Assessment of the mixed origin of the gastric epithelial extracellular vesicles in acellular transfer of Helicobacter pylori toxins and a systematic review.

BACKGROUND: H. pylori are generally considered as extracellular organisms, with exclusive colonization of the gastric milieu. Yet, several extra gastric manifestations are associated with this infection. The aim of the present study was to investigate the feasibility of toxin transfer by extracellular vesicles, from bacterial and epithelial origins. METHODS: Tox-positive H. pylori and its two cagA and vacA mutant strains were used to produce bacterial vesicles (BVs) and to infect AGS cells. The produced BVs and the infected cell vesicles (ICVs) were collected by ultracentrifugation and evaluated by western blotting, DLS and electron microscopy. These two sets of vesicles were applied to a second set of recipient AGS cells, in which the acellular transfer of toxins, IL-8 production and downstream morphologic changes were assessed, by western blotting, ELISA and light microscopy, respectively. RESULTS: The BVs were positive for H. pylori membrane markers (BabA and UreB), VacA and CagA toxins, except for from the corresponding mutant strains. The ICVs were larger in size and positive for bacterial markers, as well as epithelial markers of CD9, LGR5, but negative for nuclear (Ki76) or cytoplasmic (ß-actin) markers. Bacteria-independent transfer of CagA and VacA into the recipient cells occurred upon treatment of cells with BVs and ICVs, followed by cellular vacuolation and elongation. IL-8 production was induced in recipient AGS cells, treated with BVs (1279.4 ± 19.79 pg/106 cells), early (8 h, 1171.4 ± 11.31 pg/106 cells) and late (48 h, 965.4 ± 36.77 pg/106 cells) ICVs (P < 0.0001). CONCLUSION: Our data indicates that ICVs, with mixed bacterial and epithelial constituents, similar to BVs, are capable of transferring bacterial toxins into the recipient cells, inducing IL-8 production and subsequent morphologic changes, in an acellular manner.

Histological assessment of gastric pseudopyloric metaplasia: Intra- and inter-observer consistency.

BACKGROUND: The histological spectrum of oxyntic mucosal atrophy (a major determinant of gastric cancer risk) includes pseudopyloric metaplasia (PPM), which histological assessment has been regarded as unreliable. PPM consistently expresses Trefoil-Factor 2 (TFF2), which is histochemically detecteble (TFF2-IHC). AIMS: Intra- and inter-observer consistency in assessing PPM was examined using both hematoxylin & eosin (H&E) and TFF2-IHC. MATERIALS AND METHODS: Seventy-four oxyntic biopsy samples obtained from autoimmune gastritis were considered. Two serial histological sections obtained from the paraffin-embedded tissue-samples were stained with H&E and TFF2-IHC. Three pathologists (Alpha, Beta, Gamma) independently scored PPM by both staining and the Intra- and inter-observer consistency (H&E versus TFF2-IHC) was calculated using k-statistics and/or Spearman's coefficient. RESULTS: Based on H&E-stain versus TFF2-IHC, intra-observer consistency in PPM assessement was ranked as consistently "good" (k-values: Alpha=0.79; Beta=0.78; Gamma=0.75). Based on H&E, the overall PPM inter-observer consistency among the 3 observers was ranked as "good" (k=0.77) (the inter-observer consistency for pairs of observers was as follows: Alpha versus Beta k=0.88; Alpha versus Gamma k=0.87; Beta versus Gamma k=0.80). Based on TFF2-IHC, the overall PPM inter-observer agreement was ranked as "excellent" (k=0.91) (the inter-observer consistency for pairs of observers was as follows: Alpha versus Beta k=1; Alpha versus Gamma k=0.91; Beta versus Gamma k=0.91). CONCLUSION: Relying on either H&E staining or TFF2-IHC, pathologists assess PPM consistently.

A simple and cost-efficient adherent culture platform for human gastric primary cells, as an in vitro model for Helicobacter pylori infection.

BACKGROUND: Most two- dimensional in vitro models for studying host- H. pylori interactions rely on tumor-derived cell lines, which harbor malignant alterations. The recent development of human gastric organoids has overcome this limitation and provides a highly sophisticated, yet costly, short-term model for H. pylori infection, with restricted use in low-budget centers. METHOD: Tissue specimens from upper, middle, and lower stomachs of H. pylori-negative volunteers were collectively dispersed and cultured on mouse embryonic fibroblast (MEF) or collagen-coated plates. Gastric primary cells (GPCs) were evaluated by light microscopy, immunostaining, qRT-PCR and ELISA analysis of cellular secretions, before and after H. pylori infection. RESULTS: The formation and long-term (up to 1 year) maintenance of GPCs was highly dependent on adherent inactivated MEF cells, cultured in enriched media. These cells were multipassageable and able to undergo stable freezer storage and subsequent revival. The cellular composition of GPCs included the combination of cytokeratin 18 (CK18) and E-cadherin (E-cad)-positive epithelial cells, MUC5AC-positive gastric cells, and leucine-rich repeat containing G protein-coupled receptor 5 (LGR5)-positive progenitor cells. These cells produced significant amounts of gastric pepsinogens I and II. GPCs also allowed for extended (up to 96 hours) H. pylori infection, during which they underwent morphological alterations (cellular vacuolation and elongation) and hyperproduction of gastric pepsinogens and inflammatory cytokines (IL-8 and TNF-α). CONCLUSION: We, hereby, present a simple, consistent, and cost-efficient gastric cell culture system, which provides a suitable model for extended in vitro infection of H. pylori. This platform can be employed for a variety of gastric-related research.

Analysis of the associations among Helicobacter pylori infection, adiponectin, leptin, and 10-year fracture risk using the fracture risk assessment tool: A cross-sectional community-based study.

Helicobacter pylori (H. pylori) infection may induce inflammatory cytokines or adipokines that influence bone turnover and bone fracture risk. This study aimed to evaluate the association among H. pylori infection, adipokines, and 10-year fracture risk using the Fracture Risk Assessment Tool scale. From August 2013 to February 2016, a community-based cohort was surveyed by Keelung Chang-Gung Memorial Hospital. Subjects were included if they were older than 40 years and not pregnant. All participants underwent a standardized questionnaire survey, physical examination, urea breath test, and blood tests. A total of 2,689 participants (1,792 women) were included in this cross-sectional study. In both sexes, participants with a high fracture risk were older and had higher adiponectin values than participants without a high fracture risk (mean age, female: 72.9 ± 5.6 vs. 55.8 ± 7.3 years, P < 0.0001; male: 78.9 ± 4.7 vs. 58.1 ± 8.9 years, P < 0.001) (adiponectin, female: 10.8 ± 6.3 vs. 8.7 ± 5.2 ng/ml, P < 0.001; male: 9.7 ± 6.1 vs. 5.5 ± 3.8 ng/ml, P < 0.001). Adiponectin was correlated with high fracture risk in both sexes, but H. pylori infection and leptin was not. In logistic regression analysis, adiponectin could not predict high fracture risk when adjusting the factor of body mass index (BMI) in men group. In conclusion, H. pylori infection and leptin could not predict 10-year fracture risk in either sex. Adiponectin was correlated with bone fracture risk in both sexes and the correlation might be from the influence of BMI.

Assessment of p21, p53 expression, and Ki-67 proliferative activities in the gastric mucosa of children with Helicobacter pylori gastritis.

BACKGROUND: Helicobacter pylori that is generally acquired in childhood and infects the gastric mucosa is considered to be responsible for many pathobiological changes that are linked to the pathogenesis of gastric cancer. Although the majority of studies on the subject have been carried out in adults, there are a limited number of studies on children that reflect the early period of infection and may be of greater significance. AIM: We aimed to determine the role of H. pylori infection and/or gastritis in several histopathological changes, p53, p21, and cell proliferation-associated Ki-67 antigen expression in the gastric mucosa. MATERIALS AND METHODS: We studied 60 patients with a mean age of 7.5 ± 4.5 years at referral. On the basis of endoscopic appearance and the evaluation of the gastric antral specimens, the patients were divided into three groups: patients without gastritis, patients with H. pylori-positive gastritis, and patients with H. pylori-negative gastritis. To determine the expression of p53, Ki-67, and p21 in gastric biopsy specimens, immunohistochemical stains were performed. RESULTS: The incidence of neutrophil activity, which was one of our histopathologic parameters, was significantly higher in the H. pylori-positive gastritis group than the other two groups. The presence of lymphoid aggregate was more frequent in H. pylori ± gastritis groups than the nongastritis group. p53 expression was found to be significantly higher in the H. pylori-positive gastritis group than the nongastritis group. Ki-67 and p21 expressions were significantly more frequent in the H. pylori-positive gastritis group than the other two groups. When we evaluated the density of H. pylori, as the density of bacteria increases, we found that the expressions of p53, p21, and Ki-67 increased significantly. CONCLUSION: Expression of the studied precancerous markers in significant amounts indicates the importance of childhood H. pylori infection in the constitution of gastric cancer in adulthood.

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

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

The assessment of carotid intima-media thickness, lipid profiles and oxidative stress markers in Helicobacter pylori-positive subjects.

BACKGROUND/AIMS: Helicobacter pylori infection has been suggested to be associated with atherosclerosis. The issue is still controversial. It is well known that abnormal lipid profile and oxidative stress are related to atherosclerosis and the measurement of carotid intima-media thickness. The aim of this study was to investigate carotid intima-media thickness and oxidative stress along with lipid parameters in Helicobacter pylori-positive and -negative subjects. MATERIALS AND METHODS: Thirty Helicobacter pylori-positive subjects and 31 Helicobacter pylori-negative subjects were enrolled. Helicobacter pylori infection was diagnosed by noninvasive tests. Serum total oxidant status and total antioxidant capacity levels were measured. Oxidative stress index was calculated based on total oxidant status/total antioxidant capacity ratio. Traditional cardiovascular risk factors were recorded, and laboratory analysis included measurement of serum triglycerides, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol. carotid intima media thickness was assessed by high-resolution ultrasound. RESULTS: We found that the mean and maximum values of right and overall carotid intima-media thickness in Helicobacter pylori-positive subjects were significantly thicker than in Helicobacter pylori-negatives (p < 0.05). Serum triglycerides levels of Helicobacter pylori-positive subjects were significantly higher than in Helicobacter pylori-negatives (p < 0.05). Total oxidant status, total antioxidant capacity and oxidative stress index values were significantly higher in Helicobacter pylori-positive subjects compared with negatives (p < 0.05). No significant correlation was observed between oxidative stress markers and carotid intima-media thickness values. CONCLUSIONS: Carotid intima-media thickness, total oxidant status, total antioxidant capacity, oxidative stress index, and triglycerides values are increased in Helicobacter pylori-positive subjects compared to Helicobacter pylori-negatives. These data indicated that Helicobacter pylori infection may have a role in atherosclerotic processes.

Molecular assessment of c-H-ras p21 expression in Helicobacter pylori-mediated gastric carcinogenesis.

Helicobacter pylori (H. pylori) infection plays a significant role in causing gastric cancer; the exact molecular mechanisms of gastric carcinogenesis have not yet been fully determined. Therefore, this study was planned to examine the role of c-H-ras p21 expression in H. pylori infection at different stages of disease progression from precursor lesions to gastric carcinoma. This study was carried out in 200 patients, consisting of normal gastric mucosa (n = 20), mucosa with chronic gastritis (n = 63), intestinal metaplasia (n = 20), dysplasia (n = 11), and gastric adenocarcinoma (n = 86), in which the H. pylori status have been analysed. The expression of c-H-ras p21 was studied at mRNA as well as protein level using RT-PCR and western blotting, respectively. The localization of c-H-ras p21 was also studied semiquantitatively by immunohistochemistry. The RT-PCR and western blotting results of c-H-ras p21 mRNA and protein expressions were significantly increased in chronic gastritis, intestinal metaplasia, dysplasia, and gastric adenocarcinoma patients, respectively. Immunohistochemical study also showed the increased expression of c-H-ras p21 in the similar way. Overexpression of c-H-ras p21 might be due to H-ras mutation at codon 12 of ras gene family in H. pylori infection. The rate of expression of ras p21 was higher in the H. pylori-infected precursor lesions, chronic gastritis 49/56 (87.5%), intestinal metaplasia 16/17 (94%), and dysplasia 9/11(82%) whereas in the case of H. pylori negative cases these groups, show 12.5, 5.9, and 18.2%, respectively. The data suggested that H. pylori infection may increase the expression of c-H-ras p21 early in the process of gastric carcinogenesis.

Assessment of interactions between mucins of gastric juice and Helicobacter pylori--preliminary study.

BACKGROUND/AIMS: Helicobacter pylori resides primarily in the gastric mucus layer composed of carbohydrate-rich glycoproteins, mucins. Carbohydrates of the secretory MUC 5AC mucin are one of the proved receptors for H. pylori adhesins. A participation of the membrane-associated MUC 1 in the mechanism of infection is also suggested. The main aim of the study was to support the participation of the membrane associated MUC 1 mucin in the mechanism of infection. METHODOLOGY: 13 gastric juices were included in the study. The presence of MUC 5AC and MUC 1 mucins as well as H. pylori bindings were performed using ELISA tests. RESULTS: MUC 1 and MUC 5AC mucins were present in all the examined juices. H. pylori adhered to both glycoproteins. CONCLUSIONS: H. pylori bind to the secretory MUC 5AC mucin as well as to the epithelial MUC 1. This supports the idea that the membrane-associated mucin is involved in the mechanism of H. pylori infection.

[Assessment of rate of hydroxyl anions production by Helicobacter pylori in stomach].

Production of hydroxyl anions by tissue samples of pylorus mucous membrane obtained from 45 patients with gastric or duodenal ulcers was investigated. The production was estimated using the recently developed method based on measurement of rate of pH change in urea-containing reaction mixture. The rate of [OH-] generation as a result of H. pylori metabolism accounted on pylorus square varied from 0.4 to 1318.9 mcmol [OH-]/min, and in 90.2% of cases it did not exceed 128.1 mcmol [OH-]/min. This rate is comparable to mean rate of [H+] generation in stomach of healthy man--114.2-238.4 mcmol [H+]/min. Obtained results allow to conclude that this bacterium may participate in regulation of stomach acid-base balance.

Trials of novel 13C-urea-containing capsule for more economic and sensitive diagnosis of Helicobacter pylori infection in human subjects.

To develop a 13C-urea-containing capsule for more economic and sensitive diagnosis of Helicobacter pylori infection, the 13C-urea-containing capsules were prepared with various additives such as polyethylene glycol, microcrystalline cellulose, sodium lauryl sulfate and citric acid. Their dissolution test and 13C-urea Breath Test in human volunteers were then performed. Polyethylene glycol increased the initial dissolution rates of urea and difference delta 13C values from 13C-urea, while microcrystalline cellulose and sodium lauryl sulfate decreased them. Irrespective of addition of citric acid, the compositions with polyethylene glycol showed higher values from 13C-urea compared to a commercial 76 mg 13C-urea-containing capsule due to higher initial dissolution rate. The capsules with 38 mg 13C-urea and 1.9 mg polyethylene glycol, which showed higher Helicobacter pylori-positive value of about 8 per thousand at 10 min, improved the sensitivity of 13C-urea in human volunteers. Thus, the 13C-urea-containing capsule with polyethylene glycol would be a more economical and sensitive preparation for diagnosis of Helicobacter pylori infection.

Amoxycillin capsules with omeprazole for the eradication of Helicobacter pylori. Assessment of the importance of antibiotic dose timing in relation to meals.

BACKGROUND: Giving antibiotics after meals prolongs their gastric residence time and improves their intragastric distribution. We aimed to see whether this would result in improved eradication of Helicobacter pylori. METHODS: Eighty patients with H. pylori infection were treated with 40 mg omeprazole in the morning for 28 days and amoxycillin 500 mg q.d.s. for days 15-28. Amoxycillin dosing was randomised to either 1 h before or 10 min after food. Good compliance was pre-defined as missing less than four doses of amoxycillin or two of omeprazole. RESULTS: Amoxycillin dosing after meals was shown not to affect H. pylori eradication rate either when results were analysed on an intention-to-treat basis [amoxycillin before meals successful in 63% (25/40), after in 65% 26/40)] or for good compliers only [before meals 81% (17/21), after 71% (20/28)]. This excludes, with 95% confidence, a benefit of greater than 18% from dosing before, or 23% from dosing after meals. Good compliance, however, was shown to be important, with H. pylori eradication in 76% (37/49) of good compliers compared with 48% (11/23) of others completing the protocol (P < 0.05). CONCLUSIONS: The timing of antibiotic administration in relation to meals is not important in the treatment of H. pylori infection with this regimen of amoxycillin capsules and omeprazole. Good compliance, is however, an important determinant of treatment success.