Sense of time is slower following exhaustive cycling exercise.

Subjective perception of time is altered during vigorous exercise. This could be due in part to the fatigue associated with physical activity at high intensities. The aim of this study was to determine the effect of fatigue, specifically, on subjective time perception. Twenty-six healthy, untrained subjects (17 men/9 women; age = 26.0 ± 4.3 years; V ˙ O 2 peak = 38.13 ± 5.62 mL/kg/min) completed a maximal aerobic exercise test on a cycle ergometer. Time perception was assessed before (PRE) and after (POST) the exercise test using a time production task wherein subjects started a stopwatch and stopped it once they believed a designated time period had passed. This time produced with the stopwatch was the estimate of the target time that was compared to the target time interval. Relative error of the timing task was significantly higher for POST (0.112 ± 0.260) than for PRE (0.028 ± 0.173), p = .032, η2 = .178. Subjects produced ~ 8.4% more time than the target intervals when fatigued, which is indicative of a slower sense of time perception. A shift in attentional focus from timing to the sensations associated with fatigue is a possible factor to explain this result. Future studies which investigate the effects of exercise on time perception should consider the impact of fatigue experienced during exercise.

Derivation, validation, and transcriptomic assessment of pediatric septic shock phenotypes identified through latent profile analyses: Results from a prospective multi-center observational cohort.

Background: Sepsis poses a grave threat, especially among children, but treatments are limited due to clinical and biological heterogeneity among patients. Thus, there is an urgent need for precise subclassification of patients to guide therapeutic interventions. Methods: We used clinical, laboratory, and biomarker data from a prospective multi-center pediatric septic shock cohort to derive phenotypes using latent profile analyses. Thereafter, we trained a support vector machine model to assign phenotypes in a hold-out validation set. We tested interactions between phenotypes and common sepsis therapies on clinical outcomes and conducted transcriptomic analyses to better understand the phenotype-specific biology. Finally, we compared whether newly identified phenotypes overlapped with established gene-expression endotypes and tested the utility of an integrated subclassification scheme. Findings: Among 1,071 patients included, we identified two phenotypes which we named 'inflamed' (19.5%) and an 'uninflamed' phenotype (80.5%). The 'inflamed' phenotype had an over 4-fold risk of 28-day mortality relative to those 'uninflamed'. Transcriptomic analysis revealed overexpression of genes implicated in the innate immune response and suggested an overabundance of developing neutrophils, pro-T/NK cells, and NK cells among those 'inflamed'. There was no significant overlap between endotypes and phenotypes. However, an integrated subclassification scheme demonstrated varying survival probabilities when comparing endophenotypes. Interpretation: Our research underscores the reproducibility of latent profile analyses to identify clinical and biologically informative pediatric septic shock phenotypes with high prognostic relevance. Pending validation, an integrated subclassification scheme, reflective of the different facets of the host response, holds promise to inform targeted intervention among those critically ill.

Elevated Plasma Interleukin-18 Identifies High-Risk Acute Respiratory Distress Syndrome Patients not Distinguished by Prior Latent Class Analyses Using Traditional Inflammatory Cytokines: A Retrospective Analysis of Two Randomized Clinical Trials.

OBJECTIVES: Interleukin-18 (IL-18) plasma level and latent class analysis (LCA) have separately been shown to predict prognosis and treatment response in acute respiratory distress syndrome (ARDS). IL-18 is a measure of inflammasome activation, a pathway potentially distinct from inflammation captured by biomarkers defining previously published LCA classes. We hypothesized that elevated IL-18 would identify distinct "high-risk" patients not captured by prior LCA classifications. DESIGN: Statins for acutely injured lungs from sepsis (SAILS) and hydroxymethylglutaryl-CoA reductase inhibition with simvastatin in acute lung injury to reduce pulmonary dysfunction trial (HARP-2) are two large randomized, controlled trials in ARDS in which both LCA assignments and IL-18 levels were shown to predict mortality. We first evaluated the overlap between high IL-18 levels (≥ 800 pg/mL) with prior LCA class assignments using McNemar's test and then tested the correlation between IL-18 and LCA biomarkers using Pearson's exact test on log-2 transformed values. Our primary analysis was the association of IL-18 level with 60-day mortality in the hypoinflammatory LCA class, which was assessed using the Fisher exact test and Cox proportional hazards modeling adjusting for age, Acute Physiology and Chronic Health Evaluation score, and gender. Secondary analyses included the association of IL-18 and LCA with mortality within each IL-18/LCA subgroup. SETTING: Secondary analysis of two multicenter, randomized controlled clinical trials of ARDS patients. SUBJECTS: Six hundred eighty-three patients in SAILS and 511 patients in HARP-2. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We found that 33% of patients in SAILS and HARP-2 were discordant by IL-18 level and LCA class. We further found that IL-18 level was only modestly correlated (0.17-0.47) with cytokines used in the LCA assignment. A substantial subset of individuals classified as hypoinflammatory by LCA (14% of SAILS and 43% of HARP-2) were classified as high risk by elevated IL-18. These individuals were at high risk for mortality in both SAILS (42% 60-d mortality, odds ratio [OR] 3.3; 95% CI, 1.8-6.1; p < 0.001) and HARP-2 (27% 60-d mortality, OR 2.1; 95% CI, 1.2-3.8; p = 0.009). CONCLUSIONS: Plasma IL-18 level provides important additional prognostic information to LCA subphenotypes defined largely by traditional inflammatory biomarkers in two large ARDS cohorts.

Right Ventricular Dysfunction Patterns among Patients with COVID-19 in the Intensive Care Unit: A Retrospective Cohort Analysis.

Rationale: Right ventricular (RV) dysfunction is common among patients hospitalized with coronavirus disease (COVID-19); however, its epidemiology may depend on the echocardiographic parameters used to define it. Objectives: To evaluate the prevalence of abnormalities in three common echocardiographic parameters of RV function among patients with COVID-19 admitted to the intensive care unit (ICU), as well as the effect of RV dilatation on differential parameter abnormality and the association of RV dysfunction with 60-day mortality. Methods: We conducted a retrospective cohort study of ICU patients with COVID-19 between March 4, 2020, and March 4, 2021, who received a transthoracic echocardiogram within 48 hours before to at most 7 days after ICU admission. RV dysfunction and dilatation, respectively, were defined by guideline thresholds for tricuspid annular plane systolic excursion (TAPSE), RV fractional area change, RV free wall longitudinal strain (RVFWS), and RV basal dimension or RV end-diastolic area. Association of RV dysfunction with 60-day mortality was assessed through logistic regression adjusting for age, prior history of congestive heart failure, invasive ventilation at the time of transthoracic echocardiogram, and Acute Physiology and Chronic Health Evaluation II score. Results: A total of 116 patients were included, of whom 69% had RV dysfunction by one or more parameters, and 36.3% of these had RV dilatation. The three most common patterns of RV dysfunction were the presence of three abnormalities, the combination of abnormal RVFWS and TAPSE, and isolated TAPSE abnormality. Patients with RV dilatation had worse RV fractional area change (24% vs. 36%; P = 0.001), worse RVFWS (16.3% vs. 19.1%; P = 0.005), higher RV systolic pressure (45 mm Hg vs. 31 mm Hg; P = 0.001) but similar TAPSE (13 mm vs. 13 mm; P = 0.30) compared with those with normal RV size. After multivariable adjustment, 60-day mortality was significantly associated with RV dysfunction (odds ratio, 2.91; 95% confidence interval, 1.01-9.44), as was the presence of at least two parameter abnormalities. Conclusions: ICU patients with COVID-19 had significant heterogeneity in RV function abnormalities present with different patterns associated with RV dilatation. RV dysfunction by any parameter was associated with increased mortality. Therefore, a multiparameter evaluation may be critical in recognizing RV dysfunction in COVID-19.

The Isolation and In Vitro Differentiation of Primary Fetal Baboon Tracheal Epithelial Cells for the Study of SARS-CoV-2 Host-Virus Interactions.

The mucociliary airway epithelium lines the human airways and is the primary site of host-environmental interactions in the lung. Following virus infection, airway epithelial cells initiate an innate immune response to suppress virus replication. Therefore, defining the virus-host interactions of the mucociliary airway epithelium is critical for understanding the mechanisms that regulate virus infection, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Non-human primates (NHP) are closely related to humans and provide a model to study human disease. However, ethical considerations and high costs can restrict the use of in vivo NHP models. Therefore, there is a need to develop in vitro NHP models of human respiratory virus infection that would allow for rapidly characterizing virus tropism and the suitability of specific NHP species to model human infection. Using the olive baboon (Papio anubis), we have developed methodologies for the isolation, in vitro expansion, cryopreservation, and mucociliary differentiation of primary fetal baboon tracheal epithelial cells (FBTECs). Furthermore, we demonstrate that in vitro differentiated FBTECs are permissive to SARS-CoV-2 infection and produce a potent host innate-immune response. In summary, we have developed an in vitro NHP model that provides a platform for the study of SARS-CoV-2 infection and other human respiratory viruses.

Effect of total cholesterol and statin therapy on mortality in ARDS patients: a secondary analysis of the SAILS and HARP-2 trials.

BACKGROUND: Two acute respiratory distress syndrome (ARDS) trials showed no benefit for statin therapy, though secondary analyses suggest inflammatory subphenotypes may have a differential response to simvastatin. Statin medications decrease cholesterol levels, and low cholesterol has been associated with increased mortality in critical illness. We hypothesized that patients with ARDS and sepsis with low cholesterol could be harmed by statins. METHODS: Secondary analysis of patients with ARDS and sepsis from two multicenter trials. We measured total cholesterol from frozen plasma samples obtained at enrollment in Statins for Acutely Injured Lungs from Sepsis (SAILS) and Simvastatin in the Acute Respiratory Distress Syndrome (HARP-2) trials, which randomized subjects with ARDS to rosuvastatin versus placebo and simvastatin versus placebo, respectively, for up to 28 days. We compared the lowest cholesterol quartile (< 69 mg/dL in SAILS, < 44 mg/dL in HARP-2) versus all other quartiles for association with 60-day mortality and medication effect. Fisher's exact test, logistic regression, and Cox Proportional Hazards were used to assess mortality. RESULTS: There were 678 subjects with cholesterol measured in SAILS and 509 subjects in HARP-2, of whom 384 had sepsis. Median cholesterol at enrollment was 97 mg/dL in both SAILS and HARP-2. Low cholesterol was associated with higher APACHE III and shock prevalence in SAILS, and higher Sequential Organ Failure Assessment score and vasopressor use in HARP-2. Importantly, the effect of statins differed in these trials. In SAILS, patients with low cholesterol who received rosuvastatin were more likely to die (odds ratio (OR) 2.23, 95% confidence interval (95% CI) 1.06-4.77, p = 0.02; interaction p = 0.02). In contrast, in HARP-2, low cholesterol patients had lower mortality if randomized to simvastatin, though this did not reach statistical significance in the smaller cohort (OR 0.44, 95% CI 0.17-1.07, p = 0.06; interaction p = 0.22). CONCLUSIONS: Cholesterol levels are low in two cohorts with sepsis-related ARDS, and those in the lowest cholesterol quartile are sicker. Despite the very low levels of cholesterol, simvastatin therapy seems safe and may reduce mortality in this group, though rosuvastatin was associated with harm.

Endoscopic surveillance alone is feasible and safe in type I gastric neuroendocrine neoplasms less than 10 mm in diameter.

PURPOSE: Type I gastric neuroendocrine neoplasms (g-NENs) have a low risk of metastasis and a generally favourable prognosis. Patients with small type I g-NENs (≤10 mm) frequently require no treatment, whereas those with larger polyps usually undergo resection. We evaluated the safety and outcomes of endoscopic surveillance after no initial treatment in selected patients with type I g-NENs. METHODS: Retrospective analysis of type I g-NEN patients across two European Neuroendocrine Tumour Society Centers of Excellence 2003-2019. RESULTS: Following initial assessment, 87 of 115 patients with type I g-NEN (75 with polyps ≤10 mm) received no initial treatment and underwent endoscopic surveillance. 79/87 (91%) demonstrated no clinically meaningful change in tumour size or grade over a median 62 month follow up. Only two patients developed NEN progression that required a change in management and two other patients developed gastric adenocarcinoma/high grade dysplasia; all four initially had ≥11 mm g-NENs. CONCLUSIONS: Patients with ≤10 mm type I g-NENs were unlikely to develop clinically significant tumour progression and in most cases, resection was not needed. The endoscopic surveillance interval could therefore potentially be safely increased to every 2-3 years in such patients. However, lifelong surveillance is still advocated due to the additional risk of developing gastric adenocarcinoma.

New Developments in Gastric Neuroendocrine Neoplasms.

PURPOSE OF REVIEW: Gastric neuroendocrine neoplasms (g-NENs) are a rare type of stomach cancer. The three main subtypes have different pathogeneses, biological behaviours and clinical characteristics, so they require different management strategies. This article will provide an overview of g-NENs and highlight recent advances in the field. RECENT FINDINGS: Molecular profiling has revealed differences between indolent and aggressive g-NENs, as well as a new somatic mutation responsible for some familial type I g-NENs. Novel biomarkers have been developed which will hopefully improve diagnosis, treatment, risk stratification and follow-up. Patient treatment is also changing, as evidence supports the use of less aggressive options (e.g. endoscopic surveillance or resection) in some patients with more indolent tumours. g-NEN heterogeneity poses challenges in understanding and managing this rare disease. More basic science research is needed to investigate molecular pathogenesis, and future larger clinical studies will hopefully also further improve treatment and patient outcomes.

Effects of Proton Pump Inhibitor Therapy, H. pylori Infection and Gastric Preneoplastic Pathology on Fasting Serum Gastrin Concentrations.

Background: Hypergastrinaemia occasionally indicates the presence of a gastrinoma. However it is much more commonly associated with various benign causes including proton pump inhibitor (PPI) use, Helicobacter pylori infection and/or atrophic gastritis. The extent to which these factors interact to influence fasting serum gastrin concentrations remains incompletely understood. Materials and Methods: Fasting serum gastrin concentrations were measured by radioimmunoassay in 1,400 patients attending for diagnostic oesophagogastro-duodenoscopy. After exclusions, 982 patients were divided into four groups and their results analysed. We compared gastrin concentrations in normal patients (no H. pylori infection, no PPI use and no histological evidence of gastric preneoplasia (n=233)), with those in patients who were taking regular PPIs (H. pylori negative with no gastric preneoplasia (n=301)), patients who had active H. pylori infection but no gastric preneoplasia (n=164) and patients with histologically confirmed gastric preneoplasia (n=284). Results: Median fasting gastrin concentration in the normal group was 20pM and was significantly increased in PPI users (46pM, p<0.0001), patients with active H. pylori infection (27pM, p<0.0001), and patients with antral (25pM, p<0.01) or corpus (48pM, p<0.0001) gastric preneoplasia. PPI use resulted in further significant increases in fasting serum gastrin concentrations in patients who were infected with H. pylori (50pM, n=56) or who had antral gastric preneoplasia (53pM, n=87), but did not significantly alter serum gastrin concentrations in patients with corpus preneoplasia (90pM, n=66). Conclusions: PPI use, H. pylori infection and atrophic gastritis all caused significant elevations of median fasting gastrin concentrations. However, several patients who had potential risk factors for hypergastrinaemia still demonstrated fasting serum gastrin concentrations within the normal range.

The NOTCH3 Downstream Target HEYL Is Required for Efficient Human Airway Basal Cell Differentiation.

Basal cells (BCs) are stem/progenitor cells of the mucociliary airway epithelium, and their differentiation is orchestrated by the NOTCH signaling pathway. NOTCH3 receptor signaling regulates BC to club cell differentiation; however, the downstream responses that regulate this process are unknown. Overexpression of the active NOTCH3 intracellular domain (NICD3) in primary human bronchial epithelial cells (HBECs) on in vitro air-liquid interface culture promoted club cell differentiation. Bulk RNA-seq analysis identified 692 NICD3-responsive genes, including the classical NOTCH target HEYL, which increased in response to NICD3 and positively correlated with SCGB1A1 (club cell marker) expression. siRNA knockdown of HEYL decreased tight junction formation and cell proliferation. Further, HEYL knockdown reduced club, goblet and ciliated cell differentiation. In addition, we observed decreased expression of HEYL in HBECs from donors with chronic obstructive pulmonary disease (COPD) vs. normal donors which correlates with the impaired differentiation capacity of COPD cells. Finally, overexpression of HEYL in COPD HBECs promoted differentiation into club, goblet and ciliated cells, suggesting the impaired capacity of COPD cells to generate a normal airway epithelium is a reversible phenotype that can be regulated by HEYL. Overall, our data identify the NOTCH3 downstream target HEYL as a key regulator of airway epithelial differentiation.

Characterization of the SARS-CoV-2 Host Response in Primary Human Airway Epithelial Cells from Aged Individuals.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), a global pandemic characterized by an exaggerated immune response and respiratory illness. Age (>60 years) is a significant risk factor for developing severe COVID-19. To better understand the host response of the aged airway epithelium to SARS-CoV-2 infection, we performed an in vitro study using primary human bronchial epithelial cells from donors >67 years of age differentiated on an air-liquid interface culture. We demonstrate that SARS-CoV-2 infection leads to early induction of a proinflammatory response and a delayed interferon response. In addition, we observed changes in the genes and pathways associated with cell death and senescence throughout infection. In summary, our study provides new and important insights into the temporal kinetics of the airway epithelial innate immune response to SARS-CoV-2 in older individuals.

Prospective validation of an 11-gene mRNA host response score for mortality risk stratification in the intensive care unit.

Several clinical calculators predict intensive care unit (ICU) mortality, however these are cumbersome and often require 24 h of data to calculate. Retrospective studies have demonstrated the utility of whole blood transcriptomic analysis in predicting mortality. In this study, we tested prospective validation of an 11-gene messenger RNA (mRNA) score in an ICU population. Whole blood mRNA from 70 subjects in the Stanford ICU Biobank with samples collected within 24 h of Emergency Department presentation were used to calculate an 11-gene mRNA score. We found that the 11-gene score was highly associated with 60-day mortality, with an area under the receiver operating characteristic curve of 0.68 in all patients, 0.77 in shock patients, and 0.98 in patients whose primary determinant of prognosis was acute illness. Subjects with the highest quartile of mRNA scores were more likely to die in hospital (40% vs 7%, p < 0.01) and within 60 days (40% vs 15%, p = 0.06). The 11-gene score improved prognostication with a categorical Net Reclassification Improvement index of 0.37 (p = 0.03) and an Integrated Discrimination Improvement index of 0.07 (p = 0.02) when combined with Simplified Acute Physiology Score 3 or Acute Physiology and Chronic Health Evaluation II score. The test performed poorly in the 95 independent samples collected > 24 h after emergency department presentation. Tests will target a 30-min turnaround time, allowing for rapid results early in admission. Moving forward, this test may provide valuable real-time prognostic information to improve triage decisions and allow for enrichment of clinical trials.

Cigarette Smoke Activates NOTCH3 to Promote Goblet Cell Differentiation in Human Airway Epithelial Cells.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the United States and is primarily caused by cigarette smoking. Increased numbers of mucus-producing secretory ("goblet") cells, defined as goblet cell metaplasia or hyperplasia (GCMH), contributes significantly to COPD pathophysiology. The objective of this study was to determine whether NOTCH signaling regulates goblet cell differentiation in response to cigarette smoke. Primary human bronchial epithelial cells (HBECs) from nonsmokers and smokers with COPD were differentiated in vitro on air-liquid interface and exposed to cigarette smoke extract (CSE) for 7 days. NOTCH signaling activity was modulated using 1) the NOTCH/γ-secretase inhibitor dibenzazepine (DBZ), 2) lentiviral overexpression of the NICD3 (NOTCH3-intracellular domain), or 3) NOTCH3-specific siRNA. Cell differentiation and response to CSE were evaluated by quantitative PCR, Western blotting, immunostaining, and RNA sequencing. We found that CSE exposure of nonsmoker airway epithelium induced goblet cell differentiation characteristic of GCMH. Treatment with DBZ suppressed CSE-dependent induction of goblet cell differentiation. Furthermore, CSE induced NOTCH3 activation, as revealed by increased NOTCH3 nuclear localization and elevated NICD3 protein levels. Overexpression of NICD3 increased the expression of goblet cell-associated genes SPDEF and MUC5AC, whereas NOTCH3 knockdown suppressed CSE-mediated induction of SPDEF and MUC5AC. Finally, CSE exposure of COPD airway epithelium induced goblet cell differentiation in a NOTCH3-dependent manner. These results identify NOTCH3 activation as one of the important mechanisms by which cigarette smoke induces goblet cell differentiation, thus providing a novel potential strategy to control GCMH-related pathologies in smokers and patients with COPD.

A "Watch and Wait" Strategy Involving Regular Endoscopic Surveillance Is Safe for Many Patients with Small, Sporadic, Grade 1, Non-Ampullary, Non-Functioning Duodenal Neuroendocrine Tumours.

INTRODUCTION: Duodenal neuroendocrine tumours (d-NETs) are rare but are increasing in incidence. Current ENETS guidelines advocate resection of all localized d-NETs. However, "watch and wait" may be appropriate for some localized, small, grade 1, non-functioning, non-ampullary d-NETs. We evaluated whether patients with such d-NETs who chose "watch and wait" involving regular endoscopic surveillance had equivalent disease-related outcomes to patients undergoing endoscopic or surgical resection. METHODS: Retrospective review of patients with histologically confirmed d-NETs at Liverpool ENETS Centre of Excellence 2007-2020. RESULTS: Sixty-nine patients were diagnosed with d-NET of which 50 were sporadic, non-functioning, non-ampullary tumours. Patient treatment groups were similar in terms of age, gender, and tumour location and grade, but unsurprisingly, larger tumours (median diameter 17 mm [p < 0.0001]) were found in the surgically treated group. Five patients underwent surgical resection with no evidence of tumour recurrence or disease-related death. Twelve patients underwent endoscopic resection (ER), with 1 local recurrence detected during follow-up. Thirty patients (28 with d-NETs ≤10 mm) underwent "watch and wait" with resection only if tumours increased in size. The d-NETs in 28/30 patients remained stable or decreased in size over a median 27 months (IQR: 15-48, R: 3-98). In 7 patients, the d-NET was completely removed by avulsion during diagnostic biopsy and was not seen at subsequent endoscopies. Only 2 patients showed increased d-NET size during surveillance, of whom only one was fit for ER. No NET-related deaths were documented during follow-up. CONCLUSIONS: All of the localized, ≤10 mm, grade 1, non-functioning, non-ampullary d-NETs in this cohort behaved indolently with very low risks of progression and no tumour-related deaths. "Watch and wait," therefore, appears to be a safe alternative management strategy for selected d-NETs.

A generalizable 29-mRNA neural-network classifier for acute bacterial and viral infections.

Improved identification of bacterial and viral infections would reduce morbidity from sepsis, reduce antibiotic overuse, and lower healthcare costs. Here, we develop a generalizable host-gene-expression-based classifier for acute bacterial and viral infections. We use training data (N = 1069) from 18 retrospective transcriptomic studies. Using only 29 preselected host mRNAs, we train a neural-network classifier with a bacterial-vs-other area under the receiver-operating characteristic curve (AUROC) 0.92 (95% CI 0.90-0.93) and a viral-vs-other AUROC 0.92 (95% CI 0.90-0.93). We then apply this classifier, inflammatix-bacterial-viral-noninfected-version 1 (IMX-BVN-1), without retraining, to an independent cohort (N = 163). In this cohort, IMX-BVN-1 AUROCs are: bacterial-vs.-other 0.86 (95% CI 0.77-0.93), and viral-vs.-other 0.85 (95% CI 0.76-0.93). In patients enrolled within 36 h of hospital admission (N = 70), IMX-BVN-1 AUROCs are: bacterial-vs.-other 0.92 (95% CI 0.83-0.99), and viral-vs.-other 0.91 (95% CI 0.82-0.98). With further study, IMX-BVN-1 could provide a tool for assessing patients with suspected infection and sepsis at hospital admission.

Netazepide Inhibits Expression of Pappalysin 2 in Type 1 Gastric Neuroendocrine Tumors.

BACKGROUND & AIMS: In patients with autoimmune atrophic gastritis and achlorhydria, hypergastrinemia is associated with the development of type 1 gastric neuroendocrine tumors (gNETs). Twelve months of treatment with netazepide (YF476), an antagonist of the cholecystokinin B receptor (CCKBR or CCK2R), eradicated some type 1 gNETs in patients. We investigated the mechanisms by which netazepide induced gNET regression using gene expression profiling. METHODS: We obtained serum samples and gastric corpus biopsy specimens from 8 patients with hypergastrinemia and type 1 gNETs enrolled in a phase 2 trial of netazepide. Control samples were obtained from 10 patients without gastric cancer. We used amplified and biotinylated sense-strand DNA targets from total RNA and Affymetrix (Thermofisher Scientific, UK) Human Gene 2.0 ST microarrays to identify differentially expressed genes in stomach tissues from patients with type 1 gNETs before, during, and after netazepide treatment. Findings were validated in a human AGSGR gastric adenocarcinoma cell line that stably expresses human CCK2R, primary mouse gastroids, transgenic hypergastrinemic INS-GAS mice, and patient samples. RESULTS: Levels of pappalysin 2 (PAPPA2) messenger RNA were reduced significantly in gNET tissues from patients receiving netazepide therapy compared with tissues collected before therapy. PAPPA2 is a metalloproteinase that increases the bioavailability of insulin-like growth factor (IGF) by cleaving IGF binding proteins (IGFBPs). PAPPA2 expression was increased in the gastric corpus of patients with type 1 gNETs, and immunohistochemistry showed localization in the same vicinity as CCK2R-expressing enterochromaffin-like cells. Up-regulation of PAPPA2 also was found in the stomachs of INS-GAS mice. Gastrin increased PAPPA2 expression with time and in a dose-dependent manner in gastric AGSGR cells and mouse gastroids by activating CCK2R. Knockdown of PAPPA2 in AGSGR cells with small interfering RNAs significantly decreased their migratory response and tissue remodeling in response to gastrin. Gastrin altered the expression and cleavage of IGFBP3 and IGFBP5. CONCLUSIONS: In an analysis of human gNETS and mice, we found that gastrin up-regulates the expression of gastric PAPPA2. Increased PAPPA2 alters IGF bioavailability, cell migration, and tissue remodeling, which are involved in type 1 gNET development. These effects are inhibited by netazepide.

Matrix metalloproteinase (MMP)-7 in Barrett's esophagus and esophageal adenocarcinoma: expression, metabolism, and functional significance.

Matrix metalloproteinase (MMP)-7, unlike many MMPs, is typically expressed in epithelial cells. It has been linked to epithelial responses to infection, injury, and tissue remodeling including the progression of a number of cancers. We have now examined how MMP-7 expression changes in the progression to esophageal adenocarcinoma (EAC), and have studied mechanisms regulating its expression and its functional significance. Immunohistochemistry revealed that MMP-7 was weakly expressed in normal squamous epithelium adjacent to EAC but was abundant in epithelial cells in both preneoplastic lesions of Barrett's esophagus and EAC particularly at the invasive front. In the stroma, putative myofibroblasts expressing MMP-7 were abundant at the invasive front but were scarce or absent in adjacent tissue. Western blot and ELISA revealed high constitutive secretion of proMMP-7 in an EAC cell line (OE33) that was inhibited by the phosphatidylinositol (PI) 3-kinase inhibitor LY294002 but not by inhibitors of protein kinase C, or MAP kinase activation. There was detectable proMMP-7 in cultured esophageal myofibroblasts but it was undetectable in media. Possible metabolism of MMP-7 by myofibroblasts studied by proteomic analysis indicated degradation via extensive endopeptidase, followed by amino- and carboxpeptidase, cleavages. Myofibroblasts exhibited increased migration and invasion in response to conditioned media from OE33 cells that was reduced by MMP-7 knockdown and immunoneutralization. Thus, MMP-7 expression increases at the invasive front in EAC which may be partly attributable to activation of PI 3-kinase. Secreted MMP-7 may modify the tumor microenvironment by stimulating stromal cell migration and invasion.

Comparison of the human gastric microbiota in hypochlorhydric states arising as a result of Helicobacter pylori-induced atrophic gastritis, autoimmune atrophic gastritis and proton pump inhibitor use.

Several conditions associated with reduced gastric acid secretion confer an altered risk of developing a gastric malignancy. Helicobacter pylori-induced atrophic gastritis predisposes to gastric adenocarcinoma, autoimmune atrophic gastritis is a precursor of type I gastric neuroendocrine tumours, whereas proton pump inhibitor (PPI) use does not affect stomach cancer risk. We hypothesised that each of these conditions was associated with specific alterations in the gastric microbiota and that this influenced subsequent tumour risk. 95 patients (in groups representing normal stomach, PPI treated, H. pylori gastritis, H. pylori-induced atrophic gastritis and autoimmune atrophic gastritis) were selected from a cohort of 1400. RNA extracted from gastric corpus biopsies was analysed using 16S rRNA sequencing (MiSeq). Samples from normal stomachs and patients treated with PPIs demonstrated similarly high microbial diversity. Patients with autoimmune atrophic gastritis also exhibited relatively high microbial diversity, but with samples dominated by Streptococcus. H. pylori colonisation was associated with decreased microbial diversity and reduced complexity of co-occurrence networks. H. pylori-induced atrophic gastritis resulted in lower bacterial abundances and diversity, whereas autoimmune atrophic gastritis resulted in greater bacterial abundance and equally high diversity compared to normal stomachs. Pathway analysis suggested that glucose-6-phospahte1-dehydrogenase and D-lactate dehydrogenase were over represented in H. pylori-induced atrophic gastritis versus autoimmune atrophic gastritis, and that both these groups showed increases in fumarate reductase. Autoimmune and H. pylori-induced atrophic gastritis were associated with different gastric microbial profiles. PPI treated patients showed relatively few alterations in the gastric microbiota compared to healthy subjects.

Netazepide, a gastrin/cholecystokinin-2 receptor antagonist, can eradicate gastric neuroendocrine tumours in patients with autoimmune chronic atrophic gastritis.

AIMS: Netazepide, a gastrin/cholecystokinin 2 receptor antagonist, once daily for 12 weeks reduced the number of tumours and size of the largest one in 16 patients with autoimmune chronic atrophic gastritis (CAG), achlorhydria, hypergastrinaemia and multiple gastric neuroendocrine tumours (type 1 gastric NETs), and normalized circulating chromogranin A (CgA) produced by enterochromaffin-like cells, the source of the tumours. The aim was to assess whether longer-term netazepide treatment can eradicate type 1 gastric NETs. METHODS: After a mean 14 months off netazepide, 13 of the 16 patients took it for another 52 weeks. Assessments were: gastroscopy; gene-transcript expression in corpus biopsies using quantitative polymerase chain reaction; blood CgA and gastrin concentrations; and safety assessments. RESULTS: While off-treatment, the number of tumours, the size of the largest one, and CgA all increased again. Netazepide for 52 weeks: cleared all tumours in 5 patients; cleared all but one tumour in one patient; reduced the number of tumours and size of the largest one in the other patients; normalized CgA in all patients; and reduced mRNA abundances of CgA and histidine decarboxylase in biopsies. Gastrin did not increase further, confirming that the patients had achlorhydria. Netazepide was safe and well tolerated. CONCLUSIONS: A gastrin/cholecystokinin 2 receptor antagonist is a potential medical and targeted treatment for type 1 gastric NETs, and an alternative to regular gastroscopy or surgery. Treatment should be continuous because the tumours will regrow if it is stopped. Progress can be monitored by CgA in blood or biomarkers in mucosal biopsies.