Preventive effects of Cladosiphon fucoidan against Helicobacter pylori infection in Mongolian gerbils

Background. Recently, the acquisition by Helicobacter pylori of resistance to antibiotics has become a serious problem. Therefore, nonantibiotic substances are required to diminish H. pylori‐induced gastric lesions. In the present study, the effects of Cladosiphon fucoidan were examined in terms of H. pylori attachment to porcine gastric mucin in vitro and Helicobacter pylori‐induced gastritis in vivo. Methods. The inhibitory effect of Cladosiphon fucoidan and other polysaccharides on H. pylori attachment to porcine gastric mucin was assayed in vitro with mucin‐coated microtiter plates. The effect of Cladosiphon fucoidan on H. pylori‐induced gastritis was examined in vivo using Mongolian gerbils. H. pylori‐inoculated gerbils were given fucoidan in drinking water. Six weeks after H. pylori‐inoculation, gerbils were sacrificed for macroscopic and microscopic examination of gastric lesions and counting of viable H. pylori in the gastric mucosa. Results. Cladosiphon fucoidan inhibited the H. pylori attachment to porcine gastric mucin at pH 2.0 and 4.0. Two other sulfated polysaccharides, Fucus fucoidan and dextran sulfate sodium, also inhibited the attachment but only at pH 2.0. Inhibitory effects of these three sulfated polysaccharides were not observed at pH 7.2 and nonsulfated polysaccharides, such as mannan and dextran, exerted no influence at any pH. In the in vivo experiment, the H. pylori‐induced gastritis and the prevalence of H. pylori infected animals were markedly reduced by fucoidan in a dose‐dependent manner, at doses of 0.05 and 0.5% in the drinking water. Conclusion. Cladosiphon fucoidan may deserve particular attention as a safe agent that can prevent H. pylori infection and reduce the risk of associated gastric cancer.     

Gastric and duodenum microflora analysis after long-term Helicobacter pylori infection in Mongolian Gerbils

Background: Long‐term Helicobacter pylori infection leads to chronic gastritis, peptic ulcer, and gastric malignancies. Indigenous microflora in alimentary tract maintains a colonization barrier against pathogenic microorganisms. This study is aimed to observe the gastric and duodenum microflora alteration after H. pylori infection in Mongolian Gerbils model. Materials and Methods: A total of 18 Mongolian gerbils were randomly divided into two groups: control group and H. pylori group that were given H. pylori NCTC J99 strain intragastrically. After 12 weeks, H. pylori colonization was identified by rapid urease tests and bacterial culture. Indigenous microorganisms in stomach and duodenum were analyzed by culture method. Histopathologic examination of gastric and duodenum mucosa was also performed. Results: Three of eight gerbils had positive H. pylori colonization. After H. pylori infection, Enterococcus spp. and Staphylococcus aureus showed occurrences in stomach and duodenum. Lactobacillus spp. showed a down trend in stomach. The levels and localizations of Bifidobacterium spp., Bacteroides spp., and total aerobes were also modified. Bacteroides spp. significantly increased in H. pylori positive gerbils. No Enterobacteriaceae were detected. Positive colonization gerbils showed a higher histopathologic score of gastritis and a similar score of duodenitis. Conclusions: Long‐term H. pylori colonization affected the distribution and numbers of indigenous microflora in stomach and duodenum. Successful colonization caused a more severe gastritis. Gastric microenvironment may be unfit for lactobacilli fertility after long‐term H. pylori infection, while enterococci, S. aureus, bifidobacteria, and bacteroides showed their adaptations.     

幽门螺杆菌感染蒙古沙鼠胃部菌群改变及病理学变化

目的观察蒙古沙鼠感染幽门螺杆菌（Helicobacter pylori,H.pylori）后胃部菌群及病理学变化。方法 5周龄蒙古沙鼠60只,随机分为实验组（30只）和对照组（30只）。所有沙鼠禁食不禁水24 h后,实验组灌喂109CFU/mLH.pylori菌液0.5 mL/只,连续3次。对照组灌喂无菌肉汤。在4、8、16、24和48周处死动物,进行胃部菌群分析和H.pylori分离培养及病理学检查。结果正常沙鼠胃中存在着以乳酸菌为主的正常菌群[（8.43±5.21）×105CFU/g],感染H.pylori后正常菌群数量显著减少;实验组沙鼠H.pylori感染率为100%,第4周可见沙鼠胃组织红肿充血,第8周有炎性细胞浸润,16周和24周出现糜烂,48周见出血、慢性活动性胃炎及溃疡。对照组沙鼠无H.pylori定植及组织学病变。结论 H.pylori感染使蒙古沙鼠胃内正常菌群发生变化,从而引起胃炎和胃溃疡发生。     

In vitro and in vivo complementation of the Helicobacter pylori arginase mutant using an intergenic chromosomal site

BACKGROUND: Gene complementation strategies are important in validating the roles of genes in specific phenotypes. Complementation systems in Helicobacter pylori include shuttle vectors, which transform H. pylori at relatively low frequencies, and chromosomally based approaches. Chromosomal complementation strategies are susceptible to polar effects and disruption of other H. pylori genes, leading to unwanted pleiotropic effects. MATERIALS AND METHODS: A new complementation strategy was developed for H. pylori by utilizing a suicide plasmid vector that contains fragments of an H. pylori intergenic region (hp0203-hp0204), a chloramphenicol acetyltransferase cassette (cat), and a multiple-cloning site. Genes of interest could be cloned into the intergenic plasmid and the genes integrated into H. pylori by homologous recombination into the intergenic chromosomal region without disrupting any annotated H. pylori gene. The complementation system was validated using the gene encoding arginase (rocF). RESULTS: A rocF mutant unable to hydrolyze or consume l-arginine regained these functions by complementation with the wild-type rocF gene. Complemented strains also had restored arginase protein as determined by Western blot analysis. The complementation system could be successfully applied to multiple H. pylori strains. The intergenic region varied in length and sequence across 17 H. pylori strains, but the flanking-3' ends of the hp0203 and hp0204 coding regions were highly conserved. Inserting a cat cassette and wild-type rocF into the intergenic region did not alter the ability of strain SS1 to colonize mice. CONCLUSIONS: This complementation strategy should greatly facilitate genetic experiments in H. pylori.     

Effect of dietary anti-urease immunoglobulin Y on Helicobacter pylori infection in Mongolian gerbils

BACKGROUND AND AIM: Helicobacter pylori is known to be a major pathogenic factor in the development of gastritis, peptic ulcer disease and gastric cancer. Recently, chicken egg yolk immunoglobulin Y (IgY) has been recognized as an inexpensive antibody source for passive immunization against gastrointestinal infections. The present study was designed to investigate the effect of anti-urease IgY on H. pylori infection in Mongolian gerbils. METHODS: H. pylori-infected Mongolian gerbils were administered a diet containing anti-urease IgY, with or without famotidine (F). After 10 weeks, bacterial culture and measurement of the gastric mucosal myeloperoxidase (MPO) activity were performed. In a second experiment, another group of gerbils was started on a diet containing F + IgY a week prior to H. pylori inoculation. After 9 weeks, these animals were examined. RESULTS: In the H. pylori-infected gerbils, there were no significant differences in the level of H. pylori colonization among the different dietary and control groups. However, the MPO activity was significantly decreased in the H. pylori group administered the F + IgY diet compared with that in the H. pylori group administered the IgY, F, or control diet. Furthermore, in the gerbils administered the F + IgY diet prior to the bacterial inoculation, inhibition of H. pylori colonization and suppression of the elevated gastric mucosal MPO activity were observed. CONCLUSIONS: Oral administration of urease-specific IgY not only inhibited H. pylori disease activity in H. pylori-infected gerbils, but also prevented H. pylori colonization in those not yet infected. These encouraging results may pave the way for a novel therapeutic and prophylactic approach in the management of H. pylori-associated gastroduodenal disease.     

Role of bacterial strain diversity of Helicobacter pylori in gastric carcinogenesis induced by N-methyl-N-nitrosourea in Mongolian gerbils

AIM: Helicobacter pylori is known to enhance gastric carcinogenesis induced by chemical carcinogens. We previously demonstrated that infection with H. pylori strain SS1 did not enhance such carcinogenesis in C57BL/6 mice. Whether this result was due to the bacterial strain SS1 or to the experimental host, C57BL/6 mice, should be addressed. Therefore, we examined whether H. pylori strains introduced to the same host (Mongolian gerbils) differed in carcinogenicity. MATERIALS AND METHODS: H. pylori TN2GF4 strain (CagA(+), VacA(+)) and SS1 strain (CagA functionally(-), VacA(-)) were infected to Mongolian gerbils (n = 126). In the first experiment (induction of gastritis), histologic change in gastric mucosa of gerbils infected by H. pylori (TN2GF4, SS1, vehicle) without N-methyl-N-nitrosourea (MNU) at 1 month or 6 months was assessed. In the second experiment (experimental carcinogenesis), H. pylori (TN2GF4, SS1, vehicle) was inoculated to the gerbils after administration of MNU for 10 weeks, and the number of cancers and histopathologic changes at week 54 were assessed. RESULTS: In the first experiment, activity and inflammation in the TN2GF4 group were significantly greater than in the SS1 group at 1 month, while no significant difference was noted at 6 months. On the other hand, intestinal metaplasia and atrophy were significantly greater with TN2GF4 than with SS1 at 6 months but not at 1 month. In studies on experimental carcinogenesis, microscopically, 47.8% (11/23), 26% (7/26), and 0% (0/26), of animals had gastric adenocarcinoma in the MNU + TN2GF4 group, MNU + SS1 group, and MNU alone group, respectively. CONCLUSION: Both H. pylori strains, TN2GF4 and SS1, promoted carcinogenesis in Mongolian gerbils. The severity of gastritis and destruction and restoration of gastric mucosa may be related to gastric carcinogenesis. That the SS1 strain significantly accelerated carcinogenesis only in Mongolian gerbils and not in C57BL/6 mice suggests the crucial role of host factors in carcinogenesis by H. pylori infection.     

Inhibitory effects of various micro-organisms on the growth of Helicobacter pylori

AIMS: To examine the in vitro influence of various bacteria species on Helicobacter pylori (Hp) growth. METHODS AND RESULTS: The effects of 29 micro-organisms on 31 Hp strains were determined using two modified 'cross streak' methods. Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Morganella morganii, Serratia marcescens, Bacteroides fragilis, Fusobacterium nucleatum and Clostridium difficile showed the strongest inhibition. The inhibitory effects varied, depending on the bacteria spp. and Hp strains, and were method dependent. The cagA status of Hp strains did not correlate with the extent of inhibition. CONCLUSIONS: Helicobacter pylori is inhibited by a significant number of commensal bacteria species as well as opportunistic human pathogens. The success and progress of Hp infection may be influenced by the bacterial flora present, while the difficulty in cultivating Hp from the oral mucosa and faeces may be the result of antagonistic bacterial interaction. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides valuable data on the sensitivity of Hp to a variety of intestinal and oral commensals as well as opportunistic human pathogens. Hp's varying pathogenicity and the specific localization of infection may be the result of these sensitivities. These results can also serve as a basis for further studies to identify the inhibitory substances and make them available for therapeutic use.     

Characteristics of a clinical isolate of urease-negative Helicobacter pylori and its ability to induce gastric ulcers in Mongolian gerbils

BACKGROUND: We clinically obtained urease-negative mutant strains of Helicobacter pylori. The goal of this study was to investigate the ability of the urease-negative strain to colonize and subsequently damage the gastric mucosa in Mongolian gerbils. In addition, the genes encoding the urease production in the test strain were analyzed, and other genes encoding the virulence factors, cytotoxin-associated protein and vacuolating-cytotoxin were evaluated. MATERIALS AND METHODS: The character of urease-negative isolates of H. pylori was defined. The identification of H. pylori was confirmed by polymerase chain reaction (PCR). The H. pylori isolate was transfected into Mongolian gerbils as previously described, which were followed up to 42 weeks, and the changes in their gastric mucosa were examined histologically. RESULTS AND CONCLUSION: Fifteen Mongolian gerbils orally infected with 10(7) colony forming units of urease-negative H. pylori were killed at 4, 12, 24, 36 and 42 weeks (n = 3) after infection. Culture medium without urease-negative H. pylori was given to the Mongolian gerbils as control. H. pylori continued to exist in the subject's stomach and gastric ulceration was observed and compared with the control. Clinically obtained urease-negative H. pylori continued to exist for at least 42 weeks in the subject's stomach and it induced gastric ulcers. These data demonstrated that the urease in H. pylori was not a necessary factor in the formation of gastric ulcers in the Mongolian gerbil model.     

Seroprevalence of Helicobacter pylori in Hispanics living in Puerto Rico: A population‐based study

Background

Helicobacter pylori is an important etiologic factor for peptic ulcers and gastric cancer, one of the top ten leading causes of cancer death in Puerto Rico. However, the prevalence of H. pylori infections in this population was previously unknown. The aim of this study was to examine the seroprevalence of H. pylori and its associated risk factors in Puerto Rico.

Materials and Methods

A cross‐sectional study was designed using an existing population‐based biorepository. Seropositivity was determined using the Premier^? H. pylori immunoassay. Helicobacter pylori seroprevalence was estimated with 95% confidence using marginal standardization following logistic regression. To assess the risk factors associated with H. pylori seropositivity, a multivariable log‐binomial model was fitted to estimate the prevalence ratio (PR) and its 95% confidence interval (95% CI).

Results

A total of 528 population‐based serum samples were analyzed. The mean age of the study population was 41 ± 12 years, of whom 55.3% were females. The overall seroprevalence of H. pylori was 33.0% (95% CI = 28.3%‐38.1%). Increasing age and having <12 years of education were significantly (P < .05) associated with H. pylori seropositivity in the multivariable model; however, residing in counties with low population density reached marginal significance (P = .085).

Conclusions

We report that H. pylori infection is common among Hispanics living in Puerto Rico. The H. pylori seroprevalence observed in Puerto Rico is similar to the seroprevalence reported in the overall population of the United States. The association between H. pylori seroprevalence and the risk factors analyzed offers insight into the epidemiology of gastric cancer in Puerto Rico and warrants further investigation.

     

Identification of Helicobacter pylori strain cagPAI+ and cagPAI- Antigens by IgG antibodies from sera of experimentally colonized meriones unguiculatus (Mongolian gerbils)

Background: Mongolian gerbils that are experimentally infected with Helicobacter pylori develop a chronic inflammation that is similar to natural infections in humans. The aim of this study was to compare the antigens of H. pylori cagPAI+ and cagPAI? strains that are expressed during Meriones unguiculatus colonization. Materials and Methods: We identified H. pylori cagPAI+ and cagPAI? strain antigens via Western blotting of samples from Mongolian gerbils that were subjected to unique, mixed, and sequential bacterial infections. Results: The antigens from the J99/CG3 (cagPAI+) strain had a lower molecular weight than the antigens from the 251F/CG3 (cagPAI?) strain. There were fewer identified antigens in the single unique infections compared with the mixed and sequential infections. The number of recognized antigens that had a frequency of recognition >60% was higher for the simultaneous and sequential infection groups compared with the single infection group. A 57‐kDa antigen was present in >60% of the samples and four of the five experimental groups. Antigens specific to each bacterial strain were identified; the 190‐ and 158‐kDa antigens appear to be specific for cagPAI?, and the 70‐kDa antigen appears to be specific for cagPAI+. Conclusions: In this study, we identified antigens that are common and specific to the H. pylori cagPAI+ and cagPAI? strains.     

In vivo expression of the 25-kDa laminin-binding protein of Helicobacter pylori

The gastroduodenal pathogen Helicobacter pylori has been shown to inhibit the interaction between the extracellular matrix protein laminin and its receptor on gastric epithelial cells, potentially contributing to a loss of mucosal integrity. As a 25-kDa outer membrane protein of H. pylori in association with the bacterial lipopolysaccharides (LPS) mediates attachment to laminin, the aim of this study was to determine whether the 25-kDa protein is produced by H. pylori in infected hosts. We examined the immune response to the 25-kDa laminin binding protein in 12 paediatric patients; samples from a H. pylori-negative healthy adult were used as controls. In immunoblotting, antibodies to a 25-kDa protein were found in the serum and saliva of H. pylori-positive individuals only, and using the positive sera and saliva, laminin binding to the 25-kDa protein was inhibited. Thus, the 25-kDa laminin-binding protein is produced by H. pylori in infected hosts.     

Antibacterial effects of the urushiol component in the sap of the lacquer tree (Rhus verniciflua Stokes) on Helicobacter pylori

Background: Urushiol is a major component of the lacquer tree which has been used as a folk remedy for the relief of abdominal discomfort in Korea. The aim of this study was to evaluate the antibacterial effects of the urushiol on Helicobacter pylori. Materials and Methods: Monomer and 2–4 polymer urushiol were used. In the in vitro study, pH‐ and concentration‐dependent antibacterial activity of the urushiol against H. pylori were investigated. In addition, the serial morphological effects of urushiol on H. pylori were examined by electron microscopy. In vivo animal study was performed for the safety, eradication rate, and the effect on gastritis of urushiol. The expression of pro‐inflammatory cytokines was checked. Results: All strains survived within a pH 6.0–9.0. The minimal inhibitory concentrations of the extract against strains ranged 0.064–0.256 mg/mL. Urushiol caused separation of the membrane and lysis of H. pylori within 10 minutes. Urushiol (0.128 mg/mL × 7 days) did not cause complications on mice. The eradication rates were 33% in the urushiol monotherapy, 75% in the triple therapy (omeprazole + clarithromycin + metronidazole), and 100% in the urushiol + triple therapy, respectively. H. pylori‐induced gastritis was not changed by urushiol but reduced by eradication. Only the expression of interleukin‐1β in the gastric tissue was significantly increased by H. pylori infection and reduced by the urushiol and H. pylori eradication (p = .014). Conclusions: The urushiol has an antibacterial effect against H. pylori infection and can be used safely for H. pylori eradication in a mouse model.     

The role of interleukin-17 in the Helicobacter pylori induced infection and immunity

Background: Helicobacter pylori infection is regarded as the major cause of various gastric diseases and induces the production of several cytokines including interleukin‐17 (IL‐17) recently recognized as an important player in the mammalian immune system. Objective: This review deals with the role of IL‐17 on the H. pylori‐induced infection and immunity in humans and experimental animals. Results: H. pylori infection increases IL‐17 in the gastric mucosa of humans and experimental animals. In humans, IL‐17 induces the secretion of IL‐8 by activating the ERK 1/2 MAP kinase pathway and the released IL‐8 attracts neutrophils promoting inflammation. IL‐23 is increased in patients with H. pylori‐related gastritis and regulates IL‐17 secretion via STAT3 pathway. Studies in H. pylori‐infected mice indicate that IL‐17 is primarily associated with gastric inflammation. The early events in the immune response of immunized and challenged mice include the recruitment of T cells and the production of IL‐17. Neutrophil attracting chemokines are released, and the bacterial load is considerably reduced. IL‐17 plays a dual role in infection and vaccination. In infection, T regulatory cells (Tregs) suppress the inflammatory reaction driven by IL‐17 thereby favoring bacterial persistence. Immunization produces Helicobacter‐specific memory T‐helper cells that can possibly alter the ratio between T‐helper 17 and Treg responses so that the IL‐17‐driven inflammatory reaction can overcome the Treg response leading to bacterial clearance. Conclusion: IL‐17 plays an important role in H. pylori‐related gastritis and in the reduction of Helicobacter infection in mice following immunization.     

幽门螺杆菌球形体回复原形的实验研究

目的对幽门螺杆菌（Helicobacter pylori,Hp）球形体进行体内、外回复原形的比较研究,揭示其潜在的传播途径。方法在布氏肉汤的基础上设计了4种Hp再生培养基,对Hp螺旋体、原生质体及球形体进行体外培养;同时采用30只蒙古沙土鼠（Mongolian gerbil）进行体内感染定植实验,对感染小鼠胃粘膜进行Hp定量培养和组织学检测。结果Hp球形体在4种再生培养基中均未能回复生长;而在感染小鼠的体内却观察到了Hp球形体的回复定植。Hp螺旋体感染组在小鼠胃内的定植密度较高,且胃粘膜下可见大量炎症细胞浸润;而球形体感染组并未见到小鼠胃粘膜组织的明显炎症损伤,且仅有少量回复的螺旋体定植。结论Hp球形体作为一种低水平代谢休眠体,代谢活性及毒力均有所减弱,但仍具有潜在的致病性。本研究支持部分Hp球形体具有活力但体外不能培养成活这一假说,提示＂粪-口＂传播应引起更多的关注。     

Interaction of Helicobacter pylori Infection and Nonsteroidal Anti‐Inflammatory Drugs in Gastric and Duodenal Ulcers

Background: Gastric (GU) and duodenal ulcers (DU) are in most instances either induced by Helicobacter pylori infection or by nonsteroidal anti‐inflammatory drugs (NSAIDs). Whether eradication of H. pylori is beneficial in NSAID users for preventing NSAID induced GU and DU has been the focus of different studies. Materials and Methods: Mechanisms shared by both H. pylori and NSAIDs for the induction of GU and DU were reviewed and randomized controlled trials on H. pylori eradication for prevention and healing of GU and DU in patients requiring NSAID therapy were identified by a PubMed search. Results: Key factors in the induction of GU and DU for both H. pylori and NSAIDs are a decrease in pH, imbalance between apoptosis and proliferation, reduction in mucosal blood flow, and recruitment of polymorphonucleates in distinct compartments. For primary ulcer prevention, H. pylori eradication before starting an NSAID therapy reduces the risk of NSAID induced GU and virtually abolishes the risk of DU. H. pylori eradication alone is not sufficient for secondary prevention of NSAID induced GU and DU. H. pylori infection appears to further increase the protective effects of proton‐pump inhibitors (PPI) to reduce the risk of ulcer relapse. H. pylori eradication does not influence the healing of both GU and DU if NSAID intake is discontinued. Conclusions: Duodenal ulcer is more closely related to H. pylori infection than GU in NSAID users. H. pylori eradication is recommended for primary prevention of GU and DU in patients requiring NSAID therapy. PPI therapy is mandatory for secondary prevention of gastroduodenal ulcers, and appears to further reduce the risk of ulcer relapse in the presence of H. pylori.