Farrugia, Gianrico

Abstract Enteroendocrine cells (EECs) are specialized sensors of luminal forces and chemicals in the gastrointestinal (GI) epithelium that respond to stimulation with a release of signalling molecules such as serotonin (5‐HT). For mechanosensitive EECs, force activates Piezo2 channels, which generate a very rapidly activating and inactivating (∼10 ms) cationic (Na + , K + , Ca 2+ ) receptor current. Piezo2 receptor currents lead to a large and persistent increase in intracellular calcium (Ca 2+ ) that lasts many seconds to sometimes minutes, suggesting signal amplification. However, intracellular calcium dynamics in EEC mechanotransduction remain poorly understood. The aim of this study was to determine the role of Ca 2+ stores in EEC mechanotransduction. Mechanical stimulation of a human EEC cell model (QGP‐1) resulted in a rapid increase in cytoplasmic Ca 2+ and a slower decrease in ER stores Ca 2+ , suggesting the involvement of intracellular Ca 2+ stores. Comparing murine primary colonic EECs with colonocytes showed expression of intercellular Ca 2+ store receptors, a similar expression of IP 3 receptors, but a >30‐fold enriched expression of Ryr3 in EECs. In mechanically stimulated primary EECs, Ca 2+ responses decreased dramatically by emptying stores and pharmacologically blocking IP 3 and RyR1/3 receptors. RyR3 genetic knockdown by siRNA led to a significant decrease in mechanosensitive Ca 2+ responses and 5‐HT release. In tissue, pressure‐induced increase in the Ussing short circuit current was significantly decreased by ryanodine receptor blockade. Our data show that mechanosensitive EECs use intracellular Ca 2+ stores to amplify mechanically induced Ca 2+ entry, with RyR3 receptors selectively expressed in EECs and involved in Ca 2+ signalling, 5‐HT release and epithelial secretion. image Key points A population of enteroendocrine cells (EECs) are specialized mechanosensors of the gastrointestinal (GI) epithelium that respond to mechanical stimulation with the release of important signalling molecules such as serotonin. Mechanical activation of these EECs leads to an increase in intracellular calcium (Ca 2+ ) with a longer duration than the stimulus, suggesting intracellular Ca 2+ signal amplification. In this study, we profiled the expression of intracellular Ca 2+ store receptors and found an enriched expression of the intracellular Ca 2+ receptor Ryr3, which contributed to the mechanically evoked increases in intracellular calcium, 5‐HT release and epithelial secretion. Our data suggest that mechanosensitive EECs rely on intracellular Ca 2+ stores and are selective in their use of Ryr3 for amplification of intracellular Ca 2+ . This work advances our understanding of EEC mechanotransduction and may provide novel diagnostic and therapeutic targets for GI motility disorders.

Ablation has recently emerged as a tool for modulating gastric electrical activation and may hold interventional potential for disorders of gastric function. However, previous studies have been limited to the acute intraoperative setting. This study now presents the safety of gastric ablation after postsurgical recovery and healing. Localized electrical conduction blocks created by ablation remained after 2 wk of healing, and no perforation or other complications were observed over the postsurgical period.

Chronic nausea and vomiting syndromes (NVSs) are prevalent and debilitating disorders. Putative mechanisms include gastric neuromuscular disease and dysregulation of brain-gut interaction, but clinical tests for objectively defining gastric motor function are lacking. A medical device enabling noninvasive body surface gastric mapping (BSGM) was developed and applied to evaluate NVS pathophysiology. BSGM was performed in 43 patients with NVS and 43 matched controls using Gastric Alimetry (Alimetry), a conformable high-resolution array (8 × 8 electrodes; 20-mm interelectrode spacing), wearable reader, and validated symptom-logging app. Continuous measurement encompassed a fasting baseline (30 minutes), 482-kilocalorie meal, and 4-hour postprandial recording, followed by spectral and spatial biomarker analyses. Meal responses were impaired in NVS, with reduced amplitudes compared to controls (median, 23.3 microvolts versus 38.0 microvolts, P < 0.001), impaired fed-fasting power ratios (1.1 versus 1.6, P = 0.02), and disorganized slow waves (spatial frequency stability, 13.6 versus 49.5; P < 0.001). Two distinct NVS subgroups were evident with indistinguishable symptoms (all P > 0.05). Most patients (62%) had normal BSGM studies with increased psychological comorbidities (43.5% versus 7.7%; P = 0.03) and anxiety scores (median, 16.5 versus 13.0; P = 0.035). A smaller subgroup (31%) had markedly abnormal BSGM, with biomarkers correlating with symptoms (nausea, pain, excessive fullness, early satiety, and bloating; all r > 0.35, P < 0.05). Patients with NVS share overlapping symptoms but comprise distinct underlying phenotypes as revealed by a BSGM device. These phenotypes correlate with symptoms, which should inform clinical management and therapeutic trial design.

US hospital quality rankings and ratings use disparate methodologies and are weakly correlated. This causes confusion for patients and hospital quality staff. At the authors’ institution, a Composite Hospital Quality Index (CHQI) was developed to combine hospital quality ratings. This approach is described and a calculator is shared here for other health systems to explore their performance. Among the US News and World Report Top 50 Hospitals, hospital-specific numeric summary scores were aggregated from the 2021 Centers for Medicare and Medicaid Services (CMS) Hospital Overall Star Rating, the Spring 2021 Leapfrog Safety Grade, and the April 2021 Hospital Consumer Assessment of Healthcare Providers and Systems Star Rating. The CHQI is the hospital-specific sum of the national percentile-rankings across these 3 ratings. In this example, mean (SD) percentiles were as follows: CMS Stars 74 (19), Hospital Consumer Assessment of Healthcare Providers and Systems 63 (19), Leapfrog 65 (24), with mean (SD) CHQI of 202 (49). The CHQI is used at the authors’ institution to identify improvement opportunities and ensure that high-quality care is delivered across the health system.

Patients with delayed and normal gastric emptying (GE) had similar symptom profiles during gastric emptying scintigraphy (GES). Idiopathic patients with symptoms of gastroparesis had less gastric retention by GES; but more symptoms after GES meal and after water load satiety test (WLST) compared with diabetic patients. In patients with symptoms of gastroparesis, symptoms after WLST increased with decreasing water consumption. Early satiety and loss of appetite were associated with decreased water consumption during WLST. Thus, impaired accommodation and perhaps visceral hypersensitivity are important in explaining postprandial symptoms in gastroparesis.

Irritable bowel syndrome is a globally prevalent disorder categorized into constipation, diarrhea or mixed (IBS‐C, IBD‐D, IBS‐M) based on the predominant stool form/frequency. While the gut microbiome is recognized as an important factor underlying the pathophysiology of IBS, the specific mechanisms remain an area of active investigation. In our recent longitudinal multi‐omics study in IBS patients, we found bacteria‐derived hypoxanthine was significantly decreased in stool from IBS‐C patients. The aim of this study was to determine if IBS‐C relevant molecular pathways are affected by hypoxanthine. We hypothesized that hypoxanthine acts on enterochromaffin (EC) cells to stimulate serotonin (5‐HT) release, where 5‐HT is an important determinant of gastrointestinal (GI) transit time. We used Ca 2+ imaging in QGP cells (EC model), T84 cells (colonocyte model), primary cells and organoids from NeuroD1‐cre;GCaMP5‐tdTomato mice [NeuroD1 is a marker for enteroendocrine cells (EECs) which express calcium indicator GCaMP in these mice] in the absence or presence of receptor antagonists, and in ex vivo colon preparations with intact innervation from E2aCre:GCaMP6s mice (GCaMP is expressed in neuronal and non‐neuronal cells), to test the effect of hypoxanthine. The physiologic relevance of hypoxanthine signaling was assessed by measuring whole gut transit time. We found hypoxanthine increased Ca 2+ influx in QGP cells [using Cal520 (qualitative) and Fura‐2AM (quantitative)], primary EECs and EECs within organoids (previous research showed that the majority of these EECs are EC cells), but not in T84 cells. Hypoxanthine‐evoked Ca 2+ response was absent in Ca 2+ ‐free medium (n=6‐8, t‐test, P <0.0001), inhibited by G αi/o ‐GPCR antagonist (pertussis toxin; n=8‐14, t‐test, P <0.01)), Transient Receptor Potential Cation Channel Subfamily C Member 4 (TRPC4) antagonist (ML204; n=14‐15, t‐test, P <0.0001) and TRPC4 siRNA (n=5‐21, t‐test, P <0.01), and was accompanied by increased 5‐HT release (measured using 5‐HT biosensor). In ex vivo experiments, luminal application of hypoxanthine activated a subset of epithelial cells (likely EC cells) followed by subsets of neurons (latency to response onset, n=4, Mann‐Whitney, P <0.0001), as well as movement of the imaging field, which indicates smooth muscle contraction/relaxation. Our in vitro findings were confirmed by in vivo experiments showing that germ‐free mice colonized with hypoxanthine‐producing bacteria had faster whole gut transit and higher stool 5‐HT level compared to hypoxanthine‐consuming bacteria (n=7‐8, t‐test, P <0.05). In summary, hypoxanthine increases Ca 2+ influx specifically in EC cells by activating a signaling cascade involving a G αi/o ‐GPCR and TRPC4 channels, which results in 5‐HT release. The delay in activation of myenteric neurons and motor activity suggests that EC cell 5‐HT release likely induces neurogenic motor patterns and may underlie the shorter GI transit time seen with increased bacterial hypoxanthine production. While the current empirically designed probiotics have proven ineffective in IBS, our findings will allow development of novel mechanism‐based probiotic therapies for IBS‐C using hypoxanthine‐producing bacteria.

Background Immortalized cell lines are conventionally used to examine bacterial virulence and host responses. These models lack heterogeneity, complexity and do not allow interrogation of host specific characteristics. Human intestinal organoids provide a model of human epithelium and allow determination of whether bacterial infections elicit host dependent responses. Objective Determine how gender and stress influence responses to Campylobacter infection using human colonic organoids. Methods Colonic biopsies were collected from 20 volunteers (10/sex), with Hospital Anxiety and Depression Score of <4 (low stress) or >14 (high stress). Crypts were isolated from biopsies. Monolayers were generated, experimentally infected with 3 different clinical isolates of C. jejuni (OD 600 1) and averaged. Bacterial virulence and transepithelial resistance (TER) was measured throughout infection. Proteomics was done 24h post‐infection, with analysis via 1D‐LC‐MS using an Orbitrap Eclipse. Data was processed with MS2 ID pipeline MyriMatch‐IDPicker and proteins FDR<0.1 considered significant. Results Colonoid cultures were generated from 75% of volunteers. C. jejuni strains attachmed and invaded all colonoids. Greater bacterial attachment was seen on high stress volunteer colonoids compared low stress (0.08 ± 0.11 vs. 0.03 ± 0.08 CFU/starting inoculum for males, 0.015 ± 0.036 vs. 0.014 ± 0.014 for females, p<0.05 for both) and on high stress males compared to females (p<0.001). Colonoids from high stress volunteers had a significant drop in TER 24h post‐infection compared to non‐infected controls (42.1 ± 30.7 vs 125.2 ± 62.4, % baseline, p<0.05). Proteomic analysis showed minimal differences between high and low stress or between female and male colonoids at baseline (4 and 9 proteins respectively). Upon infection 195 proteins were differentially abundant with 161 increased in stressed organoids (FDR<0.1). Increased apoptotic pathway activation and cellular reorganization; pro‐apoptotic death‐associated protein‐kinase 3 (DAPK3), and DNA damage response serine/threonine‐protein kinase protein (TAOK3) were seen. Gene set enrichment analysis (GSEA) showed increased interferon alpha and beta signaling, IL‐1 and IL‐17 responses and MHC1/2 antigen processing in high stress infected monolayers (FDR<0.1). No differences between male high and low stress organoids post infection. In contrast, 1289 proteins were differentially abundant between high and low stress females with 84% up in stressed organoids. IL‐1 and IL‐12 signaling was up in high stress female organoids (FDR<0.1). Conclusions Colonoid monolayers can be developed from human volunteers and used to evaluate host pathogen interactions. Colonoid cultures highlight variability in C. jejuni infectivity and host responses; with robust C. jejuni virulence seen with high stress male individuals and the greatest change in proteomic profile in response to infection in high stress females. These findings demonstrate utility of colonoids and provide a platform to study personalized host responses to infection.

AbstractBackgroundPatients with gastroparesis (GP) and functional dyspepsia (FD) have similar symptoms, but the pathophysiology of postprandial symptoms remains uncertain.AimsTo compare symptoms and gastric myoelectrical activity (GMA) after liquid and solid test meals in patients with GP and FD.MethodsPatients enrolled in the Gastroparesis Clinical Research Consortium Registry were studied. Clinical characteristics were measured with standard questionnaires. GP was determined by 4‐h solid‐phase gastric scintigraphy. GMA was measured using electrogastrography before and after ingestion of a water load or nutrient bar on separate days. Symptoms were measured on visual analog scales. GMA responses to the water load for individual patients were also determined.Results284 patients with GP and 113 with FD were identified who ingested both test meals. Patients with GP and FD had similar maximal tolerated volumes of water [mean (SD) 378 (218) ml vs. 402 (226) ml,p = 0.23] and reported similar intensity of fullness, nausea, bloating, and abdominal discomfort after the test meals. Twenty‐six percent and 19% of the patients with GP and FD, respectively, ingested subthreshold (<238 ml) volumes of water (p = 0.15). Gastric dysrhythmias were recorded in 66% of the GP and 65% of the FD patients after the water load. Symptoms and GMA were similar in both groups after ingestion of the nutrient bar.ConclusionThe similarity in GMA responses and symptoms after ingestion of solid or liquid test meals suggests GP and FD are closely related gastric neuromuscular disorders.

This study presents gastric ablation as a novel tool for modulating gastric bioelectrical activation, including eliminating the normal gastric pacemaker site as well as abnormal ectopic pacemaker sites underlying gastric dysrhythmias. Targeted application of radiofrequency ablation was able to eliminate these pacemaker sites without disrupting surrounding conduction capability or tissue structure. Gastric ablation presents a powerful new research tool for modulating gastric electrical activation and may likely hold therapeutic potential for disorders of gastric function.