Induction of premalignant host responses by cathepsin x/z-deficiency in Helicobacter pylori-infected mice.

Helicobacter pylori are responsible for the induction of chronic gastric inflammation progressing to atrophy, metaplasia, and gastric cancer. The overexpression of Cathepsin X/Z (Ctsz) in H. pylori-infected mucosa and gastric cancer is mediated predominantly by an augmented migration of ctsz(-/-)positive macrophages and the up-regulation of Ctsz in tumor epithelium. To explore the Ctsz-function in the context of chronic inflammation and the development of preneoplastic lesions, we used Ctsz-deficient mice in a H. pylori gastritis model. Ctsz (-/-) and wild-type (wt) mice were infected with H. pylori strain SS1. The mice were sacrificed at 24, 36, and 50 weeks post infection (wpi). The stomach was removed, and gastric strips were snap-frozen or embedded and stained with H&E. Tissue sections were scored for epithelial lesions and inflammation. Ki-67 and F4/80 immunostaining were used to measure epithelial cell proliferation and macrophage infiltration, respectively. The upregulation of compensating cathepsins and cytokines were confirmed by Western blotting and quantitative RT-PCR. SS1-infected wt and ctsz (-/-) mice showed strong inflammation, foveolar hyperplasia, atrophy, and cystically-dilated glands. However, at 50 wpi, ctsz (-/-) mice developed significantly more severe spasmolytic polypeptide-expressing metaplasia (SPEM), showed enhanced epithelial proliferation, and higher levels of infiltrating macrophages. Induction of cytokines was higher and significantly prolonged in ctsz (-/-) mice compared to wt. Ctsz deficiency supports H. pylori-dependent development of chronic gastritis up to metaplasia, indicating a protective, but not proteolytic, function of Ctsz in inflammatory gastric disease.

Gastric high-risk gist and retroperitoneal liposarcoma--a challenging combination of two mesenchymal tumor lesions with regard to diagnosis and treatment.

Both gastrointestinal stromal tumors (GIST) and liposarcoma originate from mesenchymal tissue. Their coincidence requires a specific expertise in the diagnostic and therapeutic management. An unusual exemplary case is described representing a 47-year old female patient with a gastric GIST and a monstrous retroperitoneal liposarcoma with infiltration of the left kidney. The gastric tumor lesion was removed with a tangential resection of the gastric wall; the retroperitoneal tumor lesion was resected including the left kidney. Both tumors were resected with no macroscopic tumor residual. The technically difficult surgical intervention did not show any postoperative complication, and the postoperative course was also uneventful. The complete tumor resection is the treatment of choice in mesenchymal tumors (aim: R0). Depending on histologic tumor classification, resection status and tumor sensitivity, a subsequent radiation and/or chemotherapy is necessary, which allowed to achieve a postoperative tumor-free survival of 6 years including a good quality of life.

Dysregulation of CDX1, CDX2 and SOX2 in patients with gastric cancer also affects the non-malignant mucosa.

The interplay between gastric and intestinal transcription factors has an important impact on gastric carcinogenesis. We compared the gene expression of CDX1, CDX2, SOX2 and related downstream genes in tumour and tumour surrounding gastric tissue of 48 gastric cancer patients with 30 healthy controls. There was no difference of gene expression of CDX1 and CDX2 between tumour or tumour-adjacent and tumour-distant mucosa, but both factors were significantly higher expressed in cancer patients compared with controls (p<0.01). SOX2 was downregulated in tumour tissue compared to controls, whereas tumour-adjacent and tumour-distant mucosa showed intermediate SOX2 expression. Laurén type and Helicobacter pylori infection had no significant impact on expression of the transcription factors. Expression of CDX1 and CDX2 was higher in the presence of intestinal metaplasia. The differential regulation of the gene expression of CDX1, CDX2 and SOX2 in patients with gastric cancer affects not only the tumour but also the non-neoplastic tumour-distant mucosa.

Differential expression of human beta defensin 2 and 3 in gastric mucosa of Helicobacter pylori-infected individuals.

BACKGROUND: Antimicrobial peptides are key players of initial innate immune responses to human pathogens. Two major representatives, the human beta defensin 2 and 3 (hBD2 and hBD3), are both known to be regulated by, and to affect viability of, Helicobacter pylori. Previously, it was demonstrated in vitro that H. pylori actively abrogates hBD3 expression during prolonged infections. Here, we comprehensively assessed hBD2 and hBD3 expression ex vivo in the gastric mucosa of healthy individuals. MATERIALS AND METHODS: Twenty volunteers (H. pylori positive and H. pylori negative: n = 10) were enrolled. Helicobacter pylori-positive subjects underwent eradication therapy and repeated the protocol. Expression of both defensins was assessed by quantitative RT-PCR and ELISA, and correlated with histopathologic degree of gastritis. RESULTS: hBD2 and hBD3 were found to be ubiquitously expressed in all three groups. In general, hBD2 levels were elevated in relation to H. pylori infection (up to 40-fold). This upregulation correlated with degree of gastritis in corpus and antrum. In contrast, hBD3 protein levels were significantly decreased, while corresponding mRNA amounts remained unchanged. Eradication therapy led to normalization of mucosal hBD2 expression, while hBD3 expression demonstrated high interindividual variations among individuals. CONCLUSIONS: Both defensins are ubiquitously but differentially expressed in gastric mucosa in relation to H. pylori infection. Ex vivo data support the notion that H. pylori infection is associated with reduced hBD3 expression in chronic active gastritis.

A chronic oral exposure of pigs with deoxynivalenol partially prevents the acute effects of lipopolysaccharides on hepatic histopathology and blood clinical chemistry.

Lipopolysaccharides (LPS), a cell wall component of gram-negative bacteria, and deoxynivalenol (DON), a prevalent Fusarium-derived contaminant of cereal grains, are each reported to have detrimental effects on the liver. A potentiating toxic effect of the combined exposure was reported previously in a mouse model and hepatocytes in vitro, but not in swine as the most DON-susceptible species. Thus, pigs were fed either a control diet (CON) or a Fusarium contaminated diet (DON, 3.1mg DON/kg diet) for 37 days. At day 37 control pigs were infused for 1h either with physiological saline (CON_CON), 100µg/kg BW DON (CON_DON), 7.5µg/kg BW LPS (CON_LPS), or both toxins (CON_DON/LPS) and Fusarium-pigs with saline (DON_CON) or 7.5µg/kg BW LPS (DON_LPS). Blood samples were taken before and after infusion (-30, +30, +60, +120, and +180min) for clinical blood chemistry. Pigs were sacrificed at +195min and liver histopathology was performed. LPS resulted in higher relative liver weight (p<0.05), portal, periportal and acinar inflammation (p<0.05), haemorrhage (p<0.01) and pathological bilirubin levels (CON_CON 1.0µmol/L vs. CON_LPS 5.4µmol/L, CON_DON/LPS 8.3µmol/L; p<0.001). DON feeding alleviated effects of LPS infusion on histopathology and blood chemistry to control levels, whereas DON infusion alone had no impact.

Role of tight junction proteins in gastroesophageal reflux disease.

BACKGROUND: Gastroesophageal reflux disease (GERD) is associated with impaired epithelial barrier function that is regulated by cell-cell contacts. The aim of the study was to investigate the expression pattern of selected components involved in the formation of tight junctions in relation to GERD. METHODS: Eighty-four patients with GERD-related symptoms with endoscopic signs (erosive: n = 47) or without them (non-erosive: n = 37) as well as 26 patients lacking GERD-specific symptoms as controls were included. Endoscopic and histological characterization of esophagitis was performed according to the Los Angeles and adapted Ismeil-Beigi criteria, respectively. Mucosal biopsies from distal esophagus were taken for analysis by histopathology, immunohistochemistry and quantitative reverse-transcription polymerase chain reaction (RT-PCR) of five genes encoding tight junction components [Occludin, Claudin-1, -2, Zona occludens (ZO-1, -2)]. RESULTS: Histopathology confirmed GERD-specific alterations as dilated intercellular spaces in the esophageal mucosa of patients with GERD compared to controls (P < 0.05). Claudin-1 and -2 were 2- to 6-fold upregulation on transcript (P < 0.01) and in part on protein level (P < 0.015) in GERD, while subgroup analysis of revealed this upregulation for ERD only. In both erosive and non-erosive reflux disease, expression levels of Occludin and ZO-1,-2 were not significantly affected. Notably, the induced expression of both claudins did not correlate with histopathological parameters (basal cell hyperplasia, dilated intercellular spaces) in patients with GERD. CONCLUSIONS: Taken together, the missing correlation between the expression of tight junction-related components and histomorphological GERD-specific alterations does not support a major role of the five proteins studied in the pathogenesis of GERD.

Influence of anatomical subsite on the incidence of microsatellite instability, and KRAS and BRAF mutation rates in patients with colon carcinoma.

There is a growing amount of data supporting the concept that cancers originating from the proximal and distal colon are distinct clinicopathological entities. The incidence of MSI and BRAF mutation is strongly associated with right sided tumor location, whereas there are conflicting results for KRAS mutation rates. However, to date, no data exist whether and to what extent defined colonic subsites influence MSI status, KRAS and BRAF mutation rates. We selected primary colon cancer from 171 patients operated on at our institution between 2007 and 2010. BRAF, KRAS mutation rates and microsatellite instability were determined and correlated with clinicopathological features and tumor location. MSI-h cancers were significantly associated with poor histological grade but a lower rate of distant metastases. KRAS-mutated tumors were linked to lower T-stage and better differentiation. Colon carcinomas with BRAF mutation were significantly associated with distant metastatic spread and poor histological grade. Furthermore, we found that MSI-h status, KRAS and BRAF mutation rates varied remarkably among the colonic subsites irrespective of right- and left-sided origin, respectively. The results of the current study provide further evidence that a simple classification into right- and left-sided colon carcinoma does not represent the complexity of this tumor entity.

Do basophile structures as age dependent phenomenon indicate small vessel wall damage?

Here we demonstrate basophile structures located in the arteriolar wall and being associated with a plasma-protein-leakage. We assume, that the structures indicate blood-brain-barrier-disturbances and degenerative small vessel wall alterations.

Assessment of desmosomal components (desmoglein 1-3, plakoglobin) in cardia mucosa in relation to gastroesophageal reflux disease and Helicobacter pylori infection.

Gastroesophageal reflux disease is associated with impaired epithelial barrier function and abnormal expression of proteins forming cell-cell contacts by tight junctions and desmosomes in distal esophageal squamous mucosa. Although gastroesophageal reflux disease and Helicobacter pylori are both associated with chronic inflammation of the adjacent cardia mucosa, it is not known whether these lead to derangements of the desmosomal complexes. Here, we assessed the expression of 4 proteins (plakoglobin and desmoglein 1, 2, and 3) forming epithelial desmosomal complexes by quantitative reverse transcription polymerase chain reaction and immunohistochemistry in biopsies from 67 patients with gastroesophageal reflux disease and 23 gastroesophageal reflux disease-negative controls. Plakoglobin and desmoglein 2 were ubiquitously expressed in all samples, whereas desmoglein 1 and 3 were not expressed in cardia mucosa. Gastroesophageal reflux disease was specifically associated with elevated transcript levels of desmoglein 2 and plakoglobin. These were significantly increased from 2.0- to 2.7-fold in patients with gastroesophageal reflux disease compared with controls (P < .01), and significantly increased immunohistochemical scores for both proteins were observed (P < .05) as well. The combined presence of gastroesophageal reflux disease and Helicobacter pylori infection had no additional effect on desmosomal gene expression. Taken together, the up-regulation of plakoglobin and desmoglein 2 in cardia mucosa of patients with gastroesophageal reflux disease supports the concept that the "transition zone" between distal esophagus and proximal stomach is affected by gastroesophageal reflux disease as well, and architectural and molecular changes in the desmosomal compartment contribute to the pathogenesis of gastroesophageal reflux disease in the cardia mucosa.

Serological assessment of gastric mucosal atrophy in gastric cancer.

BACKGROUND: Non-invasive tools for gastric cancer screening and diagnosis are lacking. Serological testing with the detection of pepsinogen 1 (PG1), pepsinogen 2 (PG2) and gastrin 17 (G17) offers the possibility to detect preneoplastic gastric mucosal conditions. Aim of this study was to assess the performance of these serological tests in the presence of gastric neoplasia. METHODS: Histological and serological samples of 118 patients with gastric cancer have been assessed for tumor specific characteristics (Laurén type, localisation), degree of mucosal abnormalities (intestinal metaplasia, atrophy) and serological parameters (PG1, PG2, PG1/2-ratio, G17, H. pylori IgG, CagA status). Association of the general factors to the different serological values have been statistically analyzed. RESULTS: Patients with intestinal type gastric cancer had lower PG1 levels and a lower PG1/2-ratio compared to those with diffuse type cancer (p = 0.003). The serum levels of PG2 itself and G17 were not significantly altered. H. pylori infection in general had no influence on the levels of PG1, PG2 and G17 in the serum of gastric cancer patients. There was a trend towards lower PG1 levels in case of positive CagA-status (p = 0.058). The degree of both intestinal metaplasia and atrophy correlated inversely with serum levels for PG1 and the PG1/2-ratio (p < 0.01). Laurén-specific analysis revealed that this is only true for intestinal type tumors. Univariate ANOVA revealed atrophy and CagA-status as the only independent factors for low PG1 and a low PG1/2-ratio. CONCLUSIONS: Glandular atrophy and a positive CagA status are determinant factors for decreased pepsinogen 1 levels in the serum of patients with gastric cancer. The serological assessment of gastric atrophy by analysis of serum pepsinogen is only adequate for patients with intestinal type cancer.

Gastro-oesophageal reflux disease is associated with up-regulation of desmosomal components in oesophageal mucosa.

AIMS: Gastro-oesophageal reflux disease (GERD) is associated with impaired epithelial barrier function. This study was aimed at investigating the role of desmosomal proteins in relation to GERD. METHODS AND RESULTS: Ninety-five patients with GERD-related symptoms (erosive, n = 51; non-erosive, n = 44) and 27 patients lacking those symptoms were included. Endoscopic and histological characterization of oesophagitis was performed according to the Los Angeles and Ismeil-Beigi criteria, respectively. Multiple biopsies were taken from the oesophageal mucosa of each patient. Gene expression analysis of plakoglobin, desmoglein-1, desmoglein-2 and desmoglein-3 was performed by quantitative real time (RT)-polymerase chain reaction and immunohistochemistry in the oesophageal mucosa. Routine histology revealed specific GERD-related alterations, such as dilatation of intercellular spaces (DIS), basal cell hyperplasia (BCH), and elongation of the papillae, in the oesophageal mucosa of patients with GERD, as compared with controls (all parameters: P < 0.05). All four genes and corresponding proteins were found to be up-regulated by between 1.7 and 8.1-fold (transcript level, P < 0.05; protein level, P < 0.05). Induced gene expression levels of plakoglobin, desmoglein-1 and desmoglein-2 correlated significantly with DIS and BCH. CONCLUSIONS: Taken together, the uniform up-regulation of desmosomal genes/proteins in the oesophageal mucosa of patients with GERD supports the concept of architectural and molecular changes in the desmosomal compartment in the pathogenesis of GERD.

Protease-activated receptor-2 (PAR2) in human gastric mucosa as mediator of proinflammatory effects in Helicobacter pylori infection.

INTRODUCTION: Protease-activated receptors (PAR) are seven transmembrane receptors that are expressed throughout the gastrointestinal tract. In vitro experiments using gastric tumor cell lines, murine models and one clinical study provided evidence for a potential role of PAR2 in Helicobacter pylori-induced gastritis. AIM: To investigate PAR2 expression in H. pylori-infected patients and correlation with proinflammatory IL-8, IL-1ß as well as histologic changes of the mucosa. Furthermore, PAR2 expression was studied in context to mucosal amounts of secretory leukocyte protease inhibitor (SLPI), a putative regulator of PAR2. METHODS: Twenty-two H. pylori-infected patients and 72 H. pylori-negative subjects underwent upper GI endoscopy. In antrum-derived mucosal biopsies, PAR2, IL-1ß, IL-8, and SLPI expression was analyzed by quantitative RT-PCR, and in part by ELISA and immunohistochemistry. Histopathologic evaluation of gastritis was performed according to the updated Sydney classification. RESULTS: IL-8 gene expression was 5-fold increased in the mucosa of H. pylori-infected patients compared with non-infected (p < .0001), whereas no differences for PAR2 and IL-1ß mRNA amounts were observed between both groups. PAR2 gene expression correlated positively with transcript levels of IL-8, IL-1ß as well mucosal SLPI levels in H. pylori-infected patients (r: 0.47-0.84; p < .0001), whereas no correlation was found with the degree of gastritis. CONCLUSIONS: PAR2 represents an additive pathway of IL-8 secretion and proinflammatory effects in H. pylori-induced gastritis. Reduced SLPI levels leading to higher serine protease activities in the mucosa of infected subjects might regulate PAR2 activation.

Kidney pathology precedes and predicts the pathological cascade of cerebrovascular lesions in stroke prone rats.

INTRODUCTION: Human cerebral small vessel disease (CSVD) has been hypothesized to be an age-dependent disease accompanied by similar vascular changes in other organs. SHRSP feature numerous vascular risk factors and may be a valid model of some aspects of human CSVD. Here we compare renal histopathological changes with the brain pathology of spontaneously hypertensive stroke-prone rats (SHRSP). MATERIAL AND METHODS: We histologically investigated the brains and kidneys of 61 SHRSP at different stages of age (12 to 44 weeks). The brain pathology (aggregated erythrocytes in capillaries and arterioles, microbleeds, microthromboses) and the kidney pathology (aggregated erythrocytes within peritubular capillaries, tubular protein cylinders, glomerulosclerosis) were quantified separately. The prediction of the brain pathology by the kidney pathology was assessed by creating ROC-curves integrating the degree of kidney pathology and age of SHRSP. RESULTS: Both, brain and kidney pathology, show an age-dependency and proceed in definite stages whereas an aggregation of erythrocytes in capillaries and arterioles, we parsimoniously interpreted as stases, represent the initial finding in both organs. Thus, early renal tubulointerstitial damage characterized by rather few intravasal erythrocyte aggregations and tubular protein cylinders predicts the initial step of SHRSPs' cerebral vascular pathology marked by accumulated erythrocytes. The combined increase of intravasal erythrocyte aggregations and protein cylinders accompanied by glomerulosclerosis and thrombotic renal microangiopathy in kidneys of older SHRSP predicts the final stages of SHRSPs' cerebrovascular lesions marked by microbleeds and thrombotic infarcts. CONCLUSION: Our results illustrate a close association between structural brain and kidney pathology and support the concept of small vessel disease to be an age-dependent systemic pathology. Further, an improved joined nephrologic and neurologic diagnostic may help to identify patients with CSVD at an early stage.

Murine models of H. pylori-induced gastritis and gastric adenocarcinoma.

Laboratory mice have become one of the best animal species for mechanistic studies in gastrointestinal research. Their abundant genetic information, the way of causing carcinogenesis easily by transgenic and gene knockout techniques, limited effort in time and costs, and their practicability provide advantages over other animal models. Meanwhile, several murine practical models have been established for the investigation of the initiation, expansion, and progression of gastritis and gastric carcinoma, for assessing the effects of bacterial, genetic and environmental factors, and for evaluating therapeutic and preventive strategies in gastric diseases. This article gives a review of murine models of gastritis and gastric cancer, placing emphasis on the models associated with Helicobacter pylori infection and techniques used in our laboratory. We discuss matters of murine gastric anatomy, as well as techniques of infection, tissue preparation, and histology.

Helicobacter pylori infection and fundic gastric atrophy are not associated with esophageal squamous cell carcinoma: a case-control study.

OBJECTIVE: Recent studies from Asia and Northern Europe suggest that apart from alcohol intake and smoking, fundic gastric atrophy (FGA) may also increase the risk of esophageal squamous cell carcinoma (OSCC). However, because of the wide geographic variation of this cancer and the changing prevalence of the Helicobacter pylori infection, these findings need to be confirmed in other ethnic groups. The aim of this case-control study was to investigate whether H. pylori infection and FGA carry an increased risk for OSCC. PATIENTS AND METHODS: FGA was evaluated, by histology and serology, in 75 patients with OSCC, and 75 sex-matched and age-matched controls. Pepsinogen (PG) I levels 70 µg/ml or less and PG I/II ratio of 3 or less were indicative for FGA. H. pylori infection was defined as positivity to at least one test among histology, rapid urease test, and serology for both general anti-IgG and anti-CagA. RESULTS: Overall, the prevalence of H. pylori infection was identically high (70.7%) in both patients with OSCC and controls. FGA diagnosed by serology and histology was not associated with an increased risk for OSCC [odds ratio (OR)=1.17; 95% confidence interval (CI)=0.54-2.56 and OR=1.91; 95% CI=0.6-5.99, respectively]. ORs (95% CI) for hazardous alcohol consumption, smoking, and the presence of both risk factors were 5.75 (2.20-15.05), 22.18 (9.41-52.28), and 31.69 (8.39-119.67), respectively. CONCLUSIONS: Hazardous alcohol consumption and smoking increase synergistically the risk for developing OSCC. In our population neither H. pylori infection nor FGA was associated with an increased risk for OSCC.

The submucosal cushion does not improve the histologic evaluation of adenomatous colon polyps resected by snare polypectomy.

BACKGROUND & AIMS: Although the "submucosal cushion" technique or injection-assisted polypectomy (IAP) is often used to resect colon polyps, little is known on the influence of this technique on histologic interpretation. We aimed to evaluate whether the use of a submucosal cushion improves the histologic and margin evaluation of colon polyps. METHODS: Consecutive patients undergoing polypectomy with and without IAP were included. An experienced blinded gastrointestinal pathologist evaluated the specimens using standardized criteria. RESULTS: One hundred eleven sessile colon adenomas were analyzed (IAP, n = 65, standard, n = 46). Two-thirds of polyps ranged in size from 10 to 20 mm; the average polyp size was 13.2 mm for IAP and 9.9 mm for standard snare polypectomy (P = .001). The cautery degree, cautery amount, and margin evaluability, did not differ substantially with regard to the resection technique. For polyps ≥10-20 mm, the overall architecture quality was better in polyps resected with standard technique as compared with IAP. CONCLUSIONS: The utilization of IAP did not result in a better margin evaluability of the resected polyp. Overall, IAP does not result in a better histologic polyp evaluability.

Mucosal Progranulin expression is induced by H. pylori, but independent of Secretory Leukocyte Protease Inhibitor (SLPI) expression.

BACKGROUND: Mucosal levels of Secretory Leukocyte Protease Inhibitor (SLPI) are specifically reduced in relation to H. pylori-induced gastritis. Progranulin is an epithelial growth factor that is proteolytically degraded into fragments by elastase (the main target of SLPI). Considering the role of SLPI for regulating the activity of elastase, we studied whether the H. pylori-induced reduction of SLPI and the resulting increase of elastase-derived activity would reduce the Progranulin protein levels both ex vivo and in vitro. METHODS: The expression of Progranulin was studied in biopsies of H. pylori-positive, -negative and -eradicated subjects as well as in the gastric tumor cell line AGS by ELISA, immunohistochemistry and real-time RT-PCR. RESULTS: H. pylori-infected subjects had about 2-fold increased antral Progranulin expression compared to H. pylori-negative and -eradicated subjects (P < 0.05). Overall, no correlations between mucosal Progranulin and SLPI levels were identified. Immunohistochemical analysis confirmed the upregulation of Progranulin in relation to H. pylori infection; both epithelial and infiltrating immune cells contributed to the higher Progranulin expression levels. The H. pylori-induced upregulation of Progranulin was verified in AGS cells infected by H. pylori. The down-regulation of endogenous SLPI expression in AGS cells by siRNA methodology did not affect the Progranulin expression independent of the infection by H. pylori. CONCLUSIONS: Taken together, Progranulin was identified as novel molecule that is upregulated in context to H. pylori infection. In contrast to other diseases, SLPI seems not to have a regulatory role for Progranulin in H. pylori-mediated gastritis.

Intercellular space volume is mainly increased in the basal layer of esophageal squamous epithelium in patients with GERD.

BACKGROUND/AIMS: At present, the dilation of esophageal intercellular spaces (ICS) is considered an early morphologic marker of acid damage in patients with GERD. Nevertheless, previous electron microscopic (EM) studies had focused only on the suprabasal layer of squamous epithelium or did not nearly specify which layer of squamous epithelium was studied. Therefore, we aimed to assess the volumetric amount of the ICS in all layers of SE in patients with GERD. METHODS: In this study, 48 patients were prospectively included (NERD = 18, ERD = 17; Barrett's esophagus = 5, controls = 8). All patients with ERD and NERD had typical reflux symptoms, as assessed by a valid GERD questionnaire. ICS volume was assessed by electron microscopy in the superficial, prickle cell, and basal layers of esophageal squamous epithelium using the method of Weibel. RESULTS: ERD was associated with increased ICS volume in the basal layer (LA-A, p = 0.038; LA-B, p = 0.005) and prickle cell layer (LA-A, p = 0.006; LA-B, p = 0.007) as compared to controls. Comparisons between NERD and ERD patients revealed more dilated ICS in the basal layer (LA-B, p = 0.007), prickle cell layer (LA-A, p = 0.008; LA-B, p = 0.001) and superficial layer (LA-B, p = 0.018) in patients with ERD. CONCLUSIONS: Not only the diameter but also the volume of the ICS is increased in patients with GERD. Furthermore, the dilation of ICS is present in all three layers of the SE, being more pronounced in the basal layer. These findings support the concept that the impairment of the esophagus begins in the deeper parts of the esophageal epithelium.