Ultrastructural study of Helicobacter pylori-associated gastritis

Endoscopic biopsies of antral mucosa from 26 patients with Helicobacter pylori-associated gastritis were studied by electron microscopy (EM). Scanning electron microscopy (SEM) showed clustering of H. pylori in the intercellular areas, being entrapped by the microvilli which were decreased at the sites where the bacilli were seen. The observations of SEM were confirmed by transmission electron microscopy (TEM), which showed adherence of the bacilli to the cell surface, producing cup-shaped depressions in the epithelial cells, and occasionally intracellular infiltration by H. pylori. There were also depletion of mucus granules, degenerative changes, and disruption of intercellular junction complexes of the epithelial cells. Post-treatment biopsies showed complete disappearance of the bacilli, and ultrastructural changes associated with H. pylori infection were resolved.     

Helicobacter heilmannii-associated gastritis: clinicopathologic findings and comparison with Helicobacter pylori-associated gastritis

The aims of this study were to evaluate the clinicopathologic features of Helicobacter heilmannii-associated gastritis and to compare H. heilmannii-associated gastritis with H. pylori-associated gastritis. We reviewed 5,985 consecutive gastric biopsy specimens. All cases of chronic gastritis with Helicobacter infection were evaluated with the Updated Sydney System, and the grades of all gastritis variables were compared between H. heilmannii-associated gastritis and H. pylori-associated gastritis groups. There were 10 cases of H. heilmannii-associated gastritis (0.17%) and 3,285 cases of H. pylori-associated gastritis (54.9%). The organisms were superficially located within the mucous layer without adhesion to epithelial cells. Interestingly, in one case many intracytoplasmic H. heilmannii organisms were observed in parietal cells with cell damage. A case of low-grade mucosa-associated lymphoid tissue (MALT) lymphoma concomitant with H. heilmannii infection was detected. Compared to H. pylori-associated gastritis, H. heilmannii-associated gastritis showed less severe neutrophilic activity (p<0.0001), mononuclear cell infiltration (p=0.0029), and endoscopic findings of chronic gastritis devoid of erosion or ulcer (p=0.0309). In conclusion, we present the detailed clinicopathologic findings of H. heilmanniiassociated gastritis compared to H. pylori-associated gastritis. H. heilmannii-associated gastritis is uncommon and milder than H. pylori-associated gastritis, however it may be noteworthy with respect to the development of MALT lymphoma.     

Analysis of cell damage in Helicobacter pylori-associated gastritis

Helicobacter pylori infection is currently considered to be a major cause of acute and chronic gastritis, and of gastric and duodenal ulcers. Superoxide dismutase (SOD) is well known for scavenging superoxide radicals such as reactive oxygen species (ROS), subsequently protecting cells from oxidative injury, and for maintaining tissue homeostasis. In this study, we therefore evaluated the level of SOD activity and protein expression, as well as various factors associated with oxidative injury, in H. pylori-positive (n = 46) and -negative (n = 28) gastric mucosa obtained from endoscopy, in order to elucidate the possible biological significance of SOD in these mucosa. Overall SOD activity was significantly higher in H. pylori-positive mucosa (15.5 +/- 7.0 U/mg protein) than in negative mucosa (9.2 +/- 10.6 U/mg protein), and decreased markedly following H. pylori eradication (8.2 +/- 4.2 U/mg protein). Enzyme-linked immunosorbent assay (ELISA) analysis of SOD revealed that the manganese SOD (Mn-SOD) level in H. pylori-positive mucosa (1166.7 +/- 435.2 ng/mg protein) was significantly higher than in control tissues (446.3 +/- 435.3 ng/mg protein) and in mucosa obtained following eradication therapy (431.9 +/- 189.9 ng/mg protein). The level of Mn-SOD protein showed a significant correlation with degree of inflammation in the gastric mucosa. Moreover, Mn-SOD immunolocalization patterns were well correlated with the activity and protein levels evaluated by ELISA. Factors presumably associated with oxidative injury in human gastric mucosa, including terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling, Ki-67, 8-hydroxydeoxyguanosine and single-stranded DNA, were all significantly higher in H. pylori-positive gastric mucosa than in control tissue and in tissue following eradication. These results all suggest that Mn-SOD, but not cytoplasmic copper-zinc SOD, plays an important role as an anti-oxidant against ROS generated in H. pylori-infected gastric mucosa and, subsequently, in the maintenance of cell turnover in gastric mucosa.     

B-Cell monoclonality in Helicobacter pylori-associated chronic atrophic gastritis

B-cell monoclonality has been reported not only in gastric lymphoma, but also in 1.3-21% of Helicobacter pylori-associated chronic gastritis (Hp-CG) cases. The aim of this study was to determine the significance of B-cell monoclonality in Hp-CG. We examined 134 gastric biopsy specimens from 99 patients with Hp-CG. The density of Hp, polymorphonuclear neutrophil activity, chronic inflammation, glandular atrophy, and intestinal metaplasia (IM) were scored according to the updated Sydney System. B-cell monoclonality was analyzed for immunoglobulin heavy chain gene rearrangement using polymerase chain reaction amplification. B-cell monoclonality was detected in 6% of informative samples. B-cell monoclonality was found in 18% of the samples from Hp-CG patients with marked glandular atrophy but in none of the samples from Hp-CG patients with none to moderate glandular atrophy. Monoclonality was also detected in 20% of the samples from Hp-CG patients with marked IM, in 11% of the samples from Hp-CG patients with moderate IM, and in none of the samples from Hp-CG patients without IM. Therefore, B-cell monoclonality was significantly more frequent in Hp-CG patients with marked glandular atrophy than in Hp-CG patients with none to moderate atrophy. It was also more significantly frequent in Hp-CG patients with moderate or marked IM than in Hp-CG patients without IM (P < 0.05). Of 35 Hp-CG patients, 26 (74%) had identical B-cell populations in the antrum and the corpus, and all were polyclonal. The remaining nine (26%) Hp-CG patients had B-cell populations that differed in the antrum and the corpus. Four of the nine (44%) showed monoclonal B-cell populations in at least one gastric biopsy specimen. There were no patients with monoclonal B-cell populations in both the antrum and the corpus. These data suggest that glandular atrophy and IM in gastric biopsy specimens may be markers for gastric mucosa-associated lymphoid tissue (MALT) lymphoma-genesis and that multiple gastric biopsy specimens from both the antrum and the corpus may be needed to assess the risk of gastric MALT lymphoma.     

Role of activated protein C in Helicobacter pylori-associated gastritis

The protein C (PC) pathway has recently been suggested to play a role in the regulation of the inflammatory response. To further extend the anti-inflammatory effect of activated PC (APC) in vivo, particularly its biological relevance to human disease, the activity of APC in the mucosa of patients with Helicobacter pylori-associated gastritis and the effect of vacuolating cytotoxin (VacA), cytotoxin-associated antigen (CagA), and H. pylori lipopolysaccharide (LPS) on PC activation were evaluated. This study comprised 35 patients with chronic gastritis. There were 20 patients with and 15 without H. pylori infection. The levels of PC and APC-PC inhibitor (PCI) complex were measured by immunoassays. The level of PC was significantly decreased and the level of APC-PCI complex was significantly increased in biopsy specimens from gastric corpus and antrum in patients with H. pylori-associated gastritis as compared to H. pylori-negative subjects. The concentrations of VacA, CagA, and LPS were significantly correlated with those of the APC-PCI complex in biopsy mucosal specimens from the gastric corpus and antrum. H. pylori LPS, VacA, and CagA induced a dose-dependent activation of PC on the surface of monocytic cells. APC inhibited the secretion of tumor necrosis factor alpha (TNF-alpha) induced by H. pylori LPS. Overall, these results suggest that H. pylori infection is associated with increased APC generation in the gastric mucosa. The inhibitory activity of APC on TNF-alpha secretion may serve to protect H. pylori-induced gastric mucosal damage.     

Helicobacter pylori-associated gastritis in mice is host and strain specific.

The vacA and cagA geno- and phenotypes of two mouse-adapted strains of Helicobacter pylori, SS1 and SPM326, were determined. The SS1 strain, which had the cagA+ and vacA s2-m2 genotype, induced neither vacuole formation in HeLa cells nor interleukin-8 (IL-8) production in KATO III cells. In contrast, H. pylori SPM326, with the cagA+ and vacA s1b-m1 genotype, induced vacuoles as well as IL-8 production in vitro. Furthermore, a spontaneous mutant of SPM326, which produced a vacuolating cytotoxin but was not able to induce IL-8 production (SPM326/IL-8(-)), was detected. C57Bl/6 and BALB/c mice were infected with these three strains to investigate the colonization pattern and the effect on the immune response in vivo. The SS1 strain colonized the stomachs of all mice in large numbers which remained constant over time. Colonization with the SPM326/IL-8(+) and SPM326/IL-8(-) strains was lesser, or even absent, and decreased over time. At 5 weeks postinoculation all three H. pylori strains induced a mild increase of neutrophil count in the gastric corpus of C57Bl/6 mice, which disappeared by 12 weeks. At both 5 and 12 weeks postinoculation C57Bl/6 mice colonized with SPM326/IL-8(+) showed an increased expression of major histocompatibility complex (MHC) class II antigen in the cardia which was accompanied by an increased number of T cells. C57Bl/6 mice that were infected with SS1 and SPM326/IL-8(-) did not show chronic inflammation. BALB/c mice colonized with SS1 and SPM326/IL-8(-) also showed an increase in neutrophil count at 5 weeks, which normalized again by 12 weeks postinoculation. At this time point SS1-infected mice showed inflammation in the corpus and antrum. At these sites an increased expression of MHC class II antigens and an increased number of T cells were observed. Although small lymphoid follicles were already observed 5 weeks after inoculation with SS1, their incidence as well as their number was increased at 12 weeks. These results show that inflammation induced by H. pylori depends both on the bacterial strain and the host.     

CTLA-4 blockade inhibits induction of Helicobacter pylori-associated gastritis in mice

The balance between Th1 and Th2 response determines the outcome of Helicobacter pylori infection. Interferon (IFN)-gamma plays an inductive role in gastric inflammation, whereas interleukin (IL)-4 counterbalances Th1 response and suppresses the development of gastritis. Th cell response is regulated by co-stimulatory factors. A co-stimulatory molecule, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), plays an inhibitory role in IL-2-dependent cell growth and mediates an optimal inhibitory signal to Th1 and Th2 cells. We administered anti-CTLA-4 monoclonal antibody (MoAb), which blocks CTLA-4 signalling, to examine the relative role for this signalling during maturation of Th1 and Th2 cells in H. pylori infection in mice. Mice treated by anti-CTLA-4 MoAb within the first week of infection showed an inhibition of gastric inflammation, accompanied by an increasing ratio of H. pylori-specific IgG1/IgG2a in serum following infection. Furthermore, the treatment resulted in the higher ratio of IL-4/IFN-gamma by splenocytes in response to H. pylori antigen at 6 weeks after infection, compared with untreated mice. These results suggest that the predominance of Th2 response by CTLA-4 blockade leads to an inhibition of the development of gastric inflammation. CTLA-4 signalling could contribute to the regulation of Th subsets and the development of gastric inflammation in H. pylori infection.     

dupA polymorphisms and risk of Helicobacter pylori-associated diseases

The dupA of Helicobacter pylori has been suggested as a virulence marker associated with the development of duodenal ulcer disease. However, the studies performed in different geographical areas have shown that there are variations in the prevalence of dupA and its association with H. pylori clinical outcomes. Our group did not observe associations between the presence of dupA and H. pylori clinical outcomes in Brazil. On the other hand, we observed 2 mutations in the sequence of dupA that lead to stop codons: a deletion of an adenine at position 1311 and an insertion of an adenine at position 1426 of the gene. Our aim was to evaluate associations of the presence of dupA with duodenal ulcer and gastric cancer, considering dupA-positive only those H. pylori strains that do not have the mutations in the gene sequence. We also evaluated the effect of infection with a strain carrying an intact dupA on the gastric mucosa histology and IL-8 gastric levels. Colonization with strains that had the intact dupA was negatively associated with gastric carcinoma (p=0.001, OR=0.32, 95% CI=0.16-0.66). The presence of dupA was also associated with an increased degree of antral mucosa inflammation (p=0.01) and with decreased corpus atrophy (p<0.01) as well as with increased gastric mucosa IL-8 levels (p=0.04). In conclusion, the infection with a H. pylori strain containing the dupA without the stop codon polymorphisms is associated with a lower risk of development of gastric carcinoma in Brazilian subjects.     

Mucosal lymphocyte subsets and HLA-DR antigen expression in paediatric Helicobacter pylori-associated gastritis

Paediatric studies may provide important insights into the immunopathology of Helicobacter pylori-associated gastritis, as mucosal changes reflect different stages of the immunoinflammatory response. We characterized, by quantitative immunohistochemistry, gastric mucosal lymphocyte phenotype and HLA-DR antigen expression and evaluated correlation with histopathology, in H. pylori-infected (Hp+ve) and uninfected children (Hp-ve). In the infected group, lamina propria CD3+ and IgA plasmocyte cell numbers were significantly higher and a trend for predominance of CD8+ over CD4+ was observed both in epithelium and lamina propria. A correlation of inflammation score with lamina propria CD3+ and CD4+ cell numbers and of CD45RO+ T lymphocytes with density of colonization was observed. The proportion of epithelial cells expressing HLA-DR antigen was significantly higher in the Hp+ve group and furthermore, glandular HLA-DR expression correlated with lamina propria CD3+ cell numbers, emphasizing the potential role of epithelial cells as antigen-presenting cells at this stage of infection.     

Inflammation as a basis for Helicobacter pylori-associated diseases

The molecular-cellular features of gastric mucosal inflammation caused by Helicobacter pylori are formulated. Chronic gastritis is considered as the morphological basis of gastric ulcer and non-cardial cancer of the stomach of intestinal type. The basic influences of the products of a pathogenicity island of bacterium (Cag A-protein) and VacA-cytotoxin on the gastric mucosa, as well as bacterial genetic polymorphism are discussed. The evolution of the phenotypes of chronic gastritis-- "ulcerative" "cancerous" is analyzed. Helicobacter pylori-associated inflammation may depend on the host's inflammatory response which in turn is determined by the allelic polymorphism of cytokine genes rather than the genetic characteristics of a bacterial strain.     

The role of apoptosis and proliferation of epitheliocytes in morphogenesis of Helicobacter pylori-associated gastritis

77 patients with chronic Helicobacter gastritis verified endoscopically and exacerbation of duodenal ulcer were examined. H. pylori infection was identified by the rapid ureasa test (CLO-test) and Giemza staining. The patients received 7-day three-component therapy for eradication of H. pylori. Apoptosis and proliferation were studied in 16 patients in serial sections with the use of monoclonal antibodies. Eradication of H. pylori resulted in relief of inflammation and transformation of active gastritis in inactive one. H. pylori-associated gastritis is associated with activation of apoptosis of gastric mucosa epithelial cells and epitheliocytes proliferation. H. pylori eradication alters correlation between apoptosis of epitheliocytes and their proliferation: successful eradication of the infection decreases apoptosis, high proliferative activity of epitheliocytes persists reflecting enhancement of regeneration in gastric mucosa.     

Simultaneous EBV-positive lymphoepithelioma-like carcinoma and EBV-negative intestinal-type adenocarcinoma in a patient with Helicobacter pylori-associated chronic gastritis

We report the case of a 72-year-old man with 2 simultaneous gastric carcinomas. The larger, ulcerated mass in the antrum was a conventional infiltrating intestinal-type adenocarcinoma. The associated antral-type mucosa showed moderate chronic gastritis, foci with complete and incomplete intestinal metaplasia, and mild to moderate Helicobacter pylori infection. The second, smaller tumor was found within fundic-type mucosa and was a lymphoepithelioma-like carcinoma associated with Epstein-Barr virus (EBV) infection shown by the EBV-encoded small RNA (EBER) test. The EBER test result was negative in the intestinal type adenocarcinoma. To our knowledge, this is the first report of simultaneous gastric carcinomas with 2 different morphologic phenotypes, in which only one tumor was associated with EBV infection, while the second tumor was related to H pylori-associated chronic gastritis. Our report demonstrates 2 different but simultaneous etiologic pathways of gastric carcinogenesis in the same patient.     

B-cell monoclonality precedes the development of gastric MALT lymphoma in Helicobacter pylori-associated chronic gastritis.

Little is known about the temporal changes in Helicobacter pylori density and B-cell clonality during the evolution from chronic gastritis to gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Biopsied specimens from 28 patients with chronic gastritis who developed gastric MALT lymphoma (group A) and from 24 similar patients who did not (group B) during an equivalent follow-up period (mean, 42 months) were retrospectively scored for histological features of MALT lymphoma (0 to 5) and H. pylori density (0 to 3). B-cell clonality was analyzed by polymerase chain reaction (PCR). During the observation period, the H. pylori density in group A decreased significantly in comparison with group B; the mean change in H. pylori density (final minus initial density) per 1000 days was -1.4 for group A and +0.2 for group B (P < 0.005). Monoclonality was detected more frequently in group A (79%) than in group B (21%; P < 0.005), and it preceded the histological evidence of malignant transformation in 64% of those patients who showed monoclonality in group A. These results suggest that H. pylori is thus more closely associated with the precursor or initial phase in the genesis of gastric MALT lymphoma than with the later phase, as its density decreases as the tumor progresses. The detection of B-cell monoclonality by PCR is thus of possible use for predicting the histological genesis of gastric lymphoma.     

Helicobacter heilmannii gastritis: association with acid peptic diseases and comparison with Helicobacter pylori gastritis.

We analyzed 2 antral and 1 corpus full-thickness random endoscopic gastric mucosal samples obtained from 946 patients with duodenal ulcers (6077 biopsies) and from 281 patients with nonsteroidal anti-inflammatory drug-associated gastric ulcers (1794 biopsies). We stained tissue sections with hematoxylin and eosin and Warthin-Starry silver stain and immunostained them with polyclonal antibodies against Helicobacter pylori. Hematoxylin- and eosin-stained sections from 6 patients with Helicobacter heilmannii (18 biopsies) and 23 randomly selected patients with H. pylori (68 biopsies) were evaluated and semiquantitated for the presence of acute inflammation, chronic inflammation, glandular atrophy, intestinal metaplasia, H. pylori, H. heilmannii, lymphoid follicles, or vasodilatation. Additional specimens were obtained for H. pylori culture, a CLO test, and serologic examination. H. heilmannii was detected in 6 (0.49%) of 1227 patients (14 [0.18%] of 7871 biopsies). Of these, 4 (0.42%) of 946 were patients with duodenal ulcers (9 [0.15%] of 6077 biopsies), and 2 (0.71%) of 281 were patients with nonsteroidal anti-inflammatory drug-associated gastric ulcers (5 [0.28%] of 1794 biopsies). We found H. heilmannii with hematoxylin and eosin stain, Warthin-Starry stain, and immunoperoxidase stain for H. pylori. Culture for H. pylori was negative in the four patients with duodenal ulcers. The CLO and serologic tests were positive in three of five and five of five patients, respectively. Our results indicate that H. heilmannii, like H. pylori, is associated with peptic ulcer disease (both active and inactive gastritis) and that it preferentially colonizes the gastric antrum. The severity of the H. heilmannii-associated gastritis is less intense and lymphoid aggregates are less common than in H. pylori-associated gastritis. Morphologic detection seems to be the method of choice for detecting H. heilmanni. Immunoperoxidase stain specific for H. pylori also stains H. heilmannii, indicating cross-reacting antigenic epitopes between H. heilmannii and H. pylori.     

Usefulness of a new serological test (Bio-Rad) to diagnose Helicobacter pylori-associated gastritis.

A semiquantitative serological test (G.A.P. test; Bio-Rad) to diagnose Helicobacter (Campylobacter) pylori-associated gastritis has recently become commercially available. This test was evaluated with sera from 72 dyspeptic patients with known H. pylori status. When the instructions provided by the manufacturer were applied strictly, the sensitivity of the test was only 32.7%. With a slight modification of the instructions, the performance characteristics were still unsatisfactory (specificity, 43.5%). When a new cutoff value was determined by a receiver operating curve, the quality of the test (positive predictive value, 95.6%; negative predictive value, 77.8%) was slightly lower than that of a noncommercial enzyme-linked immunosorbent assay (positive predictive value, 95.7%; negative predictive value, 84.0%). It is concluded that the Bio-Rad G.A.P. test has low diagnostic yield, and it is stressed that commercial tests should be evaluated extensively before being applied in daily practice.     

Association of Helicobacter pylori with gastritis and peptic ulcer diseases

The occurrence of Helicobacter pylori(H.pylori) and its relationship with gastric mucosa were studied by light and electron microscopy and culture of biopsy specimens from gastric mucosa of 160 patients with upper gastrointestinal symptoms. H. pylori were present in 96.6% of patients with active chronic gastritis, 100% of patients with duodenal ulcer and 76.9% of patients with gastric ulcer, while present in only 6.3% of individuals with histologically normal gastric mucosa. The bacteria colonized the antral mucosa more frequently than the body or than the duodenal cap mucosa. The bacteria were rarely seen in the intestinalized epithelium per se, but there was no significant difference in prevalence of H. pylori between gastritis with intestinal metaplasia and gastritis without intestinal metaplasia. H. pylori could be seen in close association with the surface of gastric epithelial cells below the mucus layer without evidence of intracellular parasitism, All of the strains tested were susceptible to penicillin, erythromycin, and most of them susceptible to tinidazole and bismuth salts. It is concluded that H. pylori are highly associated with gastritis and peptic ulcer diseases and its prevalence rates in patients with those diseases is higher than in developed countries. This strong association of H. pylori infection with gastritis and peptic ulcer diseases suggest a possible etiologic role for the bacterium in those diseases.