Terry Nails: A New Look at an Old Finding and its Association with Liver Disease

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AGA Abstracts

ORIGINS OF THE ANAEROBIC GUT LUMEN: MICROBES VS. CHEMISTRY Elliot S. Friedman, Tatiana V. Esipova, Kyle Bittinger, Likai Hou, Lillian Chau, Jack Jiang, Clementina Mesaros, Peder Lund, Xue Liang, Garret FitzGerald, Daeyeon Lee, Ian A. Blair, Benjamin Garcia, Sergei Vinogradov, Gary D. Wu Background: The succession from aerobic and facultative anaerobic bacteria to obligate anaerobes in the infant gut microbiota, as well as the differences in the composition of the mucosally-adherent and the luminal gut microbiota, provide indirect evidence in humans that the gut microbiota consumes oxygen diffusing from intestinal tissue to maintain anaerobic conditions in the lumen of the gut. However, the distribution of oxygen between the host and its gut microbiota remain poorly characterized due to technical limitations. We investigated the role of the microbiota and fecal chemistry in establishing and maintaining anaerobic conditions in the lumen of the gut. Methods: The phosphorescence quenching method was used to quantify oxygen levels in both the intestinal tissue and the gut lumen of conventional and germ-free mice. In conventional mice, oxygen levels were correlated with the biomass and composition of both mucosally-adherent and luminal microbes along the length of the intestinal tract. In vitro experiments using the feces of conventional and germ-free mice were used to examine differences in oxygen depletion kinetics. Lipidomic and proteomic profiling was performed to identify oxygen-consuming chemical reactions. Results: The luminal oxygen levels along the length of the gut were found to be the same in both conventional and germ-free mice; these include high oxygen levels in the proximal small intestine and anaerobic conditions in the distal gut. In vitro oxygen measurements revealed that germ-free feces effectively consume oxygen, although at a rate significantly slower than feces from conventional mice. Lipidomic and proteomic analyses of germ-free feces from in vitro experiments showed significant oxidation of unsaturated phospholipids and moderate oxidation of proteins. In conventional mice, we show that the taxonomic composition of the gut microbiota adherent to the gut mucosa and in the lumen throughout the length of the gut paralleled levels of luminal oxygen determined by our empirical measurements. Consistent with the notion that the gut microbiota can consume oxygen, there was an increase in biomass of the gut microbiota in the distal gut corresponding to the decrease in luminal oxygen. Conclusions: The remarkable finding that germ-free feces can consume oxygen provides an explanation for observations in gnotobiotic mice where individual obligate anaerobes can effectively colonize the gut of germ-free mice and demonstrate fermentative metabolism. In total, these results demonstrate the dynamic oxygen interaction of the mammalian host with its gut microbiota. Both the microbiota and the chemistry of feces regulate luminal oxygen levels that, in turn, shape the distinctive compositions of the community in different regions of the gut.

120e COMPARATIVE GENOMICS OF RUMINOCOCCUS BROMII, A KEYSTONE SPECIES FOR THE DEGRADATION OF RESISTANT STARCH IN THE HUMAN COLON, REVEALS CONSERVATION OF AMYLOSOME ORGANISATION Indrani Mukhopadhya, Paul O. Sheridan, Jenny Laverde-Gomez, Sylvia H. Duncan, William Kelly, Athol Klieve, Nicole Koropatkin, Sarah Morais, Emmanuelle Crost, Alan W. Walker, Edward A. Bayer, Nathalie Juge, Harry J. Flint

120d SACCHAROMYCES BOULARDII CNCM I-745 LOWERS FECAL CHOLIC ACID CONCENTRATIONS DURING ANTIBIOTHERAPY IN HEALTHY VOLUNTEERS: A NEW POTENTIAL MECHANISM IN THE PROTECTION AGAINST CLOSTRIDIUM DIFFICILE INFECTION Henri Duboc, Toufic Kabbani, Caroline Chong Nguyen, Kumar Pallav, Scot E. Dowd, Lydie Humbert, Philippe Seksik, Andre Bado, Benoit Coffin, Dominique Rainteau, Ciaran P. Kelly

Introduction Ruminococcus bromii is one of the most prevalent keystone bacterial species that can ferment dietary starches that resist digestion by host enzymes (resistant starch, RS) in the human colon. Degradation of the prebiotic RS has important implications in health by providing sources of energy that can be utilized by other symbiotic short-chain fattyacid-producing bacteria in the colon. RS fermentation has been shown to reduce insulin resistance, reverse infective diarrhoea and prevent colorectal cancer. Comparative genome analysis of 4 human (L2-63, L2-36, 5AMG, and the type strain ATCC27255) and one rumen (YE282) R. bromii strains was conducted to understand the pivotal role of this species in health and disease. Results The genomic sequences of R. bromii human strains (isolated from volunteers in Scotland, Ecuador and the USA) were found to be highly similar, with 95-100% average nucleotide identity, whereas the similarity with the rumen strain was 86%. The majority of the glycoside hydrolase (GH) enzymes identified belonged to the GH13 family that is involved in starch degradation, and significant conservation was noted for the GH13 genes among the human isolates (90-100% identity at the amino acid level). The human R. bromii genomes analysed had 17 GH13s (Amy1-17) while the rumen strain lacked Amy 3 and 7. The unique organization of the amylolytic enzyme system of R. bromii involving cohesin-dockerin interactions between component proteins was largely conserved among all strains. All R. bromii strains had the maltose, galactose and fructose phosphotransferase operons and a likely oligosaccharide phosphorylase (malP) for carbohydrate transport and metabolism. The ability of the human strains to grow well on galactose as a sole source of energy was investigated and corroborated for the first time, while only the L2-36 strain grew on glucose. All human strains were able to degrade RS and utilise soluble potato starch. A full set of conserved core genes linked to sporulation and germination was detected in the R. bromii genomes. Sporulation was further confirmed in L2-63 strain by transmission electron microscopy and by the recovery of viable cultures following heat inactivation (80°C for 20 minutes) and subsequent exposure to atmospheric oxygen. Conclusion R. bromii is highly nutritionally specialized for RS and has developed an extracellular amylosome complex dedicated to the degradation of RS that is conserved between strains. The galactose utilisation operon and its activity may point towards an evolutionary advantage gained by R. bromii strains to colonize the infant gut where lactose is a predominant substrate. Sporulation was documented in this species for the first time and would underpin the hardiness of these symbiotic bacteria to survive in the human colon and to be transmitted successfully between hosts.

Background : Saccharomyces boulardii (SB) demonstrated clinical efficacy in the secondary prevention of post-antibiotic Clostridium difficile infection (CDI), but the mechanism remains unclear. Cholic acid (CA) is a primary bile acid (BA), synthesized by the liver, which triggers the germination of C. difficile spores in the intestine. Physiologically, the gut microbiota transforms primary BAs (cholic acid and chenodeoxycholic) into secondary (deoxycholic and lithocholic). CA loses its germinating properties after transformation and appears to become protective against CDI. The goal of this work in healthy volunteers (HV) was to: 1) analyze the effect of antibiotics on the presence of ‘pro-germination' primary BA including CA in the stools 2) investigate whether SB enhances transformation to ‘protective' secondary BA. Patients and Methods: this work was an ancillary of a study conducted in 4 groups of HV at, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts. Previous results of this work showed that SB CNCM I-745 modulates shifts in the microbiota and reduces diarrhea during antibiotic treatment. Group 1 (n=12) received SB CNCM I745 500 mg twice daily for 14 days. Group 2 (n=12) received Amoxicillin-Clavulanate (AC) (875/125 mg, twice daily) for 7 days. Group 3 (n=12) received AC for 7 days and SB for 14 days. Group 4 (n=12) was untreated. Groups 1, 2, 3 had successive stool samples at D28, 0, 3, 7, 10, 13, 21, and group 4 at D0, 7 and 21. Fecal concentrations of 28 BAs were measured by HPLC/MS and expressed as % of total BA concentration. Results: AC alone (group 2) significantly reduced the rate of fecal secondary BA at day 7 compared to control (group 4) (54.8±10.1 vs 83.1±7.4 %, p=0.017) (Fig 1A). In group 3 (AC plus SB), the decrease in secondary BA rate was significantly less than for AC (71,23±7,4 versus 54,20±9 % , p=0.04), and this difference was prolonged over time (Fig 1B). Similarly, the AC plus SB group showed a significantly lower (and sustained) increase in CA than in the AC alone group (Fig 1C). Conclusion: Antibiotics alter the transformation of BA by microbial enzymes in the intestine. This increases primary BA while reducing fecal secondary BA. CA, a primary BA which facilitates C. difficile spore germination, increases in stool during antibiotic therapy. The concomitant use of SB during AC treatment significantly reduces this CA peak. These results highlight new human data on a potential mechanism for post-antibiotic CDI: alteration of the microbiota can encourage germination of C. difficile spores via increased CA concentrations and reduced concentrations of secondary BAs. The effectiveness of SB in preventing recurrent CDI may be explained, in part, through modulation of microbiota changes that influence the balance of pro- and anti-germination BA concentrations.

121 TERRY NAILS: A NEW LOOK AT AN OLD FINDING AND ITS ASSOCIATION WITH LIVER DISEASE James Roat, Stephen D. Zucker Background: Terry nails are a distinct fingernail finding characterized by ground-glass opacity of the entire nail bed, excluding a 1 to 2 mm transverse band adjacent to the free edge of the nail. In the seminal article by Richard Terry in 1954, this abnormality was reported to be present in 82% of 100 patients with liver cirrhosis. The only other large cohort study published in 1984 examined 512 hospitalized patients and identified Terry nails in 25%. A significant association was found with congestive heart failure, adult-onset

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diabetes mellitus, and cirrhosis, although the latter correlation was based on a total of 8 patients. Methods: To clarify the prevalence of Terry nails in patients with liver disease and to establish potential correlations with specific liver conditions, we prospectively evaluated 300 consecutive patients seen in the ambulatory gastroenterology, hepatology, and liver transplant clinics at the University of Cincinnati Medical Center. Data regarding the presence or absence of Terry nails, age, gender, ethnicity, etiology and severity of liver disease (if present), and any history of adult-onset diabetes mellitus or congestive heart failure was recorded. Univariate analyses were performed using Mann-Whitney and Pearson Product Moment tests. Results: Of the 300 subjects analyzed, 238 (79.3%) had liver disease and 104 (34.7%) had cirrhosis. The median age was 54 (range: 19 - 81). 51.7% were male, 69.7% were Caucasian and 25.3% were African American. Individuals with any liver disease manifested a 13.4% prevalence of Terry nails, as compared with a 2.6% prevalence in those without liver disease. In patients with cirrhosis, 17.3% exhibited Terry nails. By univariate analysis, the finding of Terry nails significantly correlated with underlying cirrhosis (p=0.01) and with the presence of any liver disease (p=0.008), most specifically when alcohol is an etiology (31%; p<0.0001). Significant associations between Terry nails and increasing age (p=0.025) and with a history of congestive heart failure (p=0.0004) also were found. No correlation was identified with sex, ethnicity, diabetes mellitus, or non-alcohol etiologies of liver disease (e.g., hepatitis C, hepatitis B, NASH/NAFL). Notably, in two patients with Terry nails who underwent liver transplantation, the finding was noted to resolve within two months of surgery. Conclusions: These findings support an association between Terry nails and underlying liver disease, most commonly when alcohol is a contributing factor. Although the presence of Terry nails is more commonly identified when advanced liver fibrosis is present, the prevalence appears to be much lower than previously reported in the literature.

123 CLUSTERING OF ESOPHAGEAL CANCER MORTALITY AMONG WHITE MEN IN THE UNITED STATES Joel H. Rubenstein Background: The incidence of esophageal adenocarcinoma in Europe is greatest in the Northwest. In Australia, the risk of esophageal adenocarcinoma has been inversely associated with lifetime exposure to ultraviolet radiation (UVR). Studies of the geographic distribution of esophageal adenocarcinoma in the United States (U.S.) has been limited to 4 broad regions. We sought to determine whether esophageal adenocarcinoma clusters in the U.S. using small areas, and whether known risk factors explain the clustering. Methods: We identified fatal cases of esophageal cancer using 1999-2014 county death certificates in the Multiple Cause of Death Files from the Centers for Disease Control and Prevention WONDER Database. The data do not distinguish between adenocarcinoma and squamous cell carcinoma; restricted the analysis to deaths of white, non-Hispanic men, for whom adenocarcinoma has been the predominant type since the 1980s. Age-adjusted mortality rates were tabulated for each county, weighted to the year 2000 U.S. standard population. In order to minimize the relative standard error in calculation of rates, data from the entire 15 year period were grouped together and analysis was limited to counties with a minimum of 20 deaths over the time period. County death rates were mapped using ArcMap v.10.4.1 (ESRI, Redlands, Ca), and the Getis-Ord Gi* statistic was used to identify statistically significant clusters (α < 0.05). Estimated county-specific UVR and age-adjusted prevalences of obesity, smoking and alcohol use for males were obtained from publicly available sources. Linear regression was performed in SAS (SAS Institute, Cary, NC) to determine the proportion of variance explained in county death rates by these factors. Results: 1,426 counties had at least 20 deaths with esophageal cancer among white non-Hispanic men. Age-adjusted mortality rates in those counties ranged from 4.8 to 17.5 deaths per 100,000 person-years (Figure 1), with data missing predominantly from the central U.S. Cluster analysis demonstrated an area of high mortality encompassing New England and the Great Lakes States (Figure 2). Alaska had average, and Hawaii had low mortality with esophageal cancer (data not shown). A regression model including the prevalences of obesity, smoking, and alcohol explained only 15.9% of the variance in county mortality rates with esophageal cancer. UVR was inversely associated with mortality rates, and explained an additional 7.0% of the variance. Discussion: Using county death certificates, we found clusters of high and low mortality rates with esophageal cancer among white non-Hispanic men in the U.S. Obesity, smoking, and alcohol minimally explain these clusters. These data suggests that UVR may be protective against esophageal cancer, and there may be additional unknown environmental factors that contribute to esophageal cancer mortality.

122 INCREASED RECOGNITION OF HGD/EAC AT INDEX ENDOSCOPY OVER THE PAST 2 DECADES: DATA FROM A MULTI-CENTER U.S. CONSORTIUM Madhav Desai, Sravanthi Parasa, Ajay Bansal, Neil Gupta, Sreekar Vennelaganti, Prashanth Vennalaganti, David A. Lieberman, Srinivas Gaddam, Patrick E. Young, Gary W. Falk, Prashanthi N. Thota, Sharad Mathur, Brooks D. Cash, Fouad J. Moawad, John J. Vargo, Kevin F. Kennedy, Richard Sampliner, Prateek Sharma Introduction: Data on time trends of dysplasia and esophageal adenocarcinoma (EAC) in Barrett's esophagus (BE) during the index endoscopy (i.e. prevalent cases) are limited. Our aim was to determine the prevalence patterns of BE associated dysplasia on index endoscopy over the past 25 years. Methods: The Barrett's esophagus study trial (BEST) is a multicenter outcome project of a large cohort of BE patients. Patients with baseline index endoscopy findings of low grade dysplasia (LGD), high grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) were extracted per year of index endoscopy and 5-yearly patient cohorts were tabulated over years 1990-2010+ (2010-current). Prevalent dysplasia and endoscopic findings were trended over the past 25 years using percentage dysplasia (LGD, HGD, EAC and HGD/EAC) to assess changes in detection of BE associated dysplasia over the last 25 years. Statistical analysis was done using software SAS version 9.4 (Cary NC). Results: A total of 3643 patients were included in the analysis with baseline index endoscopy showing: IM alone (non dysplastic BE) 2513 (70.1%), LGD 412 (11.5%), HGD 193 (5.4%), and EAC 181 (5.1%). Overall, there was an increase in the mean age of BE diagnosis (51.7 ± 29 years vs 62.6 ± 11.3 years) and proportion of males (84 % vs 92.6%) diagnosed with BE and a decrease in the mean BE length (4.4±4.3 cm vs 2.9±3.0 cm) as time progressed (1990-1994 to 2010-2016 time periods). Presence of LGD on index endoscopy remained stable over years 1990-2016. However, a significant increase (HGD 148% increase and EAC 112% increase) in the diagnosis of HGD, EAC and HGD/EAC were noted on index endoscopy over last 25 years (p<0.001) (table 1). It is notable that there was a increase in the detection of visible lesion on index endoscopy (1990-1994: 5.1% to 2005-2009: 6.3% and 2010+: 16.3%) during last 25 years as shown in figure 1 which parallels with HGD/EAC in this time period. Conclusion: Our results suggest that the prevalence of not only EAC but the precursor stage of HGD in BE has significantly increased in parallel over the past 25 years despite a decrease in BE length during the same time period. This increase parallels an increase in detection of visible lesions suggesting that a careful examination at the index examination is crucial in this patient population. These results support the stepwise progression model of BE that provides opportunities for chemoprevention, early detection and intervention. Table 1. 5-year baseline prevalence of LGD, HGD, EAC and HGD/EAC on index endoscopy

Figure 1: Age-adjusted mortality rates with esophageal cancer among white non-Hispanic men per 100,000 person-years. Gray counties have missing data due to fewer than 20 deaths.

Figure 2. Cluster analysis of mortality rates with esophageal cancer among white nonHispanic men.

124 ASSOCIATION BETWEEN CIRCULATING LEVELS OF SEX STEROID HORMONES AND ESOPHAGEAL/GASTRIC CARDIA ADENOCARCINOMA Jessica Petrick, Paula L. Hyland, Patrick Carron, Roni T. Falk, Ruth Pfeiffer, Sanford M. Dawsey, Christian C. Abnet, Philip R. Taylor, Stephanie J. Weinstein, Demetrius Albanes, Neal D. Freedman, Chantal Guillemette, Peter T. Campbell, Michael B. Cook Male predominance of esophageal adenocarcinoma (EA) and gastric cardia adenocarcinoma (GCA) has been noted worldwide, with the male-to-female ratio of approximately 3-4. However, established risk factors that vary in prevalence by sex, such as smoking and obesity, cannot fully explain the male predominance. Sex steroid hormones have been

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