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

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

Helicobacter 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.     

Diagnostic value of detection of IgM antibodies to Helicobacter pylori.

This report describes the diagnostic value of anti-Helicobacter pylori IgM detection. Serum samples from 9043 symptomatic and asymptomatic individuals were evaluated with ELISA for the presence of anti-H. pylori IgG, IgM, and IgA. The specificity of detected IgM was confirmed by inhibition and cross-reactivity assays. Treatment of IgM-positive specimens with 1% 2-mercaptoethanol resulted in approximately 90% inhibition. Our data suggest a low level of cross-reactivity (5%) between H. pylori and four different enteropathogenic bacteria tested. The specificity of anti-H. pylori IgM was also demonstrated by Western blot and linearity studies. Data show that the detected IgM is highly specific. Western blot analysis revealed a variable IgM response to H. pylori antigens among patients, with the most reactive antigenic fractions being in the range of 55- to 100-kDa. Overall, the data confirm the diagnostic value of anti-H. pylori IgM detection. The prevalence of IgM antibodies to H. pylori in tested sera was significantly higher in symptomatic patients (10.4%) than in asymptomatic individuals (1.1%). Likewise, the percentage of sera positive for IgM alone was higher in symptomatic than in asymptomatic groups (3.8 vs 0.22%). About 5% of sera were positive only for IgA. We concluded that ELISA can be used for the detection of specific IgM to H. pylori and that the presence or absence of IgM antibodies to H. pylori may reflect whether or not an acute infection exists.     

Intramucosal Helicobacter pylori in the human and murine stomach: its relationship to the inflammatory reaction in human Helicobacter pylori gastritis

Intramucosal Helicobacter pylori (H. pylori) has been described in biopsy tissues and culture systems. However, the association of intramucosal H. pylori with histopathologic features has not been evaluated. The purpose of this study is to investigate the relationship between intramucosal H. pylori and inflammatory reactions in H. pylori infection. In 113 randomly selected human gastric biopsies and 20 murine stomachs, which were inoculated with SSI every day for a week, immunohistochemical analysis for intramucosal H. pylori was done and correlated with histologic parameters. Electron microscopic examination was done on murine stomachs. H. pylori infection was present in 104 gastric biopsies and 17 murine stomachs. Intraepithelial immunopositivity for H. pylori was detected in 27 of 104 (26%) biopsies and in 11 of 17 (65%) murine stomachs. Lamina proprial immunopositivity for H. pylori was present in 51 of 104 (48%) biopsies. Neutrophil-associated immunopositivity for H. pylori was observed in 22 of 90 (24%) biopsies with H. pylori chronic active gastritis. Lamina proprial and neutrophil-associated immunopositivity for H. pylori correlated significantly with the density of H. pylori and the grade of acute inflammatory reaction in H. pylori gastritis. Intramucosal location of H. pylori itself or its antigen is closely associated with acute inflammatory reactions and may play an important role in establishing a persistent infection in chronic H. pylori gastritis. Furthermore, lamina proprial and/or neutrophil-associated H. pylori appears to be more important than intraepithelial H. pylori in acute inflammatory reactions of H. pylori gastritis.     

Comparison of enzyme immunoassays detecting Helicobacter pylori specific IgG in serum and saliva with endoscopic and biopsy findings in patients with dyspepsia

Purpose: To compare the performance of two indirect enzyme-linked immunosorbent assays (ELISA) detecting Helicobacter pylori (HP)-specific IgG antibodies in serum and saliva with endoscopic observations and histologic findings of biopsies from dyspeptic patients, in an area of high HP prevalence. Materials and Methods: Sera, saliva and antral biopsies were obtained from 55 dyspeptic patients. IgG antibodies against HP were assayed in sera and saliva utilizing two indirect ELISAs. Biopsies were processed according to standard procedures in order to detect histological changes and the presence or absence of Helicobacter pylori. Laboratory data thus obtained were compared and statistically analyzed. Results: Forty-two (76.36%) biopsies were positive for HP. The organisms were detected in 4 of 16 (25%) cases with normal endoscopic findings, in all 16 cases of gastritis and in 22 of the 23 (95.6%) cases of duodenal ulcers (DU). Serum and saliva HP-specific IgG antibodies were detected in 4 normal cases with positive biopsies, in 12 and 14 cases of gastritis, respectively, and in all 22 (100%) biopsy positive cases of DU. The sensitivities of the serum and saliva tests were 90.5% and 95%, respectively, while the specificities were 84.5% and 70%, respectively. Conclusion: Due to their high sensitivity and specificity in diagnosing HP-associated DU and gastritis, serum and saliva antibody testing seems to offer a valuable alternative to invasive procedures especially in areas of high HP prevalence such as ours; saliva antibody testing is simple and practical especially in children and in difficult patients who resent venipuncture.     

Interleukin-6 polymorphism and Helicobacter pylori infection in Brazilian adult patients with chronic gastritis

Helicobacter pylori is recognised as the most common cause of chronic active gastritis and this bacterium is also an important pathogenic factor in peptic ulcer disease. The biological factors that influence clinical outcome in H. pylori infection have been extensively studied. In addition to immunological factors in the host, bacterial virulence determinants in H. pylori strains are likely to play a crucial role in gastric cancer development. Singlenucleotide polymorphisms at the 5' flanking region of the interleukin (IL)-6 gene promoter (G or C at -174 base) have been identified and individuals with the G allele at position -174 have been shown to produce higher levels of IL-6 than those with the C/C genotype. The mucosal levels of IL-6 were reported to be increased in H. pylori-associated gastritis. The present study was conducted to examine any relationship between inflammatory cytokine polymorphisms and the inflammatory process in mucosa infected by H. pylori. In our study we did not find any association between the C and G alleles in adult patients with chronic gastritis and inflammatory process in gastric mucosa.     

Relationship between Helicobacter pylori virulence genes and clinical outcomes in Saudi patients

Helicobacter pylori has been strongly associated with gastritis, gastric and duodenal ulcers, and it is a risk factor for gastric cancer. Two major virulence factors of H. pylori have been described: the cytotoxin-associated gene product (cagA) and the vacuolating toxin (vacA). Since considerable geographic diversity in the prevalence of H. pylori virulence factors has been reported, the aim of this work was to determine if there is a significant correlation between different H. pylori virulence genes (cagA and vacA) in 68 patients, from Saudi Arabia, and gastric clinical outcomes. H. pylor was recognized in cultures of gastric biopsies. vacA and cagA genes were detected by polymerase chain reaction (PCR). The cagA gene was obtained with 42 isolates (61.8%). The vacA s- and m- region genotypes were determined in all strains studied. Three genotypes were found: s1/m1 (28%), s1/m2 (40%) and s2/m2 (26%). The s2/m1 genotype was not found in this study. The relation of the presence of cagA and the development of cases to gastritis and ulcer was statistically significant (P < 0.05). The study showed a significant correlation between the vacAs1/m2 genotype and gastritis cases, and a significant correlation between vacAs1/m1 genotype and peptic ulcer cases. The results of this study might be used for the identification of high-risk patients who are infected by vacAs1/m1 genotype of H. pylori strains. In conclusion, H. pylori strains of vacA type s1 and the combination of s1/m1 were associated with peptic ulceration and the presence of cagA gene.     

Antigastric autoantibodies in Helicobacter pylori gastritis: prevalence, in-situ binding sites and clues for clinical relevance

Colonization of human gastric mucosa with Helicobacter pylori leads to chronic active gastritis and induces the occurrence of an acquired mucosa-associated lymphoid tissue (MALT) in the stomach. This remodelling of the gastric mucosa together with chronic antigen persistence may induce autoimmune reactions. The aim of this study was to investigate humoral autoimmune reactions to human gastric mucosa in H. pylori gastritis and their clinical relevance. Sera from patients with dyspeptic symptoms were tested for presence of IgG immunoglobulins against H. pylori. Gastric infection with H. pylori and alterations of gastric mucosa were demonstrated by histological examination of gastric biopsy specimens. All sera were tested for reactivity against human gastric mucosa by immunohistochemistry. Two different in-situ binding sites of antigastric autoantibodies were observed. Binding to canalicular structures within parietal cells was significantly correlated with antibodies to H. pylori, elevated basal gastrin levels and atrophy of gastric corpus glands. Our data indicate that autoimmune reactions to antigens in the human gastric mucosa occur in H. pylori gastritis and that they may play a role in the pathogenesis of the disease.     

Association of Salivary Helicobacter pylori Infection with Oral Diseases: a Cross-sectional Study in a Chinese Population

Objective: The aim of this study was to detect the prevalence of oral H.pylori among adults and to investigate the correlation between H.pylori infection and common oral diseases.Study design: A cross-sectional study was performed among adults Chinese who took their annual oral healthy examination at The First Affiliated Hospital, Zhejiang University School of Medicine, China.Results: The study included 1050 subjects in total and oral H.pylori infection occurred in 60.29% of the subjects. The prevalence rates of oral H.pylori in patients with periodontal diseases (63.42%) and caries (66.91%) were significantly increased than those without oral diseases (54.07%), respectively (P < 0.05), while the difference between subjects with recurrent aphthous stomatitis and controls was not significant. In addition, the differences of positive rates of H.pylori with or without history of gastric ulcer were statistically significant (69.47% vs 58.26%, P<0.05). Presenting with periodontal diseases (OR 1.473;95% CI 1.021 to 2.124), caries (OR 1.717; 1.127 to 2.618), and having history of gastric ulcer (OR 1.631; 1.164 to 2.285) increased the risk of H.pylori infection.Conclusions: Oral H.pylori infection is common in adult Chinese, which is significantly associated with oral diseases including periodontal diseases and caries.     

Nodular gastritis and pathologic findings in children and young adults with Helicobacter pylori infection

PURPOSE: The aim of this study was to investigate the pathologic characteristics of nodular gastritis in children and young adults infected with Helicobacter pylori (H. pylori). MATERIALS AND METHODS: A total of 328 patients were enrolled in this study, and the diagnosis of H. pylori infection was done with gastroduodenal endoscopy concomitant with a CLO(TM) test and pathologic analysis of the biopsy specimens. Diagnoses of normal, superficial gastritis, nodular gastritis, and peptic ulcer disease were made from the gastroduodenal endoscopic findings. The density of H. pylori organisms in the gastric mucosa was rated as normal, mild, moderate, or marked. The pathologic findings of nodular gastritis were based on the histopathologic findings of inflammation, immune activity, glandular atrophy and intestinal metaplasia. Each of these findings was scored as either normal (0), mild (1), moderate (2), or marked (3) according to the updated Sydney system and using visual analog scales. The gastritis score was the sum of the four histopathologic scores. RESULTS: In this study, nodular gastritis (50.6%) was most common, and mild density (51.5%) H. pylori infection was also common upon microscopic examination. Intestinal metaplasia occurred in 9 patients (2.7%). CONCLUSION: Logistic regression revealed a significant increase in the incidence of nodular gastritis with gastritis score (p=0.008), but not an association with sex, age, or H. pylori density. Gastritis score was the only significant factor influencing the occurrence of nodular gastritis. Intestinal metaplasia, which was originally thought to be a pre-malignant lesion, occurred in 2.7% of the patients with H. pylori infection.     

Effect of Helicobacter pylori eradication in Iranian patients with functional dyspepsia: a prospective,randomized, placebo-controlled trial

Introduction

Whether patients with functional dyspepsia (FD) should receive Helicobacter pylori (H. pylori) eradication therapy remains controversial. The objective of this trial was to evaluate the effect of H. pylori eradication therapy on dyspeptic symptoms of patients with FD.

Material and methods

A prospective, randomized, placebo-controlled trial of H. pylori eradication for FD was conducted. A total of 720 FD patients diagnosed by Rome III criteria were consecutively enrolled. We randomly assigned 186 H. pylori infected patients with FD to receive quadruple therapy for 14 days and 173 such patients to receive identical-appearing placebos. Severity of abdominal symptoms was assessed with the Glasgow Dyspepsia Severity Score (GDSS), and eradication of H. pylori by ¹³C-urea breath test was evaluated during one year.

Results

The rate of eradication of H. pylori infection was 87.1% in the treatment group and 2.9% in the placebo group at 6 weeks (p = 0.001). The mean GDSS at 12 months was 4.9 ±2.8 in the treatment group, as compared to 5.2 ±3.4 in the placebo group (p = 0.064). The scores in both groups were lower than those at baseline. According to the intention-to-treat analysis, at 12 months, there was no significant difference between groups in the rate of successful treatment (48.6% in the treatment group and 51.2% in the placebo group; p = 0.84). There was no significant difference in mean symptom scores between the two treatment groups at any point during follow-up.

Conclusions

The results of our study provide no evidence that H. pylori eradication leads to relief of symptoms 12 months after treatment, and there is a need for further studies.     

Interleukin-6 polymorphism and Helicobacter pylori infection in Brazilian adult patients with chronic gastritis

Abstract Helicobacter pylori is recognised as the most common cause of chronic active gastritis and this bacterium is also an important pathogenic factor in peptic ulcer disease. The biological factors that influence clinical outcome in H. pylori infection have been extensively studied. In addition to immunological factors in the host, bacterial virulence determinants in H. pylori strains are likely to play a crucial role in gastric cancer development. Singlenucleotide polymorphisms at the 5' flanking region of the interleukin (IL)-6 gene promoter (G or C at -174 base) have been identified and individuals with the G allele at position -174 have been shown to produce higher levels of IL-6 than those with the C/C genotype. The mucosal levels of IL-6 were reported to be increased in H. pylori-associated gastritis. The present study was conducted to examine any relationship between inflammatory cytokine polymorphisms and the inflammatory process in mucosa infected by H. pylori. In our study we did not find any association between the C and G alleles in adult patients with chronic gastritis and inflammatory process in gastric mucosa.     

Antimicrobial susceptibility of Helicobacter pylori isolates from Lower Silesia,Poland

Introduction

In recent years the failure of standard therapy for Helicobacter pylori infections has been observed, which results primarily from the increasing resistance of H. pylori strains to antibiotics. The aim of the study was to estimate the prevalence of antimicrobial resistance of H. pylori strains isolated from adult symptomatic patients with primary infection in the Lower Silesia Region in Poland.

Material and methods

One hundred and seventy-eight adults aged 19–89 years with dyspeptic symptoms suggesting gastroduodenal pathology were enrolled in the study. The study was performed in the years 2008–2011. Fifty H. pylori strains were isolated from gastric biopsy samples of examined patients. Antimicrobial susceptibility to 6 drugs (amoxicillin (AM), clarithromycin (CH), metronidazole (MZ), tetracycline (TC), levofloxacin (LEV), and rifabutin (RB)) was tested by the gradient-diffusion method (E-test method).

Results

The incidence of H. pylori infection among examined patients was 35%. From 50 isolated H. pylori strains, 24% showed resistance to CH, 42% to MZ and 8% to LEV alone. Multidrug resistance was detected in 26% of strains, whereas 20% of isolates were resistant to MZ and CH. Examined strains were fully susceptible to AM, TC and RB.

Conclusions

Resistance to clarithromycin strains isolated from adults of the Lower Silesia Region in Poland is high and is almost always associated with resistance to metronidazole (CH + MZ). It is necessary to continuously monitor H. pylori resistance to drugs used in therapy, especially to clarithromycin. Verification of the existing recommendations of eradication therapy is also needed.     

Toll-like receptors TLR4, TLR5 and TLR9 on gastric carcinoma cells: an implication for interaction with Helicobacter pylori

In the human stomach Toll-like receptors (TLRs) expressed by the gastric epithelium interact with Helicobacter pylori and mediate production of proinflammatory cytokines and chemokines during H. pylori infection. This results in chronic active gastritis, the background from which gastric carcinoma arises via the epithelial precursor lesions, intestinal metaplasia and dysplasia. Therefore, the question is arising whether gastric carcinoma cells are also able to interact with H. pylori. In this study, TLR4, TLR5 and TLR9 expression was investigated on tumor cells of gastric carcinoma and on its precursor lesions, intestinal metaplasia and dysplasia, by immunohistochemistry. Gastric epithelium with intestinal metaplasia (n=10) and dysplasia (n=3) expressed TLR4 and TLR5. TLR4 was strongly expressed by tumor cells of 17 out of 22 and TLR5 by tumor cells of all 22 patients with gastric carcinoma. TLR9, however, was not detectable in intestinal metaplasia or dysplasia and only focally in 6 out of 22 gastric carcinomas. In contrast to H. pylori gastritis, epithelial TLR expression in intestinal metaplasia, dysplasia and gastric carcinoma was diffusely distributed without subcellular polarization as demonstrated by confocal microscopy. This is the first study describing TLR expression on tumor cells of gastric carcinoma and its precursor lesions. Expression of TLRs enables gastric carcinoma cells to interact with H. pylori. As H. pylori can induce gastric carcinoma-promoting factors, such as IL-8, via epithelial TLR expression, TLR expression by gastric carcinoma cells may have a dangerous potential.     

Effects of a Th1- versus a Th2-biased immune response in protection against Helicobacter pylori challenge in mice

The roles that T helper type 1 (Th1) and T helper type 2 (Th2) Helicobacter pylori-specific immune responses play in protection from H. pylori challenge are poorly understood. It is expected that Th2 immune responses are required for protection against extracellular bacteria, such as H. pylori. However, recent studies have suggested that Th1 immunity is required for protection. The mechanisms by which this might occur are unknown. Our goal in this study was to more clearly define the effects of a Th1- versus a Th2-promoting H. pylori vaccine on immunity and protection. Therefore, we tested a Th1 vaccine consisting of an H. pylori sonicate and CpG oligonucleotides (CpG) and a Th2 vaccine consisting of a lipopolysaccharide (LPS)-depleted H. pylori sonicate combined with cholera toxin (CT). We demonstrate that although the Th2-promoting vaccine induced stronger systemic and local immune responses, only the Th1-promoting vaccine was protective.     

Cyclooxygenase-2 expression and its association with angiogenesis, Helicobacter pylori, and clinicopathologic characteristics of gastric carcinoma

Cyclooxygenase-2 (COX-2) is upregulated in gastric carcinoma, and its increased levels were found to have a prognostic significance in some studies. Both angiogenesis and Helicobacter pylori infection have been reported to be associated with COX-2 expression of gastric cancer in recent studies. In this study, COX-2 expression and its association with CD31 staining, H.-pylori infection, and well-known clinicopathological factors were investigated in 65 gastric cancer patients. COX-2 and CD31 expression assessment was done by immunohistochemical methods. Whartin Starry stain was performed for H.-pylori infection. Of 65 patients, 32 (49%) revealed intense COX-2 immunostaining. Among various clinicopathologic characteristics, COX-2 expression was inversely correlated with tumor size, TNM stage, and lymph node status. Thirty-two (49%) patients revealed intense CD31 immunostaining. Among various clinicopathologic characteristics, CD31 expression was associated only with lymph node metastasis. COX-2 expression was not correlated with CD31 staining and H.-pylori infection. Both COX-2 and CD31 staining had no prognostic significance. In conclusion, we found that COX-2 expression was significantly higher in earlier stages of gastric cancer. It can be suggested that COX-2 expression may be important in the initial development of gastric cancer but not in progression of the disease. Other factors which may be associated with COX-2 in gastric cancer, including angiogenesis and H.-pylori infection, should be investigated in further studies.     

Cyanidin 3-O-Glucoside Reduces Helicobacter pylori VacA-Induced Cell Death of Gastric KATO III Cells through Inhibition of the SecA Pathway

Two key virulence factors of Helicobacter pylori are the secreted virulent proteins of vacuolating toxin A (VacA) and cytotoxin associated protein A (CagA) which lead to damages of gastric epithelial cells. We previously identified that the cyanidin 3-O-glucoside (C3G) inhibits the secretion of both VacA and CagA. In the current report, we show that C3G inhibits VacA secretion in a dose-dependent manner by inhibiting secretion system subunit protein A (SecA) synthesis. As SecA is involved in translocation of bacterial proteins, we predicted that inhibition of the SecA pathway by C3G should decrease H. pylori-induced cell death. To test this hypothesis, the human gastric cell line KATO III cells were co-cultured with H. pylori 60190 (VacA⁺/CagA⁺) and C3G. We found that C3G treatment caused a decrease in activation of the pro-apoptotic proteins caspase-3/-8 in H. pylori-infected cells leading to a decrease in cell death. Our data suggest that consumption of foods containing anthocyanin may be beneficial in reducing cell damage due to H. pylori infection.     

Experimental Helicobacter pylori infection of rhesus macaques (Macaca mulatta)

The aim of the present study was to establish an animal model for Helicobacter pylori (H. pylori) infection at the German Primate Centre in rhesus monkeys (Macaca mulatta). During the experiments the susceptibility of three animals to different H. pylori strains of human origin was tested. In a follow-up study gastric biopsies from three different sites were investigated in regular intervals using microbiological, histological, electron microscopical and molecular biological methods to evaluate the presence of bacterial colonization and the occurrence of gastritis. It was possible to establish a persistent experimental infection. The rather long follow-up period of 18 months offered the possibility to demonstrate a permanent H. pylori infection in the gastric mucosa of the test animals. The three animals have now been successfully colonized with H. pylori for 18 months and presented a chronic active gastritis confirmed by microbiological and histological methods. By molecular typing, the identity of the isolates recovered from the animals was shown. It was possible to demonstrate that one infection strain outcompeted the second one. Taken together, prerequisites exist for making use of an attractive and useful animal model in rhesus monkeys especially for long term observations.