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.     

Enterocromaffin-like cell tumor induced by Helicobacter pylori infection in Mongolian gerbils

Background. Gastric carcinoids are strongly associated with chronic atrophic gastritis A, and it is suggested that hypergastrinemia plays a critical role in development of gastric carcinoids. Since Helicobacter pylori infection causes hypergastrinemia, it is held that H. pylori infection produces gastric carcinoids. We followed the histological changes of H. pylori‐infected stomachs of Mongolian gerbils for a long time. Materials and Methods. Five‐week‐old‐male Mongolian gerbils were infected with H. pylori ATCC 43504 with cagA gene, expressing vacuolating cytotoxin. Determination of the serum gastrin and histopathological examination of the stomach at 6, 12, 18, and 24 months after H. pylori inoculation was studied and compared with uninfected animals . Results In infected animals, the gastric carcinomas appeared 18 and 24 months after infection. Endocrine cell dysplasias and carcinoids with marked atrophic gastritis of the oxyntic mucosa were observed in the infected animals 24 months after H. pylori inoculation. The serum gastrin level in the infected group increased from an average of 86.2 pg/ml at the beginning of the study to an average of 498 pg/ml and 989 pg/ml at 18 and 24 months after infection, respectively. These changes in the serum gastrin levels were significant compared with uninfected controls that showed no changes. Conclusions. H. pylori infection caused not only gastric carcinomas but also enterochromaffin‐like cell tumors in Mongolian gerbils, due to hypergastrinemia. This model is thought to be useful to study the relationship between hypergastrinemia and gastric carcinoids.     

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。     

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.     

NMR-based metabonomics for detection of Helicobacter pylori infection in gerbils: which is more descriptive

Background:  Helicobacter pylori , the human pathogenic gram-negative microaerophilic bacterium, causes chronic gastric infection in more than half of the human population regardless of race. The infection of microbe is not yet controllable to pose a substantial public health impact and a growing social burden. The management of H. pylori infection primarily necessitates accurate and timely diagnosis at case level, on-demand supervision of pathologic progression, and reliable evaluation of the impact of pharmacologic interventions on the patients' population.
Methods:  The characterization of H. pylori infection on gerbils model was performed by metabolic profiling, employing ¹H NMR spectroscopy compounding multivariate pattern recognition strategies. In the same manner, urine samples were individually collected from 10 gerbils infected with H. pylori GS13, and from 10 uninfected control animals equally accessible to feed and water.
Results:  The resultant metabolic profiles indicate that H. pylori infection disturbs carbohydrate metabolism to elevate the levels of α- and β-glucose, and cis -aconitate (a TCA cycle intermediate). In addition to the energy metabolism alteration, the colonization of H. pylori in gerbil stomach generates a remarkable deviation of amino acid metabolism as indicated by depletion of taurine and arginine, and elevation of proline and glutamine in the animal urine. Moreover, the H. pylori infection modifies the gut microbiota as highlighted by a range of microbial-related metabolites such as indoxyl sulfate and hippurate.
Conclusions:  These findings demonstrate that the ¹H NMR-based urine metabolic profiling is a promising technique capable of providing an accurate, noninvasive, and rapid diagnosis of H. pylori infection.     

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.     

Prophylactic immunization to Helicobacter pylori infection using spore vectored vaccines

Background

Helicobacter pylori infection remains a major public health threat leading to gastrointestinal illness and increased risk of gastric cancer. Mostly affecting populations in developing countries no vaccines are yet available and the disease is controlled by antimicrobials which, in turn, are driving the emergence of AMR.

Materials and Methods

We have engineered spores of Bacillus subtilis to display putative H. pylori protective antigens, urease subunit A (UreA) and subunit B (UreB) on the spore surface. Following oral dosing of mice with these spores, we evaluated immunity and colonization in animals challenged with H. pylori.

Results

Oral immunization with spores expressing either UreA or UreB showed antigen-specific mucosal responses (fecal sIgA) including seroconversion and hyperimmunity. Following challenge, colonization by H. pylori was significantly reduced by up to 1-log.

Conclusions

This study demonstrates the utility of bacterial spores for mucosal vaccination to H. pylori infection. The heat stability and robustness of Bacillus spores coupled with their existing use as probiotics make them an attractive solution for either protection against H. pylori infection or potentially for therapy and control of active infection.     

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.     

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。     

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.     

Profiling and identification of eubacteria in the stomach of Mongolian gerbils with and without Helicobacter pylori infection

Background. Mongolian gerbils are frequently used to study Helicobacter pylori‐induced gastritis and its consequences. The presence of an indigenous bacterial flora with suppressive effect on H. pylori may cause difficulties with establishing this experimental model. Aim. The aim of the present study was to determine bacterial profiles in the stomach of Mongolian gerbils with and without (controls) H. pylori infection. Methods. Gastric tissue from H. pylori ATCC 43504 and CCUG 17874 infected and control animals were subjected to microbial culturing and histology. In addition, gastric mucosal samples from H. pylori ATCC 43504 infected and control animals were analyzed for bacterial profiling by temporal temperature gradient gel electrophoresis (TTGE), cloning and pyrosequencing of 16S rDNA variable V3 region derived PCR amplicons. Results. Oral administration of H. pylori ATCC 43504, but not CCUG 17874, induced colonization and gastric inflammation in the stomach of Mongolian gerbils. Temporal temperature gradient gel electrophoresis (TTGE) and partial 16S rDNA pyrosequencing revealed the presence of DNA representing a mixed bacterial flora in the stomach of both H. pylori ATCC 43504 infected and control animals. In both cases, lactobacilli appeared to be dominant. Conclusion. These findings suggest that indigenous bacteria, particularly lactobacilli, may have an impact on the colonization and growth of H. pylori strains in the stomach of Mongolian gerbils.     

Polaprezinc attenuates Helicobacter pylori-associated gastritis in Mongolian gerbils

Background. The ammonia‐monochloramine system plays an important role in Helicobacter pylori‐associated gastric mucosal injury. Polaprezinc, a new antiulcer agent, has a scavenging action against monochloramine. The aim of the experiment was to investigate the inhibitory effects of polaprezinc on the H. pylori‐induced gastritis in Mongolian gerbils. Materials and Methods. Mongolian gerbils fasting for 24 hours were orally given culture broth containing 2–4 × 10⁸ colony‐forming units of H. pylori ATCC 43054 per milliliter. From 4 hours after inoculation until the end of the experiment, gerbils were given chow pellets with or without 0.02% polaprezinc. All gerbils were killed 12 weeks later. The grades of H. pylori density and histologic features of gastritis were evaluated in accordance with the Updated Sydney System. The scavenging effect of polaprezinc on monochloramine was investigated spectrophotometrically. Results. Polaprezinc had little or no influence on the H. pylori density in both pyloric and fundic mucosae. However, it significantly attenuated the development of polymorphonuclear neutrophil activity, mononuclear infiltration, and surface epithelial erosion in both pyloric and fundic mucosae compared with those of the control group. H. pylori inoculation significantly increased the heights of both pyloric and fundic mucosae (mainly due to the increased height of foveolar hyperplasia), but polaprezinc inhibited the increase of mucosal thickness in both pyloric and fundic mucasae. No intestinal metaplasia was detected in this study. Spectrophotometric examination revealed that polaprezinc scavenged monochloramine. Conclusions. Polaprezinc inhibited the development of H. pylori‐induced gastritis through its scavenging action against monochloramine.     

Suppression of Helicobacter pylori-induced gastritis by green tea extract in Mongolian gerbils

Since urease of Helicobacter pylori is essential for its colonization, we focused attention on foodstuffs which inhibit the activity of this enzyme. Among plant-derived 77 foodstuff samples tested, some tea and rosemary extracts were found to clearly inhibit H. pylori urease in vitro. In particular, green tea extract (GTE) showed the strongest inhibition of H. pylori urease, with an IC(50) value of 13 microg/ml. Active principles were identified to be catechins, the hydroxyl group of 5(')-position appearing important for urease inhibition. Furthermore, when H. pylori-inoculated Mongolian gerbils were given GTE in drinking water at the concentrations of 500, 1000, and 2000 ppm for 6 weeks, gastritis and the prevalence of H. pylori-infected animals were suppressed in a dose-dependent manner. Since the acquisition by H. pylori of resistance to antibiotics has become a serious problem, tea and tea catechins may be very safe resources to control H. pylori-associated gastroduodenal diseases.     

Placental acquisition of maternal specific IgG and Helicobacter pylori colonization in infancy

Background. Colonization with Helicobacter pylori generally occurs in infancy, and the microorganism is often acquired from close family members. Rate of infant colonization may be affected by maternal immune status. Methods. To investigate the potential protective effect of anti‐H. pylori immunoglobulin G (IgG) acquired via the placenta, 65 mothers and their infants were studied from the infant's birth for 1 year. Circulating IgG antibodies were measured by enzyme‐linked immunosorbent assay (ELISA) in cord blood and every 8 weeks. Immunoblotting was performed on sera from infants with significant increases in IgG levels. Rate of infant H. pylori colonization was measured by ¹³C urea breath tests every 4 weeks from the age of 12 weeks. Results. Maternal and infant cord blood specific IgG levels were correlated (R² = .747, p < .001). Infant H. pylori specific IgG fell 5‐fold compared to maternal levels over the first 6 months of life, and rose subsequently in many cases, with the development of novel immunoblot patterns. There were no significant associations between the age at first positive urea breath test and maternal or infant cord specific H. pylori IgG levels. Conclusions. Transplacentally acquired specific IgG antibody does not protect infants from colonization by H. pylori.     

Reduction of Overall Helicobacter pylori Colonization Levels in the Stomach of Mongolian Gerbil by Lactobacillus johnsonii La1 (LC1) and Its in Vitro Activities against H. pylori Motility and Adherence

The effects of Lactobacillus johnsonii La1 (LC1) on Helicobacter pylori colonization in the stomach were investigated. H. pylori colonization and gastritis in LC1-inoculated Mongolian gerbils were significantly less intense than those in the control animals. LC1 culture supernatant (>10-kDa fraction) inhibited H. pylori motility and induced bacterial aggregation in human gastric epithelial cells, suggesting the potential of clinical use of LC1 product.     

高盐溶液预处理幽门螺杆菌对蒙古沙土鼠胃黏膜损伤作用

目的 探讨高盐预处理的幽门螺杆菌(H.pylori)对胃黏膜的损伤作用。 方法 将30%高盐预处理前后的胃癌来源的H.pylori菌株(4854)灌胃蒙古沙土鼠(MGs),在灌胃后13、26和73周解剖动物,通过组织病理学检查、免疫组化染色和黏膜厚度测量,探讨高盐预处理的H.pylori对胃黏膜的损伤作用。 结果 与未加盐预处理的相应菌株相比,高盐预处理组小鼠的慢性炎症、黏膜变性/坏死、腺体萎缩伴肠上皮化生的发生率较低,黏膜糜烂/溃疡和黏膜上皮增生的发生率较高,差异均有统计学意义(t=8.325 6,P=0.040 8)。第73周,高盐预处理4854菌株组胃体和胃窦黏膜增生显著高于未加盐预处理组(t=12.802 4,P=0.035 1;t=16.536 0,P=0.043 8)。 结论 高盐预处理改变了H.pylori的体内致病性,有助于阐明H.pylori感染与高盐饮食在胃病中的相互作用模式。