Cholera Vaccine Use Is Associated with a Reduced Risk of Death in Patients with Colorectal Cancer: A Population-based Study Gastroenterology (IF 18.392) Pub Date : 2017-09-18 Jianguang Ji, Jan Sundquist, Kristina Sundquist
Background & Aims Cholera toxin can act as a modulator of the immune response with anti-inflammatory effects; it reduces development of colon polyps in mouse models of colorectal cancer (CRC). We performed a population-based study to determine whether, in patients with a diagnosis of CRC, subsequent administration of the cholera vaccine (killed Vibrio cholerae O1 whole cells and recombinant cholera toxin B subunit) affects mortality. Methods We identified patients from the Swedish Cancer Register who were diagnosed with CRC from July 2005 through December 2012. These patients were linked to the Swedish Prescribed Drug Register to retrieve cholera vaccine use. We used Cox regression analysis to calculate the hazard ratio (HR) of death from CRC and overall mortality in patients with post-diagnostic use of cholera vaccine compared to matched controls. Results A total of 175 patients were diagnosed with CRC and given a prescription for the cholera vaccine after their cancer diagnosis. Compared to propensity score-matched controls and adjusted for confounding factors, patients with CRC who received the cholera vaccine had a decreased risk of death from CRC (HR, 0.53; 95% CI, 0.29–0.99) and a decreased risk of death overall (HR, 0.59; 95% CI, 0.37–0.94). The decrease in mortality with cholera vaccination was largely observed, irrespective of patient age or tumor stage at diagnosis or sex. Conclusions In a population-based study, we associated administration of the cholera vaccine after CRC diagnosis with decreased risk of death from CRC and overall mortality.
CRISPR/Cas9 Engineering of Adult Mouse Liver Demonstrates That the Dnajb1–Prkaca Gene Fusion is Sufficient to Induce Tumors Resembling Fibrolamellar Hepatocellular Carcinoma Gastroenterology (IF 18.392) Pub Date : 2017-09-18 Lars H. Engelholm, Anjum Riaz, Denise Serra, Frederik Dagnæs-Hansen, Jens V. Johansen, Eric Santoni-Rugiu, Steen H. Hansen, Francesco Niola, Morten Frödin
Background & Aims Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene (DNAJB1) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1–PRKACA gene fusion has not been shown to induce liver tumorigenesis. We used the CRISPR/Cas9 technique to delete in mice the syntenic region on chromosome 8 to create a Dnajb1–Prkaca fusion and monitored the mice for liver tumor development. Methods We delivered CRISPR/Cas9 vectors designed to juxtapose exon 1 of Dnajb1 with exon 2 of Prkaca to create the Dnajb1–Prkaca gene fusion associated with FL-HCC, or control Cas9 vector, via hydrodynamic tail vein injection to livers of 8 week-old female FVB/N mice. These mice did not have any other engineered genetic alterations and were not exposed to liver toxins or carcinogens. Liver tissues were collected 14 months after delivery; genomic DNA was analyzed by PCR to detect the Dnajb1–Prkaca fusion, and tissues were characterized by histology, immunohistochemistry, RNA sequencing, and whole-exome sequencing. Results Livers from 12 of the 15 mice given the vectors to induce the Dnajb1–Prkaca gene fusion, but none of the 11 mice given the control vector, developed neoplasms. The tumors contained the Dnajb1–Prkaca gene fusion and had histologic and cytologic features of human FL-HCCs: large polygonal cells with granular, eosinophilic, and mitochondria-rich cytoplasm, prominent nucleoli, and markers of hepatocytes and cholangiocytes. In comparing expression levels of genes between the mouse tumor and non-tumor liver cells, we identified changes similar to those detected in human FL-HCC, which included genes that affect cell cycle and mitosis regulation. Genomic analysis of mouse neoplasms induced by the Dnajb1–Prkaca fusion revealed a lack of mutations in genes commonly associated with liver cancers, as observed in human FL-HCC. Conclusions Using CRISPR/Cas9 technology, we found generation of the Dnajb1–Prkaca fusion gene in wild-type mice to be sufficient to initiate formation of tumors that have many features of human FL-HCC. Strategies to block DNAJB1–PRKACA might be developed as therapeutics for this form of liver cancer.
Risk of Metachronous High-risk Adenomas and Large Serrated Polyps in Individuals With Serrated Polyps on Index Colonoscopy: Data from the New Hampshire Colonoscopy Registry Gastroenterology (IF 18.392) Pub Date : 2017-09-18 Joseph C. Anderson, Lynn F. Butterly, Christina M. Robinson, Julia E. Weiss, Christopher Amos, Amitabh Srivastava
Background & Aims Surveillance guidelines for serrated polyps (SPs) are based on limited data on longitudinal outcomes of patients. We used the New Hampshire Colonoscopy Registry to evaluate risk of clinically important metachronous lesions associated with SPs, detected during index colonoscopies. Methods We collected data from a population-based colonoscopy registry that has been collecting and analyzing data on colonoscopies across the state of New Hampshire since 2004, including rates of adenoma and serrated polyp detection. Patients completed a questionnaire to determine demographic characteristics, health history, and risk factors for CRC, and were followed from index colonoscopy through all subsequent surveillance colonoscopies. Our analyses included 5433 participants (median age 61 years; 49.7% male) with 2 colonoscopies (median time to surveillance, 4.9 years). We used multivariable logistic regression models to assess effects of index serrated polyps (SP, n=1016), high-risk adenomas (HRA, n=817), low-risk adenomas (n=1418), and no adenomas (n=3198) on subsequent HRA or large SPs (>1 cm) on surveillance colonoscopy (metachronous lesions). Synchronous SPs, within each index risk group, were assessed for size and by histology. Serrated polyps comprise hyperplastic polyps, sessile serrated adenomas/polyps (SSA/Ps), and traditional serrated adenomas. In this study, SSA/Ps and traditional serrated adenomas are referred to collectively as STSAs. Results HRA and synchronous large SP (odds ratio [OR], 5.61; 95% CI, 1.72–18.28), HRA with synchronous STSA (OR, 16.04; 95% CI, 6.95–37.00), and HRA alone (OR, 3.86; 95% CI, 2.77–5.39) at index colonoscopy significantly increased the risk of metachronous HRA compared to the reference group (no index adenomas or serrated polyps). Large index SPs alone (OR=14.34; 95% CI, 5.03–40.86) or index STSA alone (OR, 9.70; 95% CI, 3.63–25.92) significantly increased the risk of a large metachronous SP. Conclusions In an analysis of data from a population-based colonoscopy registry, we found index large SP or index STSA with no index HRA increased risk of metachronous large SPs but not metachronous HRA. HRA and synchronous SPs at index colonoscopy significantly increased risk of metachronous HRA. Individuals with HRA and synchronous could therefore benefit from close surveillance.
Deficiency of the Mitochondrial NAD Kinase Causes Stress-induced hepatic steatosis in mice Gastroenterology (IF 18.392) Pub Date : 2017-09-17 Kezhong Zhang, Hyunbae Kim, Zhiyao Fu, Yining Qiu, Zhao Yang, Jiemei Wang, Deqiang Zhang, Xin Tong, Lei Yin, Jing Li, Jianmei Wu, Nathan R. Qi, Sander M. Houten, Ren Zhang
Background & Aims The mitochondrial nicotinamide adenine dinucleotide (NAD) kinase (NADK2, also called MNADK) catalyzes phosphorylation of NAD to yield NADP. Little is known about the functions of mitochondrial NADP and MNADK in liver physiology and pathology. We investigated the effects of reduced mitochondrial NADP by deleting MNADK in mice. Methods We generated MNADK-knockout (KO) mice on a C57BL/6NTac background; mice with a wild-type Mnadk gene were used as controls. Some mice were placed on an atherogenic high-fat diet (16% fat, 41% carbohydrate and 1.25% cholesterol supplemented with 0.5% sodium cholate) or given methotrexate intraperitoneally. We measured rates of fatty acid oxidation in primary hepatocytes using radiolabeled palmitate and in mice using indirect calorimetry. We measured levels of reactive oxygen species in mouse livers and primary hepatocytes. Metabolomic analyses were used to quantify serum metabolites, such as amino acids and acylcarnitines. Results KO mice had metabolic features of MNADK-deficient patients, such as increased serum concentrations of lysine and C10:2 carnitine. When placed on the atherogenic high-fat diet, the KO mice developed features of non-alcoholic fatty liver disease and had increased levels of reactive oxygen species in livers and primary hepatocytes, compared with control mice. During fasting, the KO mice had a defect in fatty acid oxidation. MNADK deficiency reduced the activation of cAMP-responsive element binding protein and hepatocyte specific and peroxisome proliferator-activated receptor alpha, which are transcriptional activators that mediate the fasting response. The activity of mitochondrial sirtuins was reduced in livers of the KO mice. Methotrexate inhibited the catalytic activity of MNADK in hepatocytes and in livers in mice with methotrexate injection. In mice given injections of methotrexate, supplementation of a diet with nicotinamide riboside, a NAD precursor, replenished hepatic NADP and protected the mice from hepatotoxicity, based on markers such as increased level of serum alanine aminotransferase. Conclusion MNADK facilitates fatty acid oxidation, counteracts oxidative damage, maintains mitochondrial sirtuin activity, and prevents metabolic stress-induced non-alcoholic fatty liver disease in mice.
Analysis of Genomes and Transcriptomes of Hepatocellular Carcinomas Identifies Mutations and Gene Expression Changes in the Transforming Growth Factor beta Pathway Short title: Prognostic significance of TGF-β signature in liver cancer Gastroenterology (IF 18.392) Pub Date : 2017-09-15 Jian Chen, Sobia Zaidi, Shuyun Rao, Jiun-Sheng Chen, Liem Phan, Patrizia Farci, Xiaoping Su, Kirti Shetty, Jon White, Fausto Zamboni, Xifeng Wu, Asif Rashid, Nagarajan Pattabiraman, Raja Mazumder, Anelia Horvath, Ray-Chang Wu, Shulin Li, Cuiying Xiao, Chu-Xia Deng, David A. Wheeler, Bibhuti Mishra, Rehan Akbani, Lopa Mishra
Background & Aims Development of hepatocellular carcinoma (HCC) is associated with alterations in the transforming growth factor beta (TGFβ) signaling pathway, which regulates liver inflammation and can have tumor suppressor or promoter activities. Little is known about the roles of specific members of this pathway at specific of HCC development. We took an integrated approach to identify and validate the effects of changes in this pathway in HCC and identify therapeutic targets. Methods We performed transcriptome analyses for a total of 488 HCCs that include data from The Cancer Genome Atlas. We also screened 301 HCCs reported in the Catalogue of Somatic Mutations in Cancer and 202 from Cancer Genome Atlas for mutations in genome sequences. We expressed mutant forms of spectrin beta, non-erythrocytic 1 (SPTBN1) in HepG2, SNU398, and SNU475 cells and measured phosphorylation, nuclear translocation, and transcriptional activity of SMAD family member 3 (SMAD3). Results We found somatic mutations in at least 1 gene whose product is a member of TGFβ signaling pathway in 38% of HCC samples. SPTBN1 was mutated in the largest proportion of samples (12/202, 6%). Unsupervised clustering of transcriptome data identified a group of HCCs with activation of the TGFβ signaling pathway (increased transcription of genes in the pathway) and a group of HCCs with inactivation of TGFβ signaling (reduced expression of genes in this pathway). Patients with tumors with inactivation of TGFβ signaling had shorter survival times than patients with tumors with activation of TGFβ signaling (P=.0129). Patterns of TGFβ signaling correlated with activation of the DNA damage response and sirtuin signaling pathways. HepG2, SNU398, SNU475 cells that expressed the D1089Y mutant or with knockdown of SPTBN1 had increased sensitivity to DNA crosslinking agents and reduced survival compared to cells that expressed normal SPTBN1 (controls). Conclusions In genome and transcriptome analyses of HCC samples, we found mutations in genes in the TGFβ signaling pathway in almost 40% of samples. These correlated with changes in expression of genes in the pathways; upregulation of genes in this pathway would contribute to inflammation and fibrosis whereas downregulation would indicate loss of TGFβ tumor suppressor activity. Our findings indicate that therapeutic agents for HCCs can be effective, based on genetic features of the TGFβ pathway; agents that block TGFβ should be used only in patients with specific types of HCCs.
Autophagy, Inflammation, and Immune Dysfunction in the Pathogenesis of Pancreatitis Gastroenterology (IF 18.392) Pub Date : 2017-09-14 Anna S. Gukovskaya, Ilya Gukovsky, Hana Algül, Aida Habtezion
Pancreatitis is a common disorder with significant morbidity and mortality, yet little is known about its pathogenesis and there is no specific or effective treatment. Its development involves dysregulated autophagy and unresolved inflammation, demonstrated by studies in genetic and experimental mouse models. Disease severity depends on whether the inflammatory response resolves or amplifies, leading to multi-organ failure. Dysregulated autophagy might promote the inflammatory response in the pancreas. We discuss the roles of autophagy and inflammation in pancreatitis, mechanisms of deregulation, and connections among disordered pathways. We identify gaps in our knowledge and delineate perspective directions for research. Elucidation of pathogenic mechanisms could lead to new targets for treating or reducing the severity of pancreatitis.
Development and Validation of a Chronic Pancreatitis Prognosis Score in 2 Independent cohorts Gastroenterology (IF 18.392) Pub Date : 2017-09-14 Georg Beyer, Ujjwal M. Mahajan, Christoph Budde, Thomas J. Bulla, Thomas Kohlmann, Louise Kuhlmann, Kerstin Schütte, Ali A. Aghdassi, Eckhard Weber, Ulrich Weiss, Asbjørn M. Drewes, Søren S. Olesen, Markus M. Lerch, Julia Mayerle
Background & Aims The clinical course of chronic pancreatitis is unpredictable. There is no model to assess disease severity or progression or predict patient outcomes. Methods We performed a prospective study of 91 patients with chronic pancreatitis; data were collected from patients seen at academic centers in Europe, from January 2011 through April 2014. We analyzed correlations between clinical, laboratory, and imaging data with number of hospital readmissions and in-hospital days over the next 12 months; the parameters with the highest degree of correlation were to develop 3-stage chronic pancreatitis prognosis score (COPPS). The predictive value was validated in 129 independent subjects identified from 2 prospective databases. Results The mean number of hospital admissions was 1.9 (95% CI, 1.39–2.44) and 15.2 for hospital days (95% CI, 10.76–19.71) for the development cohort and 10.9 for the validation cohort (95% CI, 7.54–14.30) (P=.08). Based on bivariate correlations, pain (numeric rating scale), level of HbA1c, level of c-reactive protein, body mass index, and platelet count were used to develop the COPPS system. The patients’ median COPPS was 8.9 points (range, 5–14). The system accurately discriminated stages of disease severity (low to high): A (5–6 points), B (7–9), and C (10–15). In Pearson correlation analysis of the development cohort, the COPPS correlated with hospital admissions (0.39; P<.01) and number of hospital days (0.33; P<.01). The correlation was validated in the validation set (Pearson correlation values of 0.36 and 0.44; P<.01). COPPS did not correlate with results from the Cambridge classification system. Conclusions We developed and validated an easy to use dynamic multivariate scoring system, similar to the Child-Pugh-Score for liver cirrhosis. The COPPS allows objective monitoring of patients with chronic pancreatitis, determining risk for readmission to hospital and potential length of hospital stay.
Intestinal Dysbiosis Featuring Abundance of Ruminococcus gnavus Associates With Allergic Diseases in Infants Gastroenterology (IF 18.392) Pub Date : 2017-09-12 Huey-Huey Chua, Hung-Chieh Chou, Ya-Ling Tung, Bor-Luen Chiang, Chien-Chia Liao, Hong-Hsing Liu, Yen-Hsuan Ni
Background & AimsDysbiosis of the intestinal microbiota has been associated with development of allergies in infants. However, it is not clear what microbes might contribute to this process. We investigated what microbe(s) might be involved in analyses of infant twins and mice.MethodsWe studied fecal specimens prospectively in a twin cohort (n=30) and age-matched singletons (n=14) born at National Taiwan University Children’s Hospital, Taipei, Taiwan, from April 2011 to March 2013. Clinical parameters (gestational age, birth body weight, mode of delivery and feeding, immunizations and medical events) were recorded. Fecal samples were collected beginning immediately after birth and for 1 year; the children were followed until 3 years of age and allergic symptoms (repetitive and continuous for at least 6 months) were noted. A skin prick test was used to ascertain atopy. Bacterial communities in fecal samples were profiled by 16S rRNA-based PCR-temporal temperature gradient gel electrophoresis and next generation sequencing. BALB/c mice without and with ovalbumin sensitization/challenge were infected with candidate bacteria by oral gauge intra-gastric intubation. Fecal, serum, lung, and colon tissue samples were collected from mice and analyzed for mechanisms of allergy development.ResultsDuring the investigation period, 20 children (45.5%) developed allergic diseases, including respiratory (allergic rhinitis and asthma) and skin (atopic dermatitis and eczema) allergies. Lachnospiraceae were detected at significantly higher frequency in allergic infants than non-allergic infants (P<.004); the high fecal count of Lachnospiraceae in allergic subjects appeared at 2 months of age and persisted until 12 months of age. The enrichment of Lachnospiraceae in allergic infants was attributed to the overgrowth of Ruminococcus gnavus, which tended to have a low frequency in non-allergic subjects (P=.0004). Increased R gnavus was observed before the onset of allergic manifestations, and associated with respiratory allergies (P<.002) or respiratory allergies coexistent with atopic eczema (P<.001). In mice, endogenous R gnavus grew rapidly following sensitization and challenge with ovalbumin. Mice gavaged with purified R gnavus developed airway hyper-responsiveness and had histologic evidence of airway inflammation (asthma). Expansion of R gnavus in mice stimulated secretion of cytokines (interleukin 25 (IL25), IL33, and thymic stromal lymphopoietin) by colon tissues, which activated type-2 innate lymphoid cells and dendritic cells to promote differentiation of T-helper (Th)2 cells and production of their cytokines (IL4, IL5, and IL13). This led to infiltration of the colon and lung parenchyma by eosinophils and mast cells.ConclusionsIn a study of a twin cohort (some infants with, some without allergies), we associated development of allergies, particularly respiratory allergies, with increased fecal abundance of R gnavus. Mice fed R gnavus developed airway inflammation, characterized by expansion of Th2 cells in the colon and lung, and infiltration of colon and lung parenchyma by eosinophils and mast cells.
Association Between Germline Mutations in BRF1, a subunit of the RNA Polymerase III Transcription Complex, and Hereditary Colorectal Cancer Gastroenterology (IF 18.392) Pub Date : 2017-09-12 Fernando Bellido, Nadine Sowada, Pilar Mur, Conxi Lázaro, Tirso Pons, Rafael Valdés-Mas, Marta Pineda, Gemma Aiza, Silvia Iglesias, José Luís Soto, Miguel Urioste, Trinidad Caldés, Milagros Balbín, Pilar Blay, Daniel Rueda, Mercedes Durán, Alfonso Valencia, Victor Moreno, Joan Brunet, Ignacio Blanco, Matilde Navarro, George A. Calin, Guntram Borck, Xose S. Puente, Gabriel Capellá, Laura Valle
Background & AimsAlthough there is a genetic predisposition to colorectal cancer (CRC), few of the genes that affect risk have been identified. We performed whole-exome sequence analysis of individuals in a high-risk family without mutations in genes previously associated with CRC risk to identify variants associated with inherited CRC.MethodsWe collected blood samples from 3 relatives with CRC in Spain (65, 62 and 40 years old at diagnosis) and perfomed whole-exome sequence analyses. Rare missense, truncating or splice-site variants shared by the 3 relatives were selected. We used targeted pooled DNA amplification followed by next-generation sequencing to screen for mutations in candidate genes in 547 additional hereditary and/or early-onset CRC cases. We carried out protein-dependent yeast-growth assays and transfection studies in the HT29 human CRC cell line to test the effects of the identified variants.ResultsA total of 42 unique or rare (population minor allele frequency below 1%) non-synonymous genetic variants in 38 genes were shared by all 3 relatives. We selected the BRF1 gene, which encodes an RNA polymerase III transcription initiation factor subunit for further analysis, based on the predicted effect of the identified variant and previous association of BRF1 with cancer. Previously unreported or rare germline variants in BRF1 were identified in 11/503 individuals in families with a history of CRC or early-onset CRC,- a significantly greater proportion than in control population (34/4,300). Seven of the identified variants (1 detected in 2 families) affected BRF1 mRNA splicing, protein stability or expression and/or function.ConclusionsIn an analysis of families with a history of CRC, we associated germline mutations in BRF1 with predisposition to CRC. We associated deleterious BRF1 variants with 1.4% of familial CRC cases, in individuals without mutations in high-penetrance genes previously associated with CRC. Our findings add additional evidence to the link between defects in genes that regulate ribosome synthesis and risk of CRC.
A Nigro-Vagal Pathway Controls Gastric Motility and is Affected in a Rat Model of Parkinsonism Gastroenterology (IF 18.392) Pub Date : 2017-09-11 Laura Anselmi, Luca Toti, Cecilia Bove, Jessica Hampton, R. Alberto Travagli
Background & Aims In most patients with Parkinson’s disease, gastrointestinal (GI) dysfunctions, such as gastroparesis and constipation, are prodromal to the cardinal motor symptoms of the disease. Sporadic Parkinson’s disease has been proposed to develop following ingestion of neurotoxicants that affect the brain–gut axis via the vagus nerve, and then travel to higher centers compromising the substantia nigra pars compacta (SNpc), and, later, the cerebral cortex. We aimed to identify the pathway that connects the brainstem vagal nuclei and the SNpc, and to determine whether this pathway is compromised in a rat model of Parkinsonism. Methods To study this neural pathway in rats, we placed tracers in the dorsal vagal complex (DVC) or SNpc; brainstem and midbrain were examined for tracer distribution and neuronal neurochemical phenotype. Rats were given injections of paraquat once weekly for 3 weeks to induce features of Parkinsonism, or vehicle (control). Gastric tone and motility were recorded following NMDA microinjection in the SNpc and/or optogenetic stimulation of nigro-vagal terminals in the DVC. Results Stimulation of the SNpc increased gastric tone and motility via activation of dopamine 1 receptors in the DVC. In the paraquat-induced model of Parkinsonism, this nigro-vagal pathway was compromised during the early stages of motor deficit development. Conclusions We identified and characterized a nigro-vagal monosynaptic pathway in rats that controls gastric tone and motility. This pathway might be involved in the prodromal gastric dysmotility observed in patients with early-stage Parkinson’s disease.
Paneth Cell Defects Induce Microbiota Dysbiosis In Mice And Promote Visceral Hypersensitivity Gastroenterology (IF 18.392) Pub Date : 2017-09-01 Ambre Riba, Maïwenn Olier, Sonia Lacroix-Lamandé, Corinne Lencina, Valérie Bacquié, Cherryl Harkat, Marion Gillet, Marine Baron, Caroline Sommer, Virginie Mallet, Christel Salvador-Cartier, Fabrice Laurent, Vassilia Théodorou, Sandrine Ménard
Background & Aims Separation of newborn rats from their mothers induces visceral hypersensitivity and impaired epithelial secretory cell lineages when they are adults. Little is known about the mechanisms by which maternal separation causes visceral hypersensitivity or its relationship with defects in epithelial secretory cell lineages. Methods We performed studies with C3H/HeN mice separated from their mothers as newborns and mice genetically engineered (Sox9flox/flox-vil-cre on C57BL/6 background) to have deficiencies in Paneth cells. Paneth cells deficiency was assessed by lysozyme staining of ileum tissues and lysozyme activity in fecal samples. When mice were 50 days old, their abdominal response to colorectal distension was assessed by electromyography. Fecal samples were collected and microbiota were analyzed using GULDA quantitative PCR. Results Mice with maternal separation developed visceral hypersensitivity and defects in Paneth cells, as reported from rats, compared to mice without maternal separation. Sox9flox/flox-vil-Cre mice also had increased visceral hypersensitivity compared to control littermate Sox9flox/flox mice. Fecal samples from mice with maternal separation and from Sox9flox/flox-vil-cre mice had evidence for intestinal dysbiosis of the microbiota, characterized by expansion of Escherichia coli. Daily gavage of conventional C3H/HeN adult mice with 109 commensal E. coli induced visceral hypersensitivity. Conversely, daily oral administration of lysozyme prevented expansion of E. coli during maternal separation and visceral hypersensitivity. Conclusions Mice with defects in Paneth cells (induced by maternal separation or genetically engineered) have intestinal expansion of E. coli leading to visceral hypersensitivity. These findings provide evidence that Paneth cell function and intestinal dysbiosis are involved in visceral sensitivity.
Anesthesia Assistance in Outpatient Colonoscopy and Risk of Aspiration Pneumonia, Bowel Perforation, and Splenic Injury Gastroenterology (IF 18.392) Pub Date : 2017-09-01 Barbara Bielawska, Lawrence C. Hookey, Rinku Sutradhar, Marlo Whitehead, Jianfeng Xu, Lawrence F. Paszat, Linda Rabeneck, Jill Tinmouth
Background & Aims The increase in use of anesthesia assistance (AA) to achieve deep sedation with propofol during colonoscopy has significantly increased colonoscopy costs without evidence for increased quality and with possible harm. We investigated the effects of AA on colonoscopy complications, specifically bowel perforation, aspiration pneumonia, and splenic injury. Methods In a population-based cohort study using administrative databases, we studied adults in Ontario, Canada undergoing outpatient colonoscopy from 2005 through 2012. Patient, endoscopist, institution, and procedure factors were derived. The primary outcome was bowel perforation, defined using a validated algorithm. Secondary outcomes were splenic injury and aspiration pneumonia. Using a matched propensity score approach, we matched persons who had colonoscopy with AA (1:1) to those who did not. We used logistic regression models under a generalized estimating equations approach to explore the relationship between AA and outcomes. Results We analyzed data from 3,059,045 outpatient colonoscopies; 862,817 of these included AA. After propensity matching, a cohort of 793,073 patients who had AA and 793,073 without AA was retained for analysis (51%, female; 78% were age 50 years or older). Use of AA did not significantly increase risk of perforation (odds ratio [OR], 0.99; 95% CI, 0.84 – 1.16) or splenic injury (OR, 1.09; 95% CI, 0.62 – 1.90]. Use of AA was associated with an increased risk of aspiration pneumonia (OR, 1.63; 95% CI, 1.11 – 2.37). Conclusions In a population-based cohort study, AA for outpatient colonoscopy was associated with a significantly increased risk of aspiration pneumonia but not bowel perforation or splenic injury. Endoscopists should warn patients, especially those with respiratory compromise, of this risk.
Association Between Inflammatory Diet Pattern and Risk of Colorectal Carcinoma Subtypes Classified by Immune Responses to Tumor Gastroenterology (IF 18.392) Pub Date : 2017-09-01 Li Liu, Reiko Nishihara, Zhi Rong Qian, Fred K. Tabung, Daniel Nevo, Xuehong Zhang, Mingyang Song, Yin Cao, Kosuke Mima, Yohei Masugi, Yan Shi, Annacarolina da Silva, Tyler Twombly, Mancang Gu, Wanwan Li, Tsuyoshi Hamada, Keisuke Kosumi, Kentaro Inamura, Jonathan A. Nowak, David A. Drew, Paul Lochhead, Katsuhiko Nosho, Kana Wu, Molin Wang, Wendy S. Garrett, Andrew T. Chan, Charles S. Fuchs, Edward L. Giovannucci, Shuji Ogino
Background & Aims Dietary patterns affect systemic and local intestinal inflammation, which have been linked to colorectal carcinogenesis. Chronic inflammation can interfere with the adaptive immune response. We investigated whether the association of a diet that promotes intestinal inflammation with risk of colorectal carcinoma was stronger for tumors with lower lymphocytic reactions than tumors with higher lymphocytic reactions. Methods We collected data from the molecular pathological epidemiology databases of 2 prospective cohort studies: the Nurses’ Health Study (since 1976) and the Health Professional Follow-up Study (since 1986). We used duplication-method time-varying Cox proportional cause-specific hazards regression to assess the association of empirical dietary inflammatory pattern (EDIP) score (derived from food frequency questionnaire data) with colorectal carcinoma subtype. Foods that contribute to high EDIP scores include red and processed meats, refined grains, carbonated beverages, and some vegetables; foods that contribute to low EDIP scores include beer, wine, coffee, tea, yellow and leafy vegetables, and fruit juice. Colorectal tissue samples were analyzed histologically for patterns of lymphocytic reactions (Crohn’s-like lymphoid reaction, peritumoral lymphocytic reaction, intratumoral periglandular reaction, and tumor-infiltrating lymphocytes). Results During follow up of 124,433 participants, we documented 1311 incident colon and rectal cancer cases with available tissue data. The association between the EDIP and colorectal cancer risk was significant (Ptrend = .02), and varied with degree of peritumoral lymphocytic reaction (Pheterogeneity < .001). Higher EDIP scores were associated with increased risk of colorectal cancer with an absent or low peritumoral lymphocytic reaction (highest vs lowest EDIP score quintile hazard ratio = 2.60; 95% CI, 1.60–4.23; Ptrend < .001) but not risk of tumors with intermediate or high peritumoral lymphocytic reaction (Ptrend > .80). Conclusions In a prospective cohort study, we associated inflammatory diets with a higher risk of colorectal cancer subtype that contains little or no peritumoral lymphocytic reaction. These findings suggest that diet-related inflammation might contribute to development of colorectal cancer, by suppressing the adaptive anti-tumor immune response.
Abnormal Responses to Local Esophageal Food Allergen Injections in Adult Patients with Eosinophilic Esophagitis Gastroenterology (IF 18.392) Pub Date : 2017-09-01 Marijn J. Warners, Ingrid Terreehorst, René M. van den Wijngaard, Jaap Akkerdaas, Betty C.A.M. van Esch, Ronald van Ree, Serge A. Versteeg, Andreas J.P.M. Smout, Albert J. Bredenoord
Skin tests and measurement of serum levels of immunoglobulin E do not accurately identify foods for elimination from the diets of patients with eosinophilic esophagitis (EoE). We investigated whether an esophageal prick test (EPT), in which the esophageal mucosa is challenged by local injection of allergen extracts, could identify individuals with esophageal sensitization. During endoscopy 6 allergens were injected in the esophagus of 8 patients with EoE and 3 patients without EoE (controls). A second endoscopy was performed after 24 hrs to evaluate delayed responses. Five of the 8 patients with EoE had evidence for an acute response (luminal obstruction and mucosal blanching); 2 other patients had a delayed wheal or flare reaction. No responses were observed in controls. We conclude that esophageal mucosal food allergen injections induce acute and/or delayed responses in patients with EoE but not controls. The EPT deserves further exploration because it might guide elimination diets.
Gastrin Induces Nuclear Export and Proteasome Degradation of Menin in Enteric Glial Cells Gastroenterology (IF 18.392) Pub Date : 2017-08-30 Sinju Sundaresan, Cameron A. Meininger, Anthony J. Kang, Amanda L. Photenhauer, Michael M. Hayes, Nirakar Sahoo, Jolanta Grembecka, Tomasz Cierpicki, Lin Ding, Thomas J. Giordano, Tobias Else, David J. Madrigal, Malcolm J. Low, Fiona Campbell, Ann-Marie Baker, Haoxing Xu, Nicholas A. Wright, Juanita L. Merchant
Background & Aims The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors (NETs) such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner’s glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia. Methods Primary enteric glial cultures were generated from the VillinCre:Men1FL/FL:Sst–/– mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from VillinCre:Men1FL/FL:Sst+/+ mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom. Results Enteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells, e.g., p75 and S100B, colocalized with gastrin in human duodenal gastrinomas. Conclusions MEN1-associated gastrinomas, which develop in the submucosa, might arise from enteric glial cells through hormone-dependent PKA signaling. This pathway disrupts nuclear menin function, leading to hypergastrinemia and associated sequelae.
Degradation of PHLPP2 by KCTD17, via a Glucagon-dependent Pathway, Promotes Hepatic Steatosis Gastroenterology (IF 18.392) Pub Date : 2017-08-30 KyeongJin Kim, Dongryeol Ryu, Paola Dongiovanni, Lale Ozcan, Shruti Nayak, Beatrix Ueberheide, Luca Valenti, Johan Auwerx, Utpal B. Pajvani
Background & Aims Obesity-induced non-alcoholic fatty liver disease (NAFLD) develops, in part, via excess insulin-stimulated hepatic de novo lipogenesis, which increases, paradoxically, in patients with obesity-induced insulin resistance. Pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2) terminates insulin signaling by dephosphorylating Akt; levels of PHLPP2 are reduced in livers from obese mice. We investigated whether loss of hepatic PHLPP2 is sufficient to induce fatty liver in mice, mechanisms of PHLPP2 degradation in fatty liver, and expression of genes that regulate PHLPP2 in livers of patients with NAFLD. Methods C57BL/6J mice (controls), obese db/db mice and mice with liver-specific deletion of PHLPP2 (L-PHLPP2) fed either normal chow or high-fat diet (HFD) were analyzed for metabolic phenotypes including glucose tolerance and hepatic steatosis. PHLPP2-deficient primary hepatocytes or CRISPR/Cas9-mediated PHLPP2-knockout hepatoma cells were analyzed for insulin signaling and gene expression. We performed mass spectrometry analyses of livers tissues from C57BL/6J mice transduced with Ad-HA-FLAG-PHLPP2 to identify post-translational modifications to PHLPP2 and proteins that interact with PHLPP2. We measured levels of mRNAs by quantitative reverse transcription PCR in liver biopsies from patients with varying degrees of hepatic steatosis. Results PHLPP2-knockout hepatoma cells and hepatocytes from L-PHLPP2 mice showed normal initiation of insulin signaling, but prolonged insulin action. Chow-fed L-PHLPP2 mice had normal glucose tolerance but hepatic steatosis. In HFD-fed C57BL/6J or db/db obese mice, endogenous PHLPP2 was degraded by glucagon and PKA-dependent phosphorylation of PHLPP2 (at Ser1119 and Ser1210), which led to PHLPP2 binding to potassium channel tetramerization domain containing 17 (KCTD17), a substrate-adaptor for Cul3-RING ubiquitin ligases. Levels of KCTD17 mRNA were increased in livers of HFD-fed C57BL/6J or db/db obese mice and in liver biopsies patients with NAFLD, compared with liver tissues from healthy control mice or patients without steatosis. Knockdown of KCTD17 with small hairpin RNA in primary hepatocytes increased PHLPP2 protein but not Phlpp2 mRNA, indicating that KCTD17 mediates PHLPP2 degradation. KCTD17 knockdown in obese mice prevented PHLPP2 degradation and decreased expression of lipogenic genes. Conclusions In mouse models of obesity, we found that PHLPP2 degradation induced lipogenesis without affecting gluconeogenesis. KCTD17, which is upregulated in liver tissues of obese mice and patients with NAFLD, binds to phosphorylated PHLPP2 to target it for ubiquitin-mediated degradation; this increases expression of genes that regulate lipogenesis to promote hepatic steatosis. Inhibitors of this pathway might be developed for treatment of patients with NAFLD.
Analysis of Liver Offers to Pediatric Candidates on the Transplant Wait List Gastroenterology (IF 18.392) Pub Date : 2017-07-13 E.K. Hsu, M.L. Shaffer, L. Gao, C. Sonnenday, M.L. Volk, J. Bucuvalas, J.C. Lai
Background & Aims Approximately 10% of children on the liver transplant wait-list in the United States (US) die every year. We examined deceased donor liver offer acceptance patterns and their contribution to pediatric wait-list mortality. Methods We performed a retrospective cohort study of children on the US liver transplant wait-list from 2007 through 2014 using national transplant registry databases. We determined the frequency, patterns of acceptance, and donor and recipient characteristics associated with deceased donor liver organ offers for children who died or were delisted compared to those who underwent transplantation. Children who died or were delisted were classified by the number of donor liver offers (0 vs 1 or more), limiting analyses to offers of livers that were ultimately transplanted into pediatric recipients. The primary outcome was death or delisting on the wait-list. Results Among 3852 pediatric liver transplant candidates, children who died or were delisted received a median 1 pediatric liver offer (inter-quartile range, 0–2) and waited a median 33 days before removal from the waitlist. Of 11328 donor livers offered to children, 2533 (12%) were transplanted into children—1179 of these (47%) were immediately accepted and 1354 (53%) were initially refused and eventually accepted for another child. Of 27831 adults, 1667 (6.0%; median 55 years) received livers from donors younger than 18 yrs (median 15 yrs), most (97%) allocated locally or regionally. Of children who died or were delisted, 173 (55%) received an offer of 1 or more liver that was subsequently transplanted into another pediatric recipient, and 143 (45%) died or were delisted with no offers. Conclusions Among pediatric liver transplant candidates in the US, children who died or were delisted received a median 1 pediatric liver offer and waited a median 33 days. Of livers transplanted into children, 47% were immediately accepted and 53% were initially refused and eventually accepted for another child. Of children who died or were delisted, 55% received an offer of 1 or more liver that was subsequently transplanted into another pediatric recipient, and 45% died or were delisted with no offers. Pediatric prioritization in allocation and development of improved risk stratification systems is required to reduce wait-list mortality among children.
Randomized Comparison of 3 High-level Disinfection and Sterilization Procedures for Duodenoscopes Gastroenterology (IF 18.392) Pub Date : 2017-07-13 Graham M. Snyder, Sharon B. Wright, Anne Smithey, Meir Mizrahi, Michelle Sheppard, Elizabeth B. Hirsch, Ram Chuttani, Riley Heroux, David S. Yassa, Lovisa B. Olafsdottir, Roger B. Davis, Anastasiou Jiannis, Vijay Bapat, Kiran Bidari, Douglas K. Pleskow, Daniel Leffler, Benjamin Lane, Alice Chen, Mandeep S. Sawhney
Background and Aims Duodenoscopes have been implicated in the transmission of multi-drug resistant bacteria (MDRO). We compared the frequency of duodenoscope contamination with MDRO or any other bacteria after disinfection or sterilization by 3 different methods. Methods We performed a single-center prospective randomized study in which duodenoscopes were randomly reprocessed by standard high-level disinfection (sHLD), double high-level disinfection (dHLD), or standard high-level disinfection followed by ethylene oxide gas sterilization (HLD/ETO). Samples were collected from the elevator mechanism and working channel of each duodenoscope and cultured before use. The primary outcome was the proportion of duodenoscopes with an elevator mechanism or working channel culture showing 1 or more MDRO; secondary outcomes included the frequency of duodenoscope contamination with more than 0 and 10 or more colony-forming units (CFU) of aerobic bacterial growth on either sampling location. Results After 3 months of enrollment, the study was closed due to the futility—we did not observe sufficient events to evaluate the primary outcome. Among 541 duodenoscope culture events, 516 were included in the final analysis. No duodenoscope culture in any group was positive for MDRO. Bacterial growth of more than 0 CFU was noted in 16.1% duodenoscopes in the sHLD group, 16.0% in the dHLD group, and 22.5% in the HLD/ETO group (P=.21). Bacterial growth or 10 or more CFU was noted in 2.3% of duodenoscopes in sHLD group, 4.1% in the dHLD group, and 4.2% in the HLD/ETO group (P=.36). MRDOs were cultured from 3.2% of pre-procedure rectal swabs and 2.5% of duodenal aspirates. Conclusions In a comparison of duodenoscopes reprocessed by sHLD, dHLD, or HLD/ETO, we found no significant differences between groups for MDRO or bacteria contamination. Enhanced disinfection methods (dHLD or HLD/ETO) did not provide additional protection against contamination. However, insufficient events occurred to assess our primary study end-point. ClinicalTrials.gov no: NCT02611648
Vasoactive Intestinal Polypeptide and Mast Cells Regulate Increased Passage of Colonic Bacteria in Patients With Irritable Bowel Syndrome Gastroenterology (IF 18.392) Pub Date : 2017-07-13 Olga Bednarska, Susanna A. Walter, Maite Casado-Bedmar, Magnus Ström, Eloísa Salvo-Romero, Maria Vicario, Emeran A. Mayer, Åsa V. Keita
Background & Aims Irritable bowel syndrome (IBS) is associated with intestinal dysbiosis and symptoms of IBS develop following gastroenteritis. We aimed to study passage of live bacteria through the colonic epithelium, and determine the role of mast cells and vasoactive intestinal polypeptide (VIP) in barrier regulation in IBS and healthy individuals. Methods Colon biopsies from 32 women with IBS and 15 age-matched healthy women (controls) were mounted in Ussing chambers; we measured numbers of fluorescently labeled Escherichia coli HS and Salmonella typhimurium that passed through from the mucosal side to the serosal side of the tissue. Some biopsies were exposed to agents that block the VIP receptors (VPAC1 and VPAC2) or mast cells. Levels of VIP and tryptase were measured in plasma and biopsy lysates. Number of mast cells and mast cells that express VIP or VIP receptors were quantified by immunofluorescence. Biopsies from an additional 5 patients with IBS and 4 controls were mounted in chambers and Salmonella were added; we studied passage routes through the epithelium by transmission electron microscopy and expression of tight junctions by confocal microscopy Results In colon biopsies from patients with IBS, larger numbers of E coli HS and Salmonella passed through the epithelium than in biopsies from controls (P<.0005). In transmission electron microscopy analyses, bacteria were found to cross the epithelium via only the transcellular route. Bacterial passage was reduced in biopsies from patients with IBS and controls after addition of antibodies against VPACs or ketotifen, which inhibits mast cells. Plasma samples from patients with IBS had higher levels of VIP than plasma samples from controls. Biopsies from patients with IBS had higher levels of tryptase, larger numbers of mast cells, and a higher percentage of mast cells that express VPAC1 than biopsies from controls. In biopsies from patients with IBS, addition of Salmonella significantly reduced levels of occludin; subsequent addition of ketotifen significantly reversed this effect. Conclusions We found that colonic epithelium tissues from patients with IBS have increased translocation of commensal and pathogenic live bacteria, compared with controls. Mechanisms of increased translocation include mast cells and VIP.
Enteric Glia Regulate Gastrointestinal Motility but Are Not Required for Maintenance of the Epithelium in Mice Gastroenterology (IF 18.392) Pub Date : 2017-07-13 Meenakshi Rao, Daniella Rastelli, Lauren Dong, Sophia Chiu, Wanda Setlik, Michael D. Gershon, Gabriel Corfas
Background & Aims When the glial fibrillary acidic protein (GFAP) promoter is used to express cellular toxins that eliminate glia in mice, intestinal epithelial permeability and proliferation increase; this led to the concept that glia are required for maintenance of the gastrointestinal epithelium. Many enteric glia, however, particularly in the mucosa, do not express GFAP. In contrast, virtually all enteric glia express proteolipid protein 1 (PLP1). We investigated whether elimination of PLP1-expressing cells compromises epithelial maintenance or gastrointestinal motility. Methods We generated mice that express tamoxifen-inducible Cre recombinase under control of the Plp1 promoter and carry the diptheria toxin subunit A (DTA) transgene in the Rosa26 locus (Plp1CreER;Rosa26DTA mice). In these mice, PLP1-expressing glia are selectively eliminated without affecting neighboring cells. We measured epithelial barrier function and gastrointestinal motility in these mice and littermate controls, and analyzed epithelial cell proliferation and ultrastructure from their intestinal tissues. To compare our findings with those from previous studies, we also eliminated glia with ganciclovir in GfapHSV-TK mice. Results Expression of DTA in PLP1-expressing cells selectively eliminated enteric glia from the small and large intestines, but caused no defects in epithelial proliferation, barrier integrity, or ultrastructure. In contrast, administration of ganciclovir to GfapHSV-TK mice eliminated fewer glia but caused considerable non-glial toxicity and epithelial cell death. Elimination of PLP1-expressing cells did not reduce survival of neurons in the intestine, but altered gastrointestinal motility in female, but not male, mice. Conclusions Using the Plp1 promoter to selectively eliminate glia in mice, we found that enteric glia are not required for maintenance of the intestinal epithelium, but are required for regulation of intestinal motility in females. Previous observations supporting the concept that maintenance of the intestinal epithelium requires enteric glia can be attributed to non-glial toxicity in GfapHSV-TK mice and epithelial-cell expression of GFAP. Contrary to widespread notions, enteric glia are therefore not required for epithelial homeostasis. However, they regulate intestinal motility in a sex-dependent manner.
Influence of Metabolic Risk Factors on Risk of Hepatocellular Carcinoma and Liver-related Death in Men with Chronic Hepatitis B: A Large Cohort Study Gastroenterology (IF 18.392) Pub Date : 2017-07-12 Ming-Whei Yu, Chih-Lin Lin, Chun-Jen Liu, Shu-Han Yang, Yu-Lin Tseng, Chih-Feng Wu
Background & Aims Little is known about the absolute risk of hepatocellular carcinoma (HCC) and liver-disease related death, in association with metabolic risk factors, for patients with hepatitis B virus (HBV) infection. Methods We collected data from 5373 male Taiwanese civil servants who visited Taiwan’s Government Employees’ Central Clinics and received routine free physical examinations from 1989 through 1992. We obtained information on liver-related morbidity and mortality in HBV carriers, 40–65 years old (n=1690), with different metabolic risk factors. We compared their medical histories with those of study participants without HBV or HCV infection in the same age range (n=1289). We used patients’ baseline data on obesity, diabetes, hypertriglyceridemia, and high blood pressure to assign them to metabolic risk categories. We then performed a case-cohort analysis of the effects of hepatitis B viral factors on risk for HCC, based on metabolic factors and insulin resistance. Results Over median follow-up period of 19 years, 158 of the 1690 HBV carriers developed HCC and 126 died from liver-related diseases. Among participants without HBV or HCV infection, only 6 developed HCC or died from liver-related disease. HBV carriers with different metabolic risk factors had significant differences in cumulative incidence of HCC and liver-related death. Patients with 3 or more metabolic risk factors had a substantially higher risk for HCC (10-year cumulative incidence, 13.60%) than patients with a low metabolic risk profile (10-year cumulative incidence, 4.83%; adjusted-hazard ratio [aHR], 2.32; 95% CI, 1.18–4.54). Smoking had a significant effect on this association (Pinteraction = .0044). Having 3 or more metabolic risk factors, compared with no factors, significantly increased the risk of HCC (aHR, 5.06; 95% CI, 2.23–11.47) and 10-year cumulative incidence of HCC (25.0% in smokers with 3 or more metabolic risk factors vs 3.87% in smokers with none; P< .0001) in smokers, but did not increase risk of HCC in nonsmokers. Metabolic risk factors and insulin resistance had the largest effect on HCC risk in patients with levels of HBV-DNA <10000 copies/mL. Conclusions In a study of men with chronic HBV infection ages 40-65 years in Taiwan, we associated a high burden of metabolic risk factors with increased risk of HCC; smoking has a significant effect on this association.
Avoidance of Cow's Milk-based Formula for At-risk Infants Does Not Reduce Development of Celiac Disease: a Randomized Controlled Trial Gastroenterology (IF 18.392) Pub Date : 2017-07-05 Mila Hyytinen, Erkki Savilahti, Suvi M. Virtanen, Taina Härkönen, Jorma Ilonen, Kristiina Luopajärvi, Raivo Uibo, Outi Vaarala, Hans K. Åkerblom, Mikael Knip,
Background & Aims Feeding during first months of life might affect risk of celiac disease. Individuals with celiac disease or type 1 diabetes have been reported to have high titers of antibodies against cow´s milk proteins. Avoidance of cow’s milk-based formula for infants with genetic susceptibility for type 1 diabetes reduced the cumulative incidence of diabetes-associated autoantibodies. We performed a randomized controlled trial, in the same population, to study whether weaning to an extensively hydrolyzed formula reduced the risk of celiac disease autoimmunity or celiac disease. Methods We performed a double-blind controlled trial of 230 infants with HLA-defined predisposition to type 1 diabetes and at least 1 family member with type 1 diabetes. The infants were randomly assigned to groups fed a casein hydrolysate formula (n=113) or a conventional formula (control, n=117) whenever breastmilk was not available during the first 6–8 months of life. Serum samples were collected over a median time period of 10 years and analyzed for antibodies to tissue transglutaminase (anti-TG2A) using a radiobinding assay, to endomysium using an immunofluorescence assay, and antibodies to a deamidated gliadine peptide using an immunofluorometry assay. Duodenal biopsies were collected if levels of anti-TG2A exceeded 20 relative units. Cow’s milk antibodies were measured during the first 2 years of life. Results Of the 189 participants analyzed for antiTG2A 25 (13.2%) tested positive. Of the 230 study participants observed 10 (4.3%) were diagnosed with celiac disease. We did not find any significant differences at the cumulative incidence of anti-TG2A positivity (hazard ratio, 1.14; 95 % CI, 0.51–2.54) or celiac disease (hazard ratio, 4.13; 95% CI, 0.81–21.02) between the casein hydrolysate and cow´s milk group. Children who developed celiac disease had increased titers of cow´s milk antibodies before the appearance of anti-TG2A or celiac disease. Conclusions In a randomized controlled trial of 230 infants with genetic risk factors for celiac disease, we did not find evidence that weaning to a diet of extensively hydrolyzed formula compared with cow’s milk-based formula would decrease the risk for celiac disease later in life. Increased titers of cow's milk antibody before anti-TG2A and celiac disease indicates that subjects with celiac disease might have increased intestinal permeability in early life.
Antibodies Against Immune Checkpoint Molecules Restore Functions of Tumor-infiltrating T cells in Hepatocellular Carcinomas Gastroenterology (IF 18.392) Pub Date : 2017-06-23 Guoying Zhou, Dave Sprengers, Patrick P.C. Boor, Michail Doukas, Hannah Schutz, Shanta Mancham, Alexander Pedroza-Gonzalez, Wojciech G. Polak, Jeroen de Jonge, Marcia Gaspersz, Haidong Dong, Kris Thielemans, Qiuwei Pan, Jan N.M. IJzermans, Marco J. Bruno, Jaap Kwekkeboom
Background & Aims Ligand binding to inhibitory receptors on immune cells, such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4), downregulates the T-cell–mediated immune response (called immune checkpoints). Antibodies that block these receptors increase anti-tumor immunity in patients with melanoma, non-small cell lung cancer, and renal cell cancer. Tumor-infiltrating CD4+ and CD8+ T cells in patients with hepatocellular carcinoma (HCC) have been found to be functionally compromised. We analyzed HCC samples from patients to determine if these inhibitory pathways prevent T-cell responses in HCCs and to find ways to restore their anti-tumor functions. Methods We collected HCC samples from 59 patients who underwent surgical resection from November 2013 through May 2017, along with tumor-free liver tissues (control tissues) and peripheral blood samples. We isolated tumor-infiltrating lymphocytes (TIL) and intra-hepatic lymphocytes. We used flow cytometry to quantify expression of the inhibitory receptors PD-1, hepatitis A virus cellular receptor 2 (TIM3), lymphocyte activating 3 (LAG3), and CTLA4 on CD8+ and CD4+ T cells from tumor, control tissue, and blood; we studied the effects of antibodies that block these pathways in T-cell activation assays. Results Expression of PD-1, TIM3, LAG3, and CTLA4 was significantly higher on CD8+ and CD4+ T cells isolated from HCC tissue than control tissue or blood. Dendritic cells, monocytes, and B cells in HCC tumors expressed ligands for these receptors. Expression of PD-1, TIM3, and LAG3 was higher on tumor-associated antigen (TAA)-specific CD8+ TIL, compared with other CD8+ TIL. Compared to TIL that did not express these inhibitory receptors, CD8+ and CD4+ TIL that did express these receptors had higher levels of markers of activation, but similar or decreased levels of granzyme B and effector cytokines. Antibodies against CD274 (PD-L1), TIM3, or LAG3 increased proliferation of CD8+ and CD4+ TIL and cytokine production in response to stimulation with polyclonal antigens or TAA. Importantly, combining antibody against PD-L1 with antibodies against TIM3, LAG3, or CTLA4 further increased TIL functions. Conclusions The immune checkpoint inhibitory molecules PD-1, TIM3, and LAG3 are up-regulated on TAA-specific T cells isolated from human HCC tissues, compared to T cells from tumor-free liver tissues or blood. Antibodies against PD-L1, TIM3, or LAG3 restore responses of HCC-derived T cells to tumor antigens, and combinations of the antibodies have additive effects. Strategies to block PD-L1, TIM3, and LAG3 might be developed for treatment of primary liver cancer.
Treatment of intestinal fibrosis in experimental inflammatory bowel disease by the pleiotropic actions of a local Rho kinase inhibitor Gastroenterology (IF 18.392) Pub Date : 2017-06-19 Tom Holvoet, Sarah Devriese, Karolien Castermans, Sandro Boland, Dirk Leysen, Yves-Paul Vandewynckel, Lindsey Devisscher, Lien Van den Bossche, Sophie Van Welden, Melissa Dullaers, Roosmarijn E. Vandenbroucke, Riet De Rycke, Karel Geboes, Arnaud Bourin, Olivier Defert, Pieter Hindryckx, Martine De Vos, Debby Laukens
Background Intestinal fibrosis resulting in (sub)obstruction is a common complication of Crohn’s disease (CD). Rho kinases (ROCKs) play multiple roles in TGFβ1-induced myofibroblast activation that could be therapeutic targets. Because systemic ROCK inhibition causes cardiovascular side-effects, we evaluated the effects of a locally acting ROCK inhibitor (AMA0825) on intestinal fibrosis. Methods Fibrosis was assessed in mouse models using dextran sulfate sodium and adoptive T cell transfer. The in vitro and ex vivo effects of AMA0825 were studied in different cell types and in CD biopsy cultures. Results ROCK is expressed in fibroblastic, epithelial, endothelial and muscle cells of the human intestinal tract and is activated in inflamed and fibrotic tissue. Prophylactic treatment with AMA0825 inhibited myofibroblast accumulation, expression of pro-fibrotic factors and accumulation of fibrotic tissue without affecting clinical disease activity and histological inflammation in two models of fibrosis. ROCK inhibition reversed established fibrosis in a chronic DSS model and impeded ex vivo pro-fibrotic protein secretion from stenotic CD biopsies. AMA0825 reduced TGFβ1-induced activation of myocardin-related transcription factor (MRTF) and p38 mitogen-activated protein kinase (MAPK), downregulating matrix metalloproteinases, collagen and IL6 secretion from fibroblasts. In these cells, ROCK inhibition potentiated autophagy, which was required for the observed reduction in collagen and IL6 production. AMA0825 did not affect pro-inflammatory cytokine secretion from other ROCK-positive cell types, corroborating the selective in vivo effect on fibrosis. Conclusions Local ROCK inhibition prevents and reverses intestinal fibrosis by diminishing MRTF and p38 MAPK activation and increasing autophagy in fibroblasts. Overall, our results show that local ROCK inhibition is promising for counteracting fibrosis as an add-on therapy for CD.
Risk of Hepatocellular Cancer in HCV Patients Treated With Direct Acting Antiviral Agents Gastroenterology (IF 18.392) Pub Date : 2017-06-19 Fasiha Kanwal, Jennifer Kramer, Steven M. Asch, Maneerat Chayanupatkul, Yumei Cao, Hashem B. El-Serag
Background and Aims The risk of hepatocellular cancer (HCC) after sustained virological response (SVR) with direct acting antivirals (DAA) is unclear. Our aim was to examine the risk and determinants of HCC in patients cured with DAA. Methods We conducted a retrospective cohort study of hepatitis C virus patients who were treated with DAA in any of the 129 Veterans Health Administration hospitals between January 1, 2015 and December 31, 2015. We calculated the annual incidence rates of HCC by SVR. We used Cox regression models to compare the risk of HCC in patients with vs those without SVR and to identify factors associated with incident HCC among patients with SVR. We reviewed a sample of HCC patients for tumor size and stage at diagnosis. Results Among 22,500 patients treated with DAA (19,518 with SVR; 2982 without SVR), the mean (standard deviation) age was 61.6 (6.1) years, and 39.0% had cirrhosis. There were 271 new cases of HCC, including 183 in patients with SVR. Compared with patients without SVR, those with SVR had a significantly reduced risk of HCC (0.90 vs 3.45 HCC/100 person-years; adjusted hazard ratio, 0.28, 95% CI=0.22–0.36). Patients with cirrhosis had the highest annual incidence of HCC after SVR (1.82 vs 0.34/100 person-years in patients without cirrhosis; adjusted hazard ratio, 4.73. 95% CI, 3.34-6.68). Most (>44.8%) HCC were classified as stage I. Maximum size of the largest lesion was ≤5 cm in over 75% of cases. Conclusions Among patients treated with DAA, SVR was associated with a considerable reduction in the risk of HCC. We did not find any evidence to suggest that DAAs promote HCC. However, in patients with SVR, the absolute risk of HCC remained high in patients with established cirrhosis. These patients should be considered for ongoing HCC surveillance.
Pharmacological Rescue of Ductal CFTR Rescue Pancreatic and Salivary glands Acinar Cells and Tissue Function in Mouse Models of Autoimmune diseases Gastroenterology (IF 18.392) Pub Date : 2017-06-19 Mei Zeng, Mitchell Szymczak, Malini Ahuja, Changyu Zheng, Hongen Yin, William Swaim, John A. Chiorini, Robert J. Bridges, Shmuel Muallem
Background & Aims Sjögren’s syndrome and autoimmune pancreatitis (AIP) are disorders with decreased function of salivary, lacrimal glands, and the exocrine pancreas. NOD/ShiLTJ mice and mice transduced with the cytokine BMP6 develop Sjögren’s syndrome and chronic pancreatitis and MRL/Mp mice are models of AIP. CFTR is a ductal Cl- channel essential for ductal fluid and HCO3- secretion. We used these models to ask: is CFTR expression altered in these diseases, does correction of CFTR correct gland function, and most notably, does correcting ductal function correct acinar function. Methods We treated the mice models with the CFTR corrector C18 and the potentiator VX770. Glandular, ductal and acinar cells damage, infiltration of immune cells, and function were measured in vivo and in isolated duct/acini. Results In the disease models, CFTR expression is markedly reduced. The salivary glands and pancreas are inflamed with increased fibrosis and tissue damage. Treatment with VX770 and, in particular C18 restored salivation, rescued CFTR expression and localization, nearly eliminated the inflammation and tissue damage. Transgenic over-expression of CFTR exclusively in the duct had similar effects. Most notably, the markedly reduced acinar cell Ca2+ signaling, Orai1, IP3 receptors, AQP5 expression and fluid secretion were restored by rescuing ductal CFTR. Conclusions Our findings reveal that correcting ductal function is sufficient to rescue acinar cell function and suggests that CFTR correctors are strong candidates for the treatment of Sjögren’s syndrome and pancreatitis.
Colorectal Cancer Cell Line Proteomes are Representative of Primary Tumors and Predict Drug Sensitivity Gastroenterology (IF 18.392) Pub Date : 2017-06-16 Jing Wang, Dmitri Mouradov, Xiaojing Wang, Robert N. Jorissen, Matthew C. Chambers, Lisa J. Zimmerman, Suhas Vasaikar, Christopher G. Love, Shan Li, Kym Lowes, Karl-Johan Leuchowius, Helene Jousset, Janet Weinstock, Christopher Yau, John Mariadason, Zhiao Shi, Yuguan Ban, Xi Chen, Oliver M. Sieber
Background and Aims Proteomics holds promise for individualizing cancer treatment. We analyzed to what extent the proteomic landscape of human colorectal cancer (CRC) is maintained in established CRC cell lines and the utility of proteomics for predicting therapeutic responses. Methods Proteomic and transcriptomic analyses were performed on 44 CRC cell lines, compared against primary CRCs (n=95) and normal tissues (n=60), and integrated with genomic and drug sensitivity data. Results Cell lines mirrored the proteomic aberrations of primary tumors, in particular for intrinsic programs. Tumor relationships of protein expression with DNA copy number aberrations and signatures of post-transcriptional regulation were recapitulated in cell lines. The 5 proteomic subtypes previously identified in tumors were represented among cell lines. Nonetheless, systematic differences between cell line and tumor proteomes were apparent, attributable to stroma, extrinsic signaling, and growth conditions. Contribution of tumor stroma obscured signatures of DNA mismatch repair identified in cell lines with a hypermutation phenotype. Global proteomic data showed improved utility for predicting both known drug-target relationships and overall drug sensitivity as compared with genomic or transcriptomic measurements. Inhibition of targetable proteins associated with drug responses further identified corresponding synergistic or antagonistic drug combinations. Our data provide evidence for CRC proteomic subtype-specific drug responses. Conclusions Proteomes of established CRC cell line are representative of primary tumors. Proteomic data tend to exhibit improved prediction of drug sensitivity as compared with genomic and transcriptomic profiles. Our integrative proteogenomic analysis highlights the potential of proteome profiling to inform personalized cancer medicine.
Epigenome-wide Association Study Identifies Methylation Sites Associated With Liver Enzymes and Hepatic Steatosis Gastroenterology (IF 18.392) Pub Date : 2017-06-15 Jana Nano, Mohsen Ghanbari, Wenshi Wang, Paul S. de Vries, Klodian Dhana, Taulant Muka, André G. Uitterlinden, Joyce B.J. van Meurs, Albert Hofman, , Oscar H. Franco, Qiuwei Pan, Sarwa Darwish Murad, Abbas Dehghan
Background & Aims Epigenetic mechanisms might be involved in the regulation of liver enzyme level. We aimed to identify CpG sites at which DNA methylation levels are associated with blood levels of liver enzymes and hepatic steatosis. Methods We conducted an epigenome-wide association study in whole blood for liver enzyme levels, including gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), among a discovery set of 731 participants of the Rotterdam Study and sought replication in a non-overlapping sample of 719 individuals. Significant DNA methylation changes were further analyzed to evaluate their relation with hepatic steatosis. Expression levels of the top identified gene were measured in 9 human liver cell lines and compared with expression profiles of its potential targets associated with lipid traits. The candidate gene was subsequently knocked down in human hepatoma cells using lentiviral vectors expressing small hairpin RNAs. Results Eight probes annotated to SLC7A11, SLC1A5, SLC43A1, PHGDH, PSORS1C1, SREBF1, ANKS3 were associated with GGT and 1 probe annotated to SLC7A11 was associated with ALT after Bonferroni correction (1.0 × 10-7). No probe was identified for AST levels. Four probes for GGT levels including cg06690548 (SLC7A11), cg11376147 (SLC43A1), cg22304262 (SLC1A5), and cg14476101 (PHGDH), and 1 for ALT cg06690548 (SLC7A11) were replicated. DNA methylation at SLC7A11 was associated with reduced risk of hepatic steatosis in participants (odds ratio, 0.69; 95% CI= 0.55–0.93; P value: 2.7 × 10-3). In functional experiments, SLC7A11 was highly expressed in human liver cells; its expression is positively correlated with expression of a panel of lipid-associated genes, indicating a role of SLC7A11 in lipid metabolism. Conclusions Our results provide new insights into epigenetic mechanisms associated with markers of liver function and hepatic steatosis, laying the groundwork for future diagnostic and therapeutic applications.
Diet Low in FODMAPs Reduces Symptoms in Patients With Irritable Bowel Syndrome and Probiotic Restores Bifidobacterium Species: A Randomized Controlled Trial Gastroenterology (IF 18.392) Pub Date : 2017-06-15 Heidi Maria Staudacher, Miranda C.E. Lomer, Freda M. Farquharson, Petra Louis, Francesca Fava, Elena Franciosi, Matthias Scholz, Kieran M. Tuohy, James O. Lindsay, Peter M. Irving, Kevin Whelan
Background & Aims Dietary restriction of fermentable carbohydrates (a low FODMAP diet) has been reported to reduce symptoms in some patients with irritable bowel syndrome (IBS). We performed a randomized, placebo-controlled study to determine its effects on symptoms and the fecal microbiota in patients with IBS. Methods We performed a 2×2 factorial trial of 104 patients with IBS (18–65 years old), based on the Rome III criteria, at 2 hospitals in the United Kingdom. Patients were randomly assigned (blinded) to groups given counselling to follow a sham diet or diet low in FODMAPs for 4 weeks, along with a placebo or probiotic supplement (VSL#3), resulting in 4 groups (27 receiving sham diet/placebo, 26 receiving sham diet/probiotic, 24 receiving low FODMAP diet /placebo, and 27 receiving low FODMAP diet/probiotic). The sham diet restricted a similar number of staple and non-staple foods as the low FODMAP diet; the diets had similar degrees of difficulty to follow. Dietary counselling was given to patients in all groups and data on foods eaten and compliance were collected. The incidence and severity of 15 gastrointestinal symptoms and overall symptoms were measured daily for 7 days before the study period; along with stool frequency and consistency. At baseline, global and individual symptoms were measured, along with generic and disease-specific health-related quality of life, using standard scoring systems. All data were collected again at 4 weeks, and patients answered questions about adequate symptom relief. Fecal samples were collected at baseline and after 4 weeks and analyzed by quantitative PCR and 16S rRNA sequencing. The co-primary endpoints were adequate relief of symptoms and stool Bifidobacterium species abundance at 4 weeks. Results There was no significant interaction between the interventions in adequate relief of symptoms (P = .52) or Bifidobacterium species (P = .68). In the intention-to-treat analysis, a higher proportion of patients in the low FODMAP diet had adequate symptom relief (57%) vs than in the sham diet group (38%), although the difference was not statistically significant (P = .051). In the per-protocol analysis, a significantly higher proportion of patients on the low FODMAP diet had adequate symptom relief (61%) than in the sham diet group (39%) (P = .043). Total mean IBS-Severity Scoring System score was significantly lower for patients on the low FODMAP diet (173 ± 95) than the sham diet (224 ± 89) (P = .001), but not different between those given probiotic (207 ± 98) or placebo (192 ± 93) (P = .721) Abundance of Bifidobacterium species was lower in fecal samples from patients on the low FODMAP diet (8.8 rRNA genes/g) than patients on the sham diet (9.2 rRNA genes/g) (P = .008), but higher in patients given probiotic (9.1 rRNA genes/g) than patients given placebo (8.8 rRNA genes/g) (P = .019). There was no effect of the low FODMAP diet on microbiota diversity in fecal samples. Conclusions In a placebo-controlled study of patients with IBS, a low FODMAP diet associates with adequate symptom relief and significantly reduced symptom scores compared with placebo. It is not clear whether changes resulted from collective FODMAP restriction or removal of a single component, such as lactose. Co-administration of the probiotic VSL#3 increased numbers of Bifidobacterium species, compared with placebo, and might be given to restore these bacteria to patients on a low FODMAP diet. Trial registration no: ISRCTN02275221.
Association Between Natural Killer Cell Activity and Colorectal Cancer in High-risk Subjects Undergoing Colonoscopy Gastroenterology (IF 18.392) Pub Date : 2017-06-15 Gilles Jobin, Roberto Rodriguez-Suarez, Katia Betito
Background & Aims Low activity of natural killer (NK) cells has been associated with increased risk of cancer and has been reported in patients with colorectal cancer (CRC). Activity of NK cells can be measured in a small volume of whole blood by a commercially available test. We investigated whether this test could be used to identify patients with CRC, using findings from colonoscopy as a reference standard. Methods We performed an open-label, prospective, cross-sectional study of 872 high-risk subjects (more than 40 years old) screened for CRC by colonoscopy at a university hospital in Montreal, Canada from October 2014 through January 2016. Blood samples were collected on the day of colonoscopy, prior to the procedure. The test involves stimulation of whole blood with cytokine that induces NK cells to secrete interferon gamma (IFNG), which is quantified by an ELISA. Tissue samples were taken from lesions during the colonoscopy and analyzed histologically; subjects were classified as having no evidence of disease, adenomatous polyps of less than 10 mm, of 10 mm or more, or CRC. We used the non-parametric Mann-Whitney test to compare NK cell activity between subjects with no evidence of CRC and subjects found to have CRC. Receiver operating characteristic curve analysis was used to assess the ability of the test to identify individuals with CRC. The primary objective was to determine the difference in NK cell activity between subjects with vs without CRC. The secondary objective was the test performance, based on receiver operating characteristic analysis, and cut-off value that most accurately identified individuals with CRC. Results We found a significant difference in NK cell activity between the 23 subjects with CRC (based on pathology analysis) and the 849 subjects without CRC: subjects found to have CRC by colonoscopy had a median level of 86.0 pg IFNG/mL (inter-quartile range, 43.3–151.0 pg IFNG/mL), whereas subjects without CRC had a median level of 298.1 pg IFNG/mL (inter-quartile range, 100.4–920.2 pg IFNG/mL) (P = .0002). The cut-off value that most accurately identified subjects with CRC was 181 pg/mL. The NK cell activity test identified subjects with CRC with 87.0% sensitivity, 60.8% specificity, a positive predictive value of 5.7%, and a negative predictive value of 99.4%. The odds ratio for detection of CRC in subjects with low NK cell activity vs subjects with higher NK cell activity was 10.3 (95% CI, 3.03–34.9). Conclusions Using colonoscopy as the reference standard, a test for NK cell activity in whole blood samples identified patients with CRC with 87.0% sensitivity and a negative predictive value of 99.4%. Subjects with low NK cell activity had a 10-fold higher risk of CRC compared with subjects with high NK cell activity. This test might be used in clinical practice to assess patients for risk of CRC. Clinicaltrials.gov number: NCT02291198.
Intestinal Fungal Dysbiosis Associates With Visceral Hypersensitivity in Patients With Irritable Bowel Syndrome and Rats Gastroenterology (IF 18.392) Pub Date : 2017-06-15 Sara Botschuijver, Guus Roeselers, Evgeni Levin, Daisy M. Jonkers, Olaf Welting, Sigrid E.M. Heinsbroek, Heleen H. de Weerd, Teun Boekhout, Matteo Fornai, Ad A. Masclee, Frank H.J. Schuren, Wouter J. de Jonge, Jurgen Seppen, Rene M. van den Wijngaard
Background & Aims Visceral hypersensitivity is one feature of irritable bowel syndrome (IBS). Bacterial dysbiosis might be involved in the activation of nociceptive sensory pathways, but there have been few studies of the role of the mycobiome (the fungal microbiome) in the development of IBS. We analyzed intestinal mycobiomes of patients with IBS and a rat model of visceral hypersensitivity. Methods We used internal transcribed spacer 1-based metabarcoding to compare fecal mycobiomes of 18 healthy volunteers with those of 39 patients with IBS (with visceral hypersensitivity or normal levels of sensitivity). We also compared the mycobiomes of Long-Evans rats separated from their mothers (hypersensitive) with non-handled (normally sensitive) rats. We investigated whether fungi can cause visceral hypersensitivity using rats exposed to fungicide (fluconazole and nystatin). The functional relevance of the gut mycobiome was confirmed in fecal transplantation experiments: adult maternally separated rats were subjected to water avoidance stress (to induce visceral hypersensitivity), then given fungicide and donor cecum content via oral gavage. Other rats subjected to water avoidance stress were given soluble β-glucans, which antagonize C-type lectin domain family 7 member A (CLEC7A or DECTIN1) signaling via spleen-associated tyrosine kinase (SYK), a SYK inhibitor to reduce visceral hypersensitivity, or vehicle (control). The sensitivity of mast cells to fungi was tested with mesenteric windows (ex vivo) and the human mast cell line HMC-1. Results α diversity (Shannon index) and mycobiome signature (stability selection) of both groups of IBS patients differed from healthy volunteers, and the mycobiome signature of hypersensitive patients differed from that of normally sensitive patients. We observed mycobiome dysbiosis in rats that had been separated from their mothers compared with non-handled rats. Administration of fungicide to hypersensitive rats reduced their visceral hypersensitivity to normal levels of sensitivity. Administration of cecal mycobiomes from rats that had been separated from their mothers (but not non-handled mycobiome) restored hypersensitivity to distension. Administration of soluble β-glucans or a SYK inhibitor reduced visceral hypersensitivity, compared with controls. Particulate β-glucan (a DECTIN-1 agonist) induced mast cell degranulation in mesenteric windows and HMC-1 cells responded to fungal antigens by release of histamine. Conclusions In an analysis of patients with IBS and controls, we associated fungal dysbiosis with IBS. In studies of rats, we found fungi to promote visceral hypersensitivity, which could be reduced by administration of fungicides, soluble β-glucans, or a SYK inhibitor. The intestinal fungi might therefore be manipulated for treatment of IBS-related visceral hypersensitivity.
Accuracy in Diagnosis of Celiac Disease Without Biopsies in Clinical Practice Gastroenterology (IF 18.392) Pub Date : 2017-06-15 K.J. Werkstetter, I.R. Korponay-Szabó, A. Popp, V. Villanacci, M. Salemme, G. Heilig, S.T. Lillevang, M.L. Mearin, C. Ribes-Koninckx, A. Thomas, R. Troncone, B. Filipiak, M. Mäki, J. Gyimesi, M. Najafi, J. Dolinšek, S. Dydensborg Sander, R. Auricchio, Iran Amir Taher Eftekhar Sadat
Background & Aims The guidelines of the European Society of Pediatric Gastroenterology, Hepatology, and Nutrition allow for diagnosis of celiac disease without biopsies in children with symptoms and levels of immunoglobulin A against tissue-transglutaminase (TGA-IgA) 10-fold or more the upper limit of normal (ULN), confirmed by detection of endomysium antibodies (EMA) and positivity for HLA-DQ2/DQ8. We performed a large, international prospective study to validate this approach. Methods We collected data from consecutive pediatric patients (18 years or younger) on a gluten-containing diet who tested positive for TGA-IgA from November 2011 through May 2014, seen at 33 pediatric gastroenterology units in 21 countries. Local centers recorded symptoms; measurements of total IgA, TGA, and EMA; and histopathology findings from duodenal biopsies. Children were considered to have malabsorption if they had chronic diarrhea, weight loss (or insufficient gain), growth failure, or anemia. We directly compared central findings from 16 antibody tests (8 for TGA-IgA, 1 for TGA-IgG, 6 for IgG against deamidated gliadin peptides, and 1 for EMA, from 5 different manufacturers) 2 HLA-DQ2/DQ8 tests from 2 manufacturers, and histopathology findings from the reference pathologist. Final diagnoses were based on local and central results. If all local and central results were concordant for celiac disease, cases were classified as proven celiac disease. Patients with only a low level of TGA-IgA (3-fold or less below the ULN) but no other results indicating celiac disease were classified as no celiac disease. Central histo-morphometry analyses were performed on all other biopsies and cases were carefully reviewed in a blinded manner. Inconclusive cases were regarded as not having celiac disease for calculation of diagnostic accuracy. The primary aim was to determine whether the non-biopsy approach identifies children with celiac disease with a positive predictive value (PPV) above 99% in clinical practice. Secondary aims included comparing performance of different serological tests and to determine whether the suggested criteria can be simplified. Results Of 803 children recruited for the study, 96 were excluded due to incomplete data, low level of IgA, or poor-quality biopsies. In the remaining 707 children (65.1% girls; median age, 6.2 years) 645 were diagnosed with celiac disease, 46 were found not to have celiac disease, and 16 had inconclusive results. Findings from local laboratories of TGA-IgA 10-fold or more the ULN, a positive result from the test for EMA, and any symptom identified children with celiac disease (n=399) with a PPV of 99.75 (95% CI, 98.61–99.99); the PPV was 100.00 (95% CI, 98.68–100.00) when only malabsorption symptoms were used instead of any symptom (n=278). Inclusion of HLA analyses did not increase accuracy. Findings from central laboratories differed greatly for patients with lower levels of antibodies, but when levels of TGA-IgA were 10-fold or more the ULN, PPVs ranged from 99.63 (95% CI, 98.67–99.96) to 100.00 (95% CI, 99.23–100.00). Conclusions Children can be accurately diagnosed with celiac disease without biopsy analysis. Diagnosis based on level of TGA-IgA 10-fold or more the ULN, a positive result from the EMA tests in a second blood sample, and the presence of at least 1 symptom could avoid risks and costs of endoscopy for more than half the children with celiac disease worldwide. HLA analysis is not required for accurate diagnosis. Clinical Trial Registration no: DRKS00003555
MAN2A1–FER Fusion Gene Is Expressed by Human Liver and Other Tumor Types and Has Oncogenic Activity in Mice Gastroenterology (IF 18.392) Pub Date : 2017-02-27 Zhang-Hui Chen, Yan P. Yu, Junyan Tao, Silvia Liu, George Tseng, Michael Nalesnik, Ronald Hamilton, Rohit Bhargava, Joel B. Nelson, Arjun Pennathur, Satdarshan P. Monga, James D. Luketich, George K. Michalopoulos, Jian-Hua Luo
Background & Aims Human tumors and liver cancer cell lines express the product of a fusion between the first 13 exons in the mannosidase α class 2A member 1 gene (MAN2A1) and the last 6 exons in the FER tyrosine kinase gene (FER), called MAN2A1−FER. We investigated whether MAN2A1−FER is expressed by human liver tumors and its role in liver carcinogenesis. Methods We performed reverse transcription polymerase chain reaction analyses of 102 non−small cell lung tumors, 61 ovarian tumors, 70 liver tumors, 156 glioblastoma multiform samples, 27 esophageal adenocarcinomas, and 269 prostate cancer samples, as well as 10 nontumor liver tissues and 20 nontumor prostate tissues, collected at the University of Pittsburgh. We also measured expression by 15 human cancer cell lines. We expressed a tagged form of MAN2A1−FER in NIH3T3 and HEP3B (liver cancer) cells; Golgi were isolated for analysis. MAN2A1−FER was also overexpressed in PC3 or DU145 (prostate cancer), NIH3T3 (fibroblast), H23 (lung cancer), and A-172 (glioblastoma multiforme) cell lines and knocked out in HUH7 (liver cancer) cells. Cells were analyzed for proliferation and in invasion assays, and/or injected into flanks of severe combined immunodeficient mice; xenograft tumor growth and metastasis were assessed. Mice with hepatic deletion of PTEN were given tail-vein injections of MAN2A1−FER. Results We detected MAN2A1−FER messenger RNA and fusion protein (114 kD) in the hepatocellular carcinoma cell line HUH7, as well as in liver tumors, esophageal adenocarcinoma, glioblastoma multiforme, prostate tumors, non−small cell lung tumors, and ovarian tumors, but not nontumor prostate or liver tissues. MAN2A1−FER protein retained the signal peptide for Golgi localization from MAN2A1 and translocated from the cytoplasm to Golgi in cancer cell lines. MAN2A1−FER had tyrosine kinase activity almost 4-fold higher than that of wild-type FER, and phosphorylated the epidermal growth factor receptor at tyrosine 88 in its N-terminus. Expression of MAN2A1−FER in 4 cell lines led to epidermal growth factor receptor activation of BRAF, MEK, and AKT; HUH7 cells with MAN2A1−FER knockout had significant decreases in phosphorylation of these proteins. Cell lines that expressed MAN2A1−FER had increased proliferation, colony formation, and invasiveness and formed larger (>2-fold) xenograft tumors in mice, with more metastases, than cells not expressing the fusion protein. HUH7 cells with MAN2A1−FER knockout formed smaller xenograft tumors, with fewer metastases, than control HUH7 cells. HUH7, A-172, and PC3 cells that expressed MAN2A1−FER were about 2-fold more sensitive to the FER kinase inhibitor crizotinib and the epidermal growth factor receptor kinase inhibitor canertinib; these drugs slowed growth of xenograft tumors from MAN2A1−FER cells and prevented their metastasis in mice. Hydrodynamic tail-vein injection of MAN2A1−FER resulted in rapid development of liver cancer in mice with hepatic disruption of Pten. Conclusions Many human tumor types and cancer cell lines express the MAN2A1−FER fusion, which increases proliferation and invasiveness of cancer cell lines and has liver oncogenic activity in mice.
Viral load affects the immune response to HBV in mice with humanized immune system and liver Gastroenterology (IF 18.392) Pub Date : 2017-08-26 Mathilde Dusséaux, Guillemette Masse-Ranson, Sylvie Darche, James Ahodantin, Yan Li, Oriane Fiquet, Elodie Beaumont, Pierrick Moreau, Lise Rivière, Christine Neuveut, Patrick Soussan, Philippe Roingeard, Dina Kremsdorf, James P. Di Santo, Helene Strick-Marchand
Background & Aims Hepatitis B virus (HBV) infects hepatocytes, but the mechanisms of the immune response against the virus, and how it affects disease progression, are unclear. Methods We performed studies with BALB/c Rag2–/–Il2rg–/–SirpaNODAlb-uPAtg/tg mice, stably engrafted with human hepatocytes (HUHEP) with or without a human immune system (HIS). HUHEP and HIS-HUHEP mice were given an intraperitoneal injection of HBV. Mononuclear cells were isolated from spleen and liver for analysis by flow cytometry. Liver was analyzed by immunohistochemistry and mRNA levels were measured by quantitative reverse transcription PCR. Plasma levels of HBV DNA was quantified by quantitative PCR, and antigen-specific antibodies were detected by immunocytochemistry of HBV transfected BHK-21 cells. Results Following HBV infection, a complete viral life cycle, with production of HBV DNA, hepatitis B e, core (HBc) and surface (HBs) antigens, and covalently closed circular DNA, was observed in HUHEP and HIS-HUHEP mice. HBV replicated unrestricted in HUHEP mice resulting in high viral titers without pathologic effects. In contrast, HBV-infected HIS-HUHEP mice developed chronic hepatitis with 10-fold lower titers and antigen-specific IgGs, (anti-HBs, anti-HBc), consistent with partial immune control. HBV-infected HIS-HUHEP livers contained infiltrating Kupffer cells, mature activated natural killer cells (CD69+), and PD-1+ effector memory T cells (CD45RO+). Reducing the viral inoculum resulted in more efficient immune control. Plasma from HBV-infected HIS-HUHEP mice had increased levels of inflammatory and immune-suppressive cytokines (C-X-C motif chemokine ligand 10 and interleukin 10), which correlated with populations of intrahepatic CD4+ T cells (CD45RO+PD-1+). Mice with high levels of viremia had HBV-infected liver progenitor cells. Giving the mice the nucleoside analogue entecavir reduced viral loads and decreased liver inflammation. Conclusion In HIS-HUHEP mice, HBV infection completes a full life cycle and recapitulates some of the immunopathology observed in patients with chronic infection. Inoculation with different viral loads led to different immune responses and levels of virus control. We found HBV to infect liver progenitor cells, which could be involved in hepatocellular carcinogenesis. This is an important new system to study anti-HBV immune responses and screen for combination therapies against hepatotropic viruses.
A Panel of Methylated MicroRNA Biomarkers for Identifying High-Risk Patients with Ulcerative Colitis-associated Colorectal Cancer Gastroenterology (IF 18.392) Pub Date : 2017-08-25 Yuji Toiyama, Yoshinaga Okugawa, Koji Tanaka, Toshimitsu Araki, Keiichi Uchida, Asahi Hishida, Motoi Uchino, Hiroki Ikeuchi, Seiichi Hirota, Masato Kusunoki, C. Richard Boland, Ajay Goel
Background&Aims Methylation of specific microRNAs (miRNAs) often occurs in an age-dependent manner, as a field defect in some instances, and may be an early event in colitis-associated carcinogenesis. We aimed to determine whether specific mRNA signature patterns (MIR1, MIR9, MIR124, MIR137, MIR34B/C) could be used to identify patients with ulcerative colitis (UC) patients who are at increased risk for colorectal neoplasia. Methods We obtained 387 colorectal tissue specimens, collected from 238 patients with UC (152 without neoplasia, 17 with dysplasia, and 69 with UC-associated colorectal cancer [UC-CRC]), from 2 independent cohorts in Japan from 2005 and 2015. We quantified methylation of miRNAs by bisulfite pyrosequencing analysis. We analyzed clinical data to determine whether miRNA methylation patterns associated with age, location, or segment of the colorectum (cecum, transverse colon, and rectum). Differences in tissue miRNA methylation and expression levels were compared among samples and associated with cancer risk using the Wilcoxon test, Mann-Whitney and Kruskal–Wallis tests as appropriately. We performed a validation study of samples from 90 patients without UC and 61 patients with UC-associated dysplasia or cancer to confirm the association between specific methylation patterns of miRNAs in non-tumor rectal mucosa from patients with UC with risk of UC-CRC. Results Among patients with UC without neoplasia, rectal tissues had significantly higher levels of methylation levels of MIR1, MIR9, MIR124, and MIR137 than in proximal mucosa; levels of methylation associated with age and duration of UC in rectal mucosa. Methylation of all miRNAs was significantly higher in samples from patients with dysplasia or CRC compared to samples from patients without neoplasia. Receiver operating characteristic analysis revealed that methylation levels of miRNAs in rectal mucosa accurately differentiated patients with CRC from those without. Methylation of MIR137 in rectal mucosa was independent risk factor for UC-CRC. Methylation patterns of a set of miRNAs (panel) could discriminated patients with UC patients with or without dysplasia or CRC in the evaluation cohort (area under the curve, 0.81) and the validation cohort (area under the curve, 0.78). Conclusions In evaluation and validation cohorts, we found specific miRNAs to be methylated in rectal mucosal samples from patients with UC with dysplasia or CRC compared with patients without neoplasms. This pattern also associated with patient age and might be used to identify patients with UC at greatest risk for developing UC-CRC. Our findings provide evidence for a field defect in rectal mucosa from patients with UC-CRC.
Combination of Alcohol and Cigarette Smoke Induces Endoplasmic Reticulum Stress and Cell Death in Pancreatic Acinar Cells Gastroenterology (IF 18.392) Pub Date : 2017-08-25 Aurelia Lugea, Andreas Gerloff, Hsin-Yuan Su, Zhihong Xu, Ariel Go, Cheng Hu, Samuel W. French, Jeremy S. Wilson, Minoti V. Apte, Richard T. Waldron, Stephen J. Pandol
Background & Aims Smoking, an independent risk factor for pancreatitis, accelerates the development of alcoholic pancreatitis. Alcohol feeding of mice induces upregulation of spliced X-box binding protein 1 (XBP1s), which regulates the endoplasmic reticulum (ER) unfolded protein response and promotes cell survival upon ER stress. We examined whether smoking affects the adaptive mechanisms induced by alcohol and accelerates disorders of the ER in pancreatic acinar cells. Methods We studied the combined effects of ethanol and cigarette smoke extract (CSE) on ER-stress and cell death responses in mouse and human primary acini and the acinar cell line AR42J. Cells were incubated with ethanol (EtOH, 50 mM), CSE (20–40 μg/ml), or both (CSE+EtOH), and analyzed by immunoblotting, quantitative reverse-transcription PCR, and cell death assays. Some cells were incubated with MKC-3946, an inhibitor of endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1) that blocks XBP1s formation. Male Sprague-Dawley rats were fed isocaloric amounts of an ethanol-containing (Lieber-DeCarli) or control diet for 11 weeks and exposed to cigarette smoke or room air in an exposure chamber for 2 hours each day. During the last 3 weeks, a subset of rats received intravenous injections of lipopolysaccharide (LPS, 3 mg/kg per weeks) to induce pancreatitis or saline (control). Pancreatic tissues were collected and analyzed by histology and immunostaining techniques. Results In AR42J and primary acini, CSE+EtOH induced cell death (necrosis and apoptosis), but neither agent alone had this effect. Cell death was associated with a significant decrease in expression of XBP1s. CSE+EtOH, but neither agent alone, slightly decreased ATP levels in AR42J cells, but induced oxidative stress and sustained activation (phosphorylation) of eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3, also called PERK) and increased protein levels of DNA damage inducible transcript 3 (DDIT3, also called CHOP). CHOP regulates transcription to promote apoptosis. Incubation of AR42J or primary mouse or human acinar cells with MKC-3946 reduced expression of XBP1s, increased levels of CHOP and induced cell death. In rats fed ethanol diet, exposure to cigarette smoke increased ER stress in acinar cells and sensitized the pancreas to LPS-induced pathology. Conclusions Cigarette smoke promotes cell death and features of pancreatitis in ethanol-sensitized acinar cells by suppressing the adaptive unfolded protein response signaling pathway. It also activates ER stress pathways that promote acinar cell death.
Effects of Mongersen (GED-0301) on Endoscopic and Clinical Outcomes in Patients With Active Crohn’s Disease Gastroenterology (IF 18.392) Pub Date : 2017-08-25 Brian G. Feagan, Bruce E. Sands, Guillermo Rossiter, Xiaobin Li, Keith Usiskin, Xiaojiang Zhan, Jean-Frédéric Colombel
GED-0301 is an antisense oligodeoxynucleotide with a sequence complementary to the Smad7 mRNA transcript. Smad7 is a negative regulator of transforming growth factor-β, which is increased in the intestinal mucosa of patients with active Crohn’s disease (CD). We randomly assigned 63 CD patients to 4-, 8-, or 12-week treatment groups receiving oral GED-0301 (160 mg/day). The primary objective was to determine GED-0301’s effect on endoscopic CD measures; secondary objectives included effects on clinical activity. Endoscopic improvement was observed in 37% of participants with evaluable endoscopy results at week 12. At week 12, 32% (4 weeks), 35% (8 weeks), and 48% (12 weeks) of patients receiving GED-0301 were in remission (CD activity index score <150); corresponding reductions from baseline in mean CD activity index scores were −124, −112, and −133 points. No new safety signals were observed. These findings support a GED-0301 benefit in active CD.
Predictors of Use of Monitored Anesthesia Care for Outpatient Gastrointestinal Endoscopy in a Capitated Payment System Gastroenterology (IF 18.392) Pub Date : 2017-08-24 Megan A. Adams, Katherine M. Prenovost, Jason A. Dominitz, Robert G. Holleman, Eve A. Kerr, Sarah L. Krein, Sameer D. Saini, Joel H. Rubenstein
Background & Aims Use of monitored anesthesia care (MAC) for gastrointestinal endoscopy has increased in the Veterans Health Administration (VHA), as in fee-for-service environments, despite the absence of financial incentives. We investigated factors associated with use of MAC in an integrated healthcare delivery system with a capitated payment model. Methods We performed a retrospective cohort study using multi-level logistic regression, with MAC use modeled as a function of procedure year, patient- and provider-level factors, and facility effects. We collected data from 2,091,590 Veterans who underwent outpatient esophagogastroduodenoscopy and/or colonoscopy during fiscal years 2000-2013 at 133 facilities. Results The adjusted rate of MAC use in the VHA increased 17% per year (odds ratio for increase, 1.17; 95% CI, 1.09–1.27) from fiscal year 2000 through 2013. The most rapid increase occurred starting in 2011. VHA use of MAC was associated with patient-level factors that included obesity, obstructive sleep apnea, higher comorbidity, and use of prescription opioids and/or benzodiazepines, though the magnitude of these effects was small. Provider-level and facility factors were also associated with use of MAC, though again the magnitude of these associations was small. Unmeasured facility-level effects had the greatest effect on the trend of MAC use. Conclusions In retrospective study of Veterans who underwent outpatient esophagogastroduodenoscopy and/or colonoscopy from fiscal year 2000 through 2013, we found that even in a capitated system, patient factors are only weakly associated with use of MAC. Facility-level effects are the most prominent factor influencing increasing use of MAC. Future studies should focus on better defining the role of MAC and facility and organizational factors that affect choice of endoscopic sedation. It will also be important to align resources and incentives to promote appropriate allocation of MAC based on clinically-meaningful patient factors.
Association of Aneuploidy and Flat Dysplasia With Development of High-Grade Dysplasia or Colorectal Cancer in Patients With Inflammatory Bowel Disease Gastroenterology (IF 18.392) Pub Date : 2017-08-24 Jia-Huei Tsai, Peter S. Rabinovitch, Danning Huang, Thomas Small, Aras N. Mattis, Sanjay Kakar, Won-Tak Choi
There is controversy over how to best manage patients with inflammatory bowel disease (IBD) and flat low-grade dysplasia (fLGD) in the colon. We performed a retrospective analysis of formalin-fixed paraffin-embedded colon tissues with fLGD from 37 patients undergoing surveillance colonoscopy for IBD, from 1990 to 2015 at the University of California at San Francisco Medical Center, to determine whether detection of aneuploidy is associated with later development of high-grade dysplasia (HGD) or colorectal cancer (CRC). Medical data were collected from the patients for a mean follow-up time of 37 months. Using flow cytometry analysis of paraffin-embedded colon tissue, we detected aneuploidy in 15 of 37 samples with fLGD (40.5%). By comparison, aneuploidy was detected in 14 of 15 samples with flat HGD (93.3%) and 2 of 45 samples that were negative for dysplasia (4.4%). The univariate hazard ratio for subsequent detection of HGD or CRC in patients with fLGD and aneuploidy was 5.3 (95% CI, 1.542-24.121) within a mean follow-up time of 37 months. The presence of aneuploidy therefore identifies patients with fLGD in colon tissue who have an increased risk for HGD or CRC and may provide supportive evidence to a morphological impression or suspicion of flat HGD.
Increased Tryptophan Metabolism is Associated With Activity of Inflammatory Bowel Diseases Gastroenterology (IF 18.392) Pub Date : 2017-08-19 Susanna Nikolaus, Berenice Schulte, Natalie Al-Massad, Florian Thieme, Dominik M. Schulte, Johannes Bethge, Ateequr Rehman, Florian Tran, Konrad Aden, Robert Häsler, Natalie Moll, Gregor Schütze, Markus J. Schwarz, Georg H. Waetzig, Philip Rosenstiel, Michael Krawczak, Silke Szymczak, Stefan Schreiber
Background & Aims Administration of tryptophan, and some of its metabolites, reduces the severity of colitis in mice, whereas removing tryptophan from the diet increases susceptibility to colitis. Transfer of the intestinal microbiome can increase susceptibility of mice to colitis. We aimed to systematically evaluate serum levels of tryptophan and its metabolites in patients with inflammatory bowel diseases (IBD), and study their association with clinical and serologic features. Methods We studied 535 consecutive patients with IBD (211 with ulcerative colitis [UC], 234 with Crohn’s disease [CD]; 236 male), enrolled in Germany from August 2013 through April 2014 and followed until July 2016. Serum samples were collected from patients and 100 matched individuals without IBD (controls); levels of tryptophan were measured using high-performance liquid chromatography. Metabolites of tryptophan were measured in serum from 148 patients and 100 controls by mass spectrometry. We measured levels of interleukin 22 (IL22) in serum from 28 patients by ELISA. Paired stool and serum samples were collected from a subset of patients with active UC (n=10) or CD (n=8) to investigate associations between serum levels of tryptophan and composition of the fecal microbiota, analyzed by 16S rDNA amplicon sequencing. We used real-time PCR to measure levels of mRNAs in colonic biopsies from 60 patients with UC, 50 with CD; and 30 controls. We collected information on patients’ disease activity scores, medications, laboratory assessments, clinical examinations during recruitment and follow-up visits. Results Serum levels of tryptophan were significantly lower in patients with IBD than controls (P=5.3x10–6) with a stronger reduction in patients with CD (vs control, P=1.1x10–10) than UC (vs control P=2.8x10–3). We found a negative correlation between serum levels of tryptophan and disease activity or level of c-reactive protein. Levels of mRNAs encoding tryptophan 2,3- dioxygenase-2 (TDO2) and solute carrier family 6 member 19 (SLC6A19, also called B0AT1) were significantly decreased in liver biopsies from patients with IBD compared with controls, whereas level of mRNA encoding indoleamine 2,3-dioxygenase-1 (IDO1) was significantly increased. The composition of the fecal microbiota associated with serum levels of tryptophan. Analysis of tryptophan metabolites revealed activation of the kynurenine pathway, based on high levels of quinolinic acid, in patients with IBD compared with controls. Serum concentration of IL22 associated with disease activity in patients with IBD; there was an inverse association between level of IL22 and serum level of tryptophan. Conclusions In an analysis of serum samples from more than 500 patients with IBD, we observed a negative correlation between serum level of tryptophan and disease activity. Increased levels of tryptophan metabolites—especially of quinolinic acid—indicated a high activity of tryptophan degradation in patients with active IBD. Tryptophan deficiency could contribute to development of IBD. Studies are needed to determine whether modification of intestinal tryptophan pathways affects the severity of IBD.
Gavage of Fecal Samples From Patients with Colorectal Cancer Promotes Intestinal Carcinogenesis in Germ-free and Conventional Mice Gastroenterology (IF 18.392) Pub Date : 2017-08-18 Sunny H. Wong, Liuyang Zhao, Xiang Zhang, Geicho Nakatsu, Juqiang Han, Weiqi Xu, Xue Xiao, Thomas NY. Kwong, Ho Tsoi, William KK. Wu, Zeng Benhua, Francis KL. Chan, Joseph JY. Sung, Hong Wei, Jun Yu
Background & Aims Altered gut microbiota is implicated in development of colorectal cancer (CRC). Some intestinal bacteria have been reported to potentiate intestinal carcinogenesis by producing genotoxins, altering the immune response and intestinal micro-environment, and activating oncogenic signaling pathways. We investigated whether stool from patients with CRC could directly induce colorectal carcinogenesis in mice. Methods We obtained stored stool samples from participants in a metagenome study performed in Hong Kong. Conventional (male C57BL/6) mice were given azoxymethane to induce colon neoplasia after receiving a course of antibiotics in drinking water. Mice were gavaged twice weekly with stool from 5 patients with CRC or 5 healthy individuals (controls) for 5 weeks. Germ-free C57BL/6 mice were gavaged once with stool from 5 patients with CRC or 5 controls. We collected intestinal tissues from mice and performed histology, immunohistochemistry, expression microarray, quantitative PCR, immunoblot, and flow cytometry analyses. We performed 16S rRNA gene sequencing analysis of feces from mice. Results Significantly higher proportions of conventional mice fed with stool from individuals with CRC than control stool developed high-grade dysplasia (P<.05) and macroscopic polyps (P<.01). We observed a higher proportion of proliferating (Ki-67-positive) cells in colons of germ-free mice fed with stool from patients with CRC vs those fed with stool from controls (P<.05). Feces from germ-free and conventional mice fed with stool from patients with CRC vs controls contained different microbial compositions, with lower richness in mice fed with stool from patients with CRC. Intestines collected from conventional and germ-free mice fed with stool from patients with CRC had increased expression of cytokines that modulate inflammation, including C-X-C motif chemokine receptor 1 (CXCR1), CXCR2, interleukin 17A (IL17A), IL22, and IL23A. Intestines from conventional and germ-free mice fed with stool from patients with CRC contained higher proportions of T-helper 1 (Th1) cells (2.25% vs 0.44%) and Th17 cells (2.08% vs 0.31%) (P<.05 for each) than mice fed with stool from controls. Real-time PCR arrays revealed upregulation of genes involved in cell proliferation, stemness, apoptosis, angiogenesis, invasiveness and metastasis in mice fed with stool from CRC patients. Conclusions We fed stool samples from patients with CRC and heathy individuals to germ-free mice and conventional mice with azoxymethane. We found stool from patients with CRC to increase the numbers of polyps, levels of intestinal dysplasia and proliferation, markers of inflammation, and proportions of Th1 and Th17 cells in colon, compared with stool from individuals without CRC. This study provides evidence that the fecal microbiota from patients with CRC can promote tumorigenesis in germ-free mice and mice given a carcinogen.
Activation of NF-κB by Tumor Necrosis Factor in Intestinal Epithelial Cells and Mouse Intestinal Epithelia Reduces Expression of the Chloride Transporter SLC26A3 Gastroenterology (IF 18.392) Pub Date : 2017-08-18 Anoop Kumar, Ishita Chatterjee, Tarunmeet Gujral, Anas Alakkam, Hayley Coffing, Arivarasu N. Anbazhagan, Alip Borthakur, Seema Saksena, Ravinder K. Gill, Waddah A. Alrefai, Pradeep K. Dudeja
Background & Aims Diarrhea associated with inflammatory bowel diseases (IBD) has been associated with increased levels of inflammatory cytokines, including tumor necrosis factor (TNF). The intestinal mucosa of patients with IBD has reduced expression of solute carrier family 26 member 3 (SLC26A3, also called DRA). We investigated whether TNF directly affects expression of DRA in human intestinal epithelial cells (IECs) and in intestines of mice, and studied the mechanisms of these effects. Methods We performed quantitative reverse transcription PCR, immunofluorescence, and immunoblot analyses of Caco-2, HT-29, and T-84 cells human IECs cultured in 2 or 3 dimensions with or without TNF (50 ng/ml for 6–24 hrs). We purified nuclear extracts and quantified NF-κB activation and DNA binding. We isolated intestinal crypts from C57/BL6 mice, cultured enteroids, incubated these with TNF (50 ng/ml, 24 hrs), and quantified mRNAs. DRA-mediated exchange of Cl– for HCO3- was measured by uptake of 125I. Expression of the NFKB inhibitor alpha (NFKBIA, also called IKBA) was knocked down in Caco-2 or HT-29 cells with small interfering RNAs. Activation of NF-κB in response to TNF was measured by luciferase reporter assays; binding of the NF-κB subunit p65 in cells was analyzed in chromatin precipitation assays. DRA promoter activity was measured in a luciferase reporter assay. C57BL/6J mice were injected with TNF (5 μg/mouse for 3–6 hrs) or vehicle (control); intestines were collected and analyzed by immunofluorescence, or RNA and protein were collected from the mucosa. Results Incubation of IECs with TNF reduced expression of DRA. Knockdown of IKBA in IECs led to nuclear translocation of the NF-κB subunit p65 and reduced levels of DRA mRNA and protein. Expression of a transgene encoding p65 or p50 in IECs led to significant reductions in the promoter activity of DRA and its expression. In chromatin precipitation assays, p65 bound directly to the promoter of DRA, at the regions of –935 of –629 and –375 to –84. Injection of mice with TNF or incubation of crypt-derived organoids with TNF reduced their expression of DRA mRNA and protein. Conclusions In human IECs and intestinal tissues from mice, we found TNF to activate NF-κB, which reduced expression of the Cl– to HCO3- exchanger DRA (SLC26A3), via direct binding to the promoter of DRA. This pathway is an important therapeutic target for IBD-associated diarrhea.
Epidemiology of Esophageal Squamous Cell Carcinoma Gastroenterology (IF 18.392) Pub Date : 2017-08-18 Christian C. Abnet, Melina Arnold, Wen-Qiang Wei
Esophageal squamous cell carcinoma (ESCC) accounts for about 90% of the 456,000 incident esophageal cancers each year. Regions of high incidence include Eastern to Central Asia, along the Rift Valley in East Africa, and into South Africa. There are many causes of ESCC, which vary among regions. Early studies in France associated smoking cigarettes and heavy alcohol consumption with high rates of ESCC, but these factors can’t explain the high incidence in other regions. We discuss other risk factors for ESCC, including polycyclic aromatic hydrocarbons from a variety of sources, high-temperature foods, diet, and oral health and the microbiome—all require further research. A growing list of defined genomic regions affect susceptibility, but large genome-wide association studies have been conducted only with ethnic Chinese subjects; more studies are called for in the rest of Asia and Africa. ESCC has been understudied, but growing infrastructure in more high-incidence countries will allow rapid progress in our understanding.
Racial Disparity in Gastrointestinal Cancer Risk Gastroenterology (IF 18.392) Pub Date : 2017-08-12 Hassan Ashktorab, Sonia S. Kupfer, Hassan Brim, John M. Carethers
Cancer from the gastrointestinal tract and its associated excretory organs will occur in over 300,000 Americans in 2017, with colorectal cancer responsible for over forty percent of that burden; there will be over 150,000 deaths from this group of cancers in the same time period. Disparities among subgroups related to these cancers' incidence and mortality exist. The epidemiology and risk factors associated with each cancer bear out differences for racial groups in the United States. Esophageal adenocarcinoma is more frequent in Non-Hispanic Whites, whereas esophageal squamous cell carcinoma with risk factors of tobacco and alcohol is more frequent among Blacks. Liver cancer has been most frequent among Asian/Pacific Islanders chiefly due to hepatitis B vertical transmission, but other racial groups show increasing rates due to hepatitis C and emergence of cirrhosis from non-alcoholic fatty liver disease. Gastric cancer incidence remains highest among Asian/Pacific Islanders likely due to gene-environment interaction. In addition to esophageal squamous cell carcinoma, cancers of the small bowel, pancreas and colorectum show the highest rates among Blacks, where the explanations for the disparity are not as obvious and are likely multifactorial, including socio-economic and health care access, treatment and prevention (vaccination and screening) differences, dietary and composition of the gut microbiome, as well as biological and genetic influences. Cognizance of these disparities in gastrointestinal cancer risk, as well as approaches that apply precision medicine methods to populations with the increased risk, may reduce the observed disparities for digestive cancers.
The Safety and Efficacy of an Alcohol-free Pancreatic Cyst Ablation Protocol Gastroenterology (IF 18.392) Pub Date : 2017-08-09 Matthew T. Moyer, Setareh Sharzehi, Abraham Mathew, John M. Levenick, Brandy D. Headlee, Jonathan T. Blandford, Heather D. Heisey, James H. Birkholz, Brooke B. Ancrile, Jennifer L. Maranki, Niraj J. Gusani, Thomas J. McGarrity, Charles E. Dye
Background & Aims Endoscopic ultrasound (EUS)-guided chemoablation with ethanol lavage followed by infusion of paclitaxel, is effective for the treatment of mucinous pancreatic cysts. However, complications arise in 3%–10% of patients, presumably linked to the inflammatory effects of ethanol. We aimed to determine whether alcohol is required for effective pancreatic cyst ablation, if removing alcohol from the ablation process would improve complication rates, and whether a multi-agent chemotherapeutic cocktail could increase the rate of complete cyst resolution compared to the findings reported from previous trials using alcohol followed by paclitaxel alone. Methods Between November 2011 and December 2016, we conducted a single center, prospective, double-blind trial of 39 patients with mucinous type pancreatic cysts. Patients were randomly assigned to 1 of 2 groups that underwent EUS-guided pancreatic cyst lavage with either 80% ethanol (control) or normal saline (alcohol-free group). Cysts in both groups were then infused with an admixture of paclitaxel and gemcitabine. Primary outcomes were the rates of complete ablation 12 months after the procedure, and rates of serious and minor adverse events within 30 days of the procedure. Results At 12 months, 67% of patients who underwent alcohol-free EUS-guided cyst chemoablation had complete ablation of cysts compared with 61% of patients in the control group. Serious adverse events occurred in 6% of patients in the control group vs none of the patients in the alcohol-free group. Minor adverse events occurred in 22% of patients in the control group and none of the patients in the alcohol-free group. The overall rate of complete ablation was 64%. Conclusions In this prospective, randomized, controlled trial, we found that alcohol is not required for effective EUS-guided pancreatic cyst ablation, and when alcohol is removed from the ablation process, there is a significant reduction in associated adverse events. A multi-agent chemotherapeutic ablation admixture did not appear to significantly improve rates of complete ablation compared to the current standard of alcohol lavage followed by paclitaxel alone. Clinical Trials.gov no.: NCT01475331
Metabolic Circuit Involving Free Fatty Acids, microRNA 122, and Triglyceride Synthesis in Liver and Muscle Tissues Gastroenterology (IF 18.392) Pub Date : 2017-08-09 Chofit Chai, Mila Rivkin, Liav Berkovits, Alina Simerzin, Elina Zorde-Khvalevsky, Nofar Rosenberg, Shiri Klein, Dayana Yaish, Ronen Durst, Shoshana Shpitzen, Shiran Udi, Joseph Tam, Joerg Heeren, Anna Worthmann, Christoph Schramm, Johannes Kluwe, Revital Ravid, Eran Hornstein, Eithan Galun
Background & Aims Effective treatments are needed for hepatic steatosis characterized by accumulation of triglycerides in hepatocytes, which leads to hepatocellular carcinoma. MicroRNA 122 (MIR122) is expressed only in the liver, where it regulates lipid metabolism. We investigated the mechanism by which free fatty acids (FFAs) regulate MIR122 expression and the effect of MIR122 on triglyceride synthesis. Methods We analyzed MIR122 promoter activity and validated its target mRNAs by transfection of luciferase reporter plasmids into Huh7, BNL-1ME, and HEK293 cultured cell lines. We measured levels of microRNAs and mRNAs by quantitative real-time PCR analysis of RNA extracted from plasma, liver, muscle, and adipose tissues of C57BL/6 mice given the FFA-inducer CL316243. MIR122 was inhibited using antogomiR-122. Metabolic profiles of mice were determined utilizing metabolic chambers and by histologic analyses of liver tissues. We performed RNA sequence analyses to identify metabolic pathways involving MIR122. Results We validated human Agpat1 and Dgat1 mRNAs, involved in triglyceride synthesis, as targets of MIR122. FFAs increased MIR122 expression in livers of mice by activating the retinoic acid-related orphan receptor alpha (RORA), and induced secretion of MIR122 from liver to blood. Circulating MIR122 entered muscle and adipose tissues of mice, reducing mRNA levels of genes involved in triglyceride synthesis. Mice injected with an inhibitor of MIR122 (antagomiR-122) and then given CL316243, accumulated triglycerides in liver and muscle tissues and had reduced rates of β-oxidation. There was a positive correlation between level of FFAs and level of MIR122 in plasma samples from 6 healthy individuals, collected before and during fasting. Conclusions In biochemical and histologic studies of plasma, liver, muscle, and adipose tissues from mice, we found that FFAs increase hepatic expression and secretion of MIR122, which regulates energy storage vs expenditure in liver and peripheral tissues. Strategies to reduce triglyceride levels, by increasing MIR122, might be developed for treatment of metabolic syndrome.
Interactions Between Platelets and Inflammatory Monocytes Affect Sickness Behavior in Mice With Liver Inflammation Gastroenterology (IF 18.392) Pub Date : 2017-08-09 Charlotte D'Mello, Wagdi Almishri, Hongqun Liu, Mark Gordon Swain
Background & Aims Patients with inflammatory liver disease commonly develop debilitating symptoms, called sickness behaviors, which arise via changes in brain function. Monocytes that produce tumor necrosis factor (TNF) interact with cerebral endothelial cells to activate microglial cells and promote sickness behavior. Platelets regulate inflammation, and aggregates of monocytes and platelets are increased in the circulation of patients with liver disease. We investigated the role of platelets in inducing inflammatory features of circulating monocytes and promoting sickness behaviors in mice with cholestatic liver injury. Methods We performed bile-duct ligations or sham surgeries on C57BL/6 or toll like receptor 4 (TLR4)-knockout mice to induce liver inflammation. Liver inflammation was also induced in a separate group of mice by administration of concanavalin A. Circulating platelets, aggregates of monocytes and platelets, and activation of microglial cells were measured by flow cytometry. To deplete platelets, mice were given anti-thrombocyte serum or normal rabbit serum (control) 4 days after surgery. Interactions between monocytes and cerebral endothelial cells were analyzed by intravital microscopy. Sickness behaviors were quantified based on time spent by adult mice engaging in social behaviors toward a juvenile mouse, compared to time spent in non-social behavior or remaining immobile. Results Aggregates of monocytes and platelets in circulation of mice increased significantly following bile-duct ligation. Platelet-monocyte interactions were required for activation of inflammatory monocytes and production of TNF. Platelet depletion greatly reduced adhesive interactions between inflammatory monocytes and adhesive interactions with cerebral endothelial cells and activation of the microglia, as well as development of sickness behavior. Furthermore, TLR4 signaling was important for aggregation of monocytes and platelets, and development of sickness behavior following bile-duct ligation. These findings were confirmed in mice with concanavalin A-induced liver injury. Conclusions In mice with liver inflammation, we found TLR4 and aggregates of monocytes and platelets to regulate microglial activation and development of sickness behavior development. These findings might lead to new therapeutic strategies for liver disease-associated symptoms.
Alcohol Use and Cardiovascular Disease Risk in Patients with Nonalcoholic Fatty Liver Disease Gastroenterology (IF 18.392) Pub Date : 2017-08-09 Lisa B. VanWagner, Hongyan Ning, Norrina B. Allen, Veeral Ajmera, Cora E. Lewis, John Jeffrey Carr, Donald M. Lloyd-Jones, Norah A. Terrault, Juned Siddique
Background & Aims Cardiovascular disease (CVD) is the leading cause of death among patients with non-alcoholic fatty liver disease (NAFLD). Moderate drinking (vs abstinence) is associated with lower risk of CVD in the general population. We assessed whether alcohol use is associated with CVD risk in patients with NAFLD. Methods We analyzed data from participants in the Coronary Artery Risk Development in Young Adults longitudinal cohort study of 5115 black and white young adults, 18–30 years old, recruited from 4 cities in the United States from 1985 through 1986. Participants self-reported alcohol use at study entry and then again after 15, 20, and 25 years. At year 25 (2010–2011), participants underwent computed tomography examination of the thorax and abdomen and tissue Doppler echocardiography with myocardial strain measured by speckle tracking. Coronary artery calcification was defined as an Agatston score above 0. NAFLD was defined as liver attenuation less than 51 Hounsfield Units after exclusions. Drinkers reported 1–21 (men) or 1–14 (women) standard drinks/week at years 15, 20, or 25. Nondrinkers reported no alcohol use at years 15, 20, and 25. Results Of the 570 participants with NAFLD (mean age 50 years; 54% black; 46% female), 332 (58%) were drinkers; significantly higher proportions of drinkers were white, male, and with higher levels of education compared with nondrinkers (P<.05 for all). Higher proportions of drinkers had obesity, diabetes, and the metabolic syndrome compared with nondrinkers (P<.01). There was no difference in liver attenuation between groups (P=.12). After multivariable adjustment, there was no association between alcohol use and CVD risk factors (diabetes, hypertension, hyperlipidemia) or subclinical CVD measures (coronary artery calcification, E/A ratio, global longitudinal strain). Conclusions In a population-based sample of individuals with NAFLD in midlife, prospectively assessed alcohol use is not associated with significant differences in risk factors for CVD or markers of subclinical CVD. In contrast to general population findings, alcohol use may not reduce risk of CVD in patients with NAFLD.
Management of Gastroesophageal Reflux Disease Gastroenterology (IF 18.392) Pub Date : 2017-08-05 C. Prakash Gyawali, Ronnie Fass
Management of GERD commonly starts with an empiric trial of proton pump inhibitor (PPI) therapy and complementary lifestyle measures, for patients without alarm symptoms. Optimization of therapy (improving compliance and timing of PPI doses), or increasing PPI dosage to twice daily in select circumstances, can reduce persistent symptoms. Patients with continued symptoms can be evaluated with endoscopy and tests of esophageal physiology, to better determine their disease phenotype and optimize treatment. Laparoscopic fundoplication, magnetic sphincter augmentation, and endoscopic therapies can benefit patients with well-characterized GERD. Patients with functional diseases that overlap with or mimic GERD can also be treated with neuromodulators (primarily antidepressants), or psychological interventions (psychotherapy, hypnotherapy, cognitive and behavioral therapy). Future approaches to treatment of GERD include potassium-competitive acid blockers, reflux-reducing agents, bile acid binders, injection of inert substances into the esophagogastric junction, and electrical stimulation of the lower esophageal sphincter.
MFSD2A Promotes Endothelial Generation of Inflammation-resolving Lipid Mediators and Reduces Colitis in Mice Gastroenterology (IF 18.392) Pub Date : 2017-08-04 Federica Ungaro, Carlotta Tacconi, Luca Massimino, Paola Antonia Corsetto, Carmen Correale, Philippe Fonteyne, Andrea Piontini, Valeria Garzarelli, Francesca Calcaterra, Silvia Della Bella, Antonino Spinelli, Michele Carvello, Angela Maria Rizzo, Stefania Vetrano, Luciana Petti, Gionata Fiorino, Federica Furfaro, Domenico Mavilio, Silvio Danese
Background & Aims Alterations in signaling pathways that regulate resolution of inflammation (resolving pathways) contribute to pathogenesis of ulcerative colitis (UC). The resolution process is regulated by lipid mediators, such as those derived from the ω-3 docosahexaenoic acid (DHA), whose esterified form is transported by the major facilitator superfamily domain containing 2A (MFSD2A) through the endothelium of brain, retina, and placenta. We investigated if and how MFSD2A regulates lipid metabolism of gut endothelial cells to promote resolution of intestinal inflammation. Methods We performed lipidomic and functional analyses of MFSD2A in mucosal biopsies and primary human intestinal microvascular endothelial cells (HIMECs) isolated from surgical specimens from patients with active, resolving UC and healthy individuals without UC (controls). MFSD2A was knocked down in HIMECss with small hairpin RNAs or overexpressed from a lentiviral vector. Human circulating endothelial progenitor cells that overexpress MFSD2A were transferred to CD1 nude mice with dextran sodium sulfate-induced colitis, with or without oral administration of DHA. Results Colonic biopsies from patients with UC had reduced levels of inflammation-resolving DHA-derived epoxy metabolites compared to healthy colon tissues or tissues with resolution of inflammation. Production of these metabolites by HIMECs required MFSD2A, which is required for DHA retention and metabolism in the gut vasculature. In mice with colitis, transplanted endothelial progenitor cells that overexpressed MFSD2A not only localized to the inflamed mucosa but also restored the ability of the endothelium to resolve intestinal inflammation, compared to mice with colitis that did not receive MFSD2A-overexpressing endothelial progenitors Conclusions Levels of DHA-derived epoxides are lower in colon tissues from patients with UC than healthy and resolving mucosa. Production of these metabolites by gut endothelium requires MFSD2A; endothelial progenitor cells that overexpress MFSD2A reduce colitis in mice. This pathway might be induced to resolve intestinal inflammation in patients with colitis.
Circulating and Tissue-resident CD4+ T Cells With Reactivity to Intestinal Microbiota Are Abundant in Healthy Individuals and Function is Altered During Inflammation Gastroenterology (IF 18.392) Pub Date : 2017-08-04 Ahmed N. Hegazy, Nathaniel R. West, Michael J.T. Stubbington, Emily Wendt, Kim I.M. Suijker, Angeliki Datsi, Sebastien This, Camille Danne, Suzanne Campion, Sylvia H. Duncan, Benjamin M.J. Owens, Holm H. Uhlig, Andrew McMichael, , Andreas Bergthaler, Sarah A. Teichmann, Satish Keshav, Fiona Powrie
Background & Aims Interactions between commensal microbes and the immune system are tightly regulated and maintain intestinal homeostasis, but little is known about these interactions in humans. We investigated responses of human CD4+ T cells to the intestinal microbiota. We measured the abundance of T cells in circulation and intestinal tissues that respond to intestinal microbes and determined their clonal diversity. We also assessed their functional phenotypes and effects on intestinal resident cell populations, and studied alterations in microbe-reactive T cells in patients with chronic intestinal inflammation. Methods We collected samples of peripheral blood mononuclear cells (PBMC) and intestinal tissues from healthy individuals (controls, n=13–30) and patients with inflammatory bowel diseases (IBD, total n=119; 59 with UC and 60 with Crohn’s disease). We used 2 independent assays (CD154 detection and carboxy-fluorescein succinimidyl ester dilution assays) and 9 intestinal bacterial species (Escherichia coli, Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis, Faecalibacterium prausnitzii, Bacteroides vulgatus, Roseburia intestinalis, Ruminococcus obeum, Salmonella typhimurium and Clostridium difficile) to quantify, expand, and characterize microbe-reactive CD4+ T cells. We sequenced T-cell receptor vβ genes in expanded microbe-reactive T-cell lines to determine their clonal diversity. We examined the effects of microbe-reactive CD4+ T cells on intestinal stromal and epithelial cell lines. Cytokines, chemokines, and gene expression patterns were measured by flow cytometry and quantitative PCR. Results Circulating and gut-resident CD4+ T cells from controls responded to bacteria at frequencies of 40–4000 per million for each bacterial species tested. Microbiota-reactive CD4+ T cells were mainly of a memory phenotype, present in PBMCs and intestinal tissue, and had a diverse T-cell receptor vβ repertoire. These cells were functionally heterogeneous, produced barrier protective cytokines, and stimulated intestinal stromal and epithelial cells via interleukin 17A (IL17A), interferon gamma, and tumor necrosis factor. In patients with IBD, microbiota-reactive CD4+ T cells were reduced in the blood compared to intestine; T-cell responses we detected had an increased frequency of IL17A production compared to responses of T cells from blood or intestinal tissues of controls. Conclusions In an analysis of PBMC and intestinal tissues from patients with IBD vs controls, we found that reactivity to intestinal bacteria is a normal property of the human CD4+ T-cell repertoire, and does not necessarily indicate disrupted interactions between immune cells and the commensal microbiota. T-cell responses to commensals might support intestinal homeostasis, by producing barrier-protective cytokines and providing a large pool of T cells that react to pathogens.
Presentation and Epidemiology of Gastroesophageal Reflux Disease Gastroenterology (IF 18.392) Pub Date : 2017-08-03 Joel E. Richter, Joel H. Rubenstein
Gastroesophageal reflux disease (GERD) is the most prevalent gastrointestinal disorder in the United States, and leads to substantial morbidity, though associated mortality is rare. The prevalence of GERD symptoms appeared to increase until 1999. Risk factors for complications of GERD include advanced age, male sex, white race, abdominal obesity, and tobacco use. Most patients with GERD presents with heartburn and effortless regurgitation. Coexistent dysphagia is considered an alarm symptom, prompting evaluation. There is substantial overlap between symptoms of GERD and those of eosinophilic esophagitis, functional dyspepsia, and gastroparesis, posing a challenge for patient management.
BMI1 and MEL18 Promote Colitis-Associated Cancer in Mice via REG3B and STAT3 Gastroenterology (IF 18.392) Pub Date : 2017-08-03 Xicheng Liu, Wendi Wei, Xiaowei Li, Pengcheng Shen, Dapeng Ju, Zhen Wang, Rukui Zhang, Fu Yang, Chunyan Chen, Kun Cao, Guoli Zhu, Hongyan Chen, Liang Chen, Jianhua Sui, Erquan Zhang, Kaichun Wu, Fengchao Wang, Liping Zhao, Rongwen Xi
Background & Aims Polycomb group proteins are epigenetic factors that silence gene expression; they are dysregulated in cancer cells and contribute to carcinogenesis by unclear mechanisms. We investigated whether BMI1 proto-oncogene, polycomb ring finger (BMI1) and polycomb group ring finger 2 (PCGF2, also called MEL18) are involved in initiation and progression of colitis-associated cancer (CAC) in mice. Methods We generated mice containing floxed alleles of Bmi1 and/or Mel18 and/or Reg3b using the villin-Cre promoter (called Bmi1ΔIEC, Mel18ΔIEC, DKO, and TKO mice). We also disrupted Bmi1 and/or Mel18 specifically in intestinal epithelial cells (IECs) using the villin-CreERT2 inducible promoter. CAC was induced in cre-negative littermate mice (control) and mice with conditional disruption of Bmi1 and/or Mel18 by intraperitoneal injection of azoxymethane followed by addition of dextran sulfate sodium (DSS) to drinking water. Colon tissues were collected from mice and analyzed by histology and immunoblots; IECs were isolated and used in cDNA microarray analyses. Results Following administration of azoxymethane and DSS, DKO mice developed significantly fewer polyps than control, Bmi1ΔIEC, Mel18ΔIEC, Reg3bΔIEC, or TKO mice. Adenomas in the colons of DKO mice were low-grade dysplasias whereas adenomas in control, Bmi1ΔIEC, Mel18ΔIEC, Reg3bΔIEC, or TKO mice were high-grade dysplasias with aggressive invasion of the muscularis mucosa. Disruption of Bmi1 and Mel18 (DKO mice) during late stages of carcinogenesis significantly reduced the numbers of large adenomas and the load of total adenomas, reduced proliferation, and increased apoptosis in colon tissues. IECs isolated from DKO mice after azoxymethane and DSS administration had increased expression of Reg3b, compared with control, Bmi1ΔIEC, or Mel18ΔIEC mice. Expression of REG3B was sufficient to inhibit cytokine-induced activation of STAT3 in IECs. The human REG3β protein, the functional counterpart of mouse REG3B, inhibited STAT3 activity in human 293T cells, and its expression level in colorectal tumors correlated inversely with pSTAT3 level and survival times of patients. Conclusions BMI1 and MEL18 contribute to development of CAC in mice by promoting proliferation and reducing apoptosis, via suppressing expression of Reg3b. REG3B negatively regulates cytokine-induced activation of STAT3 in colon epithelial cells. This pathway might be targeted in patients with colitis to reduce carcinogenesis.
The Epidemiology of Esophageal Adenocarcinoma Gastroenterology (IF 18.392) Pub Date : 2017-08-03 Helen G. Coleman, Shao-Hua Xie, Jesper Lagergren
The incidence of esophageal adenocarcinoma (EAC) has increased in many Western countries, and is higher in men than women. Some risk factors for EAC have been identified—mainly gastroesophageal reflux disease (GERD), Barrett’s esophagus, obesity, and tobacco smoking. It is not clear whether interventions to address these factors can reduce risk of EAC, although some evidence exists for smoking cessation. Although consumption of alcohol is not associated with EAC risk, other exposures, such as physical activity, nutrition, and medication use, require further study. Genetic variants have been associated with risk for EAC, but their overall contribution is low. Studies are needed to investigate associations between risk factors and the molecular subtypes of EAC. The prognosis for patients with EAC has slightly improved, but remains poor—screening and surveillance trials of high-risk individuals are needed.
Endoscopic Management of Early Adenocarcinoma and Squamous Cell Carcinoma of the Esophagus—Screening, Diagnosis, and Therapy Gastroenterology (IF 18.392) Pub Date : 2017-08-02 Massimiliano di Pietro, Marcia I. Canto, Rebecca C. Fitzgerald
Since the esophagus is easily accessible with endoscopy, early diagnosis and curative treatment of esophageal cancer is possible. However, diagnosis is often delayed because symptoms are not specific during early stages of tumor development. The onset of dysphagia is associated with advanced disease, and fewer than 15% of patients survive for 5 years. Population screening by endoscopy is not cost effective, but a number of alternative imaging and cell analysis technologies are under investigation. The ideal screening test should be inexpensive, well tolerated, and applicable to primary care. Over the last 10 years, significant progress has been made in endoscopic diagnosis and treatment of dysplasia (squamous and Barrett’s), and early esophageal cancer using resection and ablation technologies supported by evidence from randomized controlled trials. We review the state-of-the-art technologies for early diagnosis and minimally invasive treatment, which together could reduce the burden of disease.
Intra-hepatic Depletion of Mucosal Associated Invariant T cells in Hepatitis C Virus-induced Liver Inflammation Gastroenterology (IF 18.392) Pub Date : 2017-08-02 Fabian J. Bolte, Ashley C. O’Keefe, Lauren M. Webb, Elisavet Serti, Elenita Rivera, T. Jake Liang, Marc Ghany, Barbara Rehermann
Background & Aims Chronic hepatitis affects phenotypes of innate and adaptive immune cells. Mucosal associated invariant T (MAIT) cells are enriched in the liver as compared to the blood, respond to intra-hepatic cytokines, and (via the semi-invariant T-cell receptor) to bacteria translocated from the gut. Little is known about the role of MAIT cells in livers of patients with chronic hepatitis C virus (HCV) infection and their fate after antiviral therapy. Methods We collected blood samples from 42 patients with chronic HCV infection who achieved a sustained virologic response after 12 weeks of treatment with sofosbuvir and velpatasvir. Mononuclear cells were isolated from blood before treatment, at weeks 4 and 12 during treatment, and 24 weeks after the end of treatment. Liver biopsies were collected from 37 of the patients prior to and at week 4 of treatment. Mononuclear cells from 56 blood donors and 10 livers that were not suitable for transplantation were used as controls. Liver samples were assessed histologically for inflammation and fibrosis. Mononuclear cells from liver and blood were studied by flow cytometry and analyzed for responses to cytokine and bacterial stimulation. Results The frequency of MAIT cells among T cells was significantly lower in blood and liver samples of patients with HCV infection than of controls (median 1.31% vs 2.32% for blood samples, P=.0048 and median 4.34% vs 13.40% for liver samples, P=.001). There was an inverse correlation between the frequency of MAIT cells in the liver and histologically determined levels of liver inflammation (r=-.5437, P=.0006) and fibrosis (r=-.5829, P=.0002). MAIT cells from the liver had higher levels of activation and cytotoxicity than MAIT cells from blood (P<.0001). Production of interferon gamma (IFNG) by MAIT cells was dependent on monocyte-derived interleukin 18 (IL18), and was reduced in patients with HCV infection in response to T-cell receptor-mediated but not cytokine-mediated stimulation, as compared to controls. Anti-viral therapy rapidly decreased liver inflammation and MAIT cell activation and cytotoxicity, and increased the MAIT cell frequency among intra-hepatic but not blood T cells. The MAIT cell response to T-cell receptor-mediated stimulation did not change during the 12 weeks of antiviral therapy. Conclusions In analyses of paired blood and liver samples from patients with chronic HCV infection before, during and after antiviral therapy with sofosbuvir and velpatasvir, we found that intrahepatic MAIT cells are activated by monocyte-derived cytokines and depleted in HCV-induced liver inflammation.
BRCA1 Associated Protein Increases Invasiveness of Esophageal Squamous-Cell Carcinoma Gastroenterology (IF 18.392) Pub Date : 2017-08-02 Yanjie Zhao, Lixuan Wei, Mingming Shao, Xudong Huang, Jiang Chang, Jian Zheng, Jiahui Chu, Qionghua Cui, Linna Peng, Yingying Luo, Wenle Tan, Wen Tan, Dongxin Lin, Chen Wu
Background & Aims We performed a screen for genes whose expression correlates with invasiveness of esophageal squamous-cell carcinoma (ESCC) cells. We studied the effects of overexpression and knockdown of these genes in cell lines and expression levels in patient samples. Methods We selected genes for analysis from 11 loci associated with risk of ESCC. We analyzed the effects of knocking down expression of 47 of these genes using RNA interference on-chip analysis in ESCC cells and HeLa cells. Cells with gene overexpression and knockdown were analyzed in migration and invasion assays or injected into nude mice and metastasis of xenograft tumors was quantified. We collected ESCC and non-tumor esophageal tissues from 94 individuals who underwent surgery in China from 2010 and 2014; clinical information was collected and survival time was measured from the date of diagnosis to the date of last follow-up or death. Levels of mRNAs were quantified by RNA sequencing, and levels of proteins were determined from immunoblot analyses. Patient survival was compared with mRNA levels using Kaplan-Meier methods and hazard ratios were calculated by Cox models. Results We identified 8 genes whose disruption increased migration and 10 genes whose disruption reduced migration. Knockdown of BRCA1 associated protein gene (BRAP) significantly reduced migration or KYSE30, KYSE150, and HeLa cells. In patient tumors, 90% of ESCCs examined had higher levels of BRAP protein than paired non-tumor tissues, and 63.8% had gains in BRAP DNA copy number. Levels of BRAP mRNA in ESCC tissues correlated with patient survival time, and high expression increased risk of death 2.4 fold compared with low expression. ESCCs that had metastasized to lymph node had significantly higher levels of BRAP mRNA than tumors without metastases. Knockdown of BRAP in ESCC and HeLa cell lines significantly reduced migration and invasiveness; these cell lines formed more metastases in mice than control cells. Nuclear translocation of the NF-kappaB P65 subunit and phosphorylation of inhibitor of NF-kappaB kinase subunit beta (IKBKB or IKKbeta) increased in cells that overexpressed BRAP and decreased in cells with BRAP knockdown. In immunoprecipitation assays, BRAP interacted directly with IKKbeta. Expression of metalloproteinase 9 (MMP9) and vascular epithelial growth factor C (VEGFC), which are regulated by NF-kappaB, was significantly reduced in cells with knockdown of BRAP and significantly increased in cells that overexpressed BRAP. Conclusion Expression of BRAP is increased in ESCC samples, compared with non-tumor esophageal tissues; increased expression correlates with reduced patient survival time and promotes metastasis of xenograft tumors in mice. BRAP overexpression leads to increased activity of NF-kappaB and expression of MMP9 and VEGFC.
Assessing Old and New Diagnostic Tests for Gastroesophageal Reflux Disease Gastroenterology (IF 18.392) Pub Date : 2017-08-01 Michael F. Vaezi, Daniel Sifrim
A detailed critique of objective measurements of gastroesophageal reflux disease (GERD) would improve management of patients suspecting of having reflux, leading to rational selection of treatment and better outcomes. Many diagnostic tests for GERD have been developed over the past decades. We analyze their development, positive- and negative-predictive values, and ability to predict response to treatment. These features are important for development of medical, surgical, and endoscopic therapies for GERD. We discuss the value of available diagnostic tests and review their role in management of patients with persistent reflux symptoms despite adequate medical or surgical treatment. This is becoming a significant health economic problem, due to the widespread use of proton pump inhibitors. GERD is believed to cause non-esophageal symptoms, such as those provoked by ear, nose, throat, or respiratory disorders. We analyze the value of GERD diagnostic tests in evaluation of these troublesome, non-esophageal symptoms.
Epidemiology and Natural History of Eosinophilic Esophagitis Gastroenterology (IF 18.392) Pub Date : 2017-08-01 Evan S. Dellon, Ikuo Hirano
Eosinophilic esophagitis (EoE) has emerged over the past 2 decades as a major cause of upper gastrointestinal morbidity. Over this time, the epidemiology of EoE has also rapidly evolved. EoE has transformed from a rare case-reportable condition to disease that is commonly encountered in the gastroenterology clinic, hospital emergency room, and endoscopy suite. The incidence and prevalence are increasing at rates that outpace increased disease recognition. Current incidence estimates range from 5 to 10 cases per 100,000, and current prevalence estimates range from 0.5 to 1 case per 1000. We review the data and potential reasons behind this increase, examine risk factors, and identify important areas for research into disease etiology. The article also discusses the progression of EoE from an inflammatory to fibrostenotic phenotype. An accurate view of the natural history of EoE is central to discussions with patients regarding disease prognosis and decisions about long-term use of medical, endoscopic, and diet therapies. Progressive remodelling appears to be gradual, but not universal, and the duration of untreated disease is the best predictor of stricture risk. Ultimately, prospective, long-term outcome studies focusing on multiple aspects of disease activity are needed to fully understand the natural history of EoE.
American Gastroenterological Association Technical Review on the Role of Therapeutic Drug Monitoring in the Management of Inflammatory Bowel Diseases Gastroenterology (IF 18.392) Pub Date : 2017-07-31 Niels Vande Casteele, Hans Herfarth, Jeffry Katz, Yngve Falck-Ytter, Siddharth Singh
Therapeutic drug monitoring (TDM), which involves measurement of drug or active metabolite levels and anti-drug antibodies, is a promising strategy that can be used to optimize inflammatory bowel disease therapeutics. It is based on the premise that there is a relationship between drug exposure and outcomes, and that considerable inter-individual variability exists in how patients metabolize the drug (pharmacokinetics) and the magnitude and duration of response to therapy (pharmacodynamics). Therefore, the American Gastroenterological Association has prioritized clinical guidelines on the role of TDM in the management of inflammatory bowel disease. To inform these clinical guidelines, this technical review was developed in accordance with the GRADE (Grading of Recommendations Assessment, Development, and Evaluation) framework for interventional and prognostic studies, and focused on the application of TDM for biologic therapy, specifically anti-tumor necrosis factor−α agents, and for thiopurines. Focused questions address the benefits and risks of a strategy of reactive TDM (in patients with active inflammatory bowel disease) to guide treatment changes compared with empiric treatment changes, and the benefits and risks of a strategy of routine proactive TDM (during routine clinical care in patients with quiescent disease) compared with no routine TDM. Additionally, the review addresses the benefits and risks of routine measurement of thiopurine methyltransferase enzyme activity or genotype before starting thiopurine therapy compared with empiric weight-based dosing and explores the performance of different trough drug concentrations for anti−tumor necrosis factor agents and thiopurines to inform clinical decision making when applying TDM in a reactive setting. Due to a paucity of data, this review does not address the role of TDM for more recently approved biologic agents, such as vedolizumab or ustekinumab.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
- Acad. Manag. Ann.
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- Appl. Phys. Lett.
- Appl. Phys. Rev.
- Arch. Pharm.
- Asian J. Org. Chem.
- CA: Cancer J. Clin.
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- Cancer Res.
- Carbohydr. Polym.
- Catal. Sci. Technol.
- Catal. Today
- Cell Chem. Bio.
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- Ceram. Int.
- Chem. Asian J.
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- Chem. Eng. J.
- Chem. Eur. J.
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- Chem. Phys.
- Chem. Phys. Lett.
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- Cryst. Growth Des.
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- Environ. Sci.: Processes Impacts
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- Eur. Heart J.
- Eur. J. Inorg. Chem.
- Eur. J. Med. Chem.
- Eur. J. Org. Chem.
- Eur. Polym. J.
- Eur. Respir. J.
- Eur. Urol.
- Ecol. Lett.
- Electrochem. Commun.
- Electrochim. Acta
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- Environ. Sci. Technol. Lett.
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- Environ. Sci.: Processes Impacts
- Environ. Sci.: Water Res. Technol.
- Eur. Heart J.
- Eur. J. Inorg. Chem.
- Eur. J. Med. Chem.
- Eur. J. Org. Chem.
- Eur. Polym. J.
- Eur. Respir. J.
- Eur. Urol.
- J Nucl. Med.
- J. Agric. Food Chem.
- J. Allergy Clin. Immunol.
- J. Alloys Compd.
- J. Am. Ceram. Soc.
- J. Am. Chem. Soc.
- J. Am. Coll. Cardiol.
- J. Anal. At. Spectrom.
- J. Antibiot.
- J. Cachexia Sarcopenia Muscle
- J. Catal.
- J. Chem. Educ.
- J. Chem. Eng. Data
- J. Chem. Inf. Model.
- J. Chem. Phys.
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- J. Chromatogr. B
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- J. Control. Release
- J. Cryst. Growth
- J. Electrochem. Soc.
- J. Eur. Ceram. Soc.
- J. Exp. Med.
- J. Fluid Mech.
- J. Fluorine Chem.
- J. Funct. Foods
- J. Hazard. Mater.
- J. Hepatol.
- J. Mater. Chem. A
- J. Mater. Chem. B
- J. Mater. Chem. C
- J. Med. Chem.
- J. Membr. Sci.
- J. Nat. Gas Sci. Eng.
- J. Nat. Prod.
- J. Natl. Cancer Inst.
- J. Org. Chem.
- J. Photochem. Photobiol. C Photochem. Rev.
- J. Phys. Chem. A
- J. Phys. Chem. B
- J. Phys. Chem. C
- J. Phys. Chem. Lett.
- J. Pineal. Res.
- J. Power Sources
- J. Proteome Res.
- J. Virol.
- JACC Cardiovasc. Imag.
- JAMA Intern. Med.
- JAMA Neurol.
- JAMA Oncol.
- JAMA Pediatr.
- JAMA Psychiatry