The chemorepellent, SLIT2, bolsters innate immunity against Staphylococcus aureus.

Neutrophils are essential for host defense against Staphylococcus aureus (S. aureus). The neuro-repellent, SLIT2, potently inhibits neutrophil chemotaxis, and might, therefore, be expected to impair antibacterial responses. We report here that, unexpectedly, neutrophils exposed to the N-terminal SLIT2 (N-SLIT2) fragment kill extracellular S. aureus more efficiently. N-SLIT2 amplifies reactive oxygen species production in response to the bacteria by activating p38 mitogen-activated protein kinase that in turn phosphorylates NCF1, an essential subunit of the NADPH oxidase complex. N-SLIT2 also enhances the exocytosis of neutrophil secondary granules. In a murine model of S. aureus skin and soft tissue infection (SSTI), local SLIT2 levels fall initially but increase subsequently, peaking at 3 days after infection. Of note, the neutralization of endogenous SLIT2 worsens SSTI. Temporal fluctuations in local SLIT2 levels may promote neutrophil recruitment and retention at the infection site and hasten bacterial clearance by augmenting neutrophil oxidative burst and degranulation. Collectively, these actions of SLIT2 coordinate innate immune responses to limit susceptibility to S. aureus.

Codevelopment and usability testing of Patient Engagement 101: a Patient-Oriented Research Curriculum in Child Health e-learning module for health care professionals, researchers and trainees.

BACKGROUND: Patient and family engagement is thought to improve the quality and relevance of child health research. We developed and evaluated the usability of Patient Engagement 101, an e-learning module designed to strengthen the patient-oriented research readiness of health care professionals, researchers, trainees and other stakeholders. METHODS: The development of Patient Engagement 101 was co-led by a parent and a researcher and overseen by a diverse multistake-holder steering committee. The module was refined and evaluated using a mixed-methods usability testing approach with 2 iterative cycles of semistructured interviews, observations and questionnaires. We collected module feedback by way of semistructured interviews, the validated System Usability Scale, and satisfaction, knowledge and confidence questionnaires. Thematic coding of transcripts and field notes, informed by team discussions, guided the module revisions. RESULTS: Thirty end-users completed usability testing (15 per cycle). In each cycle, we modified the module with respect to its content, learner experience, learner-centred design and aesthetic design. Participants were highly satisfied, and System Usability Scale scores indicated the module had the best imaginable usability. Substantial increases in the participants' knowledge test scores and the confidence to engage in patient-oriented research, but not self-rated knowledge, were observed after module completion. INTERPRETATION: Codevelopment with patients and caregivers, and refinement through comprehensive end-user testing, resulted in a training resource with exceptional usability that improved knowledge and confidence to engage in patient-oriented research in child health. Patient Engagement 101 is openly available online, and the methods used to develop and evaluate it may facilitate the creation and evaluation of similar capacity-building resources.

A Fluorescence-based Assay of Membrane Potential for High-throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines.

Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery campaigns targeting recombinant ion channel proteins overexpressed in cells such as HEK-293 cells. However, there is increasing emphasis on the use of tissue-relevant cell lines for studying the effects of small molecule interventions. The following protocol describes the adaptation of a fluorescence-based membrane potential assay for the study of ion channels endogenously expressed in epithelial cell lines. The membrane potential assay details a high-throughput assay for chloride channel activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in two commonly studied epithelial cell lines, Caco-2 and Calu-3. In addition, this paper describes a novel application of this system to measure the activity of the Epithelial Sodium Channel (ENaC) in a high-throughput format in the same epithelial cell lines. Together, these fluorescence-based assays provide a robust and flexible platform for studying small molecule modulators, targeting two epithelial channels in a relevant cellular context.

Co-development and Usability Testing of Research 101: A Patient-Oriented Research Curriculum in Child Health (PORCCH) E-Learning Module for Patients and Families.

Background: Engaging patients and families as research partners increases the relevance, quality, and impact of child health research. However, those interested in research engagement may feel underequipped to meaningfully partner. We sought to co-develop an online learning (e-learning) module, "Research 101," to support capacity-development in patient-oriented child health research amongst patients and families. Methods: Module co-development was co-led by a parent and researcher, with guidance from a diverse, multi-stakeholder steering committee. A mixed-methods usability testing approach, with three iterative cycles of semi-structured interviews, observations, and questionnaires, was used to refine and evaluate the e-learning module. Module feedback was collected during testing and a post-module interview, and with the validated System Usability Scale (SUS), and satisfaction, knowledge, and self-efficacy questionnaires. Transcripts and field notes were analyzed through team discussion and thematic coding to inform module revisions. Results: Thirty participants fully tested Research 101, and another 15 completed confirmatory usability testing (32 caregivers, 6 patients, and 7 clinician-researchers). Module modifications pertaining to learner-centered design, content, aesthetic design, and learner experience were made in each cycle. SUS scores indicated the overall usability of the final version was "excellent." Participants' knowledge of patient-oriented research and self-efficacy to engage in research improved significantly after completing Research 101 (p < 0.01). Conclusions: Co-development and usability testing facilitated the creation of an engaging and effective resource to support the scaling up of patient-oriented child health research capacity. The methods and findings of this study may help guide the integration of co-development and usability testing in creating similar resources.

Positive role of continuous positive airway pressure for intensive care unit patients with severe hypoxaemic respiratory failure due to COVID-19 pneumonia: A single centre experience.

OBJECTIVES: Continuous positive airway pressure (CPAP) may be a useful treatment strategy for patients with severe COVID-19 pneumonia but its effectiveness in preventing mechanical ventilation is unknown. We aimed to evaluate the outcomes of COVID-19 patients treated with CPAP and determine predictors of CPAP response. DESIGN: This was a retrospective observational cohort study. SETTING: The study took place in the intensive care unit (ICU) at Royal Papworth Hospital (RPH) in Cambridge, UK. PATIENTS: We included all consecutive patients with confirmed COVID-19 pneumonia who were transferred from neighbouring hospitals between 14th March and 6th May, 2020 for consideration of ventilatory support. INTERVENTION: We instituted the use of CPAP for all patients who arrived in RPH not intubated and were not making satisfactory progress on supplemental oxygen alone. MEASUREMENTS AND MAIN RESULTS: Of 33 self-ventilating patients included in this study, 22 (66.7%) were male and the mean age was 54 ± 13.23 patients received CPAP. They were more hypoxaemic than those treated with oxygen alone (PaO2/FiO2 ratio; 84.3 ± 19.0 vs 170.0 ± 46.0 mmHg, p = 0.001). There was a significant improvement in PaO2/FiO2 ratio 1-2 hours after CPAP initiation (167.4 ± 49.0 from 84.3 ± 19.0 mmHg, p = 0.001). 14 (61%) patients responded to CPAP and 9 required intubation. There was no difference between these two groups in terms of the severity of baseline hypoxaemia (PaO2/FiO2 ratio; 84.5 ± 16.0 vs 83.9 ± 23.0 mmHg, p = 0.94) but CPAP responders had significantly lower C-reactive protein (CRP) (176 ± 83 vs 274 ± 63 mg/L, p = 0.007), interleukin-6 (IL-6) (30 ± 47 vs 139 ± 148 pg/mL, p = 0.037), and D-dimer (321 ± 267 vs 941 + 1990 ng/mL, p = 0.003). CT pulmonary angiogram was performed in 6 out of 9 intubated patients and demonstrated pulmonary emboli in 5 of them. All patients were discharged from ICU and there were no fatalities. CONCLUSIONS: In this cohort, CPAP was an effective treatment modality to improve hypoxaemia and prevent invasive ventilation in a substantial proportion of patients with severe respiratory failure. Accepting the small sample size, we also found raised biomarkers of inflammation (CRP and IL-6) and coagulopathy (D-Dimer) to be more useful predictors of CPAP responsiveness than the severity of hypoxaemia, and could help to guide intubation decisions in this clinical setting.

Under the Radar: Strategies Used by Helicobacter pylori to Evade Host Responses.

The body depends on its physical barriers and innate and adaptive immune responses to defend against the constant assault of potentially harmful microbes. In turn, successful pathogens have evolved unique mechanisms to adapt to the host environment and manipulate host defenses. Helicobacter pylori (Hp), a human gastric pathogen that is acquired in childhood and persists throughout life, is an example of a bacterium that is very successful at remodeling the host-pathogen interface to promote a long-term persistent infection. Using a combination of secreted virulence factors, immune subversion, and manipulation of cellular mechanisms, Hp can colonize and persist in the hostile environment of the human stomach. Here, we review the most recent and relevant information regarding how this successful pathogen overcomes gastric epithelial host defense responses to facilitate its own survival and establish a chronic infection.

High-Throughput Functional Analysis of CFTR and Other Apically Localized Proteins in iPSC-Derived Human Intestinal Organoids.

Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis (CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease-relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. Specifically, we show how this assay can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This phenotypic platform promises to expand CF therapy discovery to include strategies that target multiple determinants of epithelial fluid transport.

Vitamin D deficiency enhances expression of autophagy-regulating miR-142-3p in mouse and "involved" IBD patient intestinal tissues.

Vitamin D deficiency is an environmental factor involved in the pathogenesis of inflammatory bowel disease (IBD); however, the mechanisms surrounding its role remain unclear. Previous studies conducted in an intestinal epithelial-specific vitamin D receptor (VDR) knockout model suggest that a lack of vitamin D signaling causes a reduction in intestinal autophagy. A potential link between vitamin D deficiency and dysregulated autophagy is microRNA (miR)-142-3p, which suppresses autophagy. In this study, we found that wild-type C57BL/6 mice fed a vitamin D-deficient diet for 5 wk had increased miR-142-3p expression in ileal tissues compared with mice that were fed a matched control diet. Interestingly, there was no difference in expression of key autophagy markers ATG16L1 and LC3II in the ileum whole tissue. However, Paneth cells of vitamin D-deficient mice were morphologically abnormal and had an accumulation of the autophagy adaptor protein p62, which was not present in the total crypt epithelium. These findings suggest that Paneth cells exhibit early markers of autophagy dysregulation within the intestinal epithelium in response to vitamin D deficiency and enhanced miR-142-3p expression. Finally, we demonstrated that treatment-naïve IBD patients with low levels of vitamin D have an increase in miR-142-3p expression in colonic tissues procured from "involved" areas of the disease. Taken together, our findings demonstrate that insufficient vitamin D levels alter expression of autophagy-regulating miR-142-3p in intestinal tissues of mice and patients with IBD, providing insight into the mechanisms by which vitamin D deficiency modulates IBD pathogenesis.NEW & NOTEWORTHY Vitamin D deficiency has a role in IBD pathogenesis, and although the mechanisms surrounding its role remain unclear, it has been suggested that autophagy dysregulation is involved. Here, we show increased ileal expression of autophagy-suppressing miR-142-3p in mice that were fed a vitamin D-deficient diet and in "involved" colonic biopsies from pediatric IBD patients with low vitamin D. miR-142-3p serves as a potential mechanism mediating vitamin D deficiency and reduced autophagy.

Helicobacter pylori Infection.

Helicobacter pylori infection remains one of the most prevalent infections worldwide, causing significant morbidity and mortality from gastric malignancies and peptic ulcers. This article provides a summary of the microbiology and pathogenesis of this bacterium, emphasizing the complex and protean effects of H pylori on gastric epithelial cells, including stem and progenitor populations, and evasion of host immune defenses. Increasing antibiotic resistance has made management more challenging. This article discusses the appropriate diagnostic modality for different clinical scenarios, and the evolving treatment of H pylori infections, including the use of antibiotic susceptibility testing to aid regimen selection.

Development of the patient-oriented research curriculum in child health (PORCCH).

BACKGROUND: The Canadian Institutes for Health Research launched a national 'Strategy for Patient-Oriented Research' (SPOR) in 2011. Patient-oriented research is defined as a continuum of research that engages patients as partners, focuses on patient-identified priorities, and improves patient outcomes. Capacity development is a core element of SPOR. Barriers to patient-oriented research include unfamiliarity with the research process for patients and families and unfamiliarity with the methods of patient and family engagement for researchers. METHODS: The aim of the Patient-Oriented Research Curriculum in Child Health (PORCCH) is to build capacity in patient-oriented research in child health among patients and families, researchers, healthcare professionals, decision-makers, and trainees through a curriculum delivered via a series of interactive online modules (e-learning). A multi-disciplinary, multi-stakeholder steering committee, which included patients and families, guided the development of the curriculum and provided feedback on individual modules. The content, design, and development of each module were co-led by a parent and researcher in an equal partnership. RESULTS: PORCCH consists of a series of five modules. All modules are interactive and include video vignettes and knowledge comprehension questions. Access to the modules is free and each module takes approximately 30 min to complete. The five modules are: Research 101 (an Introduction to Patient-Oriented Research, parts 1 and 2), Patient Engagement 101 (an Introduction to Patient Engagement in Child Health Research, parts 1 and 2), and Research Ethics 101. CONCLUSIONS: PORCCH was developed specifically to overcome recognized barriers to the engagement of patients and families in child health research. The aim of the curriculum is to build capacity in patient-oriented research in child health. The goal is for PORCCH to be a useful resource for all stakeholders involved in patient-oriented research: patients and families, researchers, healthcare professionals, decision-makers, and trainees.

Canada launched a national 'Strategy for Patient-Oriented Research' in 2011. The aim of the strategy is to improve patients' health and well being by focusing on research that is important to patients, and by involving patients and families as equal partners in research. Our group developed the Patient-Oriented Research Curriculum in Child Health (PORCCH) to help patients and families, and other individuals involved in research, learn about patient-oriented research. Patients and families will learn about the research process, who is involved, and the timelines of a research study. Researchers will learn about the methods used in patient-oriented research and some practical ways to meaningfully engage patients and families in research. The online curriculum includes five modules. The design and development of each module was co-led by a parent and researcher in an equal partnership. Access to the modules is free ( www.porcch.ca ) and each module takes approximately 30 min to complete. The five modules are: a) Research 101 part 1, What is Health Research and Who is Involved? b) Research 101 part 2, Timeline of a Research Study, c) Patient Engagement 101 part 1, Foundations of Patient Engagement, d) Patient Engagement 101 part 2, Patient Engagement in Practice, and e) Research Ethics 101. It is hoped that PORCCH will be a useful resource for patients and families and other stakeholders involved in patient-oriented research in child health.

Review: Helicobacter pylori infection in children.

Helicobacter pylori infection in children and adolescents differs in comparison to adults with respect to epidemiology, host responses, and disease manifestations. Furthermore, treatment options are limited in this population and antibiotic resistance rates continue to increase. Therefore, ongoing research is vital to understand disease pathogenesis and provide optimal management of children with infection. This review summarizes relevant publications from April 2019 to March 2020. Similar to adults, recent studies show a decreasing prevalence of infection in the pediatric population. Studies of pathogenesis investigated serum immune responses and the potential inverse association of infection and allergy. Several studies investigated the effect of H pylori and related inflammation on the gut microbiome. The recommendation of endoscopy-based testing to identify the cause of symptoms and not just H pylori, reserving noninvasive UBT or stool antigen tests for post-eradication follow-up, was supported by the current literature.

MCOLN1/TRPML1 inhibition - a novel strategy used by Helicobacter pylori to escape autophagic killing and antibiotic eradication therapy in vivo.

Inhibition of host macroautophagy/autophagy is one of the strategies used by several intracellular pathogens, including H. pylori, to escape killing. Here we discuss our recent work that revealed the novel mechanism by which the vacuolating cytotoxin A (VacA) produced by H. pylori inhibits lysosomal and autophagic killing. We discovered that VacA impairs the activity of the lysosomal calcium channel MCOLN1/TRPML1 leading to the formation of enlarged, dysfunctional lysosomes and autophagosomes that serve as an intracellular niche, which allows the bacteria to escape eradication therapy.

VacA generates a protective intracellular reservoir for Helicobacter pylori that is eliminated by activation of the lysosomal calcium channel TRPML1.

Helicobacter pylori infection is a proven carcinogen for gastric cancer. Its virulence factor vacuolating cytotoxin A (VacA) promotes more severe disease and gastric colonization. VacA, by an unknown mechanism, usurps lysosomal and autophagy pathways to generate a protected reservoir for H. pylori that confers bacterial survival in vitro. Here, we show the existence of a VacA-generated intracellular niche in vivo that protects the bacteria from antibiotic treatment and leads to infection recrudescence after therapy. Furthermore, we report that VacA targets the lysosomal calcium channel TRPML1 to disrupt endolysosomal trafficking and mediate these effects. Remarkably, H. pylori that lack toxigenic VacA colonize enlarged dysfunctional lysosomes in the gastric epithelium of trpml1-null mice, where they are protected from eradication therapy. Furthermore, a small molecule agonist directed against TRPML1 reversed the toxic effects of VacA on endolysosomal trafficking, culminating in the clearance of intracellular bacteria. These results suggest that TRPML1 may represent a therapeutic target for chronic H. pylori infection.

VacA promotes CagA accumulation in gastric epithelial cells during Helicobacter pylori infection.

Helicobacter pylori (H. pylori) is the causative agent of gastric cancer, making it the only bacterium to be recognized as a Class I carcinogen by the World Health Organization. The virulence factor cytotoxin associated gene A (CagA) is a known oncoprotein that contributes to the development of gastric cancer. The other major virulence factor vacuolating cytotoxin A (VacA), disrupts endolysosomal vesicular trafficking and impairs the autophagy pathway. Studies indicate that there is a functional interplay between these virulence factors by unknown mechanisms. We show that in the absence of VacA, both host-cell autophagy and the proteasome degrade CagA during infection with H. pylori. In the presence of VacA, CagA accumulates in gastric epithelial cells. However, VacA does not affect proteasome function during infection with H. pylori suggesting that VacA-disrupted autophagy is the predominant means by which CagA accumulates. Our studies support a model where in the presence of VacA, CagA accumulates in dysfunctional autophagosomes providing a possible explanation for the functional interplay of VacA and CagA.

Joint ESPGHAN/NASPGHAN Guidelines for the Management of Helicobacter pylori in Children and Adolescents (Update 2016).

BACKGROUND: Because of the changing epidemiology of Helicobacter pylori infection and low efficacy of currently recommended therapies, an update of the European Society for Paediatric Gastroenterology Hepatology and Nutrition/North American Society for Pediatric Gastroenterology, Hepatology and Nutrition recommendations for the diagnosis and management of H pylori infection in children and adolescents is required. METHODS: A systematic review of the literature (time period: 2009-2014) was performed. Representatives of both societies evaluated the quality of evidence using GRADE (Grading of Recommendation Assessment, Development, and Evaluation) to formulate recommendations, which were voted upon and finalized using a Delphi process and face-to-face meeting. RESULTS: The consensus group recommended that invasive diagnostic testing for H pylori be performed only when treatment will be offered if tests are positive. To reach the aim of a 90% eradication rate with initial therapy, antibiotics should be tailored according to susceptibility testing. Therapy should be administered for 14 days, emphasizing strict adherence. Clarithromycin-containing regimens should be restricted to children infected with susceptible strains. When antibiotic susceptibility profiles are not known, high-dose triple therapy with proton pump inhibitor, amoxicillin, and metronidazole for 14 days or bismuth-based quadruple therapy is recommended. Success of therapy should be monitored after 4 to 8 weeks by reliable noninvasive tests. CONCLUSIONS: The primary goal of clinical investigation is to identify the cause of upper gastrointestinal symptoms rather than H pylori infection. Therefore, we recommend against a test and treat strategy. Decreasing eradication rates with previously recommended treatments call for changes to first-line therapies and broader availability of culture or molecular-based testing to tailor treatment to the individual child.

Protein kinase C δ signaling is required for dietary prebiotic-induced strengthening of intestinal epithelial barrier function.

Prebiotics are non-digestible oligosaccharides that promote the growth of beneficial gut microbes, but it is unclear whether they also have direct effects on the intestinal mucosal barrier. Here we demonstrate two commercial prebiotics, inulin and short-chain fructo-oligosaccharide (scFOS), when applied onto intestinal epithelia in the absence of microbes, directly promote barrier integrity to prevent pathogen-induced barrier disruptions. We further show that these effects involve the induction of select tight junction (TJ) proteins through a protein kinase C (PKC) δ-dependent mechanism. These results suggest that in the absence of microbiota, prebiotics can directly exert barrier protective effects by activating host cell signaling in the intestinal epithelium, which represents a novel alternative mechanism of action of prebiotics.

The Toronto Consensus for the Treatment of Helicobacter pylori Infection in Adults.

BACKGROUND & AIMS: Helicobacter pylori infection is increasingly difficult to treat. The purpose of these consensus statements is to provide a review of the literature and specific, updated recommendations for eradication therapy in adults. METHODS: A systematic literature search identified studies on H pylori treatment. The quality of evidence and strength of recommendations were rated according to the Grading of Recommendation Assessment, Development and Evaluation (GRADE) approach. Statements were developed through an online platform, finalized, and voted on by an international working group of specialists chosen by the Canadian Association of Gastroenterology. RESULTS: Because of increasing failure of therapy, the consensus group strongly recommends that all H pylori eradication regimens now be given for 14 days. Recommended first-line strategies include concomitant nonbismuth quadruple therapy (proton pump inhibitor [PPI] + amoxicillin + metronidazole + clarithromycin [PAMC]) and traditional bismuth quadruple therapy (PPI + bismuth + metronidazole + tetracycline [PBMT]). PPI triple therapy (PPI + clarithromycin + either amoxicillin or metronidazole) is restricted to areas with known low clarithromycin resistance or high eradication success with these regimens. Recommended rescue therapies include PBMT and levofloxacin-containing therapy (PPI + amoxicillin + levofloxacin). Rifabutin regimens should be restricted to patients who have failed to respond to at least 3 prior options. CONCLUSIONS: Optimal treatment of H pylori infection requires careful attention to local antibiotic resistance and eradication patterns. The quadruple therapies PAMC or PBMT should play a more prominent role in eradication of H pylori infection, and all treatments should be given for 14 days.