Reactive nitrogen oxide species induce dilatation of the intercellular space of rat esophagus.

OBJECTIVE: Dilatation of the intercellular space (DIS) of the esophageal epithelium is recognized as one of the earliest histological changes in gastroesophageal reflux disease patients. At the human gastroesophageal junction, reactive nitrogen oxide species (RNOS) are generated luminally through the entero-salivary re-circulation of dietary nitrate. In cases with gastroesophageal reflux, the site of luminal RNOS generation may shift to the distal esophagus. The aim of this study was to investigate whether luminal RNOS exposure could be involved in the pathogenesis of DIS. MATERIAL AND METHODS: Rat esophageal mucosa was studied with an Ussing chamber model. On the luminal side of the chamber, RNOS were generated by the acidification of physiologic concentrations of sodium nitrite (1.0 or 5.0 mM). Esophageal barrier function was assessed by means of electrophysiological transmembrane resistance and membrane permeability by means of (3)H-mannitol flux. The dimensions of the intercellular spaces were assessed by using transmission electron microscopy. RESULTS: Administration of acid plus sodium nitrite induced DIS of the esophageal epithelium, and this ultrastructural morphological change was accompanied by a concomitant decrease in the transmembrane resistance and an increase in the epithelial permeability. The DIS induced by luminal RNOS was also confirmed in an in vivo exposure model. CONCLUSIONS: The present animal study indicates that the RNOS generated by the acidification of salivary nitrite in the presence of refluxed gastric acid in the esophagus could be a luminal factor that is responsible for the induction of DIS. Further studies are warranted to investigate the clinical relevance of the present findings to the human situation.

Gastric hyposecretion in esophageal squamous-cell carcinomas.

Recently, gastric fundic atrophy is reported to be an independent risk factor for esophageal squamous-cell carcinoma (ESCC). The aim of this study is to investigate the acid secretory level in ESCC in a case-control study. From April 2004 to March 2008, 100 consecutive subjects with early ESCC and 100 age- and sex-matched asymptomatic controls were prospectively enrolled. Gastrin-stimulated acid output was assessed by endoscopic gastrin test. Conditional regression analyses were used to adjust for other potential confounders. Multivariate analyses revealed a strong association between profound hypochlorhydria and ESCC with odds ratio (95% confidence interval): 6.0 (1.9-18.4). The association remained significant after adjusting for the effect of gastric atrophy as a covariate. The association became stronger as the ESCC developed more distal site of the esophagus. This study indicates that profound hypochlorhydria is a strong independent risk factor for ESCC even after adjusting for the influence of gastric atrophy.

Identification of a novel NOTCH-4/INT-3 RNA species encoding an activated gene product in certain human tumor cell lines.

Ectopic expression of the intracellular domain of NOTCH-4/INT-3 leads to tumorigenesis in the mouse mammary gland. This results from a gain-of-function mutation. To evaluate gain-of-function NOTCH-4/INT-3 activity in human cancers we have surveyed human breast, lung, and colon carcinoma tissue culture cell lines for evidence of increased NOTCH-4/INT-3 RNA expression. High levels of a 1.8 Kb NOTCH-4/INT-3 RNA species are detected in normal human testis but not in other tissues where a 6.5 Kb species is prevalent. Transformed human cancer cell lines express the 1.8 Kb NOTCH-4/INT-3 RNA species. We show that this RNA species encodes a truncated form of the NOTCH-4/INT-3 intracellular domain (ICD). This novel NOTCH-4/INT-3 protein includes the CDC10 repeats and amino acid residues C-terminal to them, but is missing the CBF-1 binding region of the NOTCH-4/INT-3 ICD. This suggests that it has a different mode of action. Furthermore, we show that a transgene which expresses the 1.8 Kb NOTCH-4/INT-3 RNA species in the 'normal' human mammary epithelial cell line MCF-10A enables these cells to grow in soft agar.

Identification of a high-risk group for low-dose aspirin-induced gastropathy by measuring serum pepsinogen in H. pylori-infected subjects.

BACKGROUND: We recently demonstrated in humans that the extent of low-dose aspirin (LDA)-induced gastropathy was directly related to the individual gastric acid secretion level. We also established reliable cutoff serum pepsinogen (PG) values to predict gastric acid secretion status. In this study, we investigated the clinical usefulness of measuring the serum pepsinogen values for identifying a high-risk group for gastric mucosal injury among chronic LDA users. METHODS: One hundred long-term LDA users were enrolled in this analysis. Serum from each subject was subjected to determination of H. pylori status and measurement of pepsinogen values. According to our recent report, a PG I value ≥ 50 ng/mL was defined as estimated hyperchlorhydria in H. pylori-negative subjects, while a PG I/II ≥ 3.3 was defined as estimated hyperchlorhydria in H. pylori-positive subjects. The grade of gastric mucosal injury was assessed endoscopically, and multiple logistic regression analyses were used to estimate the risk. RESULTS: Estimated hyperchlorhydria was a strong independent risk for intensive gastric mucosal injury with an OR (95% CI): 34.0 (4.5-259) and for gastric ulcer with an OR (95% CI): 10.2 (1.8-58.3) in H. pylori-positive subjects, while it was not a significant risk in H. pylori-negative subjects. The association persisted even after excluding those with conventional risks for LDA-gastropathy such as ulcer histories. CONCLUSION: Using simple serum measurement of H. pylori antibody and pepsinogen concentrations, an extremely high-risk group for LDA-induced gastropathy could be extracted, and these patients should become a therapeutic target for prevention of LDA-induced gastropathy.

Characterization of the Drosophila ortholog of mouse eIF-3p48/INT-6.

The mouse mammary tumor virus (MMTV) has been shown to integrate frequently into INT-6 in MMTV-induced mouse mammary tumors. The INT6 gene has been highly conserved through evolution and has recently been shown to encode the p48 component of the eucaryotic translation initiation factor 3 (eIF-3) complex. We report here the isolation of the Drosophila eIF-3p48/INT-6. The gene comprises three exons within 1.8kb of genomic DNA located at cytogenetic position 73C2 in the Drosophila genome. The 1.5kb eIF-3p48/INT-6 RNA species encodes a protein composed of 364 amino-acid residues whose sequence is 71% similar to that of the mouse/human eIF-3/INT-6 amino-acid sequence. eIF-3p48/INT-6 RNA is expressed throughout development in Drosophila and the encoded protein is associated with the microsomal subcellular fraction.

Long-term effect of Helicobacter pylori eradication on the reversibility of acid secretion in profound hypochlorhydria.

BACKGROUND: Although profound hypochlorhydria is considered to be an important risk factor for development of gastric cancer, long-term effect of Helicobacter pylori eradication on its reversibility remains uncertain. AIM: To clarify the change in acid secretion after eradication in a long-term follow-up over 5 years in patients with profound hypochlorhydria. METHODS: Twenty-three H. pylori-positive patients with hypochlorhydria (<0.6 mmol/10 min) were enrolled prospectively. Assessment of gastrin-stimulated acid output and histologic evaluation of biopsy specimens were performed prior to, and 1, 7 months after eradication. Subsequently, gastric acid secretion was assessed for long-term period over 5 years after eradication in 12 patients. RESULTS: Gastric acid secretion was reversed to normal range in nine of 23 patients (39%) at 7 months after eradication. In the long-term follow-up, gradual and significant recovery in gastric acid secretion was observed up to 2 years post-therapy. However, there was no additional increase during the last 3 years of 5-year follow-up period, leaving the acid secretory levels subnormal in the majority of the patients. CONCLUSIONS: This long-term follow-up study suggests that the pathologic process has already progressed to an irreversible stage in the majority of H. pylori-positive patients with marked body atrophy and profound hypochlorhydria.

Double immunostaining for c-erbB-2 and p53 in human stomach cancer cells.

We examined 32 cases of surgically resected stomach cancer for the immunohistochemical expression of p53 and c-erB-2 protein and correlated the findings with clinical outcome and established prognostic factors. p53 Was observed in the nuclei of tumor cells in 11 of 32 cases (33%) and c-erbB-2 immunoreactivity was observed in nine of 32 cases (27%). In eight cases both p53 and c-erbB-2 were positive; however, double immunostaining revealed that less than a third of the same tumor cells were positive for both p53 and c-erbB-2 in these cases. The immunohistochemical localization patterns of p53 and c-erbB-2 were not related to the histopathologic differentiation of the carcinoma cells. No correlation was observed between expression of p53 and/or c-erbB-2 and the clinical outcome and established prognostic factors, including clinical stage and histologic types of the tumor examined. These results indicate that abnormalities of p53 and c-erbB-2 may not play major roles in the biologic behavior of human stomach cancer and that abnormalities in p53 and c-erbB-2 do not necessarily occur simultaneously in the development of stomach cancer.

Analysis of p53 abnormalities in endoscopic gastric biopsies.

We performed immunohistochemical staining for p53 in 152 endoscopic gastric biopsy specimens, including 39 adenomas, 80 carcinomas, and 33 cases of regenerative atypia. Direct DNA sequencing of exons 5,7 and 8 of the p53 gene was performed on 8 specimens. Nuclear p53 immunoreactivity was observed in 36 of 80 carcinomas, 7 of 39 adenomas and none of the cases of regenerative atypia. Three of the adenomas demonstrated diffuse or focal p53 immunoreactivity. All three cases were associated with severe degrees of epithelial dysplasia, and two of them harbored carcinoma on subsequent polypectomy. Among 6 p53 positive carcinomas and adenomas sequenced, 3 showed DNA mutations, all G:C to A:T transitions. These results indicate that positive p53 immunoreactivity in intraepithelial atypical cells on gastric biopsies represents true dysplasia or carcinoma rather than regenerative atypia.

The polymorphism interleukin 8 -251 A/T influences the susceptibility of Helicobacter pylori related gastric diseases in the Japanese population.

BACKGROUND: Helicobacter pylori infection is associated with variable clinical outcomes, including gastroduodenal diseases, and genetic factors may be relevant in this process. AIMS: We investigated the effects of an interleukin 8 (IL-8) gene polymorphism on the risk of gastroduodenal diseases, the degree of H pylori induced gastritis, and IL-8 gene transcription. SUBJECTS: The study was performed in 244 healthy control subjects and 690 H pylori positive patients with non-cardia gastric cancer, gastric ulcer, duodenal ulcer, or gastritis. METHODS: We identified the IL-8 -251 A/T polymorphism by direct sequence analysis, and measured the gastritis score and serum pepsinogen (PG). The transcriptional promoter activity of the IL-8 gene was assessed by luciferase assay. RESULTS: IL-8 -251A was associated with a higher risk of gastric cancer and gastric ulcer. Patients carrying IL-8 -251A showed an increased risk of gastric cancer (odds ratios (OR) 2.01 (95% confidence interval (CI) 1.38-2.92)) and gastric ulcer (OR 2.07 (95% CI 1.37-3.12)). Compared with patients younger than 49 years, atrophy and metaplasia scores in the antrum were significantly higher and the PG I/II ratio significantly lower in -251A carriers than in T/T carriers. In the in vitro assay, IL-8 -251A showed enhanced promoter activity in response to IL-1beta or tumour necrosis factor alpha. CONCLUSIONS: The IL-8 -251A allele may be associated with progression of gastric atrophy in patients with H pylori infection, and may increase the risk of gastric cancer and gastric ulcer in Japanese people.

Cell proliferation and apoptosis in normal and pathologic human adrenal.

Cell proliferation and programmed cell death play important roles in the maintenance of tissue dynamics. The adrenal cortex has been known as a cell renewal tissue. We studied cell proliferation by Ki67 immunostaining and programmed cell death or apoptosis by a recently developed 3'-OH nick end labeling technique. Fifteen cases of normal human adrenal; 22 cases of adrenocortical adenoma including Cushing's adenoma (five cases), aldosteronoma (nine cases), and nonfunctioning adenoma (eight cases); and six cases of adrenocortical carcinoma were examined. In normal adrenal cortex, Ki67 immunoreactivity was predominantly observed in the zona fasciculata in all the cases examined, whereas cortical cells positive for nick end labeling were present in the zona reticularis in all cases and in the zona glomerulosa in five cases. These results suggest that the "cell migration" or "centripetal" theory is also applicable in cell turnover of normal human adrenal cortex, and cortical cells may move in two directions, from fasciculata to reticularis and from fasciculata to glomerulosa in some instances. CD68-positive sinusoidal lining cells, which are considered to ingest the cells undergoing apoptosis, were present throughout the cortex. In adrenocortical adenoma, Ki67 immunoreactivity was observed in all cases, and tumor cells positive for nick end labeling were observed in 12 cases (Cushing's adenoma in three cases, aldosteronoma in four cases, and nonfunctioning adenoma in five cases). In adrenocortical carcinoma, Ki67 immunoreactivity was observed in all the cases, and its labeling index was significantly higher than that of normal adrenal and adrenocortical adenoma.(ABSTRACT TRUNCATED AT 250 WORDS)

In situ analysis of tissue dynamics and p53 expression in human gastric mucosa.

In situ tissue dynamics were studied in 12 cases of human gastric mucosa, including normal gastric body mucosa and gastric glands with intestinal metaplasia, obtained from gastrectomy specimens of adenocarcinoma. Cell proliferation was determined by Ki67 immunoreactivity. DNA fragmentation was studied in situ by TdT-mediated dUTP-biotin nick end labelling (TUNEL). In addition, p53 expression was examined by both immunohistochemistry and mRNA in situ hybridization. In the oxyntic gastric glands, Ki67 immunoreactivity was observed exclusively in the proliferative zone and TUNEL-positive cells were present predominantly in the surface foveolar epithelium. In the gastric glands with complete intestinal metaplasia, Ki67-positive cells were present in the lower portion of the glands and TUNEL-positive cells in the superficial epithelium. In the gastric glands with incomplete intestinal metaplasia, TUNEL-positive cells were detected in the lower gastric glands adjacent to cells immunoreactive for Ki67; the proportion of these gastric glands with TUNEL-positive cells (40 out of 108 glands) was significantly higher than for oxyntic glands (94 out of 620 glands) or for glands with complete metaplasia (31 out of 254 glands). Relatively strong p53 immunoreactivity and mRNA hybridization were also observed in the proliferative and apoptotic areas of gastric glands with incomplete intestinal metaplasia. These results indicate that incomplete intestinal metaplasia is associated with increased cell turnover and p53 overexpression, possibly in response to various noxious or DNA-damaging stimuli.

The chromosome location of the human homolog of the mouse mammary tumor-associated gene INT6 and its status in human breast carcinomas.

The INT6 gene is a common integration site for the mouse mammary tumor virus in mouse mammary tumors. We have determined that the human homolog of INT6 is located on chromosome region 8q22-q23. A processed INT6 pseudogene is located on chromosome 6q. INT6 is composed of 13 exons that span 45 kb of genomic DNA. The deduced amino acid sequence of the gene product is identical to the mouse protein and contains three potential translation start signals. We have examined 100 primary breast carcinoma DNAs for evidence of genetic alteration affecting INT6. Loss of heterozyosity (LOH) was detected in 11 of 39 (28%) of the tumor samples informative for a polymorphic sequence in intron 7 of INT6. Since single-strand conformation and hybrid mismatch analysis of the remaining allele in these tumor DNAs failed to detect any mutations, we conclude that the target gene for LOH must be closely linked to INT6.

Analysis of cell damage and proliferation in Helicobacter pylori-infected human gastric mucosa from patients with gastric adenocarcinoma.

Helicobacter pylori (HP) infection, a cause of multifocal atrophic gastritis, is considered an important factor related to the evolution of the human gastric mucosa from normal to intestinal-type adenocarcinoma. We examined cell proliferation and both double and single strand DNA damage in situ in 35 patients undergoing gastrectomy for adenocarcinoma with HP-infected gastric mucosa by immunolocalization of Ki-67, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, and in situ nick translation. We also studied the distribution of intraepithelial neutrophils by elastase immunolocalization. HP infection was confirmed in all cases by serum anti-HP antibodies, ureas testing, and histopathological examination. HP-infected gastric mucosa was classified according to the degree of inflammation and intestinal metaplasia. Ki-67, terminal deoxynucleotidyl transferase-mediated labeling, in situ nick translation, and intraepithelial neutrophil indices all increased with the progression of gastritis and were highest in glands with incomplete intestinal metaplasia. All indices were lowest in gastric glands with complete intestinal metaplasia. Significant positive correlations were observed among these markers. Increased proliferative activity in HP-associated chronic gastritis in response to cell damage or injury was clearly demonstrated, suggesting that both HP-associated toxins and intraepithelial neutrophils are important in HP-related gastric epithelial injury. Increased cell turnover associated with incomplete intestinal metaplasia may result in DNA instability and subsequent development of intestinal-type gastric adenocarcinoma in HP-infected mucosa.