Broad transcriptional response of the human esophageal epithelium to proton pump inhibitors.

BACKGROUND: Proton pump inhibitors (PPIs) have been recognized as a primary treatment of eosinophilic esophagitis (EoE), an allergic inflammatory disease of the esophageal mucosa. The mechanisms underlying esophageal epithelial responses to PPIs remain poorly understood. OBJECTIVE: We hypothesized that PPIs can counteract IL-13-mediated esophageal epithelial responses that are germane for EoE pathogenesis. METHODS: Transcriptional responses of human esophageal cells to IL-13 and the PPIs omeprazole and esomeprazole were assessed by RT-PCR and RNA sequencing. Cytokine secretion was measured by multiplex analysis and ELISA. RESULTS: Human esophageal epithelial cells robustly responded to PPI stimulation by inducing a set of 479 core genes common between omeprazole and esomeprazole treatments. The transcriptional response to PPIs was partially mediated through the aryl hydrocarbon receptor signaling pathway, as the aryl hydrocarbon receptor antagonist GNF-351 modified approximately 200 genes, particularly those enriched in metabolic processes and regulation of cell death. PPI treatment reversed approximately 20% of the IL-13 transcriptome. Functional analysis of the PPI-responsive, upregulated genes revealed enrichment in metabolic and oxidation processes, and the unfolded protein response. In contrast, downregulated genes were overrepresented in functional terms related to cell division and cytoskeletal organization, which were also enriched for the genes in the EoE transcriptome reversed by PPIs. Furthermore, PPI treatment decreased the IL-13-induced proliferative response of esophageal epithelial cells. CONCLUSIONS: These results demonstrate broad effects of PPIs on esophageal epithelium, including their ability to curtail transcriptomic processes involved in cellular proliferation and IL-13-induced responses, and they highlight the importance of AHR signaling in mediating these responses.

Notch Signaling Mediates Differentiation in Barrett's Esophagus and Promotes Progression to Adenocarcinoma.

BACKGROUND & AIMS: Studies are needed to determine the mechanism by which Barrett's esophagus (BE) progresses to esophageal adenocarcinoma (EAC). Notch signaling maintains stem cells in the gastrointestinal tract and is dysregulated during carcinogenesis. We explored the relationship between Notch signaling and goblet cell maturation, a feature of BE, during EAC pathogenesis. METHODS: We measured goblet cell density and levels of Notch messenger RNAs in BE tissues from 164 patients, with and without dysplasia or EAC, enrolled in a multicenter study. We analyzed the effects of conditional expression of an activated form of NOTCH2 (pL2.Lgr5.N2IC), conditional deletion of NOTCH2 (pL2.Lgr5.N2fl/fl), or loss of nuclear factor κB (NF-κB) (pL2.Lgr5.p65fl/fl), in Lgr5+ (progenitor) cells in L2-IL1B mice (which overexpress interleukin 1 beta in esophagus and squamous forestomach and are used as a model of BE). We collected esophageal and stomach tissues and performed histology, immunohistochemistry, flow cytometry, transcriptome, and real-time polymerase chain reaction analyses. Cardia and forestomach tissues from mice were cultured as organoids and incubated with inhibitors of Notch or NF-kB. RESULTS: Progression of BE to EAC was associated with a significant reduction in goblet cell density comparing nondysplastic regions of tissues from patients; there was an inverse correlation between goblet cell density and levels of NOTCH3 and JAG2 messenger RNA. In mice, expression of the activated intracellular form of NOTCH2 in Lgr5+ cells reduced goblet-like cell maturation, increased crypt fission, and accelerated the development of tumors in the squamocolumnar junction. Mice with deletion of NOTCH2 from Lgr5+ cells had increased maturation of goblet-like cells, reduced crypt fission, and developed fewer tumors. Esophageal tissues from in pL2.Lgr5.N2IC mice had increased levels of RelA (which encodes the p65 unit of NF-κB) compared to tissues from L2-IL1B mice, and we found evidence of increased NF-κB activity in Lgr5+ cells. Esophageal tissues from pL2.Lgr5.p65fl/fl mice had lower inflammation and metaplasia scores than pL2.Lgr5.N2IC mice. In organoids derived from pL2-IL1B mice, the NF-κB inhibitor JSH-23 reduced cell survival and proliferation. CONCLUSIONS: Notch signaling contributes to activation of NF-κB and regulates differentiation of gastric cardia progenitor cells in a mouse model of BE. In human esophageal tissues, progression of BE to EAC was associated with reduced goblet cell density and increased levels of Notch expression. Strategies to block this pathway might be developed to prevent EAC in patients with BE.

Pathogenesis and Cells of Origin of Barrett's Esophagus.

In patients with Barrett's esophagus (BE), metaplastic columnar mucosa containing epithelial cells with gastric and intestinal features replaces esophageal squamous mucosa damaged by gastroesophageal reflux disease. This condition is estimated to affect 5.6% of adults in the United States, and is a major risk factor for esophageal adenocarcinoma. Despite the prevalence and importance of BE, its pathogenesis is incompletely understood and there are disagreements over the cells of origin. We review mechanisms of BE pathogenesis, including transdifferentiation and transcommitment, and discuss potential cells of origin, including basal cells of the squamous epithelium, cells of esophageal submucosal glands and their ducts, cells of the proximal stomach, and specialized populations of cells at the esophagogastric junction (residual embryonic cells and transitional basal cells). We discuss the concept of metaplasia as a wound-healing response, and how cardiac mucosa might be the precursor of the intestinal metaplasia of BE. Finally, we discuss shortcomings in current diagnostic criteria for BE that have important clinical implications.

Characterizing caspase-1 involvement during esophageal disease progression.

Barrett's esophagus (BE) is an inflammatory condition and a neoplastic precursor to esophageal adenocarcinoma (EAC). Inflammasome signaling, which contributes to acute and chronic inflammation, results in caspase-1 activation leading to the secretion of IL-1ß and IL-18, and inflammatory cell death (pyroptosis). This study aimed to characterize caspase-1 expression, and its functional importance, during disease progression to BE and EAC. Three models of disease progression (Normal-BE-EAC) were employed to profile caspase-1 expression: (1) a human esophageal cell line model; (2) a murine model of BE; and (3) resected tissue from BE-associated EAC patients. BE patient biopsies and murine BE organoids were cultured ex vivo in the presence of a caspase-1 inhibitor, to determine the importance of caspase-1 for inflammatory cytokine and chemokine secretion.Epithelial caspase-1 expression levels were significantly enhanced in BE (p < 0.01). In contrast, stromal caspase-1 levels correlated with histological inflammation scores during disease progression (p < 0.05). Elevated secretion of IL-1ß from BE explanted tissue, compared to adjacent normal tissue (p < 0.01), confirmed enhanced activity of caspase-1 in BE tissue. Caspase-1 inhibition in LPS-stimulated murine BE organoids caused a significant reduction in IL-1ß (p < 0.01) and CXCL1 (p < 0.05) secretion, confirming the importance of caspase-1 in the production of cytokines and chemokines associated with disease progression from BE to EAC. Targeting caspase-1 activity in BE patients should therefore be tested as a novel strategy to prevent inflammatory complications associated with disease progression.

Immunoglobulin G4-Related Disease Manifesting as Isolated, Typical, and Nontypical Gastroesophageal Lesion: A Research of Literature Review.

BACKGROUND: Immunoglobulin G4-related disease (IgG4-RD) is an autoimmune inflammatory and fibrotic condition. The disease is characterized by tissue infiltration with dense lymphoplasmacytes and IgG4-positive plasma cells. SUMMARY: The aim of this study was to provide gastroenterologists with novel insights into evaluating the gastroesophageal involvement with IgG4-RD or mimickers of this condition and to give special attention to clinicopathological features. A literature review was performed using the PubMed database. A total of 39 studies presenting cases in the form of isolated, typical, and nontypical gastroesophageal involvement with IgG4-RD published between 2010 and 2018 were included. These studies were thoroughly reviewed for symptoms, lesion location, lesion type, lesion size, immune-histopathology, associated diseases, treatment, and follow-up. Of the 39 studies reviewed, 9 were esophageal IgG4-RD lesions, isolated esophageal IgG4-RD 66.66% (6/9), a typical form of esophageal IgG4-RD 11.11% (1/9), and nontypical form esophageal IgG4-RD 22.22% (2/9). The 30 gastric IgG4-RD that include isolated gastric IgG4-RD 46.66% (14/30), typical gastric IgG4-RD 40% (12/30), and nontypical gastric IgG4-RD 13.33% (4/30). The majority of lesions were inflammatory tumors, ulceration, nodular lesions, chronic gastritis, and malignant lesions. Key Messages: IgG4-RD may be manifested by isolated, typical and nontypical forms of gastroesophageal lesions and should be taken into consideration in the differential diagnosis. Corticosteroids may be the sole diagnostic treatment for this condition.

One-Hour Esophageal String Test: A Nonendoscopic Minimally Invasive Test That Accurately Detects Disease Activity in Eosinophilic Esophagitis.

OBJECTIVES: Eosinophilic esophagitis (EoE), a chronic food allergic disease, lacks sensitive and specific peripheral biomarkers. We hypothesized that levels of EoE-related biomarkers captured using a 1-hour minimally invasive Esophageal String Test (EST) would correlate with mucosal eosinophil counts and tissue concentrations of these same biomarkers. We aimed to determine whether a 1-hour EST accurately distinguishes active from inactive EoE or a normal esophagus. METHODS: In a prospective, multisite study, children and adults (ages 7-55 years) undergoing a clinically indicated esophagogastroduodenoscopy performed an EST with an esophageal dwell time of 1 hour. Subjects were divided into 3 groups: active EoE, inactive EoE, and normal esophageal mucosa. Eosinophil-associated protein levels were compared between EST effluents and esophageal biopsy extracts. Statistical modeling was performed to select biomarkers that best correlated with and predicted eosinophilic inflammation. RESULTS: One hundred thirty-four subjects (74 children, 60 adults) with active EoE (n = 62), inactive EoE (n = 37), and patient controls with a normal esophagus (n = 35) completed the study. EST-captured eosinophil-associated biomarkers correlated significantly with peak eosinophils/high-power field, endoscopic visual scoring, and the same proteins extracted from mucosal biopsies. Statistical modeling, using combined eotaxin-3 and major basic protein-1 concentrations, led to the development of EoE scores that distinguished subjects with active EoE from inactive EoE or normal esophagi. Eighty-seven percent of children, 95% of parents, and 92% of adults preferred the EST over endoscopy if it provided similar information. DISCUSSION: The 1-hour EST accurately distinguishes active from inactive EoE in children and adults and may facilitate monitoring of disease activity in a safe and minimally invasive fashion.

Can Eosinophilic Esophagitis Cause Achalasia and Other Esophageal Motility Disorders?

Eosinophilic esophagitis (EoE), a disorder identified by its esophageal mucosal features, often is associated with esophageal motility abnormalities, which are manifestations of esophageal muscle dysfunction. Those motility abnormalities sometimes normalize with treatments that reduce esophageal eosinophilia, suggesting that eosinophils can cause reversible esophageal motility disturbances, perhaps by releasing myoactive and neuroactive eosinophil products. Although achalasia uncommonly is associated with EoE as currently defined, most achalasia patients have evidence of an abnormal accumulation of eosinophils and/or their degranulation products in the esophageal muscularis propria, a location inaccessible to routine endoscopic evaluation. Achalasia is an idiopathic condition resulting from destruction of neurons in the myenteric plexus of the esophagus, and degranulating eosinophils release toxic proteins capable of destroying those neurons, thereby causing the irreversible motility abnormalities of achalasia. This report reviews data on the association of esophageal eosinophilia with achalasia and other esophageal motility abnormalities. Based on this review, we propose that EoE, like eosinophilic gastroenteritis, might have mucosal-predominant and muscle-predominant forms with different clinical manifestations. A muscle-predominant form of EoE could underlie a variety of reversible and irreversible esophageal motility disorders, including achalasia. The concept that esophageal motility abnormalities might develop from a muscle-predominant form of EoE warrants serious consideration and further investigation.

Exposure to bile acids alters the intracellular location and function of MnSOD in Barrett's esophagus.

BACKGROUND: Oxidative stress secondary to bile-acid exposure has been associated with metaplastic degeneration of normal esophageal mucosa into Barrett's esophagus (BE) cells and eventually esophageal adenocarcinoma. We previously reported that the macromolecular response of BE cells to this stress was largely regulated by the expression of manganese-dependent mitochondrial superoxide dismutase (MnSOD). As the mitochondrion plays a vital role in MnSOD activation, this study sought to determine the location and activity of MnSOD within BE cells after exposure to oxidative stress. METHODS: A human BE cell line, BAR-T cell, was exposed 0.4 mM concentrations of taurocholic acid (Tau) or a 0.4 mM 1:1 mixture of bile salts for 4 h. Cell viability was performed with 3-(4, 5-dimthyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays. Proteins were extracted and separated into mitochondrial, nuclear, and cytoplasmic fractions followed by analysis by a western blot and enzymatic activities. RESULTS: BAR-T cell showed resistance to the bile-salt insults. Expression of MnSOD was significantly increased in the cells exposed to a mixture of bile acids and Tau versus control. Mitochondria MnSOD is abundant and highly active. Nuclear fraction displayed presence of both MnSOD and Cu/zinc superoxide dismutase secondary to bile-acid exposure; however, the MnSOD was inactive in nuclear fraction. CONCLUSIONS: This is the first study to specifically evaluate cellular fraction MnSOD expression, increased in BE cells in response to the oxidative stress of bile exposure. Mitochondrial MnSOD contributes to resistance of BAR-T cells to the bile-salt insults. Further investigation is required to determine the potential correlation between bile exposure and BE to adenocarcinoma progression via MnSOD-mediated cell signaling.

Mucosal Two-Step Pathogenesis in Gastroesophageal Reflux Disease: Repeated Weakly Acidic Stimulation and Activation of Protease-Activated Receptor-2 on Mucosal Interleukin-8 Secretion.

BACKGROUND: Activation of protease-activated receptor-2 (PAR2) is involved in the mucosal immune pathogenesis of gastroesophageal reflux disease (GERD) that is characterized by proinflammatory cytokines such as interleukin-8 (IL-8). PAR2 activation on epithelial cells induces epithelial IL-8 secretion and initiates mucosal inflammation. METHODS: A human primary esophageal epithelial cell model was established to investigate the effects of repeated stimulation with weakly acidic solutions and subsequent PAR2 activation. After creating a monolayer, cells were incubated under weakly acidic conditions for 7 h followed by 17 h at pH 7.4. This short-term exposure was repeated once. After weakly acidic stimulation, PAR2 activation was achieved by a synthetic agonist at pH 7.4. RESULTS: After repeated weakly acidic incubation, PAR2 transcript levels were 3.6-fold upregulated (p = 0.001) and IL-8 transcripts were 2.4-fold enhanced (p = 0.034) compared to nonstimulated controls, while IL-8 protein in the cell pellet and supernatant was not increased. Only the additional PAR2 activation upon pH stimulation led to increased IL-8 secretion into the supernatant. CONCLUSIONS: We propose a 2-step mechanism in which repeated weakly acidic exposure leads to the upregulation of epithelial PAR2 expression. The subsequent activation of upregulated PAR2 contributes to the initiation of mucosal inflammation, which underlies the important role of esophageal epithelium in GERD pathogenesis.

Phosphorylation of Smad2/3 at the specific linker threonine residue indicates slow-cycling esophageal stem-like cells before re-entry to the cell cycle.

UNLABELLED: The stem cell compartment in the esophageal epithelium is possibly located in the basal layer. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in the epithelial cells of murine stomach and intestine, and have suggested that these cells are epithelial stem cells. In this study, we explore whether pSmad2/3L-Thr could serve as a biomarker for esophageal stem cells. We examined esophageal tissues from normal C57BL/6 mice and those with esophagitis. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, CDK4, p63, or CK14 was performed. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. We used the 5-bromo-2-deoxyuridine (BrdU) labeling assay to examine label retention of pSmad2/3L-Thr immunostaining-positive cells. We collected specimens 5, 10, 15 and 20 days after repeated BrdU administrations and observed double immunofluorescent staining of pSmad2/3L-Thr with BrdU. In the esophagus, pSmad2/3L-Thr immunostaining-positive cells were detected in the basal layer. These cells were detected between Ki67 immunostaining-positive cells, but they were not co-localized with Ki67. pSmad2/3L-Thr immunostaining-positive cells showed co-localization with CDK4, p63, and CK14. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features. Until 20 days follow-up period, pSmad2/3L-Thr immunostaining-positive cells were co-localized with BrdU. pSmad2/3L-Thr immunostaining-positive cells significantly increased in the regeneration phase of esophagitis mucosae, as compared with control mice (esophagitis vs. CONTROL: 6.889 ± 0.676/cm vs. 4.293 ± 0.659/cm; P < 0.001). We have identified significant expression of pSmad2/3L-Thr in the specific epithelial cells of murine esophagi. We suggest that these cells are slow-cycling epithelial stem-like cells before re-entry to the cell cycle.

Defining the contribution of Troy-positive progenitor cells to the mouse esophageal epithelium.

Progenitor cells adapt their behavior in response to tissue demands. However, the molecular mechanisms controlling esophageal progenitor decisions remain largely unknown. Here, we demonstrate the presence of a Troy (Tnfrsf19)-expressing progenitor subpopulation localized to defined regions along the mouse esophageal axis. Lineage tracing and mathematical modeling demonstrate that Troy-positive progenitor cells are prone to undergoing symmetrical fate choices and contribute to esophageal tissue homeostasis long term. Functionally, TROY inhibits progenitor proliferation and enables commitment to differentiation without affecting fate symmetry. Whereas Troy expression is stable during esophageal homeostasis, progenitor cells downregulate Troy in response to tissue stress, enabling proliferative expansion of basal cells refractory to differentiation and reestablishment of tissue homeostasis. Our results demonstrate functional, spatially restricted progenitor heterogeneity in the esophageal epithelium and identify how dynamic regulation of Troy coordinates tissue generation.

Autophagy Contributes to Homeostasis in Esophageal Epithelium Where High Autophagic Vesicle Level Marks Basal Cells With Limited Proliferation and Enhanced Self-Renewal Potential.

BACKGROUND & AIMS: Autophagy plays roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelial homeostasis. METHODS: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histologic and biochemical analyses. We fluorescence-activated cell sorted esophageal basal cells based on fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID and then subjected these cells to transmission electron microscopy, image flow cytometry, three-dimensional organoid assays, RNA sequencing, and cell cycle analysis. Three-dimensional organoids were subjected to passaging, single-cell RNA sequencing, cell cycle analysis, and immunostaining. RESULTS: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells under homeostatic conditions and also was associated with significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Esophageal basal cells with high AV level (Cyto-IDHigh) displayed limited organoid formation capability on initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-IDLow). RNA sequencing suggested increased autophagy in Cyto-IDHigh esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. Single-cell RNA sequencing of three-dimensional organoids generated by Cyto-IDLow and Cyto-IDHigh cells identified expansion of 3 cell populations and enrichment of G2/M-associated genes in the Cyto-IDHigh group. Ki67 expression was also increased in organoids generated by Cyto-IDHigh cells, including in basal cells localized beyond the outermost cell layer. CONCLUSIONS: Autophagy contributes to maintenance of the esophageal proliferation-differentiation gradient. Esophageal basal cells with high AV level exhibit limited proliferation and generate three-dimensional organoids with enhanced self-renewal capacity.

Perfusion decellularization of porcine esophagus: Study of two processing factors affecting the folded mucosal structure of the esophageal scaffold.

Acellular scaffolds from decellularized donor organs are showing promising clinical results in tissue and organ repair and regeneration. A successful decellularization process is determined by (a) its capability to decellularize complete organs of large animals, (b) retention of the extracellular matrix (ECM) structures and morphologies, and (c) minimal loss of ECM proteins. In this study, porcine esophagi were perfused in full thickness with 0.25% w/v sodium dodecyl sulfate at perfusion rates 0.1-0.2 ml/min for up to 5 days. Decellularized tissues were characterized for their residual DNA, histological staining for their matrix structures, immunohistochemical staining for collagen type IV and laminin, and scanning electron microscopy for structural integrity. Our results showed that full thickness esophageal tissues treated using the horizontal perfusion setup were decellularized with good structural and biochemical integrity in the ECM. Residual DNA content in decellularized tissues was found to be 36 ± 12 ng/mg of tissues (n = 6) which was significantly lower than that of native tissues (p = .00022). Our study showed that the organ must be decellularized in full thickness and perfusion pressure must be controlled to minimize radial expansion. These factors were found to be critical in preserving the folded mucosa in the decellularized tissues.

Morphologic changes in the cytoskeleton and adhesion apparatus during the conversion from pseudostratified single columnar to stratified squamous epithelium in the developing mouse esophagus.

The apico-basal (AB) polarity of epithelial cells is maintained by organized arrays of the cytoskeleton and adhesion apparatus. We previously reported that mouse embryonic esophageal epithelium exhibits interkinetic nuclear migration (INM), an AB-polarity-based regulatory mechanism of stem-cell proliferation, and suggested that the pseudostratified single columnar epithelium, a hallmark of INM, is converted to stratified squamous epithelium via rearrangement of the cytoskeleton and cell-adhesion apparatus. Here, we chronologically examined morphological changes in the cytoskeleton and adhesion apparatus in the mouse esophageal epithelium at embryonic day (E) 11.5, E13.5, E14.5, and E15.5, during which epithelial conversion has been suggested to occur. We used phalloidin to examine the apical terminal web (ATW), immunofluorescent anti-zonula occludens protein (ZO-1) antibody to reveal ZO-1, and anti-gamma tubulin antibody to detect primary cilia (PC). At E11.5, a thick ATW, apically oriented ZO-1 and apical PC were observed, indicating a pseudostratified single columnar structure. At E13.5 and E14.5, the phalloidin-staining, ZO-1, and PC distribution patterns were not apically localized, and the epithelial cells appeared to have lost the AB polarity, suggesting conversion of the epithelial structure and cessation of INM. At E15.5, light and transmission electron microscope observations revealed the ATW, ZO-1, PC, and tight junction which were localized into two-1ayers: the apical and subapical layers of the epithelium. These findings suggest that dynamic remodeling of the cytoskeleton and adhesion apparatus is involved in the conversion from pseudostratified single columnar to stratified squamous morphology and is closely related with temporal perturbation of the AB-polarity and cessation of INM.

Neutrophils, eosinophils, and intraepithelial lymphocytes in the squamous esophagus in subjects with and without gastroesophageal reflux symptoms.

Whilst intraepithelial lymphocytes (IELs) are considered normal within the distal esophageal mucosa, they have an increasingly recognised role in the pathogenesis of reflux esophagitis, and IEL quantification establishes the diagnosis of lymphocytic esophagitis. Knowledge regarding the upper limit of a normal IEL count in health is lacking. We studied 117 non-healthcare seeking adult volunteers from a random community sample (the Kalixanda study) with esophageal biopsies 2 cm above the gastroesophageal junction. Subjects were divided into four groups based on the presence or absence of gastro-esophageal reflux symptoms and/or esophagitis on endoscopy. Asymptomatic subjects with no endoscopic esophagitis were selected as controls, and the cell counts in this group were used to define the upper limit of normal of IELs, eosinophils and neutrophils. The entire sample was used to identify independent predictors of increased cellular counts by logistic regression analysis. None of the healthy controls had an IEL count of more than three per five high power fields (HPF), and therefore this was considered as the upper limit of normal; no controls had eosinophils or neutrophils in esophageal biopsies. Independent predictors of an elevated IEL count were spongiosis on histology (OR 11.17, 95% CI 3.32-37.58, P < 0.01) and current smoking (OR 4.84, 95% CI 1.13-2.71, P = 0.03). A receiver operating characteristics analysis concluded that a threshold of 3 IELs/5HPFs performs best in predicting reflux symptoms when a normal esophageal mucosa is visualized on endoscopy (sensitivity = 100.0%, specificity = 35.2%). The healthy esophageal mucosa does not contain more than three IELs per five HPF in the distal esophagus.

Desmoplakin and periplakin genetically and functionally contribute to eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic allergic inflammatory disease with a complex underlying genetic etiology. Herein, we conduct whole-exome sequencing of a multigeneration EoE pedigree (discovery set) and 61 additional multiplex families with EoE (replication set). A series of rare, heterozygous, missense variants are identified in the genes encoding the desmosome-associated proteins DSP and PPL in 21% of the multiplex families. Esophageal biopsies from patients with these variants retain dilated intercellular spaces and decrease DSP and PPL expression even during disease remission. These variants affect barrier integrity, cell motility and RhoGTPase activity in esophageal epithelial cells and have increased susceptibility to calpain-14-mediated degradation. An acquired loss of esophageal DSP and PPL is present in non-familial EoE. Taken together, herein, we uncover a pathogenic role for desmosomal dysfunction in EoE, providing a deeper mechanistic understanding of tissue-specific allergic responses.

Isolation of primary immune cells from fibrotic skin, esophageal, and gut tissue.

Understanding the interplay between immune and structural cells is important for studying fibrosis and inflammation; however, primary immune cell isolation from organs that are typically enriched in stromal cells, like the lung, esophagus, or gut, proves to be an ongoing challenge. In fibrotic conditions, this challenge becomes even greater as infiltrating cells become trapped in the robust extracellular matrix (ECM). This protocol details a method to isolate cells at high yield from stroma-rich organs that can be used for further analyses via flow cytometry, stimulation, or culturing. Validation of this method is confirmed by flow cytometry data assessing immune cell populations of interest. This protocol can be completed in approximately 5-6 h.

Deletion of TRPV4 enhances in vitro wound healing of murine esophageal keratinocytes.

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel that is widely expressed in different body tissues and plays several physiological roles. This channel is highly expressed in esophageal keratinocytes where its activation mediates ATP release. However, whether TRPV4 has a role in wound healing of esophageal keratinocytes is unclear. In this study, we demonstrated that both cell migration and proliferation were slower in wild-type esophageal keratinocytes compared to cells having TRPV4 knockout. Our results suggest that TRPV4-mediated release of ATP from esophageal keratinocytes contributes to a decrease in the rate of in vitro wound healing via the ATP degradation product adenosine, which acts on A2B adenosine receptors.

Novel Insights Into Tissue-Specific Biochemical Alterations in Pediatric Eosinophilic Esophagitis Using Raman Spectroscopy.

INTRODUCTION: Elucidating esophageal biochemical composition in eosinophilic esophagitis (EoE) can offer novel insights into its pathogenesis, which remains unclear. Using Raman spectroscopy, we profiled and compared the biochemical composition of esophageal samples obtained from children with active (aEoE) and inactive EoE (iEoE) with non-EoE controls, examined the relationship between spectral markers and validated EoE activity indices. METHODS: In vitro Raman spectra from children with aEoE (n = 8; spectra = 51) and iEoE (n = 6; spectra = 48) and from non-EoE controls (n = 10; spectra = 75) were acquired. Mann-Whitney test was used to assess the differences in their Raman intensities (median [interquartile range]) and identify spectral markers. Spearman correlation was used to evaluate the relationship between spectral markers and endoscopic and histologic activity indices. RESULTS: Raman peaks attributable to glycogen content (936/1,449 cm) was lower in children with aEoE (0.20 [0.18-0.21]) compared with that in non-EoE controls (0.24 [0.23-0.29]). Raman intensity of proteins (1,660/1,209 cm) was higher in children with aEoE compared with that in non-EoE controls (3.20 [3.07-3.50] vs 2.91 [2.59-3.05]; P = 0.01), whereas that of lipids (1,301/1,260 cm) was higher in children with iEoE (1.56 [1.49-1.63]) compared with children with aEoE (1.40 [1.30-1.48]; P = 0.02). Raman peaks attributable to glycogen and lipid inversely correlated with eosinophilic inflammation and basal zone hyperplasia. Raman mapping substantiated our findings. DISCUSSION: This is the first study to identify spectral traits of the esophageal samples related to EoE activity and tissue pathology and to profile tissue-level biochemical composition associated with pediatric EoE. Future research to determine the role of these biochemical alterations in development and clinical course of EoE can advance our understanding of EoE pathobiology.

High Content Imaging of Barrett's-Associated High-Grade Dysplasia Cells After siRNA Library Screening Reveals Acid-Responsive Regulators of Cellular Transitions.

BACKGROUND & AIMS: Esophageal adenocarcinoma (EAC) develops from within Barrett's esophagus (BE) concomitant with gastroesophageal reflux disease (GERD). Wound healing processes and cellular transitions, such as epithelial-mesenchymal transitions, may contribute to the development of BE and the eventual migratory escape of metastatic cancer cells. Herein, we attempt to identify the genes underlying esophageal cellular transitions and their potential regulation by the low pH environments observed in GERD and commonly encountered by escaping cancer cells. METHODS: Small interfering RNA library screening and high-content imaging analysis outlined changes in BE high-grade dysplasia (HGD) and EAC cell morphologies after gene silencing. Gene expression microarray data and low pH exposures studies modeling GERD-associated pulses (pH 4.0, 10 min) and tumor microenvironments (pH 6.0, constant) were used. RESULTS: Statistical analysis of small interfering RNA screening data defined 207 genes (Z-score >2.0), in 12 distinct morphologic clusters, whose suppression significantly altered BE-HGD cell morphology. The most significant genes in this list included KIF11, RRM2, NUBP2, P66BETA, DUX1, UBE3A, ITGB8, GAS1, GPS1, and PRC1. Guided by gene expression microarray study data, both pulsatile and constant low pH exposures were observed to suppress the expression of GPS1 and RRM2 in a nonoverlapping temporal manner in both BE-HGD and EAC cells, with no changes observed in squamous esophageal cells. Functional studies uncovered that GPS1 and RRM2 contributed to amoeboid and mesenchymal cellular transitions, respectively, as characterized by differential rates of cell motility, pseudopodia formation, and altered expression of the mesenchymal markers vimentin and E-cadherin. CONCLUSIONS: Collectively, we have shown that low pH microenvironments associated with GERD, and tumor invasive edges, can modulate the expression of genes that triggered esophageal cellular transitions potentially critical to colonization and invasion.