkunshito had no significant effect on the motor activity. Intragastric administration of Rikkunshito accelerated gastric emptying. Atropine and hexamethonium significantly diminished Rikkunshito-induced contractions. After administration of Rikkunshito, plasma acylated-ghrelin gradually increased, and the increase was much more significant than in the control at 3h after administration. Conclusions: Intragastric administration of Rikkunshito stimulated gastrointestinal contractions in the interdigestive state and accelerated gastric emptying. Moreover, Rikkunshito increased plasma acylated-ghrelin levels. Rikkunshito, a traditional herbal medicine, may alleviate gastrointestinal tract disorders through its prokinetic effect.