Emily 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.

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.

Mo1870 Measuring Anti-Vinculin and Anti-CdtB Antibodies in Two Animal Models of Post-Infectious IBS

Diverticulosis is a common GI condition among older adults in the United States. However, limited information exists on the etiologic risk factors for diverticulosis. We hypothesized that the gut microbiome and chronic inflammation contribute to the development of diverticulosis.We evaluated the association between gut bacteria, mucosal inflammation and diverticulosis in a case-control study. Methods:Participants (n=196) were consented patients who underwent screening colonoscopy at UNC Hospitals. Subjects were classified as case if diverticula were present or control if no diverticula were present. The abundance of specific bacteria in the sigmoid mucosa was determined by qPCR based on the bacterial 16s rRNA gene for Bacteroides, Bifidobacterium, Clostridium, E. coli, Lactobacillus, and Eubacteria.Mucosal mRNA gene expression of cytokines of IL-6, IL-10, IL-17, and TNF-α and housekeeping gene HMBS were assessed by qRT-PCR. Immune cells in the lamina propria were stained for CD4, CD8, CD57 and Mast cell tryptase (MCT) by immunohistochemistry (IHC). The IHC stained sections were digitally imaged using the Aperio ScanScope XT. Analysis of biomarkers was performed using Definiens Tissue Studio software with the Composer_ Nuclei_and_Simulated_Cells algorithm. The percent cells with strong (+3), medium (+2) and weak (+1) positive signal, was used to compare biomarker levels among tissue samples. Statistical analysis included t-test and logistic regression to compare cases and controls as well as Spearman correlation to assess association between bacteria and inflammation. Results:We observed a higher relative abundance of Clostridium, Bacteroides, Bifidobacterium, E. coli, and Eubacteria in cases compared to controls. Cases had lower density of CD8 compared to controls. Although the cytokine gene expression levels of IL-6, IL-10, IL-17, and TNF-α did not differ between cases and controls, there was a significant positive correlation between CD57 expression and IL-6 (r=0.23, p=0.03), CD57 and TNFα (r0.33, p=0.001) and CD57 and IL-10 (r=0.22, p= 0.03) among cases. The association between bacterial abundance and immune cell density also varied depending on case-control status. Among cases, MCT expression showed positive correlations with Bacteroides (r=0.21, p= 0.05), Clostridium (r=0.18, p=0.08) and inverse correlation with Bifidobacteria (r=-0.26, p= 0.01). Among controls, CD57 expression was inversely correlated with Lactobacillus (r=0.19, p=0.06). Conclusions: Our findings suggest that gut bacteria and inflammation are associated with diverticulosis. This data combined with further research could have clinical implications for the prevention and treatment of diverticulosis.

847 Evaluation of Antibodies to Cytolethal Distending Toxin B (CdtB) and Vinculin in Celiac Disease

Background: Patients with refractory gastroesophageal reflux disease (GERD) may have undiagnosed celiac disease. These patients often undergo esophagogastroduodenoscopy to determine the etiology and severity of GERD. Because a duodenal biopsy is the gold standard for diagnosing celiac disease and a gluten-free diet is an effective treatment, performing routine duodenal biopsy during endoscopy may result in early diagnosis and symptom improvement, allowing for discontinuation of proton pump inhibitor therapy. We aimed to evaluate the cost-effectiveness of performing routine duodenal biopsy during endoscopy for diagnosing celiac disease in patients with refractory GERD. Methods: We constructed a decision tree using data from published literature to calculate cost-effectiveness of endoscopy with and without duodenal biopsy in a cohort of 10,000 40-year-old patients. Results: We found that the biopsy strategy would detect 70 out of 100 celiac disease patients in a cohort of 10,000 GERD patients undergoing endoscopy if the prevalence of celiac disease was 1% in this cohort. Up-front biopsy would increase the remaining quality-adjusted life years (QALYs) by 0.0032. Testing for celiac disease using this approach would increase the lifetime cost by

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

389 per patient. Compared with no biopsy, the biopsy strategy cost

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

55,693 per celiac case detected, and

Autoimmunity Links Vinculin to the Pathophysiology of Chronic Functional Bowel Changes Following Campylobacter jejuni Infection in a Rat Model.

121,875 per QALY gained. The incremental cost-effectiveness ratio (ICER) met the threshold of 1.8%, the specificity of biopsy was >98%, the cost of gluten-free diet was

Tu2056 Antibodies to Cytolethal Distending Toxin B and Auto-Antibodies to Human Vinculin Are Elevated in IBS Subjects

5,874 per year. Increasing the sensitivity of duodenal biopsy to 100% did not affect the cost-effectiveness threshold. Conclusion: Esophagogastroduodenoscopy with duodenal biopsy for refractory GERD patients can be a cost-effective approach for screening when the prevalence of celiac disease in this patient population is 1.8% or greater.

Tu2110 Circulating Antibodies to Cytolethal Distending Toxin B Correlate With the Development of Small Intestinal Bacterial Overgrowth in a Rat Model of Post-Infectious 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.