A Novel Hypomorphic Apex1 Mouse Model Implicates Apurinic/Apyrimidinic Endonuclease 1 in Oxidative DNA Damage Repair in Gastric Epithelial Cells.

Aims: Though best known for its role in oxidative DNA damage repair, apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that regulates multiple host responses during oxidative stress, including the reductive activation of transcription factors. As knockout of the APE1-encoding gene, Apex1, is embryonically lethal, we sought to create a viable model with generalized inhibition of APE1 expression. Results: A hypomorphic (HM) mouse with decreased APE1 expression throughout the body was generated using a construct containing a neomycin resistance (NeoR) cassette knocked into the Apex1 site. Offspring were assessed for APE1 expression, breeding efficiency, and morphology with a focused examination of DNA damage in the stomach. Heterozygotic breeding pairs yielded 50% fewer HM mice than predicted by Mendelian genetics. APE1 expression was reduced up to 90% in the lungs, heart, stomach, and spleen. The HM offspring were typically smaller, and most had a malformed tail. Oxidative DNA damage was increased spontaneously in the stomachs of HM mice. Further, all changes were reversed when the NeoR cassette was removed. Primary gastric epithelial cells from HM mice differentiated more quickly and had more evidence of oxidative DNA damage after stimulation with Helicobacter pylori or a chemical carcinogen than control lines from wildtype mice. Innovation: A HM mouse with decreased APE1 expression throughout the body was generated and extensively characterized. Conclusion: The results suggest that HM mice enable studies of APE1's multiple functions throughout the body. The detailed characterization of the stomach showed that gastric epithelial cells from HM were more susceptible to DNA damage. Antioxid. Redox Signal. 38, 183-197.

Survey Finds Gender Disparities Impact Both Women Mentors and Mentees in Gastroenterology.

INTRODUCTION: Gastroenterologists at all levels of practice benefit from formal mentoring. Much of the current literature on mentoring in gastroenterology is based on expert opinion rather than data. In this study, we aimed to identify gender-related barriers to successful mentoring relationships from the mentor and mentee perspectives. METHODS: A voluntary, web-based survey was distributed to physicians at 20 academic institutions across the United States. Overall, 796 gastroenterology fellows and faculty received the survey link, with 334 physicians responding to the survey (42% response rate), of whom 299 (90%; 129 women and 170 men) completed mentorship questions and were included in analysis. RESULTS: Responses of women and men were compared. Compared with men, more women preferred a mentor of the same gender (38.6% women vs 4.2% men, P < 0.0001) but less often had one (45.5% vs 70.2%, P < 0.0001). Women also reported having more difficulty finding a mentor (44.4% vs 16.0%, P < 0.0001) and more often cited inability to identify a mentor of the same gender as a contributing factor (12.8% vs 0.9%, P = 0.0004). More women mentors felt comfortable advising women mentees about work-life balance (88.3% vs 63.8%, P = 0.0005). Nonetheless, fewer women considered themselves effective mentors (33.3% vs 52.6%, P = 0.03). More women reported feeling pressured to mentor because of their gender (39.5% vs 0.9% of men, P < 0.0001). Despite no gender differences, one-third of respondents reported negative impact of the COVID-19 pandemic on their ability to mentor and be mentored. DISCUSSION: Inequities exist in the experiences of women mentees and mentors in gastroenterology, which may affect career advancement and job satisfaction.

Functional Restoration of Bacteriomes and Viromes by Fecal Microbiota Transplantation.

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection (rCDI). However, the overall mechanisms underlying FMT success await comprehensive elucidation, and the safety of FMT has recently become a serious concern because of the occurrence of drug-resistant bacteremia transmitted by FMT. We investigated whether functional restoration of the bacteriomes and viromes by FMT could be an indicator of successful FMT. METHODS: The human intestinal bacteriomes and viromes from 9 patients with rCDI who had undergone successful FMT and their donors were analyzed. Prophage-based and CRISPR spacer-based host bacteria-phage associations in samples from recipients before and after FMT and in donor samples were examined. The gene functions of intestinal microorganisms affected by FMT were evaluated. RESULTS: Metagenomic sequencing of both the viromes and bacteriomes revealed that FMT does change the characteristics of intestinal bacteriomes and viromes in recipients after FMT compared with those before FMT. In particular, many Proteobacteria, the fecal abundance of which was high before FMT, were eliminated, and the proportion of Microviridae increased in recipients. Most temperate phages also behaved in parallel with the host bacteria that were altered by FMT. Furthermore, the identification of bacterial and viral gene functions before and after FMT revealed that some distinctive pathways, including fluorobenzoate degradation and secondary bile acid biosynthesis, were significantly represented. CONCLUSIONS: The coordinated action of phages and their host bacteria restored the recipients' intestinal flora. These findings show that the restoration of intestinal microflora functions reflects the success of FMT.

Food Allergies and Intolerances: A Clinical Approach to the Diagnosis and Management of Adverse Reactions to Food.

Food allergy is an aberrant immunological response to food antigen, which can result in potentially life-threatening reactions. It is often challenging to differentiate food allergy from other adverse reactions to food because their presentations can be indistinguishable. The purpose of this article is to give an overview of the classification, evaluation, and management of adverse food reactions, key differentiating features of food allergy, roles and limitations of various food allergy testing, and promising areas of emerging research. Case studies are used to highlight some of the clinical pearls in diagnosing and managing food-related diseases.

Educating the Next Generation of Undergraduate URM Cancer Scientists: Results and Lessons Learned from a Cancer Research Partnership Scholar Program.

To improve cancer disparities among under-represented minority (URM) populations, better representation of URM individuals in cancer research is needed. The San Diego State University and University of California San Diego Moores Cancer Center Partnership is addressing cancer disparities through an educational program targeting undergraduate URM students. The Partnership provides a paid intensive summer research internship enriched with year-round activities that include educational sessions, a journal club, mentorship, social activities, and poster sessions and presentations. Program evaluation through follow-up surveys, focus groups, and other formal and informal feedback, including advisory and program steering committees, are used to improve the program. Long-term follow-up among scholars (minimum of 10 years) provides data to evaluate the program's long-term impact on scholars' education and career path. Since 2016, 63 URM undergraduate students participated in the scholar program. At the year-2 follow-up (2016 cohort; n = 12), 50% had completed their Graduate Record Examination (GRE) and/or applied to graduate or medical school. Lessons learned during the course of the program led to implementation of changes to provide a better learning experience and increase overall program satisfaction. Updates were made to recruitment timeline, improvements of the recruitment processes, refinement of the program contracts and onboarding meetings, identification of essential program coordinator skills and responsibilities, adjustments to program components, and establishment of a well-mapped and scheduled evaluation plan. The Partnership identified best practices and lessons learned for implementing lab-based internship scholar programs in biomedical and public health fields that could be considered in other programs.

Metagenome Data on Intestinal Phage-Bacteria Associations Aids the Development of Phage Therapy against Pathobionts.

The application of bacteriophages (phages) is proposed as a highly specific therapy for intestinal pathobiont elimination. However, the infectious associations between phages and bacteria in the human intestine, which is essential information for the development of phage therapies, have yet to be fully elucidated. Here, we report the intestinal viral microbiomes (viromes), together with bacterial microbiomes (bacteriomes), in 101 healthy Japanese individuals. Based on the genomic sequences of bacteriomes and viromes from the same fecal samples, the host bacteria-phage associations are illustrated for both temperate and virulent phages. To verify the usefulness of the comprehensive host bacteria-phage information, we screened Clostridioides difficile-specific phages and identified antibacterial enzymes whose activity is confirmed both in vitro and in vivo. These comprehensive metagenome analyses reveal not only host bacteria-phage associations in the human intestine but also provide vital information for the development of phage therapies against intestinal pathobionts.

Celiac Disease.

Gluten-related disorders, including celiac disease, wheat allergy, and nonceliac gluten sensitivity (NCGS), are increasingly reported worldwide. Celiac disease is caused by an immune-mediated reaction to ingested gluten in genetically susceptible persons. NCGS is largely a diagnosis of exclusion when other causes of symptoms have been ruled out. All patients with celiac disease should be referred to a registered dietitian nutritionist with expertise in celiac disease and a gastroenterologist who specializes in celiac disease and malabsorptive disorders, and they should remain on a strict gluten-free diet indefinitely. This article provides an overview of gluten- and wheat-related disorders.

Apurinic/Apyrimidinic Endonuclease 1 Restricts the Internalization of Bacteria Into Human Intestinal Epithelial Cells Through the Inhibition of Rac1.

Pathogenic intestinal bacteria lead to significant disease in humans. Here we investigated the role of the multifunctional protein, Apurinic/apyrimidinic endonuclease 1 (APE1), in regulating the internalization of bacteria into the intestinal epithelium. Intestinal tumor-cell lines and primary human epithelial cells were infected with Salmonella enterica serovar Typhimurium or adherent-invasive Escherichia coli. The effects of APE1 inhibition on bacterial internalization, the regulation of Rho GTPase Rac1 as well as the epithelial cell barrier function were assessed. Increased numbers of bacteria were present in APE1-deficient colonic tumor cell lines and primary epithelial cells. Activation of Rac1 was augmented following infection but negatively regulated by APE1. Pharmacological inhibition of Rac1 reversed the increase in intracellular bacteria in APE1-deficient cells whereas overexpression of constitutively active Rac1 augmented the numbers in APE1-competent cells. Enhanced numbers of intracellular bacteria resulted in the loss of barrier function and a delay in its recovery. Our data demonstrate that APE1 inhibits the internalization of invasive bacteria into human intestinal epithelial cells through its ability to negatively regulate Rac1. This activity also protects epithelial cell barrier function.

Safety and efficacy of AMG 714 in patients with type 2 refractory coeliac disease: a phase 2a, randomised, double-blind, placebo-controlled, parallel-group study.

BACKGROUND: Refractory coeliac disease type 2 is a rare subtype of coeliac disease with high mortality rates; interleukin 15 (IL-15) is strongly implicated in its pathophysiology. This trial aimed to investigate the effects of AMG 714, an anti-IL-15 monoclonal antibody, on the activity and symptoms of refractory coeliac disease type 2. METHODS: This was a randomised, double-blind, placebo-controlled, phase 2a study of adults with a confirmed diagnosis of refractory coeliac disease type 2. Patients were randomly assigned at a 2:1 ratio to receive seven intravenous doses over 10 weeks of AMG 714 (8 mg/kg) or matching placebo. Biopsy samples were obtained at baseline and week 12 for cellular analysis and histology. The change in the proportion of aberrant intraepithelial lymphocytes from baseline to week 12 with respect to all intraepithelial lymphocytes was the primary endpoint and was quantified using flow cytometry. Secondary endpoints were the change in aberrant intraepithelial lymphocytes with respect to intestinal epithelial cells; intestinal histological scores (villous height-to-crypt depth ratio; VHCD); intraepithelial lymphocyte counts; Marsh score; and patient-reported symptom measures, including the Bristol stool form scale (BSFS) and gastrointestinal symptom rating scale (GSRS). Main analyses were done in the per-protocol population of patients who received their assigned treatment, provided evaluable biopsy samples, and did not have major protocol deviations; only patients with non-atypical disease were included in the analyses of aberrant intraepithelial lymphocytes, including the primary analysis. Safety was assessed in all patients who received at least one dose of study drug. This study is registered at ClinicalTrials.gov (NCT02633020) and EudraCT (2015-004063-36). FINDINGS: From April 13, 2016, to Jan 19, 2017, 28 patients were enrolled and randomly assigned to AMG 714 (n=19) and placebo (n=9). Six patients were not included in the primary analysis because of protocol deviation (one in the AMG 714 group), insufficient biopsy samples (one in the AMG 714 group), and atypical intraepithelial lymphocytes (three in the AMG 714 group and one in the placebo group). At 12 weeks, the least square mean difference between AMG 714 and placebo in the relative change from baseline in aberrant intraepithelial lymphocyte percentage was -4·85% (90% CI -30·26 to 20·56; p=0·75). The difference between the AMG 714 and placebo groups in aberrant intraepithelial lymphocytes with respect to epithelial cells at 12 weeks was -38·22% (90% CI -95·73 to 19·29; nominal p=0·18); the difference in change in Marsh score from baseline was 0·09% (95% CI -1·60-1·90; nominal p=0·92); the difference in VHCD ratio was 10·67% (95% CI -38·97 to 60·31; nominal p=0·66); and the difference in change in total intraepithelial lymphocyte count was -12·73% (95% CI -77·57-52·12); nominal p=0·69). Regarding symptoms, the proportion of patients with diarrhoea per the BSFS score decreased from ten (53%) of 19 at baseline to seven (37%) of 19 at week 12 in the AMG 714 group and increased from two (22%) of nine at baseline to four (44%) of nine at week 12 in the placebo group (nominal p=0·0008); and the difference between the groups in change in GSRS score was -0·14 (SE 0·19; nominal p=0·48). Eight (89%) patients in the placebo group and 17 (89%) in the AMG 714 group had treatment-emergent adverse events, including one (11%) patient in the placebo group and five (26%) in the AMG 714 group who had serious adverse events. The most common adverse event in the AMG 714 group was nasopharyngitis (eight [42%] patients vs one [11%] in the placebo group). INTERPRETATION: In patients with refractory coeliac disease type 2 who were treated with AMG 714 or placebo for 10 weeks, there was no difference between the groups in terms of the primary endpoint of aberrant intraepithelial lymphocyte reduction from baseline. Effects on symptoms and other endpoints suggest that further research of AMG 714 may be warranted in patients with refractory coeliac disease type 2. FUNDING: Celimmune and Amgen.

Antigen-Specific Mucosal Immunity Regulates Development of Intestinal Bacteria-Mediated Diseases.

BACKGROUND & AIMS: Dysregulation of the microbiome has been associated with development of complex diseases, such as obesity and diabetes. However, no method has been developed to control disease-associated commensal microbes. We investigated whether immunization with microbial antigens, using CpG oligodeoxynucleotides and/or curdlan as adjuvants, induces systemic antigen-specific IgA and IgG production and affects development of diseases in mice. METHODS: C57BL/6 mice were given intramuscular injections of antigens (ovalbumin, cholera toxin B-subunit, or pneumococcal surface protein A) combined with CpG oligodeoxynucleotides and/or curdlan. Blood and fecal samples were collected weekly and antigen-specific IgG and IgA titers were measured. Lymph nodes and spleens were collected and analyzed by enzyme-linked immunosorbent assay for antigen-specific splenic T-helper 1 cells, T-helper 17 cells, and memory B cells. Six weeks after primary immunization, mice were given a oral, nasal, or vaginal boost of ovalbumin; intestinal lamina propria, bronchial lavage, and vaginal swab samples were collected and antibodies and cytokines were measured. Some mice were also given oral cholera toxin or intranasal Streptococcus pneumoniae and the severity of diarrhea or pneumonia was analyzed. Gnotobiotic mice were gavaged with fecal material from obese individuals, which had a high abundance of Clostridium ramosum (a commensal microbe associated with obesity and diabetes), and were placed on a high-fat diet 2 weeks after immunization with C ramosum. Intestinal tissues were collected and analyzed by quantitative real-time polymerase chain reaction. RESULTS: Serum and fecal samples from mice given injections of antigens in combination with CpG oligodeoxynucleotides and curdlan for 3 weeks contained antigen-specific IgA and IgG, and splenocytes produced interferon-gamma and interleukin 17A. Lamina propria, bronchial, and vaginal samples contained antigen-specific IgA after the ovalbumin boost. This immunization regimen prevented development of diarrhea after injection of cholera toxin, and inhibited lung colonization by S pneumoniae. In gnotobiotic mice colonized with C ramosum and placed on a high-fat diet, the mice that had been immunized with C ramosum became less obese than the nonimmunized mice. CONCLUSIONS: Injection of mice with microbial antigens and adjuvant induces antigen-specific mucosal and systemic immune responses. Immunization with S pneumoniae antigen prevented lung infection by this bacteria, and immunization with C ramosum reduced obesity in mice colonized with this microbe and placed on a high-fat diet. This immunization approach might be used to protect against microbe-associated disorders of intestine.

Statement on Best Practices in the Use of Pathology as a Diagnostic Tool for Celiac Disease: A Guide for Clinicians and Pathologists.

Small intestinal biopsy interpretation has been the cornerstone for the diagnosis of celiac disease for over 50 years. Despite the existence of sensitive and specific serological tests, duodenal mucosal biopsies continue to be obtained in the vast majority of patients in whom a diagnosis of celiac disease is being considered. The accurate evaluation of these biopsies requires coordination and information sharing between the gastroenterologist, laboratory, and pathologist in order to optimize tissue sampling, preparation and interpretation. This document, a collaboration between the Rodger C. Haggitt Gastrointestinal Pathology Society and the North American Association for the Study of Celiac Disease, is intended to provide clinicians and pathologists with a summary of best practices in the use of endoscopy and biopsy for patients with suspected celiac disease. The authors present a comprehensive and critical appraisal of the literature with respect to the topics of endoscopic findings, best methods for the obtaining biopsies, completing the pathology form and pathologic assessment, including evaluating intraepithelial lymphocytes and villous architecture. A discussion of conditions with overlapping pathologic findings in duodenal mucosal biopsies is presented. In order to provide additional guidance for challenging situations, the authors include an appendix containing practical suggestions. This review may be utilized in interdisciplinary discussions to optimize care for patients with possible celiac disease.

Houston Consensus Conference on Testing for Helicobacter pylori Infection in the United States.

Despite guidelines for detection and treatment of Helicobacter pylori infection, recommendations to test patients before and after therapy are commonly not followed in the United States. At the Houston Consensus Conference, 11 experts on management of adult and pediatric patients with H pylori, from different geographic regions of the United States, met to discuss key factors in diagnosis of H pylori infection, including identification of appropriate patients for testing, effects of antibiotic susceptibility on testing and treatment, appropriate methods for confirmation of infection and eradication, and relevant health system considerations. The experts divided into groups that used a modified Delphi panel approach to assess appropriate patients for testing, testing for antibiotic susceptibility and treatment, and test methods and confirmation of eradication. The quality of evidence and strength of recommendations were evaluated using the GRADE system. The results of the individual workshops were presented for a final consensus vote by all panel members. After the Expert Consensus Development meeting, the conclusions were validated by a separate panel of gastroenterologists, who assessed their level of agreement with each of the 29 statements developed at the Expert Consensus Development. The final recommendations are provided, on the basis of the best available evidence, and provide consensus statements with supporting literature to implement testing for H pylori infection at health care systems across the United States.