Shear stress, temperature, and inoculation concentration influence the adhesion of water-stressed Helicobacter pylori to stainless steel 304 and polypropylene

Although molecular techniques have identified Helicobacter pylori in drinking water-associated biofilms, there is a lack of studies reporting what factors affect the attachment of the bacterium to plumbing materials. Therefore, the adhesion of H. pylori suspended in distilled water to stainless steel 304 (SS304) coupons placed on tissue culture plates subjected to different environmental conditions was monitored. The extent of adhesion was evaluated for different water exposure times, using epifluorescence microscopy to count total cell numbers. High shear stresses-estimated through computational fluid dynamics-negatively influenced the adhesion of H. pylori to the substrata (P < 0.001), a result that was confirmed in similar experiments with polypropylene (P < 0.05). However, the temperature and inoculation concentration appeared to have no effect on adhesion (P > 0.05). After 2 hours, H. pylori cells appeared to be isolated on the surface of SS304 and were able to form small aggregates with longer exposure times. However, the formation of a three-dimensional structure was only very rarely observed. This study suggests that the detection of the pathogen in well water described by other authors can be related to the increased ability of H. pylori to integrate into biofilms under conditions of low shear stress. It will also allow a more rational selection of locations to perform molecular or plate culture analysis for the detection of H. pylori in drinking water-associated biofilms.     

Failure To Detect Helicobacter pylori DNA in Drinking and Environmental Water in Dhaka,Bangladesh, Using Highly Sensitive Real-Time PCR Assays

The main transmission pathway of Helicobacter pylori has not been determined, but several reports have described detection of H. pylori DNA in drinking and environmental water, suggesting that H. pylori may be waterborne. To address this possibility, we developed, tested, and optimized two complementary H. pylori-specific real-time PCR assays for quantification of H. pylori DNA in water. The minimum detection level of the assays including collection procedures and DNA extraction was shown to be approximately 250 H. pylori genomes per water sample. Using our assays, we then analyzed samples of drinking and environmental water (n = 75) and natural water biofilms (n = 21) from a high-endemicity area in Bangladesh. We could not identify H. pylori DNA in any of the samples, even though other pathogenic bacteria have been found previously in the same water samples by using the same methodology. A series of control experiments were performed to ensure that the negative results were not falsely caused by PCR inhibition, nonspecific assays, degradation of template DNA, or low detection sensitivity. Our results suggest that it is unlikely that the predominant transmission route of H. pylori in this area is waterborne.Helicobacter pylori is the most common human bacterial pathogen in the world (15), and it has been estimated that 50% of the world''s population is infected. The prevalence of H. pylori infection varies greatly worldwide, with infection rates of more than 80% in some developing countries and below 20% in some developed countries (29). H. pylori causes peptic ulcers in 10 to 15% and stomach cancer in another 1 to 2% of those infected (29).H. pylori naturally resides in the human stomach, and except for some primate species, no other host has been identified. Outside its host, H. pylori is fastidious and can grow only under microaerophilic conditions at 34 to 40°C in nutrient-rich media (29). Under suboptimal conditions, H. pylori transforms into nonculturable spherical or coccoid forms. To date, it is not clear if this process is reversible or if the coccoid form is infectious or even viable, but it has been reported to retain some metabolic activity, its genome, and an intact membrane (1, 6, 12, 28, 38, 47).Transmission of H. pylori has been proposed to occur via gastric-oral, oral-oral, or fecal-oral routes, with studies suggesting transmission through saliva and dental plaque (14, 23), normal and diarrheal stools (18, 23, 41, 43), and vomitus (30, 41). Infected mothers or older siblings, low standards of living, and crowded households have been shown to be major risk factors for contracting H. pylori (25, 35, 50). Other studies have shown a relation between infection, water sanitation, and drinking water sources (24, 26, 39), further supported by reports of H. pylori DNA in drinking, river, lake, or seawater (3, 7, 16, 19-22, 25, 33, 34, 37, 40, 43, 49).Since none of the latter group of studies have shown a causative relation between traces of H. pylori in water and new infections, our original aim was to perform a 2-year prospective study tracing H. pylori in water in a high-endemicity area and relate the findings with new infections in children. For this purpose, we developed highly sensitive and specific quantitative real-time PCR assays for detecting H. pylori DNA in water or human samples while allowing analysis of clonal relatedness between samples of different origins by sequencing of recovered DNA. Using these assays, we conducted a study in a slum area in Dhaka, Bangladesh, where we have recently shown a very high rate of H. pylori infections, i.e., that 60% of the children were infected by the age of 2 years (4). Drinking, waste, and environmental water samples and natural drinking water biofilm samples were collected and analyzed, with rigorous controls for falsely positive or negative results.     

Effect of Chlorine on Incorporation of Helicobacter pylori into Drinking Water Biofilms

The use of a specific peptide nucleic acid (PNA) probe demonstrated that Helicobacter pylori persisted inside biofilms exposed to low concentrations of chlorine (0.2 and 1.2 mg liter⁻¹) for at least 26 days, although no culturable cells were recovered. Coupled with data obtained using viability stains in pure culture, this result suggests that H. pylori can survive chlorination but remain undetectable by culture methods, which can be effectively replaced by PNA hybridization.Helicobacter pylori is a Gram-negative microorganism that colonizes the human stomach and can cause gastric ulcers that can degenerate into gastric carcinoma (5). The route of transmission for this pathogen is not well known, and even though culturable H. pylori has never been isolated from drinking water distribution systems (DWDS), molecular techniques such as PCR have detected the presence of H. pylori DNA in potable water (6, 15, 17), indicating that this environment could act as a reservoir for this bacterium.Chlorine is the most commonly used disinfectant worldwide to ensure the safe distribution of water to the consumer (19). Although studies conducted by Johnson et al. (14) and Baker et al. (4) have shown that H. pylori is inactivated by chlorine, their conclusions were based on the lack of recovery using standard culture plating methods which fail to consider cells that have entered a viable but nonculturable (VBNC) state. Recently, Moreno et al. (16) applied molecular techniques to demonstrate that H. pylori can survive in low concentrations of chlorine in a VBNC state. However, all these studies were performed with pure cultures using suspended cells, and until now, there have been no studies reporting on the effect of chlorination on H. pylori when associated with heterotrophic biofilms where, as is well known, microorganisms become more resistant to the biocide effect of chlorine (10).In a recent study, we demonstrated that H. pylori can be incorporated into drinking water biofilms and remain viable in the lower layers of these structures (11). It is therefore important to understand the ability of this pathogen to be incorporated and survive in heterotrophic biofilms formed in chlorinated waters. If this pathogen can remain viable under these conditions, it might therefore represent a risk to public health when released into the bulk fluid.The aim of this work was to study the effect of low concentrations of chlorine on H. pylori cells both when associated with heterotrophic biofilms and when suspended in pure culture, to assess whether the biofilm can provide protection against disinfection.     

Higher glucose level can enhance the H. pylori adhesion and virulence related with type IV secretion system in AGS cells

Background

Hyperglycemia increases the risk of gastric cancer in H. pylori-infected patients. High glucose could increase endothelial permeability and cancer-associated signaling. These suggest high glucose may affect H. pylori or its infected status.We used two strains to investigate whether H. pylori growth, viability, adhesion and CagA-phosphorylation level in the infected-AGS cells were influenced by glucose concentration (100, 150, and 200 mg/dL).

Results

The growth curves of both strains in 200 mg/dL of glucose were maintained at the highest optimal density after 48 h and the best viability of both strains were retained in the same glucose condition at 72 h. Furthermore, adhesion enhancement of H. pylori was significantly higher in 200 mg/dL of glucose as compared to that in 100 and 150 mg/dL (p < 0.05). CagA protein also increased in higher glucose condition. The cell-associated CagA and phosphorylated-CagA was significantly increased in 150 and 200 mg/dL of glucose concentrations as compared to that of 100 mg/dL (p < 0.05), which were found to be dose-dependent.

Conclusion

Higher glucose could maintain H. pylori growth and viability after 48 h. H. pylori adhesion and CagA increased to further facilitate the enhancement of cell-associated CagA and phosphorylated CagA in higher glucose conditions.     

Genetic Polymorphisms of miR-146a and miR-27a,H. pylori Infection,and Risk of Gastric Lesions in a Chinese Population

Background

MicroRNAs (miRNAs) have been implicated in various human diseases. Single nucleotide polymorphisms (SNPs) in inflammation-related miRNA may play an important role in Helicobacter pylori (H. pylori)-induced gastric lesions. To evaluate the associations between miRNA SNPs, H. pylori and gastric lesions, a population-based study was conducted in Linqu County, China.

Methodology/Principal Findings

Based on serum miRNA array conducted in this population, two SNP loci (miR-146a rs2910164: G>C and miR-27a rs895819: T>C) were determined by polymerase chain reaction-restriction fragment length polymorphism in 2,380 participants with diverse gastric lesions. Using participants with superficial gastritis and mild chronic atrophic gastritis as the reference group, we found that rs2910164 CC carriers had a significantly increased risk of intestinal metaplasia [adjusted odds ratio (OR), 1.42; 95% confidence interval (CI), 1.03–1.97] and dysplasia (OR, 1.54; 95% CI, 1.05–2.25) compared to GG carriers, whereas no significant association was observed for rs895819. Stratified analysis by H. pylori infection indicated that rs2910164 C allele was associated with an increased risk of intestinal metaplasia and dysplasia only among individuals infected with H. pylori (CC vs. GG: OR, 1.53; 95% CI, 1.12–2.08, P for trend = 0.004). Participants who simultaneously carried variant alleles and H. pylori infection were more likely to develop intestinal metaplasia and dysplasia, although the interaction between genetic variants and H. pylori infection was not significant (P for interaction = 0.35 for rs2910164 and 0.92 for rs895819).

Conclusions/Significance

These findings suggest that miR-146a rs2910164 polymorphism may contribute to the evolution of H. pylori-associated gastric lesions in this high-risk population.     

In Vitro and In Vivo Inhibition of Helicobacter pylori by Lactobacillus casei Strain Shirota

We studied the potential inhibitory effect of Lactobacillus casei strain Shirota (from the fermented milk product Yakult [Yakult Ltd., Tokyo, Japan]) on Helicobacter pylori by using (i) in vitro inhibition assays with H. pylori SS1 (Sydney strain 1) and nine H. pylori clinical isolates and (ii) the in vivo H. pylori SS1 mouse model of infection over a period of 9 months. In vitro activity against H. pylori SS1 and all of the clinical isolates was observed in the presence of viable L. casei strain Shirota cells but not in the cell-free culture supernatant, although there was profound inhibition of urease activity. In vivo experiments were performed by oral administration of L. casei strain Shirota in the water supply over a period of 9 months to 6-week-old C57BL/6 mice previously infected with H. pylori SS1 (study group; n = 25). Appropriate control groups of H. pylori-infected but untreated animals (n = 25) and uninfected animals given L. casei strain Shirota (n = 25) also were included in the study. H. pylori colonization and development of gastritis were assessed at 1, 2, 3, 6, and 9 months postinfection. A significant reduction in the levels of H. pylori colonization was observed in the antrum and body mucosa in vivo in the lactobacillus-treated study group, as assessed by viable cultures, compared to the levels in the H. pylori-infected control group. This reduction was accompanied by a significant decline in the associated chronic and active gastric mucosal inflammation observed at each time point throughout the observation period. A trend toward a decrease in the anti-H. pylori immunoglobulin G response was measured in the serum of the animals treated with lactobacillus, although this decrease was not significant.     

CpG Island Methylator Phenotype,Helicobacter pylori,Epstein-Barr Virus,and Microsatellite Instability and Prognosis in Gastric Cancer: A Systematic Review and Meta-Analysis

Background

The controversy of CpG island methylator phenotype (CIMP) in gastric cancer persists, despite the fact that many studies have been conducted on its relation with helicobacter pylori (H. pylori), Epstein-Barr virus (EBV), and microsatellite instability (MSI) and prognosis. To drive a more precise estimate of this postulated relationship, a meta-analysis was performed based on existing relevant studies.

Methods

We combined individual patient data from 12 studies which involved 1000 patients with gastric cancer, which met the criteria. We tabulated and analyzed parameters from each study, including H. pylori, EBV, MSI, and clinical information of patients.

Results

The overall OR for H. pylori infection in CIMP positive group vs. negative group revealed that significantly elevated risks of positive H. pylori infection in the former were achieved (OR 2.23 95% CI, 1.25–4.00; P = 0.007, P_{heterogeneity} = 0.05). Similarly, strong relation between EBV infection and CIMP was achieved by OR 51.27 (95% CI, 9.39–279.86; P<0.00001, P_{heterogeneity} = 0.39). The overall OR for MSI in CIMP positive group vs. negative group was 4.44 (95% CI, 1.17–16.88; P = 0.03, P_{heterogeneity} = 0.01). However, there did not appear to be any correlations with clinical parameters such as tumor site, pathological type, cell differentiation, TNM stage, distant metastasis, lymph node metastasis, and 5-year survival.

Conclusions

The meta-analysis highlights the strong relation of CIMP with H. pylori, EBV, and MSI, but CIMP can not be used as a prognostic marker for gastric cancer.     

Age-Dependent Association among Helicobacter pylori Infection,Serum Pepsinogen Levels and Immune Response of Children to Live Oral Cholera Vaccine CVD 103-HgR

Background

Through its effects on gastric secretion, we hypothesized that Helicobacter pylori infection may influence oral immunization. Accordingly, we examined the association between H. pylori infection, serum pepsinogen (PG) (measures for H. pylori gastritis) and vibriocidal antibody (a correlate of protection) seroconversion following oral immunization with CVD 103-HgR live cholera vaccine among children of different ages.

Methods

Sera from 422 Chilean children who were vaccinated with a single dose of CVD 103-HgR were tested by ELISA for serum IgG antibodies to H. pylori, PG I and PG II levels and antibodies to Shigella flexneri 2a lipopolysaccharide and hepatitis A virus (as markers of low socioeconomic status and exposure to enteric pathogens).

Results

The likelihood of vibriocidal antibody seroconversion following vaccination with CVD 103-HgR was significantly decreased in H. pylori-seropositive children age 6 months to 4 years with PG II>8 µg/L (adjusted OR 0.14 (95% CI 0.03–0.61; P = 0.009), and also in H. pylori seropositives with lower PG II level (adjusted OR 0.34, 95% CI 0.14–0.83; P = 0.017), compared to H. pylori-seronegatives. H. pylori-seropositive children aged 5–9 years with serum PG I>30 µg/L (indicating more severe gastritis) had higher odds of vibriocidal seroconversion than those with lower PG I levels (adjusted OR 4.41, 95%CI 1.26–15.38; P = 0.02). There was no significant association between exposures to S. flexneri 2a or hepatitis A virus and vibriocidal seroconversion.

Conclusions

As H. pylori gastritis progresses with increasing pediatric age in developing country venues, changes in gastric secretion ensue that we believe explain the observed differences in age-related immune responses to immunization with live oral cholera vaccine. The effect of H. pylori and changes of gastric acid secretion on the immunogenicity of various oral vaccines should be studied in different developing, transitional and industrialized country settings.     

H. pylori CagL-Y58/E59 Prime Higher Integrin α5β1 in Adverse pH Condition to Enhance Hypochlorhydria Vicious Cycle for Gastric Carcinogenesis

Background/Aims

H. pylori CagL amino acid polymorphisms such as Y58/E59 can increase integrin α5β1 expression and gastric cancer risk. Hypochlorhydria during chronic H. pylori infection promotes gastric carcinogenesis. The study test whether CagL-Y58/E59 isolates may regulate integrin α5β1 to translocate CagA via the type IV secretory system even under adverse pH conditions, and whether the integrin α5β1 expression primed by H. pylori is a pH-dependent process involving hypochlorhydria in a vicious cycle to promote gastric carcinogenesis.

Methods

The expressions of integrin α5 and β1, CagA phosphorylation, IL-8, FAK, EGFR, and AKT activation of AGS cells exposed to CagL-Y58/E59 H. pylori, isogenic mutants, and different H. pylori CagL amino acid replacement mutants under different pH values were determined. Differences in the pepsinogen I/II ratio (indirectly indicating gastric acidity) and gastric integrin α5β1 expression were compared among the 172 H. pylori-infected patients with different cancer risks.

Results

Even under adversely low pH condition, H. pylori CagL-Y58/E59 still keep active integrin β1 with stronger binding affinity, CagA translocation, IL-8, FAK, EGFR, and AKT activation than the other mutants (p<0.05). The in vitro assay revealed higher priming of integrin α5β1 by H. pylori under elevated pH as hypochlorhydria (p<0.05). In the H. pylori-infected patients, the gastric integrin α5β1 expressions were higher in those with pepsinogen I/II ratio <6 than in those without (p<0.05).

Conclusions

H. pylori CagL-Y58/E59 prime higher integrin under adverse pH and may involve to enhance hypochlorhydria vicious cycle for gastric carcinogenesis, and thus require an early eradication.     

Eradication of Helicobacter pylori Infection Improves Levodopa Action,Clinical Symptoms and Quality of Life in Patients with Parkinson's Disease

Background

Previous studies have demonstrated a higher prevalence of Helicobacter pylori (H. pylori) infection in patients with Parkinson''s disease (PD) compared to controls. H. pylori infection affects levodopa absorption and its eradication significantly improves clinical response to levodopa. Here, we studied the prevalence of H. pylori infection and its eradication effects among our PD patients.

Methods

A prospective study involving idiopathic PD patients on levodopa therapy. ¹³C-urea breath test (UBT) was used to detect H. pylori. UBT-positive patients were given standard eradication therapy and followed up at 6 and 12 weeks in an open label single arm design. Repeat UBT was performed at 12 weeks. The UPDRS, PD NMQ, PD NMSS and PDQ-39 were administered at baseline and post-eradication (6 and 12 weeks). Levodopa ‘onset’ time and ON-duration were recorded.

Results

Of 82 patients recruited, 27 (32.9%) had positive UBT. H. pylori-positive patients had significantly poorer total UPDRS (p = 0.005) and PDQ39 (p<0.0001) scores compared to H. pylori-negative patients. At 12 weeks post-eradication, the mean levodopa onset time shortened by 14 minutes (p = 0.011). The mean ON duration time increased by 56 minutes at week 6 (p = 0.041) and 38 minutes at week 12 (p = 0.035). The total UPDRS scores (p<0.0001), scores for parts II (p = 0.001), III (p<0.0001) and IV (p = 0.009) were significantly better. The total PDQ-39 scores (p = 0.001) and subdomains mobility (p = 0.002), ADL (p = 0.001), emotional well being (p = 0.026) and stigma (p = 0.034) significantly improved. The PD NMSQ did not show significant improvement.

Conclusions

H. pylori eradication improved levodopa onset time, ON duration, motor severity and quality of life parameters. Screening and eradication of H. pylori is inexpensive and should be recommended in PD patients, particularly those with erratic response to levodopa.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02112812","term_id":"NCT02112812"}}NCT02112812     

The Association between Cultural Orientation and Drinking Behaviors among University Students in Wuhan,China

Objectives

This study examines the association between cultural orientation and drinking behaviors among university students. Cultural orientation is the measure of how the cultural values of individuals living in their own society are influenced by cultural values introduced from the outside.

Methods

In 2011, a cross-sectional survey collected data from 1279 university students from six universities in central China. Participants used a likert scale to rank a series of statements reflecting cultural values from the previously validated Chinese Cultural Orientation Scale and answered questions about their drinking behaviors and socio-demographic characteristics.

Results

Statistically significant differences in cultural orientation were observed for gender, hometown and type of university attendance. Traditional-oriented students were more likely to be occasional drinkers or nondrinkers, while marginal-oriented students, bicultural-oriented students and western-oriented students were more likely to be regular drinkers. Bicultural orientation (OR = 1.80, P<0.05) and marginal orientation (OR = 1.64, P<0.05) increased the likelihood of the student being regular drinking, compared to students with traditional orientations. Males (OR = 4.40, P<0.05) had a higher likelihood of regular drinking than females, graduate students (OR = 2.59, P<0.05) had a higher likelihood of regular drinking than undergraduates, students from urban areas (OR = 1.79, P<0.05) had a higher likelihood of regular drinking than those from towns/rural areas, and students attending key universities (OR = 0.48, P<0.05) had a lower likelihood of regular drinking than those attending general universities.

Conclusions

Cultural orientation influences drinking behaviors. Traditional cultural orientation was associated with less drinking while western cultural orientation, marginal cultural orientation and bicultural orientation were associated with more drinking. The role of gender, hometown and university attendance is partially moderated through the influence of cultural orientation. The relationship between a traditional cultural orientation and alcohol drinking suggests that traditional Chinese cultural values should be examined for their role in possibly reducing alcohol-related risks through education and policy initiatives.     

Comparison of lipopolysaccharides composition of two different strains of <Emphasis Type="Italic">Helicobacter pylori</Emphasis>

Background

Helicobacter pylori (H. pylori) is a Gram-negative, microaerophilic bacterium that is recognized as a major cause of chronic gastritis, peptic ulcers, and gastric cancer. Comparable to other Gram-negative bacteria, lipopolysaccharides (LPS) are an important cellular component of the outer membrane of H. pylori. The LPS of this organism plays a key role in its colonization and persistence in the stomach. In addition, H. pylori LPS modulates pathogen-induced host inflammatory responses resulting in chronic inflammation within the gastrointestinal tract. Very little is known about the comparative LPS compositions of different strains of H. pylori with varied degree of virulence in human. Therefore, LPS was analyzed from two strains of H. pylori with differing potency in inducing inflammatory responses (SS1 and G27). LPS were extracted from aqueous and phenol layer of hot-phenol water extraction method and subjected for composition analysis by gas chromatography – mass spectrometry (GC-MS) to sugar and fatty acid compositions.

Results

The major difference between the two strains of H. pylori is the presence of Rhamnose, Fucose and GalNAc in the SS1 strain, which was either not found or with low abundance in the G27 strain. On the other hand, high amount of Mannose was present in G27 in comparison to SS1. Fatty acid composition of lipid-A portion also showed considerable amount of differences between the two strains, phenol layer of SS1 had enhanced amount of 3 hydroxy decanoic acid (3-OH-C10:0) and 3-hydroxy dodecanoic acid (3-OH-C12:0) which were not present in G27, whereas myristic acid (C14:0) was present in G27 in relatively high amount.

Conclusion

The composition analysis of H. pylori LPS, revealed differences in sugars and fatty acids composition between a mouse adapted strain SS1 and G27. This knowledge provides a novel way to dissect out their importance in host-pathogen interaction in further studies.

     

Helicobacter pylori Infection Induces Anemia,Depletes Serum Iron Storage,and Alters Local Iron-Related and Adult Brain Gene Expression in Male INS-GAS Mice

Iron deficiency anemia (IDA) affects > 500 million people worldwide, and is linked to impaired cognitive development and function in children. Helicobacter pylori, a class 1 carcinogen, infects about half of the world’s population, thus creating a high likelihood of overlapping risk. This study determined the effect of H. pylori infection on iron homeostasis in INS-GAS mice. Two replicates of INS-GAS/FVB male mice (n = 9-12/group) were dosed with H. pylori (Hp) strain SS1 or sham dosed at 6–9 weeks of age, and were necropsied at 27–29 weeks of age. Hematologic and serum iron parameters were evaluated, as was gene expression in gastric and brain tissues. Serum ferritin was lower in Hp SS1-infected mice than uninfected mice (p < 0.0001). Infected mice had a lower red blood cell count (p<0.0001), hematocrit (p < 0.001), and hemoglobin concentration (p <0.0001) than uninfected mice. Relative expression of gastric hepcidin antimicrobial peptide (Hamp) was downregulated in mice infected with Hp SS1 compared to sham-dosed controls (p<0.001). Expression of bone morphogenic protein 4 (Bmp4), a growth factor upstream of hepcidin, was downregulated in gastric tissue of Hp SS1-infected mice (p<0.001). Hp SS1-infected mice had downregulated brain expression of tyrosine hydroxylase (Th) (p = 0.02). Expression of iron-responsive genes involved in myelination (myelin basic protein (Mbp) and proteolipid protein 2 (Plp2)) was downregulated in infected mice (p = 0.001 and p = 0.02). Expression of synaptic plasticity markers (brain derived neurotrophic factor 3 (Bdnf3), Psd95 (a membrane associated guanylate kinase), and insulin-like growth factor 1 (Igf1)) was also downregulated in Hp SS1-infected mice (p = 0.09, p = 0.04, p = 0.02 respectively). Infection of male INS-GAS mice with Hp SS1, without concurrent dietary iron deficiency, depleted serum ferritin, deregulated gastric and hepatic expression of iron regulatory genes, and altered iron-dependent neural processes. The use of Hp SS1-infected INS-GAS mice will be an appropriate animal model for further study of the effects of concurrent H. pylori infection and anemia on iron homeostasis and adult iron-dependent brain gene expression.     

Cathepsin X-deficient Gastric Epithelial Cells in Co-culture with Macrophages: CHARACTERIZATION OF CYTOKINE RESPONSE AND MIGRATION CAPABILITY AFTER HELICOBACTER PYLORI INFECTION*

Our previous studies have shown an association between Helicobacter pylori infection, the strong up-regulation of cathepsin X (CTSX, also called cathepsin Z/P), and the development of gastric cancer. In the present study, we analyzed primary and conventional gastric epithelial cell lines to establish an optimal in vitro mouse model system for the examination of H. pylori-induced overexpression of Ctsx in a functional way. Gastric epithelial cells were isolated from stomachs of wild-type C57BL6/N and Ctsx^−/− mice and compared with the gastric cancer cell line CLS103. Indirect co-cultures of epithelial cells and macrophages were infected with H. pylori strain SS1 and analyzed for the expression of cathepsins, cytokines, and adhesion factors. Cellular interactions, migration capability, and adherence of H. pylori were assessed using time-lapse video microscopy and colony-forming assays. Isolated primary cells from wild-type and transgenic mice revealed qualities and expression profiles similar to those of corresponding tissue samples. Adherence of H. pylori was significantly higher in primary compared with commercially cells. Thus, induction of cathepsins, cytokines, and adhesion proteins was detected solely in primary cells and co-cultured macrophages. Microarray and migration experiments indicated that Ctsx is involved in B/T-cell proliferation/migration and adhesion of macrophages. Primary epithelial cells from stomach of Ctsx^−/− mice represent an excellent model of H. pylori gastritis to elaborate the special functions of Ctsx in regulating the immune response to H. pylori.     

TLR4 and TLR2 expression in biopsy specimens from antral and corporal stomach zones in Helicobacter pylori infections

Background:

It is not yet known which types of Toll-like receptors (TLRs) are most effective in Helicobacter pylori (H. pylori) recognition. It is also not known which gastric zones have the most prominent roles in TLR-mediated bacterial recognition. The aim of this work was to analyze the expression of TLR2 and TLR4 in biopsy specimens from H. pylori-infected patients.

Methods:

Thirty-eight patients with gastrointestinal disorders were divided into four groups in this study. The groups were: (A) H. pylori infection and peptic ulcer (n=15), (B) peptic ulcer only (n=5), (C) H. pylori infection only (n=10) and (D) control, with neither H. pylori infection nor peptic ulcer (n=8). Biopsy specimens from sites of redness or atrophic mucosa from gastric antrum and body in patients with gastritis were collected. RNAs from the antrum and body specimens were isolated. TLR2 and TLR4 mRNA expression was assessed by RT-PCR and quantified as densitometric ratios of TLR2 and TLR4/β-actin mRNA.

Results:

In the antral zones of H. pylori-infected patients (Groups A and C) TLR2 and TLR4 expression was significantly greater than in uninfected patients (Groups B and D) regardless of peptic ulcers (p < 0.05). In the gastric body samples TLR2 expression was significantly greater in Group C (H. pylori infection only) than in Group B (peptic ulcer only) and TLR4 expression was significantly greater in group A (H. pylori infection and peptic ulcer) than in Group B (peptic ulcer only) (p < 0.05). No significant differences in expression of TLR4 and TLR2 were observed between samples from the antrum and body in same groups.

Conclusions:

We conclude that H. pylori infection leads to significant increase in TLR2 and TLR4 molecules expression in antral region related to the control group. Considering the stimulatory effect of H. pylori on TLRs expression in the gastric tissue, we assume that colonization of H. pylori infection might occurs more in the gastric antral region than in the gastric body.Key Words: Helicobacter pylori, Toll-like receptors, TLR4; TLR2, Peptic ulcer     

The role of integrating conjugative elements in <Emphasis Type="Italic">Helicobacter pylori</Emphasis>: a review

The genome of Helicobacter pylori contains many putative genes, including a genetic region known as the Integrating Conjugative Elements of H. pylori type four secretion system (ICEHptfs). This genetic regions were originally termed as “plasticity zones/regions” due to the great genetic diversity between the original two H. pylori whole genome sequences. Upon analysis of additional genome sequences, the regions were reported to be extremely common within the genome of H. pylori. Moreover, these regions were also considered conserved rather than genetically plastic and were believed to act as mobile genetic elements transferred via conjugation. Although ICEHptfs(s) are highly conserved, these regions display great allele diversity, especially on ICEHptfs4, with three different subtypes: ICEHptfs4a, 4b, and 4c. ICEHptfs were also reported to contain a novel type 4 secretion system (T4SS) with both epidemiological and in vitro infection model studies highlighting that this novel T4SS functions primarily as a virulence factor. However, there is currently no information regarding the structure, the genes responsible for forming the T4SS, and the interaction between this T4SS and other virulence genes. Unlike the cag pathogenicity island (PAI), which contains CagA, a gene found to be essential for H. pylori virulence, these novel T4SSs have not yet been reported to contain genes that contribute significant effects to the entire system. This notion prompted the hypothesis that these novel T4SSs may have different mechanisms involving cag PAI.     

Multiple Pathways of Plasmid DNA Transfer in Helicobacter pylori

Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species.     

Effects of interleukin-10 polymorphisms, Helicobacter pylori infection, and smoking on the risk of noncardia gastric cancer

Objective

Both variations in the interleukin-10 (IL10) gene and environmental factors are thought to influence inflammation and gastric carcinogenesis. Therefore, we investigated the associations between IL10 polymorphisms, Helicobacter pylori (H. pylori) infection, and smoking in noncardia gastric carcinogenesis in Koreans.

Methods

We genotyped three promoter polymorphisms (-1082A>G, -819T>C, and -592 A>C) of IL10 in a case-control study of 495 noncardia gastric cancer patients and 495 sex- and age-matched healthy controls. Multiple logistic regression models were used to detect the effects of IL10 polymorphisms, H. pylori infection, and smoking on the risk of gastric cancer, which was stratified by the histological type of gastric cancer.

Results

The IL10-819C and -592C alleles were found to have complete linkage disequilibrium, and all three IL10 polymorphisms were associated with an increased risk of intestinal-type noncardia gastric cancer. These associations were observed only in H. pylori-positive subjects and current smokers. A statistically significant interaction between the IL10-592 genotype and H. pylori infection on the risk of intestinal-type gastric cancer was observed (P for interaction  = 0.047). In addition, H. pylori-positive smokers who were carriers of either the IL10-1082G (OR [95% CI]  = 17.76 [6.17−51.06]) or the -592C (OR [95% CI]  = 8.37 [2.79−25.16]) allele had an increased risk of intestinal-type gastric cancer compared to H. pylori-negative nonsmokers homozygous for IL10-1082A and -592A, respectively. The interaction between the IL10-1082 polymorphism and the combined effects of H. pylori infection and smoking tended towards significance (P for interaction  = 0.080).

Conclusions

Inflammation-related genetic variants may interact with H. pylori infection and smoking to increase the risk of noncardia gastric cancer, particularly the intestinal-type. These findings may be helpful in identifying individuals at an increased risk for developing noncardia gastric cancer.     

Helicobacter pylori's Unconventional Role in Health and Disease

The discovery of a bacterium, Helicobacter pylori, that is resident in the human stomach and causes chronic disease (peptic ulcer and gastric cancer) was radical on many levels. Whereas the mouth and the colon were both known to host a large number of microorganisms, collectively referred to as the microbiome, the stomach was thought to be a virtual Sahara desert for microbes because of its high acidity. We now know that H. pylori is one of many species of bacteria that live in the stomach, although H. pylori seems to dominate this community. H. pylori does not behave as a classical bacterial pathogen: disease is not solely mediated by production of toxins, although certain H. pylori genes, including those that encode exotoxins, increase the risk of disease development. Instead, disease seems to result from a complex interaction between the bacterium, the host, and the environment. Furthermore, H. pylori was the first bacterium observed to behave as a carcinogen. The innate and adaptive immune defenses of the host, combined with factors in the environment of the stomach, apparently drive a continuously high rate of genomic variation in H. pylori. Studies of this genetic diversity in strains isolated from various locations across the globe show that H. pylori has coevolved with humans throughout our history. This long association has given rise not only to disease, but also to possible protective effects, particularly with respect to diseases of the esophagus. Given this complex relationship with human health, eradication of H. pylori in nonsymptomatic individuals may not be the best course of action. The story of H. pylori teaches us to look more deeply at our resident microbiome and the complexity of its interactions, both in this complex population and within our own tissues, to gain a better understanding of health and disease.Common wisdom circa 1980 suggested that the stomach, with its low pH, was a sterile environment. Then, endoscopy of the stomach became common and, in 1984, pathologist Robin Warren and gastroenterologist Barry Marshall saw an extracellular, curved bacillus, often in dense sheets, lining the stomach epithelium of patients with gastritis (inflammation of the stomach) and ulcer disease [1]. Soon, the medical community understood that the gram-negative bacterium Helicobacter pylori, not stress, is the major cause of stomach inflammation, which, in some infected individuals, precedes peptic ulcer disease (10%–20%), distal gastric adenocarcinoma (1%–2%), and gastric mucosal-associated lymphoid tissue (MALT) lymphoma (<1%) [2]–[5]. Thus, H. pylori gained distinction as the only known bacterial carcinogen [6]. It is believed that half of the world''s population is infected with H. pylori; however, the burden of disease falls disproportionately on less-developed countries. The incidence of infection in developed countries has fallen dramatically, for unknown reasons, with a corresponding decrease in gastric cancer [7]. This public health success is tempered by the recent demonstration of an inverse relationship between H. pylori infection and esophageal adenocarcinoma, Barrett''s esophagus, and reflux esophagitis [8]. H. pylori has been with humans since our earliest days, thus it is not surprising that its relationship is that of both a commensal bacterium and a pathogen, causing some diseases and possibly protecting against others. In addition, it is genetically diverse, likely as a result of constant exposure to both environmental and immunological selection, suggesting that genetic diversification is a strategy for long-term colonization.     

Association of MTOR and AKT Gene Polymorphisms with Susceptibility and Survival of Gastric Cancer

Background

The phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB, AKT)/mammalian target of rapamycin (mTOR) signaling pathway plays a critical role in angiogenesis and cell growth, proliferation, metabolism, migration, differentiation, and apoptosis. Genetic diversity in key factors of this pathway may influence protein function and signal transduction, contributing to disease initiation and progression. Studies suggest that MTOR rs1064261 and AKT rs1130233 polymorphisms are associated with risk and/or prognosis of multiple cancer types. However, this relationship with gastric cancer (GC) remains unclear. The aim of this study was to investigate the role of MTOR and AKT polymorphisms in the risk and prognosis of GC.

Methods

The Sequenom MassARRAY platform was used to genotype 1842 individuals for MTOR rs1064261 T→C and AKT rs1130233 G→A polymorphisms. ELISA was used to detect Helicobacter pylori antibodies in serum. Immunohistochemical analysis was used to detect total and phosphorylated MTOR and AKT proteins.

Results

The MTOR rs1064261 (TC+CC) genotype and the AKT rs1130233 (GA+AA) genotype were associated with increased risk of GC in men (P = 0.049, P = 0.030). In H. pylori-negative individuals, the AKT rs1130233 GA and (GA+AA) genotypes were related to increased risk of atrophic gastritis (AG; P = 0.012, P = 0.024). Notably, the AKT rs1130233 (GA+AA) genotype demonstrated significant interactions with H. pylori in disease progression from healthy controls (CON) to AG (P = 0.013) and from AG to GC (P = 0.049). Additionally, for individuals with the AKT rs1130233 variant, those in the H. pylori-positive group had higher levels of phosphorylated AKT (p-AKT) expression. The AKT rs1130233 genotype was found to be associated with clinicopathological parameters including lymph node metastasis and alcohol drinking (P<0.05).

Conclusion

MTOR rs1064261and AKT rs1130233 polymorphisms were associated with increased GC risk in males and increased AG risk in H. pylori-negative individuals. A significant interaction existed between the AKT rs1130233 genotype and H. pylori infection in CON→AG→GC disease progression. The AKT rs1130233 genotype influenced p-AKT protein expression in H. pylori-infected individuals.