Presence Of Dilated Intercellular Spaces Research Articles

Epithelial Thickness is a Marker of Gastroesophageal RefluxDisease.

Histologic criteria have been refined for the diagnosis of gastroesophageal reflux disease (GERD). We aimed to evaluate these criteria for the assessment of GERD and to measure interassessor agreement. We performed a post hoc analysis of data from the Diamond study (NCT 00291746), conducted in Europe and Canada on adults with frequent upper gastrointestinal symptoms who had not taken a proton pump inhibitor in the previous 2 months. GERD was diagnosed based on the presence of 1 or more of the following: reflux esophagitis, pathologic esophageal acid exposure, and/or positive symptom-acid association probability. Nonerosive reflux disease was defined as the presence of pathologic esophageal acid exposure and/or a positive symptom-acid association probability, but no reflux esophagitis. Biopsies collected from 336 patients from 0.5 cm and 2.0 cm above the Z line were evaluable; they were analyzed independently at pathology centers in Germany and Italy (biopsies from 258 and 195 patients, respectively). The primary outcomes were the accuracy of histologic criteria for the diagnosis of GERD, defined by endoscopy and pH monitoring, and interassessor agreement on histologic criteria. At the assessment site for basal cell layer thickness, total epithelial thickness was the best-performing criterion for diagnosis of investigation-defined GERD; it also identified nonerosive reflux disease, reflux esophagitis, and pathologic esophageal acid exposure at 0.5 cm and 2.0 cm above the Z line. Basal cell layer thickness and presence of dilated intercellular spaces did not identify patients with GERD. Among the criteria tested, the best agreement between assessments carried out at the 2 pathology centers was for total epithelial thickness at 0.5 cm and 2.0 cm above the Z line. Based on an analysis of 336 patients with frequent upper gastrointestinal symptoms, total epithelial thickness is a robust histologic marker for GERD.

Bile Salts at Low pH Cause Dilation of Intercellular Spaces in In Vitro Stratified Primary Esophageal Cells, Possibly by Modulating Wnt Signaling

BackgroundThe presence of dilated intercellular spaces in the stratified squamous lining of the esophagus is the pathognomonic feature of reflux esophagitis secondary to gastroesophageal reflux disease (GERD). In addition to stomach acid, bile salts are major constituents of gastroesophageal refluxate. The aim of our study was to determine the effect of bile salts cocktail at different pHs on epithelial junctions in an in vitro transwell model of stratified esophageal squamous epithelium. DiscussionHuman telomerase reverse transcriptase (hTERT) immortalized primary esophageal EPC1 cells were grown on polyester transwell surfaces in calcium-enriched media. The cells exhibited gradual stratification into an 11-layered squamous epithelium over 7 days, together with epithelial barrier function as indicated by increased transepithelial electrical resistance (TEER). This stratified epithelium demonstrated well-formed tight junctions, adherens junctions, and desmosomes as visualized by immunofluorescence and electron microscopy. When exposed to short pulses of bile salts at pH 5, but not either condition alone, there was loss of stratification and decrease in TEER, concomitant with disruption of adherens junctions, tight junctions, and desmosomes, leading to the appearance of dilated intercellular spaces. At the cellular level, bile salts at pH 5 activated the Wnt pathway (indicated by increased β-catenin Ser552 phosphorylation). ConclusionIn conclusion, in our in vitro transwell model bile salts at pH 5, but not bile salts or media at pH 5 alone, modulate Wnt signaling, disrupt different junctional complexes, and cause increased permeability of stratified squamous esophageal epithelium. These changes approximate the appearance of dilated intercellular space similar to that found in GERD patients.

Erratum to: Acaricidal effect and histological damage induced by Bacillus thuringiensis protein extracts on the mite Psoroptes cuniculi

The mite Psoroptes cuniculi is a common worldwide ectoparasite and the most frequently found in rabbit farms. It causes significant economic losses on commercial rabbit breeding associated with poor leather quality, reduced conception rates, weight loss, poor growth and death. Several strategies have been proposed for the treatment of mange caused by this mite, ranging from the use of acaricides, entomopathogenic fungi, essential oils and vaccines. However, therapy and control of both human scabies and animal mange are still based mainly on the use of drugs and chemicals such as ivermectin, which involves disadvantages including genotoxic and cytotoxic effects, resistance and environmental damage. Bacillus thuringiensis is a bacterium, innocuous for human being, domestic animals and plants that produces highly biodegradable proteins, and has been used worldwide for biological control. The aim of this work was to find an alternative treatment based on biological control for scabies caused by Psoroptes cuniculi, using protein extracts from strains of Bacillus thuringiensis. P. cuniculi mites were obtained from naturally infected New Zealand rabbits, and different doses of protein from B. thuringiensis were added to the mites. We measured mortality and obtained the median lethal concentration and median lethal times. For histological analysis, the mites were fixed in 10 % formalin, processed according to the paraffin embedded tissue technique. Sections were stained with hematoxylin-eosin to observe the general histological structure. We report here for the first time evidence about the in vitro acaricidal effect caused by the strain GP532 of B. thuringiensis on the mite Psoroptes cuniculi, with an LC50 of 1.3 mg/ml and a LT50 of 68 h. Histological alterations caused by B. thuringiensis on this mite, included the presence of dilated intercellular spaces in the basal membrane, membrane detachment of the peritrophic matrix and morphological alterations in columnar cells of the intestine. Since this mite is an obligate ectoparasite that affects rabbits, goats, horses, cows and sheep, B. thuringiensis protein extracts are proposed as a potential treatment for biological control of mange in farm animals.

PTU-162 Oesophageal Mucosal Dilated Intercellular Spaces (Dis), Transepithelial Electrical Resistance and Perception of Heartburn

IntroductionIt is proposed that the presence of oesophageal mucosa dilated intercellular spaces (DIS) underlies heartburn in patients with NERD. However, 20% of asymptomatic subjects display DIS in distal oesophageal biopsies....

Tu1848 Distribution of the Acid Sensing Channels Asic-1, Asic-2, 5Enac and PAR-2 Receptors in the Human Esophageal Mucosa

Background It is proposed that the presence of esophageal mucosa dilated intercellular spaces (DIS) underlies heartburn in patients with NERD. However, 20% of asymptomatic subjects display DIS in distal esophageal biopsies. Furthermore, esophageal experimental acidification in healthy volunteers provokes DIS, but often without heartburn despite ongoing acid perfusion. These observations suggest that mucosal impairment above and beyond DIS may be relevant in heartburn perception. Functional mucosal integrity in response to acid challenge can be tested in esophageal mucosal biopsies "in vitro" by measuring changes in transepithelial electrical resistance (TER).We aimed to further assess the relationship between presence of DIS, changes in functional esophageal mucosal integrity (TER) and heartburn perception. Methods We took distal esophageal biopsies from patients with and without heartburn. Histological examination for epithelial intercellular space diameter (ISD) was done using light microscopy by taking 50 random measurements at several levels from the basal epithelial layers for each biopsy. DIS was declared at above the 95% CI for normal values as previously published (.0.72μm). We identified 11 subjects with DIS (4 with predominant daily troublesome heartburn, 7 with predominant dyspepsia and no heartburn). Biopsies were placed in mini-Ussing chambers, and baseline TER was measured. The luminal aspect of the biopsy was then exposed for 30min to an acidic solution (pH2+1mg/ml pepsin+1mM taurodeoxycholate). During exposures, % changes in TER relative to baseline were analyzed. Results The mean ISD in all subjects was 0.94μm (range 0.74-1.18μm). There was no significant difference in ISD (0.95 vs. 0.91μm) or baseline TER (144 vs. 165V.cm2) between predominant heartburn and predominant dyspepsia groups. Despite the pre-existing DIS in all subjects, 30 minutes acid exposure was able to induce a further reduction in TER (-23.0% change from baseline, p,0.01). The reduction in TER was greater in subjects with predominant reflux vs. those with predominant dyspepsia (-40.4±10.3% vs. -11.2±3.7%, p=0.01). Conclusion In subjects with pre-existing DIS with and without hearburn, trans-epithelial electrical resistance can be further impaired with in vitro acid exposure, suggesting that the mechanism for acid-induced mucosal integrity impairment is not limited to DIS. Furthermore, acid-induced functional integrity impairment was greater in patients with heartburn. This difference in mucosal behaviour in the presence of acid suggests that other mechanisms beyond DIS might be needed to further stimulate afferent nerves in heartburn generation.

Tu1847 Esophageal Mucosa Dilated Intercellular Spaces (DIS), Transepithelial Electrical Resistance and Perception of Heartburn

Introduction It is proposed that the presence of oesophageal mucosa dilated intercellular spaces (DIS) underlies heartburn in patients with NERD. However, 20% of asymptomatic subjects display DIS in distal oesophageal biopsies. Furthermore, oesophageal experimental acidification in healthy volunteers provokes DIS, but often without heartburn despite ongoing acid perfusion. These observations suggest that mucosal impairment above and beyond DIS may be relevant in heartburn perception. Functional mucosal integrity in response to acid challenge can be tested in oesophageal mucosal biopsies “in vitro” by measuring changes in transepithelial electrical resistance (TER). We aimed to further assess the relationship between presence of DIS, changes in functional oesophageal mucosal integrity (TER) and heartburn perception. Methods We took distal oesophageal biopsies from patients with and without heartburn. Histological examination for epithelial intercellular space diameter (ISD) was done using light microscopy by taking 50 random measurements at several levels from the basal epithelial layers for each biopsy. DIS was declared at above the 95% CI for normal values as previously published (> 0.72 μm). We identified 11 subjects with DIS (4 with predominant daily troublesome heartburn, 7 with predominant dyspepsia and no heartburn). Biopsies were placed in mini-Ussing chambers, and baseline TER was measured. The luminal aspect of the biopsy was then exposed for 30min to an acidic solution (pH2 + 1 mg/ml pepsin + 1 mM taurodeoxycholate). During exposures, % changes in TER relative to baseline were analysed. Results The mean ISD in all subjects was 0.94 μm (range 0.74–1.18 μm). There was no significant difference in ISD (0.95 vs. 0.91 μm) or baseline TER (144 vs. 165 Ω.cm 2 ) between predominant heartburn and predominant dyspepsia groups. Despite the pre-existing DIS in all subjects, 30 minutes acid exposure was able to induce a further reduction in TER (–23.0% change from baseline, p The reduction in TER was greater in subjects with predominant reflux vs. those with predominant dyspepsia (–40.4 ± 10.3% vs. –11.2 ± 3.7%, p = 0.01). Conclusion In subjects with pre-existing DIS with and without heartburn, trans-epithelial electrical resistance can be further impaired with in vitro acid exposure, suggesting that the mechanism for acid-induced mucosal integrity impairment is not limited to DIS. Furthermore, acid-induced functional integrity impairment was greater in patients with heartburn. This difference in mucosal behaviour in the presence of acid suggests that other mechanisms beyond DIS might be needed to further stimulate afferent nerves in heartburn generation. Disclosure of Interest None Declared

Mo1837 Beneficial Effects of Diazoxide on Hepatic Ischemia/Reperfusion Injury

The pathognomonic feature of reflux esophagitis secondary to gastro-esophageal reflux disease is the presence of dilated intercellular spaces in the stratified squamous lining of the esophagus. Bile acid is a major constituent of gastro-esophageal refluxate. In our present study, we developed a novel in vitro transwell culture model for stratified esophageal squamous epithelium. We grew h-TERT transformed primary esophageal cell line EPC1 on polyester transwell surfaces, apically and basally supplemented with calcium enriched media, and observed that the EPC1 cells gradually stratify into a 11-layered squamous epithelium in 7 days. This epithelium also demonstrated well-formed cell junctions, essential for formation of the stratified epithelium. When the EPC1 cells on transwells were treated with a combination of bile acid and pH5, there was loss of epithelial barrier function. Electron microscopy and confocal imaging of the cell junctions showed disruption of adherens junction, tight junction and desmosomes, thus leading to dilated intercellular spaces. At the cellular level, the combination of bile acid and pH5 induced β-catenin phosphorylation and reduced SMAD-1/5/8 phosphorylation, both of which can lead to loss of cell junction proteins. In conclusion, combination of bile acid at low pH in our trasnwell culture model mimicked the effects of gastro-esophageal reflux in vivo, possibly by modulating WNT and BMP signaling pathways.

Evaluation of Histologic Criteria for the Diagnosis of Gastroesophageal Reflux Disease

Background. Standardized criteria for the histologic assessment of gastroesophageal reflux disease (GERD) were recently developed and evaluated by an international working group, and showed promising levels of intraand inter-assessor agreement1. To gain clinical acceptance these criteria require appropriate validation2.Aim. To evaluate new criteria for histologic markers of GERD in a well characterized primary care population. Methods. The study included primary care patients from the Diamond study3 (ClinicalTrials.gov: NCT00291746) who had not previously taken proton pump inhibitors and who had upper gastrointestinal symptoms ≥ 2 times a week for ≥ 4 weeks, and at least mild symptoms on ≥ 3 days in the week before biopsy. GERD was diagnosed as any of the following: Los Angeles grade reflux esophagitis; distal esophageal pH 5.5% of 24 hours; ≥ 95% symptom association probability. Biopsies were collected at 0.5 cm and 2.0 cm above the Z-line and data were analyzed in a single-blind manner by one pathologist (MV). Variables assessed included total epithelial thickness (TET), papillary length (PL), basal cell thickness (all in μm), presence of dilated intercellular spaces (severity score: 0-2)1, number of inflammatory cells (in one high powered field), presence of necrosis, and presence of active or healed erosions. Logistic regression analysis of the histologic variables was performed for GERD versus non-GERD. Cut-off values for sensitivity, specificity, and predictive power (positive and negative) were calculated, and the receiver operating characteristic (ROC) curve was analyzed. Results. The analysis included 258 subjects: 138 (53%) GERD and 120 (47%) non-GERD. TET and PL (μm) at 0.5 cm and 2.0 cm were both significant predictors of GERD (p < 0.005), as was the presence of eosinophils at 2.0 cm (p < 0.05). The optimal efficiency based on the ROC analysis was observed for TET at 0.5 cm, with sensitivity and specificity values of 77% and 52%, and positive and negative predictive values of 65% and 66%, based on a cut-off of 390 μm. TET and PL were significant (p < 0.05) predictors of both reflux esophagitis and of pathologic esophageal pH. TET and PL were also significant predictors (p < 0.05) of non-erosive reflux disease versus non-GERD. Conclusions. Potential new criteria for the histologic evaluation of GERD showed good diagnostic properties when assessed in a well-characterized primary care population with upper gastrointestinal symptoms.

Nonerosive reflux disease: A pathophysiologic perspective

Nonerosive reflux disease (NERD) is the most common phenotype of gastroesophageal reflux disease. By definition, patients with NERD have typical reflux symptoms caused by the intraesophageal reflux of gastric contents but have no visible esophageal mucosal injury. This is in contrast to patients with reflux esophagitis, also known as erosive reflux disease, and Barrett's esophagus, who have obvious esophageal mucosal injury on endoscopy. Only 50% of patients with NERD have pathologic esophageal acid contact time (ACT) as detected on 24-hour pH monitoring (ie, NERD-positive). NERD patients with physiologic esophageal ACT and good temporal correlation of symptoms with reflux events (symptom index > 50% or symptom-association probability > 95%) are considered to have esophageal hypersensitivity (ie, NERD-negative). Finally, patients with physiologic esophageal ACT but poor symptom-reflux correlation are now considered to have functional heartburn and not NERD. NERD-positive patients have motor dysfunction and acidic reflux abnormalities that are similar to patients with reflux esophagitis and Barrett's esophagus, whereas NERD-negative patients have minimal abnormalities that are not much different than healthy controls. The histopathologic feature most indicative of NERD is the presence of dilated intercellular spaces within squamous epithelium, an ultrastructural abnormality readily identified on transmission electron microscopy and on light microscopy.

Radial Distribution of Dilated Intercellular Spaces of the Esophageal Squamous Epithelium in Patients with Reflux Disease Exhibiting Discrete Endoscopic Lesions

Introduction: Dilatation of intercellular spaces of the esophageal squamous epithelium has been suggested as a marker of early acid reflux-induced damage. This change is a potentially useful addition to histomorphological changes that represent so called minimal endoscopic lesions. We have assessed dilatation of intercellular spaces with regard to: (1) interobserver variability, and (2) whether the incidence of this varies between ‘red streaks’ and the adjacent normal looking squamous epithelium. Methods: Esophageal biopsies from 44 patients with chronic gastro-esophageal reflux (GERD) were evaluated. At endoscopy, these patients had one or more red streaks on the tops of the mucosal folds in the distal esophagus. Biopsies were taken from the red streaks and from the normal-appearing mucosa 1 cm lateral to the red streaks. Biopsies were assessed in a blinded fashion by two independent pathologists (MV & RF). Criteria for assessing intercellular space dilatation were evaluated and agreed on prior to the study. Results: Good interobserver agreement was recorded (kappa = 0.82 at the streaks and 0.77 for the control tissues) for absence/presence of intercellular space dilatation. Red streak and control biopsies differed significantly (p = 0.0001), with respect to presence of dilated intercellular spaces, with 90.5 % of the former demonstrating this as present compared to 56.1% in the controls. Conclusion: This study supports the concept that esophageal mucosal minimal changes due to reflux is localised and that dilatation of intercellular spaces is an early sign of reflux-induced epithelial damage. The low interobserver variability in the assessment of intercellular space dilatation suggests that this may be a useful variable for assessment of early signs of acid-reflux induced damage to the squamous epithelium of the esophagus by use of light microscopy.