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Unlabelled: Deregulated cell turnover in Helicobacter pylori (H. pylori)-colonized gastric mucosa has been suggested to be linked to the gastric carcinogenesis pathway. We previously reported attenuation of apoptosis and enhancement of cellular proliferation in the H. pylori-colonized gastric mucosa of Mongolian gerbils as compared to that in mice, which might reflect a specific link between H. pylori colonization and carcinogenesis in the Mongolian gerbils; the difference between the two strains could be attributable to differences in the host genetic background. Inducible-type nitric oxide synthase (iNOS) is thought to participate in not only the inflammatory response, but also in the regulation of gastric mucosal cell turnover in H. pylori-colonized gastric mucosa. Thus, the present study was designed to examine gastric leukocyte activation and epithelial cell apoptosis in the gastric mucosa following H. pylori inoculation in iNOS-knockout mice., Methods: iNOS-knockout mice (iNOS(-/-)) and their iNOS(+/+) littermates were orally inoculated with the Sydney strain of H. pylori (SS1, 10(8) colony-forming units [CFU]). H. pylori infection was confirmed by microaerobic bacterial culture. The stomach of each mouse was evaluated 14 weeks and 30 weeks after the inoculation. Gastric mucosal accumulation of polymorphonuclear leukocytes (PMN) was assessed by determining the myeloperoxidase (MPO) activity and histological score based on the updated Sydney system. The level of apoptosis was determined by estimation of the cytoplasmic levels of mono- and oligonucleosomes and by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method., Results: The SS1-inoculated mice showed persistent H. pylori colonization for 12 weeks. While gastric mucosal PMN infiltration increased following SS1 inoculation in both iNOS(+/+) and iNOS(-/-)strains, enhanced DNA fragmentation was observed in only SS1-colonized iNOS(+/+) mice, and not in the iNOS(-/-) mice. In conclusion, although the recruitment of PMN in response to H. pylori was evoked even in the gastric mucosa of iNOS(-/-) mice, epithelial cell apoptosis induced by H. pylori was attenuated in this strain. These data suggest that iNOS may play an important role in promoting apoptosis in the H. pylori-infected inflamed gastric mucosa, and that persistent inflammation without apoptosis in iNOS(-/-) mice with H. pylori infection may be linked to preneoplastic transformation.

Objective: The aim of this pharmacogenomics study was to investigate the influence of different cytochrome P450 (CYP) genotypes in Helicobacter pylori eradication therapy., Method: The study involved 143 consecutive Italian Caucasian patients with H. pylori infection diagnosed and treated with 1-wk triple therapy according to European Helicobacter Pylori Study Group guidelines. Using human genomic DNA, CYP2C19 (*2 and *3) and CYP3A4 alleles (*1B, *2, and *3) were evaluated by polymerase chain reaction-restriction fragment length polymorphism assays and confirmed by sequencing the amplicons., Result: According to the endoscopy-based gold standard, 93 patients achieved H. pylori eradication. Regarding CYP2C19 genotype, the 50 patients who remained infected were all homozygous or heterozygous extensive metabolizers (homEM or hetEM). Carriers of homEM fared significantly less well than those of hetEM; homEM genotype was also predictive of failure at univariate/multivariate analysis. Carriers of CYP3A4 polymorphisms achieved favorable eradication rates similar to patients bearing CYP2C19. All four patients with single CYP3A4*2 polymorphism achieved eradication, and only 29% (5/17) of all CYP3A4*1B carriers did not achieve eradication. All nine patients carrying CYP3A4 polymorphisms in the CYP2C19 hetEM subgroup were cured, suggesting the possibility of a positive synergism between CYP3A4 and CYP2C19., Conclusions: This first pharmacogenomics study on the influence of different CYP genotypes on H. pylori therapy suggests that, as in Asian populations, CYP2C19 genotype patterns are probably also relevant in Caucasians receiving H. pylori eradication regimens that include omeprazole. The possibility of a favorable drug interaction mediated by CYP2C19 and CYP3A4 requires investigation.

Serum levels of pepsinogen and gastrin are parameters that can be used as biomarkers for gastric mucosa. The aim of this study was to validate these serum biomarkers, that is pepsinogen A (PGA), pepsinogen C (PGC), PGA/PGC ratio, and gastrin, as screening tests for precancerous lesions: atrophic chronic gastritis (ACG) or Helicobacter pylori-related corpus-predominant or multifocal atrophy. The study population was comprised of a subsample of 284 patients from the 451 included in the Eurohepygast cohort, between 1995 and 1997. The concentrations of PGA, PGC, and gastrin were measured by radioimmunoassays. Histological diagnosis was the gold standard. Cut-off points were calculated using receiving operator characteristics (ROC) curves. Factors linked to variation of biomarkers were identified using multivariate linear regression. The mean of each biomarker in the sample was: PGA, 77.4 microg x l(-1); PGC, 13.2 microg x l(-1); PGA/PGC, 6.7; and gastrin, 62.4 ng x l(-1). For ACG patients, the areas under the PGA, PGC, PGA/PGC, and gastrin ROC curves were 0.55, 0.62, 0.73, and 0.58, respectively. The best cut-off point for PGA/PGC was 5.6, with sensitivity 65% and specificity 77.9%. For H. pylori-related corpus-predominant or multifocal atrophy, the areas under the respective ROC curves were 0.57, 0.67, 0.84, and 0.69. The best cut-off point for PGA/PGC was 4.7, with sensitivity 77.1% and specificity 87.4%. The results suggested that only the PGA/PGC ratio can be considered as a biomarker for precancerous lesions of the stomach, and may be useful as a screening test.

Helicobacter pylori gastric infection has been associated with various digestive and extradigestive diseases. In liver disease bacterial infections have been associated with impairment of cytochrome P-450 liver metabolic activity. Moreover, infection by Helicobacter spp. seems to be linked with the development of hepatocellular carcinoma (HCC) in mice. Our aims were to evaluate the influence of H. pylori infection on cytochrome P-450 liver metabolic activity as assessed by means of monoethylglycinexylidide (MEGX) test and to assess the prevalence of H. pylori infection in patients with HCC. Ninety-six hepatitis C virus (HCV) -positive cirrhotic patients, 36 of whom had HCC, were tested for H. pylori infection by means of anti-H. pylori IgG. Patients underwent the MEGX test. Characteristics of the patients were then analyzed on the basis of the presence of H. pylori infection. Seroprevalence of H. pylori infection was similar between cirrhotic patients without (68%) or with (63.8%) HCC. Mean MEGX values were significantly (P < 0.0001) lower in H. pylori infected patients (18.2 +/- 13.9 ng/ml) as compared to the noninfected ones (46.9 +/- 17.1 ng/ml), independently of Child-Pugh's classification. These differences persisted even after subdividing patients according to the presence of HCC. In conclusion, in anti-HCV positive cirrhotic patients H. pylori infection is associated to an impairment of cytochrome P-450 liver metabolic activity. Seroprevalence of H. pylori infection in HCC patients is similar to that observed in tumor-free cirrhotics.

Background & Aims: Matrix metalloproteinases (MMPs), enzymes capable of degrading extracellular matrix components, are believed to be active in connective tissue remodeling associated with various physiologic processes and in pathologic conditions. The aim of this study was to analyze the molecular mechanism responsible for Helicobacter pylori-mediated MMP expression., Methods: Expression of MMP messenger RNA and MMP activity were assessed by reverse-transcription polymerase chain reaction and zymography, respectively. Chloramphenicol acetyltransferase assay was used to monitor activation of the MMP-9 gene promoter, and electrophoretic mobility shift assay was used to explore the binding of transcription factors to this promoter. Gastric tissue samples were immunohistochemically stained for MMP-9., Results: H. pylori induced MMP-9 expression in 2 gastric epithelial cell lines but had no effect on MMP-2. Induction of MMP-9 was dependent on an intact cag pathogenicity island. Activation of the MMP-9 promoter by H. pylori occurred through the action of nuclear factor kappaB. Transfection of kinase-deficient mutants of IkappaB kinase and nuclear factor kappaB-inducing kinase inhibited H. pylori-mediated activation of MMP-9. MMP-9 expression was higher in epithelial cells of H. pylori-positive tissue compared with those of H. pylori-negative tissue., Conclusions: H. pylori induced activation of nuclear factor kappaB through an intracellular signaling pathway that involved IkappaB kinase and nuclear factor kappaB-inducing kinase, leading to MMP-9 gene transcription. MMP-9 induction by H. pylori may play an important role in gastric inflammation, ulcer formation, and carcinogenesis.

Overproduction of nitric oxide by inducible nitric oxide synthase (iNOS) acts cytotoxically and contributes to inflammation. We explored the roles of iNOS in the pathogenesis of Helicobacter pylori-associated diseases. Using reverse-transcribed PCR, we examined topographical patterns of iNOS mRNA expression in the gastroduodenal mucosa in H. pylori-negative controls and H. pylori-positive patients with duodenal ulcer (DU), gastric ulcer (GU), and ulcer-free gastritis. iNOS expression showed topographical variations among the tested disorders. As compared to controls, DU had a significantly higher expression of iNOS mRNA in the duodenum, GU in the antrum and duodenum, and gastritis in the antrum and corpus. H. pylori eradication yielded a significant reduction of iNOS mRNA in the duodenum of DU and in the antrum of GU. Diverse topographical patterns of H. pylori-induced iNOS expression may contribute to mechanisms by which H. pylori elicits different clinical disorders.

It has long been thought that duodenal reflux induces residual gastritis after distal gastrectomy. H. pylori infection appears to be another factor in residual gastritis; and H. pylori induced gastritis may exist preoperatively or may have been introduced postoperatively. Up until now, the surgical effect itself and H. pylori infection have not been well differentiated as causes of residual gastritis. Our aim in this study was to clarify the relationship between the surgical effect and H. pylori infection in residual gastritis. A residual gastritis model using the Mongolian gerbil has been established with microsurgical technique. Residual gastritis with and without H. pylori infection has been studied by histopathological examination and quantitated by Rauws' score. The expression of cyclooxygenase (both COX-1 and COX-2) has also been examined immunohistologically. Elevation of pH in gastric juice after surgery was confirmed. H. pylori infection led to deterioration after surgery. The postoperative Rauws' score with infection is higher than without infection. Levels of COX-1 were higher after surgery in both animals. COX-2 was not expressed in the animals without infection and only a little was expressed in the animals with infection. COX-2 was strongly expressed in the operated animals with infection, but the surgical effect was minute in the animals without infection. Residual gastritis consisted of both surgical gastritis and H. pylori gastritis. H. pylori gastritis is curable with eradication of the organism, but surgical gastritis is not. The COX inhibitor can be a good candidate in preventing residual gastritis after eradication of the H. pylori organism. The clinical implications of COX expression for patients with residual gastritis might deserve further study in the point of treatment of surgical and H. pylori gastritis.

Human autoimmune gastritis (AIG) is an organ-specific inflammatory disorder leading to gastric atrophy and pernicious anemia. Gastric H+,K(+)-ATPase was identified as the autoantigen in both human disease and experimental murine AIG (EAIG). Studies of EAIG significantly contributed to current knowledge of human AIG, but to what extent EAIG mimics AIG is still debated, and the autoantigenic epitopes in AIG are yet unknown. This study aimed to identify the H+,K(+)-ATPase epitopes recognized by gastric T cell clones from AIG patients, to define their TCR Vbeta usage and epitope-induced cytokine response. Sixteen H+,K(+)-ATPase-reactive CD4+ gastric T cell clones of four AIG patients were tested for proliferation to overlapping 15-mer peptides spanning the a and beta chains of H+,K(+)-ATPase. We identified 6 epitopes in the a chain and 5 in the beta chain; TCR Vbeta usage was not restricted. Four (36%) of the 11 H+,K(+)-ATPase epitopes recognized in AIG were found to overlap with epitopes that are relevant in EAIG, including a previously described gastritogenic epitope. Gastric T cell recognition of the peptide epitopes resulted in secretion of Th1 cytokines. Our data suggest a striking similarity between human AIG and EAIG, at the epitope level, with regard to cytokine secretion and likely also with regard to pathogenic mechanisms.

Background: Adenosine deaminase is an enzyme which is postulated to have a role in the generation of gastric mucosal inflammation. The aim of our study was to determine and compare adenosine deaminase activity in the gastric mucosa of patients with chronic gastritis developed in partially resected and intact stomachs., Material/methods: 182 patients were studied, 102 non-operated and 80 after distal gastric resection. Biopsy specimens were taken endoscopically from the gastric mucosa 2 cm proximal to the stoma or corresponding upper third of the intact stomach. Gastritis was classified according to the Sydney system. The activity of adenosine deaminase in the mucosal homogenates was measured by determination of ammonia liberated from the substrate and expressed in nmol NH3/mg protein/min., Results: Adenosine deaminase activity was lower in partially resected than in intact stomachs, regardless of Helicobacter pylori infection. While no difference was found in adenosine deaminase activity between Billroth I and Billroth II procedures in subjects without H. pylori infection, the activity was lower in those with Billroth II procedure in the presence of H. pylori infection. As the severity of gastritis increased, enzyme activity decreased in the mucosa of the intact stomach, but was not significantly altered in the mucosa of the gastric remnant., Conclusions: Adenosine deaminase activity differs in intact and partially resected stomachs, but it does not appear to be a factor promoting chronic gastritis.

Pepsin K (chicken pepsin) in the test for hemoglobin proteolysis is 1.7 times superior to abomin (porcine pepsin) in the medium pH 2 and 44 times more potent in the medium pH 3. Even in pH 4 chicken pepsin retains its enzymatic activity while porcine pepsin in pH 4 looses its activity. These biochemical findings show that pepsin K is more effective in the treatment of enzymatic insufficiency of the stomach. Intake of 1-3 pepsin K tablets after meal for 4 weeks by 82 patients having chronic gastritis with secretory insufficiency was effective in 75% of patients.

Serum pepsinogen levels were measured in 137 stomach cancer patients and compared with those of 288 normal cancer-free subjects. The serum pepsinogen levels of stomach cancer patients, especially pepsinogen I and the pepsinogen I/pepsinogen II ratio were significantly lower than those of normal controls and correlated well with the extent of chronic gastritis associated with the cancerous stomach. These results were in good accordance with the results of previous studies indicating that the cancer derived from the stomach where chronic gastritis/intestinal metaplasia is extensive. The initial step of the screening procedure can be completed rapidly. Our results indicate that serum pepsinogen screening(pepsinogen test method) is a valuable method to detect gastric cancers in the Japanese population.

A CD4(+) T cell response to the gastric H/K ATPase beta-subunit (H/Kbeta) is required for the onset of experimental autoimmune gastritis in BALB/c mice. The extent to which endogenous H/Kbeta contributes toward the tolerance of the H/Kbeta-specific T cell repertoire in normal individuals is not known. By comparison of T cell responses in H/Kbeta-deficient (o/o) and H/Kbeta-expressing BALB/c mice, in this work we show that the endogenous H/Kbeta autoantigen plays a major role in the tolerance of pathogenic H/Kbeta-specific T cells. First, T cell-dependent Ab responses to the H/Kbeta Ag were enhanced in H/K ATPase-immunized H/Kbeta-deficient mice compared with wild-type mice. Second, peptide immunization experiments indicated that immune responses to the major gastritogenic epitope of the H/K ATPase, namely H/Kbeta(253-277), were significantly more vigorous in H/Kbeta-deficient mice compared with wild-type mice. Third, unfractionated splenocytes from H/Kbeta-deficient mice, but not H/Kbeta-expressing mice, induced autoimmune gastritis after adoptive transfer to BALB/c nude mice. The enhanced responses to H/Kbeta in H/Kbeta-deficient mice were shown to be intrinsic to CD4(+)CD25(-) T cells rather than a change in status of CD4(+)CD25(+) regulatory T cells. We conclude from these studies that the H/Kbeta-specific T cells in wild-type mice represent the residue of a T cell repertoire, directed toward a single determinant, that has been subjected to partial tolerance induction.

Helicobacter pylori infection induces innate immune responses in macrophages, contributing to mucosal inflammation and damage. Macrophage apoptosis is important in the pathogenesis of mucosal infections but has not been studied with H. pylori. NO derived from inducible NO synthase (iNOS) can activate macrophage apoptosis. Arginase competes with iNOS by converting L-arginine to L-ornithine. Since we reported that H. pylori induces iNOS in macrophages, we now determined whether this bacterium induces arginase and the effect of this activation on apoptosis. NF-kappa B-dependent induction of arginase II, but not arginase I, was observed in RAW 264.7 macrophages cocultured with H. pylori. The time course of apoptosis matched those of both arginase and iNOS activities. Surprisingly, apoptosis was blocked by the arginase inhibitors N(omega)-hydroxy-L-arginine or N(omega)-hydroxy-nor-L-arginine, but not by the iNOS inhibitor N-iminoethyl-L-lysine. These findings were confirmed in peritoneal macrophages from iNOS-deficient mice and were not dependent on bacterial-macrophage contact. Ornithine decarboxylase (ODC), which metabolizes L-ornithine to polyamines, was also induced in H. pylori-stimulated macrophages. Apoptosis was abolished by inhibition of ODC and was restored by the polyamines spermidine and spermine. We also demonstrate that arginase II expression is up-regulated in both murine and human H. pylori gastritis tissues, indicating the likely in vivo relevance of our findings. Therefore, we describe arginase- and ODC-dependent macrophage apoptosis, which implicates polyamines in the pathophysiology of H. pylori infection.

Modifications of mucosal phospholipids have been detected in samples from patients with Helicobacter pylori-positive gastritis. These alterations appear secondary to increased phospholipase A2 activity (PLA2). The cytosolic form of this enzyme (cPLA2), normally involved in cellular signaling and growth, has been implicated in cancer pathogenesis. The aim of this study was to investigate cPLA2 expression and PLA2 activity in the gastric mucosae of patients with and without H. pylori infection. In gastric biopsies from 10 H. pylori-positive patients, cPLA2 levels, levels of mRNA as determined by reverse transcriptase PCR, levels of protein as determined by immunohistochemistry, and total PLA2 activity were higher than in 10 H. pylori-negative gastritis patients. To clarify whether H. pylori had a direct effect on the cellular expression of cPLA2, we studied cPLA2 expression in vitro with different human epithelial cell lines, one from a patient with larynx carcinoma (i.e., HEp-2 cells) and two from patients with gastric adenocarcinoma (i.e., AGS and MKN 28 cells), incubated with different H. pylori strains. The levels of cPLA2, mRNA, and protein expression were unchanged in Hep-2 cells independently of cellular adhesion or invasion of the bacteria. Moreover, no change in cPLA2 protein expression was observed in AGS or MKN 28 cells treated with wild-type H. pylori. In conclusion, our study shows increased cPLA2 expression and PLA2 activity in the gastric mucosae of patients with H. pylori infection and no change in epithelial cell lines exposed to H. pylori.

Background: Human gastric mucosa contains three alcohol dehydrogenase (ADH) isozymes (classes I, III, and IV). Various factors such as Helicobacter pylori infection, sex, age, and the part of the stomach involved have been suggested to affect alcohol dehydrogenase activities, although these views are controversial. In this study, these unsettled issues were reexamined., Methods: Activities of class I and IV ADHs were evaluated in the cytosolic fraction of human gastric mucosa samples by reduction of their preferred substrates, namely acetaldehyde and m-nitrobenzaldehyde, and activities of class III were evaluated by oxidation of its preferred substrate, formaldehyde. Then, effects of Helicobacter pylori infection, sex, age, and the part of the stomach involved were examined., Results: Class I, III, and IV ADH activities were 17.5 +/- 8.4, 4.2 +/- 2.5, and 8.9 +/- 3.9 nmol of nicotinamide adenine dinucleotide oxidation per minute per milligram of protein, respectively, for the entire population. Helicobacter pylori infection significantly reduced class I and IV ADH activities but did not affect activity of class III. In the samples without Helicobacter pylori infection and severe gastritis, sex did not affect class I, III, or IV ADH activities. In the same series, class IV ADH activity significantly decreased with age (p = 0.006), whereas no correlation was found between age and ADH activity of class I and III ADHs. The level of class IV ADH activity was significantly higher in the upper body than in the lower regions, whereas no such heterogeneity was observed in class I and III ADH., Conclusions: Various factors affect human gastric ADH activities, such that careful interpretation of their significance is necessary.

Background: Knowledge about the possible role of phospholipase C (PLC) activity of microbial pathogens in the development of disease is increasing. Recently attention has focused on investigating PLC activity elaborated by Helicobacter pylori, but the role of this enzyme in H. pylori pathogenesis is still unknown. The aim of this study was to correlate PLC-activity of H. pylori on the basis of the cagA status with the clinical diagnosis of the patients., Materials and Methods: Helicobacter pylori was isolated from patients with gastritis (G; n = 38), duodenal ulcer (DU; n = 15), gastric ulcer (GU; n = 11) and gastric cancer (GC; n = 12). Polymerase chain reaction primers DZ3/R009 which amplified a 1350-bp fragment were used to detect the cagA gene. PLC activity was determined using p-nitrophenylphosphorylcholine as substrate., Results: Of the strains, 60% were cagA(+) and 40% were cagA(-). All strains showed PLC activity (2.20 +/- 0.91 U mg(-1) protein). PLC activity showed no association with the cagA status: cagA(+) (2.21 +/- 1.03 U mg(-1) protein), cagA(-) (2.18 +/- 0.79 U mg(-1) protein). Patients with GU had the highest PLC activity (2.77 +/- 1.26 U mg(-1) protein) and patients with GC had the lowest activity (1.8 +/- 0.57 U mg(-1) protein)., Conclusions: Although PLC activity was present in all strains tested, it may only have pathological importance in patients with GU. However, the extent of PLC activity was independent of the presence of the cagA gene.

Background: Nitric oxide (NO) is synthesized enzymatically from L-arginine by NO synthase, which is measured by inducible NO synthase (iNOS). Helicobacter pylori (H. pylori) infection produces a state of chronic immunostimulation in the gastric epithelium. Infection with cagA+ H. pylori has greater degree of gastric inflammation and epithelial cell damage. Therefore, we compared the levels of iNOS in patients with H. pylori infection in relation to cagA status and H. pylori-related disease., Materials and Methods: One hundred and seven patients, including 51 patients with gastric cancer, 12 patients with gastric ulcer, 18 patients with duodenal ulcer and 26 patients with chronic gastritis, were enrolled in this study. Biopsies from the antrum and body were obtained for histologic examination, culture and reverse transcriptionase-PCR (RT-PCR) for detection of iNOS gene expression. The presence of H. pylori was confirmed by Giemsa staining or culture and the gene expression of cagA in H. pylori isolates was confirmed by PCR., Results: H. pylori infection was detected in 70.1% (75/107) and cagA was detected in 84.8% (28/33). iNOS expression was detected in 49.5% (53/107) and there was no significant difference in iNOS expression according to H. pylori infection nor the cagA status in the gastroduodenal diseases. However, iNOS expression was more frequently detected in gastric cancer than the other H. pylori-related diseases (64.7% vs. 35.7%, p <.05)., Conclusion: Although NO was thought be involved in the gastric carcinogenesis, the level of NO production was not related to H. pylori infection or cagA status.

An increased expression of inducible nitric oxide synthase (iNOS) has been observed in the inflamed human gastric mucosa as well as in some tumors. This observation suggests a pathobiological role of elevated NO production. The purpose of this study was to compare the immunohistochemical iNOS expression in the different kinds of gastritis before and after the eradication of Helicobacter pylori. We performed iNOS and H. pylori immunohistochemical staining and counted iNOS positive cells. We detected elevated expression of iNOS around sites infected with H. pylori. iNOS expression in chemical gastritis was strongly elevated in mucosal glands. After treatment, we found a significant difference in iNOS expression in patients with classical H. pylori-induced antrum predominant gastritis and in patients with active autoimmune gastritis. In the special case of progressed gastritis with intestinal metaplasia we found persistence of intestinal metaplasia, and we could not find a significant difference in the number of positive iNOS cells before and after treatment. The persistence of IM as a possibly precancerous lesion is probably at least in the antrum a source of continuous iNOS induction even after H. pylori eradication.

Mechanisms leading to breakdown of immunological tolerance and initiation of autoimmunity are poorly understood. Experimental autoimmune gastritis is a paradigm of organ-specific autoimmunity arising from a pathogenic autoimmune response to gastric H/K ATPase. The gastritis is accompanied by autoantibodies to the gastric H/K ATPase. The best characterized model of experimental autoimmune gastritis requires neonatal thymectomy. This procedure disrupts the immune repertoire, limiting its usefulness in understanding how autoimmunity arises in animals with intact immune systems. Here we tested whether local production of GM-CSF, a pro-inflammatory cytokine, is sufficient to break tolerance and initiate autoimmunity. We generated transgenic mice expressing GM-CSF in the stomach. These transgenic mice spontaneously developed gastritis with an incidence of about 80% after six backcrosses to gastritis-susceptible BALBc/CrSlc mice. The gastritis is accompanied by mucosal hypertrophy, enlargement of draining lymph nodes and autoantibodies to gastric H/K ATPase. An infiltrate of dendritic cells and macrophages preceded CD4 T cells into the gastric mucosa. T cells from draining lymph nodes specifically proliferated to the gastric H/K ATPase. CD4 but not CD8 T cells transferred gastritis to nude mouse recipients. CD4(+) CD25(+) T cells from the spleen retained anergic suppressive properties that were reversed by IL-2. We conclude that local expression of GM-CSF is sufficient to break tolerance and initiate autoimmunity mediated by CD4 T cells. This new mouse model should be useful for studies of organ-specific autoimmunity.

Earlier reports suggested that adenosine deaminase activity in the gastric corpus mucosa depends upon either gastric acid secretion or severity of chronic gastritis. Knowing that gastric acid secretion corresponds well with histological status of the gastric mucosa, the aim of this study was to determine the enzyme activity in relation to these two factors evaluated simultaneously. The study was conducted on Helicobacter pylori positive duodenal ulcer patients treated for two weeks with either omeprazole alone or omeprazole in combination with amoxycillin and tinidazole. It was found that these two therapeutic regimens decreased adenosine deaminase activity only in the gastric corpus mucosa and a decline was more deeper in patients on omeprazole monotherapy. Moreover, omeprazole monotherapy inhibited completely basal gastric acid secretion and did not change the severity of chronic gastritis, while omeprazole-based eradication therapy decreased both the gastric acid secretion and severity of chronic gastritis. One month after completion of eradication therapy adenosine deaminase activity returned to the pretreatment values but severity of chronic gastritis decreased further. The results of the present study indicate that adenosine deaminase activity in the gastric corpus mucosa depends primarily upon gastric acid secretion but not upon the severity of Helicobacter pylori associated chronic gastritis.

FR167653 was discovered as a cytokine production inhibitor, but its target molecule has remained unclear. We examined the effect of FR167653 on activities of purified protein kinases. FR167653 dose dependently inhibited p38alpha mitogen-activated protein kinase activity without affecting the activities of other kinases. FR167653 had no effect on cyclooxygenase (COX)-1 or COX-2 activities, whereas SB203580 inhibited them. FR167653 suppressed endogenous p38 kinase activity in interleukin-1-stimulated NRK-F cells. These results indicate that FR167653 is a p38 kinase-selective inhibitor without affecting COX activity. To evaluate the role of p38 kinase in Helicobacter pylori gastritis, we therefore examined the effect of FR167653 on H. pylori-induced gastritis in Mongolian gerbils. H. pylori infection activated p38 kinase in the gastric mucosa and caused neutrophil infiltration from 2 and 3 weeks of infection, respectively. At 4 weeks, severe mucosal inflammation with erosive injury was observed. When FR167653 was administered to H. pylori-infected gerbils from 2 weeks, both neutrophil infiltration and mucosal injury at 4 weeks were significantly prevented. FR167653 markedly reduced the H. pylori-induced increase in endogenous p38 kinase activity in the gastric mucosa, and also significantly inhibited neutrophil chemokine production. In contrast, the drug did not affect H. pylori colonization or acid secretion. FR167653 did not cause any pathological change in the gastric mucosa of normal animals. These results indicate that p38 kinase plays a crucial role in H. pylori-induced gastritis in Mongolian gerbils.

We compared immunohistochemical and silver stains of pediatric gastric biopsy sections for the identification of Helicobacter pylori infection with chronic inflammation and a negative urease screening test. Thirty-seven patients (age range 10 months to 21 years) whose gastric antral biopsies were negative for the rapid urease test (CLO(R)) but positive for lymphocytic infiltration were selected for a retrospective study. Specimens had been subjected to a rapid urease test (CLO(R)) and hematoxylin and eosin staining, and Dieterle silver staining and immunohistochemical staining specific for H. pylori were also performed. Twelve additional patients with urease-positive biopsies were used as controls. With Dieterle staining, 8/37 (22%) urease-negative biopsies contained organisms morphologically compatible with H. pylori, 21/37 (56%) contained organisms not compatible with H. pylori, and 8/37 (22%) were negative for organisms. Immunostaining confirmed 6/8 (75%) Dieterle-positive cases as being H. pylori, was negative in 2/8 (25%) Dieterle-positive cases, and was positive in 2/8 (25%) Dieterle-negative cases. Biopsies from 8/12 (67%) urease-positive specimens contained organisms seen with both Dieterle and immunohistochemical stains, and 4/12 (33%) were negative with both stains. Although both stains yielded comparable results with H. pylori-positive biopsies, Dieterle staining was potentially confusing because of nonspecific staining of other organisms. A significant proportion of (CLO(R))-negative biopsies was positive for H. pylori with special stains. We therefore recommend the use of immunohistochemical staining rather than silver staining in the evaluation of urease-negative gastric biopsies demonstrating chronic inflammation in children.

Background and Aims: Constitutive cyclooxygenase (COX) 1 is believed to mediate prostaglandin dependent gastric protection. However, gastric mucosa contains cells capable of expressing inducible COX-2. We therefore investigated COX-1 and COX-2 expression, localisation, and activity in normal and abnormal human gastric mucosa., Methods: COX-1 and COX-2 distribution was investigated by light and electron microscopic immunohistochemistry and by western blot analysis, and their contribution to prostaglandin (PG)E(2) synthesis using selective enzyme inhibitors., Results: There was strong parietal cell COX-1 and COX-2 immunoreactivity in all sections and isolated cells, with macrophage and myofibroblast reactivity in some sections. Immunostaining was specifically abolished by antigen absorption. Western blot analysis confirmed COX-1 and 2 expression. COX-1 and COX-2 immunostaining was increased in Helicobacter pylori gastritis, particularly the mid glandular zone and lamina propria inflammatory cells. This was associated with increased ex vivo PGE(2) synthesis (62.4 (13.5) pg/mg v 36.3 (15.5) pg/mg in uninflamed mucosa; p=0. 017) which was significantly inhibited by COX-1 but not COX-2 inhibition. Increased COX-2 immunostaining in macrophages, endothelial cells, and myofibroblasts (with reduced epithelial expression) was seen at the rim of ulcers., Conclusion: COX-2, as well as COX-1, is expressed by normal human gastric mucosa and is increased at the rim of ulcers. Although both are increased with H pylori, COX-1 contributes more than COX-2 to gastric PGE(2) production.

Cyclooxygenase (COX)-2 expression is induced in the gastric mucosa of Helicobacter pylori-infected patients, but its role remains unclear. We examined the effects of NS-398 and indomethacin on gastric pathology in H. pylori-infected Mongolian gerbils. COX-1 was detected in both normal and H. pylori-infected mucosa, whereas COX-2 was expressed only in the infected mucosa. PGE(2) production was elevated by H. pylori infection, and the increased production was reduced by NS-398, which did not affect PGE(2) production in normal mucosa. Indomethacin inhibited PGE(2) production in both normal and infected mucosa. Hemorrhagic erosions, neutrophil infiltration, lymphoid follicles, and epithelium damage were induced by H. pylori infection. NS-398 and indomethacin aggravated these pathological changes but did not increase viable H. pylori number. H. pylori-increased production of neutrophil chemokine and interferon-gamma was potentiated by NS-398 and indomethacin. Neither NS-398 nor indomethacin caused any pathological changes or cytokine production in normal animals. These results indicate that COX-2 as well as COX-1 might play anti-inflammatory roles in H. pylori-induced gastritis.

Telomerase adds hexameric repeats of 5'-TTAGGG-3' to the ends of chromosomal DNA (telomere) and has been implicated in cell immortalization and cellular senescence. The aim of this study was to measure quantitatively the telomerase activity and human telomerase RNA component (hTR) content in gastric cancer and to examine the relation between these values and histologic factors including Helicobacter pylori as a risk factor for gastric cancer. Telomerase activity was measured by a modified telomeric repeat amplification protocol in cancerous and noncancerous tissues (intestinal metaplasia, chronic gastritis, normal mucosa) from 27 gastric cancer patients; hTR expression was examined by the quantitative reverse transcriptase-polymerase chain reaction using fluorescent probes. Telomerase activity was higher in cancers (total product generated: 33.7) than in noncancerous tissues. Telomerase activity was higher in intestinal metaplasia (16.7) and chronic gastritis (10.6) than in normal mucosa (3.5). In patients with intestinal-type gastric cancer, telomerase activity was higher in intestinal metaplasia with H. pylori infection than in that without infection. hTR expression was not correlated with telomerase activity. H. pylori infection may influence telomerase activity in cancer and noncancerous tissues.

Background: Prostaglandin endoperoxide synthase/cyclooxygenase (COX) is the key enzyme in gastric mucosal protection and repair but its cellular localisation in the human stomach is still unclear., Aims: To investigate immunohistochemically the cellular distribution of COX-1 and COX-2 proteins in the human stomach with or without gastritis or ulceration., Patients and Methods: Tissues were obtained by surgical resection of gastric ulcers associated with perforation (n = 9) or by biopsy from Helicobacter pylori positive patients with gastric ulcers (n = 45) and H pylori negative healthy subjects (n = 15). COX expression was detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and light and electron microscopic immunohistochemistry., Results: COX-2 mRNA and protein were detected in gastric ulcer tissues but not in intact gastric mucosa. COX-1 mRNA and protein were detected in the intact mucosa. COX-2 immunostaining was exclusively localised in macrophages and fibroblasts between necrotic and granulation tissues of the ulcer bed. The percentage of COX-2 expressing cells was significantly higher in open than in closed ulcers, and in gastritis than in gastric mucosa without H pylori infection. COX-1 immunoreactivity localised in lamina propria mesenchymal cells was similar in various stages of ulcer disease and in intact gastric mucosa. Electron microscopic immunohistochemistry revealed both COX-1 and COX-2 on the luminal surfaces of the endoplasmic reticulum and nuclear envelope of macrophages and fibroblasts., Conclusions: Our results showed that COX-2 protein was induced in macrophages and fibroblasts in gastric ulcers and H pylori related gastritis, suggesting its involvement in the tissue repair process.

Endothelin-1 (ET-1) is a vasoactive peptide produced from a biologically inactive big ET-1 by the action of endothelin-converting enzyme-1 (ECE-1). We investigated gastric mucosal expression of ECE-1 during a 10-day course of inflammatory responses associated with acute gastritis elicited by Helicobacter pylori lipopolysaccharide. The ECE-1 activity was associated with microsomal fraction and the level of its expression reflected the extent of mucosal inflammatory involvement. The histologic pattern of inflammation reached a maximum on the 4th day following the lipopolysaccharide and was accompanied by a 4.1-fold enhancement in the expression of ECE-1 activity and a significant elevation in ET-1 (3.1-fold), TNF-alpha (8.8-fold), and apoptosis (11.6-fold). A 41.5% decrease in the severity of mucosal inflammation by the 10th day following the lipopolysaccharide was reflected in a 62.3% reduction in the mucosal expression of ECE-1 and a decline in TNF-alpha, ET-1, and apoptosis. Thus, H. pylori infection causes up-regulation of gastric mucosal ECE-1 expression, which leads to the enhancement of ET-1 production, induction of TNF-alpha, and triggering the apoptotic events that exacerbate the inflammatory process., (Copyright 2000 Academic Press.)

Blood serum in stomach cancer and chronic gastritis has been compared. A sharp decrease in pepsinogen 1 level both in cancer and gastritis patients was found as compared with healthy subjects. Pepsinogen 1 level in poorly-differentiated tumor (37.4 ng/ml) was lower than in well-differentiated one (58.2 ng/ml).

Glutathione S-transferase (GST) enzymes are involved in detoxification of many potentially carcinogenic compounds. The homozygous deletions or null genotypes of GSTT1 (theta class) and GSTM1 (mu class) genes may be associated with an increased risk of cancer. Few studies have evaluated the relationship between GSTT1, GSTM1 and the risk of gastric cancer, as well as the potential interactions between these genetic markers and other risk factors of gastric cancer in the Chinese population. We conducted a case-control study with 143 cases with gastric cancer, 166 chronic gastritis (CG) cases and 433 cancer-free population controls from Yangzhong County, China. The epidemiological data were collected by a standard questionnaire for all of the subjects, and blood samples were obtained from 91 gastric cancer cases, 146 CG cases, and 429 controls. GSTT1 and GSTM1 genotypes were assayed by the PCR method, and Helicobacter pylori infection was measured by the ELISA method. Using logistic regression model in SAS, we assessed the independent effects of GSTT1 and GSTM1 null genotypes on the risk of gastric cancer and their potential interactions with other factors. The prevalence of GSTM1 null genotype was 48% in gastric cancer cases, 60% in CG patients, and 51% in controls. The prevalence of GSTT1 null genotype was 54% in gastric cancer cases, 48% in CG patients, and 46% in controls. After controlling for age, gender, education, pack-years of smoking, alcohol drinking, body mass index, H. pylori infection, and fruit and salt intake, the adjusted odds ratio (OR) for GSTT1 and gastric cancer was 2.50 (95% confidence interval (CI), 1.01-6.22). When gastric cancer cases were compared with CG patients, the adjusted OR for GSTT1 was 2.33 (95% CI, 0.75-7.25). However, GSTT1 null genotype was not associated with the risk of CG when using population controls. No obvious association was found between GSTM1 and the risk of both gastric cancer and CG. Our results suggest that GSTT1 null genotype may be associated with an increased risk of gastric cancer in a Chinese population.

Immunoglobulin A and IgM are subjected to epithelial transport only when they are produced as polymers with incorporated J chain. Immunocytes containing various Ig isotypes and associated J chain in gastric mucosa, as well as IgA-degrading protease activity in Helicobacter pylori cultures, were examined. Gastric body specimens from 15 H. pylori-positive and 14 H. pylori-negative patients were studied by paired immunofluorescence for IgA, IgA1, IgA2, IgG, or IgM and concurrent cellular J chain. H. pylori isolates were incubated with IgA1 or secretory IgA and examined by immunoelectrophoresis for cleavage products. A substantial increase of Ig-producing cells occurred in chronic gastritis, particularly in the IgA1 isotype, but H. pylori was shown to possess neither IgA1-specific nor nonspecific IgA-degrading protease activity. Regardless of infection status, reduced J chain expression was observed for all immunocyte isotypes (except for IgM) in inflamed compared with normal gastric body mucosa, the median positivity for IgA1 cells being reduced to 58.7% versus 87.9% (P = 0.0002), and for IgA2 cells to 48.9% versus 87.8% (P = 0.0002). This down-regulation of the J chain suggested that a large fraction of IgA monomers is produced in gastritis.

For a better understanding of the regulation of prostaglandin and nitric oxide (NO) synthesis in circumstances in which the gastric mucosa is inflamed, we have examined the ex vivo production of NO and prostaglandin E2 and the protein expression of inducible nitric oxide synthase (iNOS) and 2 cyclo-oxygenase (COX) isoforms in gastric biopsies from nine Helicobacter pylori-infected patients with active gastritis and six Helicobacter pylori (HP)-negative patients. The results indicate a significant increased of NO and PGE2 in patients with HP infection compared with uninfected samples. These findings were paralleled by marked increases in iNOS and in COX-1 and COX-2 protein expression. Expression of iNOS and COX-2 protein was absent in the mucosa of HP-negative controls. We have demonstrated that iNOS protein is expressed in the gastric mucosa of patients with HP infection. It is likely that iNOS expression and the corresponding high release of NO may play an important role in gastric inflammation associated with HP infection. However, the expression of COX-1 and COX-2 and the parallel increase of prostaglandin E2 could imply that these factors could limit the extend of mucosal damage. In previous reports NO has been shown to stimulate the COX activity, so we think that the role of NO could be both in the regulation of normal function and in the genesis of diseases.

We applied the animal model of H. pylori lipopolysaccharide-induced gastritis to assess the effect of antiulcer agent, sulglycotide, on the mucosal inflammatory responses by analyzing the interplay between the activity of a key apoptotic caspase, caspase-3, epithelial cell apoptosis, and the expression of constitutive (cNOS) and inducible (NOS-2) nitric oxide synthase. H. pylori lipopolysaccharide applied intragastrically elicited within 4 days a pattern of mucosal responses resembling that of acute gastritis. This was accompanied by an 11.2-fold increase in epithelial cell apoptosis, a 6.5-fold induction in mucosal expression of NOS-2 and a 2.2-fold decline in cNOS, and a 5.4-fold increase in caspase-3 activity. Treatment with sulglycotide led to a 56.7% reduction in the extent of mucosal inflammatory changes elicited by H. pylori lipopolysaccharide and an 88.3% decrease in the epithelial cells apoptosis. Furthermore, this effect of sulglycotide was associated with a 51% decrease in mucosal expression of caspase-3 activity, a 73.7% decline in NOS-2, and a 64.1% increase in cNOS. The findings suggest that sulglycotide suppresses the H. pylori-induced mucosal inflammatory responses by up-regulating cNOS and interfering with the events propagated by NOS-2 and caspase-3., (Copyright 1999 Academic Press.)

Background & Aims: Inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 are important regulators of mucosal inflammation and epithelial cell growth. To determine the role of iNOS and COX-2 in Helicobacter pylori-induced tissue injury, we compared their gene expression in H. pylori-induced gastritis with that in normal gastric mucosa and in non-H. pylori gastritis., Methods: In 43 patients, we assessed H. pylori infection status, histopathology, messenger RNA (mRNA) and protein expression, and cellular localization of iNOS and COX-2., Results: By reverse-transcription polymerase chain reaction (RT-PCR), antral iNOS and COX-2 mRNA expression was absent to low in normal mucosa (n = 10), significantly increased in H. pylori-negative gastritis (n = 13), and even more markedly increased in H. pylori-positive gastritis (n = 20). Increased iNOS and COX-2 levels were confirmed by Northern and Western blot analysis and were both greater in the gastric antrum than in the gastric body of infected patients. Immunohistochemistry also showed increased expression of both genes in H. pylori gastritis: iNOS protein was detected in epithelium, endothelium, and lamina propria inflammatory cells, and COX-2 protein localized to mononuclear and fibroblast cells in the lamina propria., Conclusions: iNOS and COX-2 are induced in H. pylori-positive gastritis and thus may modulate the inflammation and alterations in epithelial cell growth that occur in this disease. Higher levels of iNOS and COX-2 in H. pylori-positive vs. -negative gastritis and in gastric antrum, where bacterial density is greatest, suggest that expression of these genes is a direct response to H. pylori infection.

It has been known that patients with chronic renal failure have elevated concentrations of serum pepsinogens, which are raised by Helicobacter pylori (H. pylori) infection of the stomach. This study was conducted to examine how renal dysfunction and H. pylori infection affect serum pepsinogen (PG) concentrations. The subjects consisted of 93 patients with renal disease (60 males and 33 females with a mean age of 55 +/- 1.3 years). Dialysis patients were not included. Twenty-four-hour urinary collection was performed, and creatinine clearance (Ccr) was calculated for all the subjects. Status of H. pylori infection was assessed by serum IgG antibody against H. pylori. Fasting serum PG I and PG II were measured by RIA. The subjects were divided into 4 groups based on Ccr: group A: Ccr > or = 71 ml/min; group B: 30-70 ml/min; group C: 11-30 ml/min; group D: Ccr < or = 10 ml/min. Regardless of the H. pylori status, serum PG I concentrations were elevated as the renal function declined; serum PG I levels of groups C and D (177.5 +/- 15.2 ng/ml and 234.0 +/- 32.2 ng/ml, respectively) were significantly higher than those of group A (66.1 +/- 9.6 ng/ml, p < 0.01); Group D also had a significantly higher concentration of PG I than group B (106.0 +/- 17.2 ng/ml, p < 0.01). The same tendency was found in serum PG II concentrations. However, the differences among the 4 groups were not statistically significant (16.2 +/- 2.3, 24.2 +/- 4.0, 28.3 +/- 3.5, 34.3 +/- 5.6 ng/ml for group A, B, C, and D, respectively). There was a negative correlation between Ccr and serum PG I concentrations (r = -0.45, p < 0.01). In comparison between H. pylori-infected patients and uninfected patients, serum levels of PG II were higher in the former patients than in the latter. This difference was significant in groups A and C. Serum PG I concentrations were the same between H. pylori-infected patients and their uninfected counterparts for the 4 groups. The present study has shown that serum PG I concentrations are raised by a loss of renal function, while PG II levels are elevated mainly by H. pylori infection of the stomach. It is concluded that renal function and H. pylori infection should be taken into account when serum PG concentrations are evaluated in patients with renal diseases.

Objective: Helicobacter pylori (H. pylori) causes chronic gastritis. The inducible prostaglandin synthetase cyclooxygenase 2 (COX-2) plays an important role in inflammatory conditions. We hypothesized that H. pylori-associated chronic gastritis would express COX-2 protein. Our aim was to evaluate the effect of eradication of H. pylori infection on COX-2 expression in the antral mucosa of patients before and after antibiotic therapy., Methods: Tissues were obtained from patients with non-ulcer dyspepia undergoing H. pylori eradication. Ten patients with proven H. pylori infection and subsequent successful eradication were studied. Three biopsies of antral mucosa were evaluated before and after H. pylori eradication. The amount of acute and chronic inflammation was quantitated. Immunohistochemical staining for COX-2 was expressed as a percentage of the total number of cells and correlated with the degree of chronic inflammation., Results: Specific immunostaining for COX-2 was observed in antral mucosa of patients infected with H. pylori. Patchy cytoplasmic staining was seen in surface epithelial cells and strong cytoplasmic staining for COX-2 was seen in parietal cells. Spotty cytoplasmic staining for COX-2 was also seen in lamina propria plasma cells, as well as there being macrophages present in the germinal centers of lymphoid aggregates. COX-2 expression could be detected both before and after eradication of H. pylori. The mean percentage of cells staining for COX-2 was significantly higher in H. pylori-infected mucosa, compared with mucosa after successful H. pylori eradication (33.4% +/- 5.4 vs 18.9% +/- 3.3, p = 0.038). COX-2 immunostaining correlated best with the chronic inflammation score (r2 = 0.78, p < 0.001). There was a strong correlation for those subjects who were H. pylori infected, as well as for those who had successful H. pylori eradication., Conclusions: H. pylori associated acute and chronic antral inflammation was associated with immunohistochemical detection of COX-2 protein in epithelial cells, in addition to associated mononuclear cells and parietal cells. Expression was reduced, but not eliminated, in the epithelium after successful eradication of H. pylori. Despite the reduction in COX-2 expression after H. pylori eradication, expression of COX-2 in epithelial cells remained and strongly correlated with the extent of the chronic inflammatory cell infiltrate. The clinical implications of H. pylori-associated induction of COX-2 expression for patients on selective COX-2 inhibitors, in addition to the role of COX-2 in gastric carcinogenesis, deserve further study.

Background: How Helicobacter pylori infection affects gastric acid secretion has not been made clear. This study aimed to elucidate the effects of H. pylori infection on H+/K+-adenosine triphosphatase (ATPase) mRNA in gastric fundic gland mucosa., Methods: Twenty patients with chronic gastritis and H. pylori infection were treated with lansoprazole and antibiotics. Before and 1 month after treatment gastroduodenoscopy was performed, and changes in the amount of H+/K+-ATPase mRNA in the fundic gland mucosa, gastric juice pH, and serum gastrin levels were determined., Results: The amount of H+/ K+-ATPase mRNA in the fundic gland mucosa was increased in patients with eradication of H. pylori, in whom significant decreases in gastric juice pH and serum gastrin levels were observed. No significant changes were observed in patients without eradication of H. pylori., Conclusions: These results suggest that one of the mechanisms by which H. pylori infection suppresses acid secretion is by the inhibition of proton pump synthesis in parietal cells.

We have previously shown that autoimmune gastritis can be elicited in mice by immunization with the gastric parietal cell H/K ATPase alpha beta heterodimer and that tolerance specifically induced to the H/K ATPase beta-subunit protects mice from the development of gastritis. Here we have identified the immunodominant gastritogenic epitope of the H/K ATPase beta-subunit (H/Kbeta). Epitope mapping was carried out with a panel of 21 overlapping peptides that spanned the entire sequence of the gastric H/K ATPase beta-subunit. T cells from gastric H/K ATPase-immunized mice responded to only one of the overlapping peptides, namely H/Kbeta253-277. Furthermore, a single subcutaneous immunization of 6-week-old BALB/c mice with the ATPase beta-subunit peptides resulted in a T-cell response to only H/Kbeta253-277. Multiple immunization with the overlapping H/K ATPase peptides demonstrated that H/Kbeta253-277 was capable of inducing a mononuclear infiltrate specifically within the gastric mucosa. We conclude that H/Kbeta253-277 is the dominant gastritogenic epitope of the gastric H/K ATPase.

Chronic inflammation induced by Helicobacter pylori infection has been associated with an increased risk of stomach cancer. We have analysed 167 stomach biopsies from 99 patients for H. pylori infection and immunohistochemically for the expression of inducible nitric oxide synthase (iNOS), catalase and superoxide dismutases (SODs) as markers of oxidative stress. Biopsies were graded as follows on the basis of histology: normal, superficial gastritis, variable severity of atrophic gastritis with or without intestinal metaplasia, and dysplasia. iNOS was detected in inflammatory cells in all types of gastritis with or without H. pylori infection and independently of its severity. In foveolar cells, iNOS was observed in approximately 25% of all biopsies showing any type of gastritis, but in a markedly higher proportion of dysplastic samples. Catalase and Mn-type SOD in inflammatory cells and catalase in foveolar cells were more frequently observed in marked atrophic gastritis biopsies than in less severe gastritis. Individual differences were found in the expression of these enzymes within groups with the same severity of gastritis. Prolonged oxidative stress in severe gastritis and dysplasia may play an important role in gastric carcinogenesis, through increased damage of DNA and tissue by reactive oxygen and nitrogen species.

Helicobacter pylori lipopolysaccharide is recognized as a primary virulence factor evoking acute mucosal inflammatory reaction associated with H. pylori infection. We investigated the activity of a key apoptotic protease, caspase-3, and the expression of inducible nitric oxide synthase (NOS-2) during H. pylori lipopolysaccharide-induced acute gastritis. The assays conducted 4 days following intragastric dose of the lipopolysaccharide revealed a pattern of acute mucosal responses characterized by an 11.2-fold increase in epithelial cells apoptosis, inflammatory infiltration of the lamina propria, hyperemia, and epithelial hemorrhage. This was accompanied by a 5.4-fold increase in caspase-3 activity, while the mucosal expression of NOS-2 showed a 6.5-fold induction. The results implicate H. pylori lipopolysaccharide in the induction of NOS-2 expression, and point to its effect on activation of the signaling cascade involving caspase-3 in the process gastric epithelial cells apoptosis.

Nitric oxide produced by inducible nitric oxide synthase (iNOS) has been claimed to be involved in gastritis; however, its characteristics are largely unknown. We assessed (1) iNOS expression in the human gastric mucosa in chronic gastritis and (2) the cytokines associated with iNOS expression. Gastric biopsy specimens were obtained from patients with chronic gastritis. Total RNA was isolated and reverse transcription-polymerase chain reaction (PCR) was performed semiquantitatively using specific primer sets for iNOS, interleukin (IL)-1beta, IL-8, IL-6, interferon-y, tumor necrosis factor-alpha (TNF-alpha), TNF receptors, IL-6 receptors, and sucrase, respectively. Helicobacter pylori infection was examined by the PCR assay. Reverse transcription-PCR analysis showed that expression of iNOS was detected in 10 of 23 samples. Expression of iNOS mRNA was closely correlated with expression of TNF-alpha and was observed frequently in subjects with intestinal metaplasia. The Helicobacter pylori gene was detected by PCR assay in all iNOS-positive cases. These results indicate that iNOS is predominantly expressed in the gastric mucosa with intestinal metaplasia and H. pylori infection. TNF-alpha is thought to be an important cytokine associated with iNOS expression.

Gastric biopsy specimens were taken from 737 patients undergoing upper gastrointestinal endoscopy and assessed for Helicobacter pylori infection. The diagnostic utilities of H. pylori culture (733 patients), detection of urease production (724 patients) and histopathological examination (469 patients) were compared. Since each of these techniques may fail to diagnose patients infected with H. pylori, an attempt was made to estimate the true rate of infection using a mathematical approach that combined the results of culture, histopathology and urease testing; 34% of the 733 patients were thought to be infected. Using this figure as a benchmark, the sensitivity, specificity, positive predictive value and negative predictive value of H. pylori culture were 73.2%, 100%, 100% and 86.3%, respectively, compared with 58.7%, 100%, 100% and 89.6%, respectively for urease production and 77.0%, 100%, 100% and 82.4%, respectively for histopathology. Thus, histopathological examination was the single most reliable test. A combination of histopathological examination and H. pylori culture diagnosed 99.5% of patients that were estimated to be truly infected. The minimum inhibitory concentrations of a number of antibiotics were measured for 135 isolates of H. pylori. All isolates were susceptible to amoxycillin and tetracycline whereas 5.2% were resistant to clarithromycin and 60% were resistant to metronidazole.

Intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LpLs) have not been well studied in gastric mucosa, particularly in lymphocytic gastritis. Therefore, they were immunohistologically characterized with antibodies recognizing CD3, CD8, CD57, T cell-restricted intracellular antigen (TIA-1), and granzyme B (GrB). The TIA-1 labels cytotoxic granules of resting and activated T-cells, whereas GrB decorates activated cytotoxic T cells. Thirty patients with celiac disease, including 20 taking gluten and 10 on a gluten-free diet, 15 patients with nonceliac disease-associated lymphocytic gastritis, and 20 controls were studied. Stained cells were counted and results were given as IELs/100 epithelial cells or percentage of lamina propria cells. Sixty percent to 90% of CD3+ IELs and up to 12% of lamina propria cells contained TIA-1-positive cytotoxic granules. The number of GrB+ IELs and LpLs was increased in Helicobacter pylori-positive controls (p < 0.03 vs. H pylori-negative controls) and celiac disease patients taking gluten (p < 0.05 vs. controls). The highest number of GrB+ IELs and LpLs was found in nonceliac disease-associated lymphocytic gastritis (p < 0.009 vs. controls, p < 0.05 vs. celiac disease). This study shows that a high proportion of gastric IELs and LpLs is potentially cytotoxic in nature. Through stimuli not yet identified, a proportion of them becomes activated after H pylori infestation and in lymphocytic gastritis.

Thymectomy of susceptible strains of mice on day 3 of life results in a spectrum of organ-specific autoimmunity that can be prevented by reconstitution of the thymectomized animals early in life with normal adult lymphocytes. The effectors and suppressors of autoimmunity in this model have been convincingly shown to be CD4+ T cells. It has been demonstrated recently that the regulatory CD4+ T cells that prevent disease coexpress CD25. We have further characterized the population of CD4+CD25+ immunoregulatory cells and demonstrated that they can suppress not only the induction of disease post-thymectomy, but can also efficiently suppress disease induced by cloned autoantigen-specific effector cells. Furthermore, the CD4+CD25+ T cells appear to be members of a unique lineage of regulatory T cells, as the induction of CD25 expression on a monospecific population of T cells derived from TCR transgenic SCID mice did not result in suppression of post-thymectomy autoimmunity. In addition, the TCR transgenic SCID mice were highly susceptible to autoimmune disease induced by the cloned line of autoantigen-specific effectors, while normal mice were relatively resistant. The capacity of the cloned line to transfer disease to nu/nu recipients could be inhibited by normal spleen cell populations containing CD4+CD25+ cells and by purified CD4+CD25+ cells. Although the target Ag(s) and mechanism of action of the CD4+CD25+ T cells remain to be determined, it is likely that they also play an important role in modulating other autoimmune diseases that are mediated by activation of "ignorant" self-reactive T cells present in the normal peripheral lymphocyte pool.

Immunohistochemistry has been critical in determining the tissue localization of inducible nitric oxide synthase (iNOS). However, this technique suffers from nonspecific staining which may lead to false-positive results and the failure of antisera to recognize iNOS from different species. We developed a technique to determine the localization of iNOS mRNA, as opposed to protein, in tissue sections using an in situ RT-PCR (IS RT-PCR) technique. Sections of inflamed gastrointestinal mucosa were used because they were known to be positive for iNOS. The IS RT-PCR technique localized iNOS mRNA to the same sites as immunoreactive iNOS in human gastritis associated with Helicobacter pylori infection, Crohn's disease, and experimental inflammatory bowel disease induced by the hapten TNBS, in rat colon and in guinea pig ileum. The detection of mRNA had an excellent signal-to-noise ratio. Preservation of tissue morphology was poorer than that with immunohistochemistry due the cycles of heating required in the PCR process. This method could be very useful in detecting iNOS gene expression in situations of excessive nonspecific staining with immunohistochemistry or of failure of antibodies to react because of species differences. This technique is also readily applicable to detect RNA and DNA markers of other disease processes.

The stomach provides some protection against the penetration of ethanol into the body by contributing to the metabolism of ethanol. The latter is attenuated by various drugs and, although the magnitude of this effect is still the subject of debate, patients should be warned of the corresponding possible increase in blood alcohol levels. Furthermore, oxidation of ethanol generates acetaldehyde, a toxic metabolite. In addition, chronic alcohol abuse seems to favor colonization by Helicobacter pylori, which produces ammonia that also contributes to the commonly associated chronic gastritis. Because antibiotics were shown over the last 4 decades to effectively eliminate gastric ammonia, they should be considered for the routine treatment of such chronic gastritis in the way they are now being used for ulcer therapy.

Background & Aims: Autoimmune gastritis is associated with gastric H+, K(+)-adenosine triphosphatase (ATPase)-specific autoantibodies (HKAb). The (auto) antigen that triggers disease and the pathogenic role of the autoantibodies are unknown. The aim of this study was to analyze when these autoantibodies are produced during autoimmune gastritis in neonatally thymectomized mice and whether a native H+, K(+)-ATPase antigen preparation can induce disease in mice., Methods: Autoantibodies were characterized by a novel assay based on immunoprecipitation of a functional H+, K(+)-ATPase expressed in Xenopus oocytes. Normal mice were injected intraperitoneally with H+, K(+)-ATPase-enriched gastric membranes in the absence of adjuvant., Results: Conformational autoantibodies recognizing both H+, K(+)-ATPase subunits appeared simultaneously with the gastric lesions 1 month after thymectomy. Immunization of neonates, but not adults, induced a persistent autoimmune gastritis in the body mucosa, characterized by lymphocytic infiltrations, loss of parietal and chief cells, metaplasia, and H+, K(+)-ATPase-specific autoantibodies. The histopathological lesions of this new model are similar to those in humans and thymectomized mice., Conclusions: The onset of gastritis and autoantibody production parallels the expression of the H+, K(+)-ATPase during ontogeny. Exposure of the neonatal immune system to organ-specific antigens expressed late after birth induces autoimmune gastritis in adult mice.

There is an accumulation of evidence to suggest that mast cells may play a key role in gastrointestinal inflammation. We have investigated the numbers and heterogeneity in staining properties of mast cells in biopsies of the duodenum of normal subjects (n = 10), and of normal duodenum from patients with Crohn's disease of the ileum and/or colon (n = 7) or with Helicobacter-associated gastritis of the antrum/corpus (n = 6). In normal donors, two subsets of mast cells, one located in the duodenal mucosa and the other in the submucosa, were clearly distinguished by their morphology and dye-binding properties. Whereas submucosal mast cells stained metachromatically with Toluidine Blue after neutral formalin fixation and emitted a yellow fluorescence after staining with Berberine sulphate, those in the mucosa were invisible using these stains. In patients with gastritis or Crohn's disease, there were marked changes in the numbers of mucosal mast cells compared with control subjects even though the duodenal biopsies were from apparently uninvolved tissue. Gastritis was associated with increased mucosal mast cell numbers (controls: 187 +/- 23 cells mm-2; gastritis: 413 +/- 139 cells mm-2; p = 0.0004), but mean mucosal mast cell counts in the uninvolved duodenum of Crohn's patients were actually decreased (34 +/- 30 cells mm-2, p = 0.0147). The clear differentiation between mucosal and submucosal mast cells on the basis of metachromasia with Toluidine Blue was not seen in biopsies from the patients with gastritis or Crohn's disease. Previous studies which have suggested that there are no distinct mucosal and submucosal mast cell subsets in the human intestine may, therefore, have been affected by the use of tissue from diseased subjects. Heterogeneity in the expression of mast cell tryptase and chymase was seen by immunohistochemistry using specific antibodies, but the relative numbers of mast cell subsets were critically dependent on the methods used. Using a sensitive staining procedure, the majority of mucosal mast cells stained positively for chymase as well as for tryptase, an observation confirmed by immunoelectron microscopy and immunoabsorption studies. Our findings suggest that early stages in intestinal inflammation may be reflected in changes in mast cell numbers and in their staining properties, and call for a reappraisal of mast cell heterogeneity in the human intestinal tract.