Effect of Weissella confusa Strain PL9001 on the Adherence and Growth of Helicobacter pylori

Viable and nonviable Weissella confusa strain PL9001 inhibited the binding of Helicobacter pylori to human gastric-cell line MKN-45 cells by more than 90%. Spent culture supernatant of PL9001 rapidly decreased the viability of H. pylori, rupturing cell walls. The results suggest that PL9001 is a probiotic that can reduce the infectivity and persistence of H. pylori.     

Helicobacter pylori utilises urea for amino acid synthesis

Abstract Helicobacter pylori has one of the highest urease activities of all known bacteria. Its enzymatic production of ammonia protects the organism from acid damage by gastric juice. The possibility that the urease activity allows the bacterium to utilise urea as a nitrogen source for the synthesis of amino acids was investigated. H. pylori (NCTC 11638) was incubated with 50 mM urea, enriched to 5 atom% excess ¹⁵N, that is the excess enrichment of ¹⁵N above the normal background, in the presence of either NaCl pH 6.0, or 0.2M citrate pH 6.0. E. coli (NCTC 9001) was used as a urease-negative control. ¹⁵N enrichment was detected by isotope ratio mass spectrometry. H. pylori showed intracellular incorporation of ¹⁵N in the presence of citrate buffer pH 6.0 but there was no significant incorporation of ¹⁵N in unbuffered saline or by E. coli in either pH 6.0 citrate buffer or unbuffered saline. The intracellular fate of the urea-nitrogen was determined by means of gas chromatography/mass spectrometry following incubation with ¹⁵N enriched 5 mM urea in the presence of either 0.2 M citrate buffer pH 6.0 or 0.2 M acetate buffer pH 6.0. After 5 min incubation in either buffer the ¹⁵n label appeared in glutamate, glutamine, phenylalanine, aspartate and alanine. It appears, therefore, that at pH and urea concentrations typical of the gastric mucosal surface, H. pylori utilises exogenous urea as a nitrogen source for amino acid synthesis. The ammonia produced by H. pylori urease activity thus facilitates the organism's nitrogen metabolism at neutral pH as well as protecting it from acid damage at low pH.     

Characterization of a culture method to recover Helicobacter pylori from the feces of infected patients

Background. Helicobacter pylori is difficult to culture from stool. Multiple efforts from multiple laboratories have been unsuccessful, and the optimal conditions to recover H. pylori from stool are still not known. Recovery of H. pylori from feces of infected individuals is important for the performance of molecular epidemiological investigations, especially in children, where their symptoms do not warrant endoscopy to recover the organism.
Methods. Fresh fecal specimens (noncathartic) were obtained from 19 known H. pylori –infected patients and were processed to recover the organism. Fresh fecal specimens (noncathartic) were also obtained from three known H. pylori –negative individuals (controls) to determine whether H. pylori could be isolated from stools seeded with known concentrations of the organism. Treatment of the fecal suspensions with cholestyramine, a basic anion exchange resin that binds bile acids, was used in an attempt to enhance recovery of H. pylori by sequestering bile acids that are inhibitory to H. pylori growth. H. pylori was identified based on colony morphology, cell morphology, Gram's stain, biochemical reactions, and polymerase chain reaction for two H. pylori genes.
Results. Among 19 patients, H. pylori was cultured at least once from 3 and three times from 2 (5 of 19). Feces that were seeded with H. pylori and obtained from three H. pylori –negative volunteer controls yielded positive recovery in all instances.
Conclusion. We have confirmed that it is possible to culture H. pylori from human stool, but the procedure for optimal recovery has still not been defined.     

Enhancement in Ex Vivo Phagocytic Capacity of Peritoneal Leukocytes in Mice by Oral Delivery of Various Lactic-Acid-Producing Bacteria

Lactic-acid-producing bacteria (LABs) are known to have immunomodulating activity. In the current study, various LABs were tested for their immunity-enhancing activity, especially the phagocytic activity of leukocytes. Viable but not heat-killed cells of Weissella kimchii strain PL9001, Lactobacillus fermentum strain PL9005, and L. plantarum strain PL9011 significantly increased the ex vivo phagocytic capacity of mouse peritoneal leukocytes to ingest fluorescein isothiocyanate (FITC)-labeled Escherichia coli in a strain-dependent manner. Results of this and previous studies suggest these LABs as candidates for new probiotics. This is the first report of the enhancement of peritoneal leukocyte activity of these species.     

Long-term follow up of Helicobacter pylori IgG serology after eradication and reinfection rate of H. pylori in South Korea

Background: Serology is widely used for epidemiologic research of Helicobacter pylori . However, there is limited information on the long-term follow up of H. pylori titers after eradication. In addition, it is presumed that the reinfection rate decreases as the H. pylori infection rate decreases. The aim of this study was to investigate the long-term follow up of H. pylori IgG, and to evaluate the reinfection rate of H. pylori in Korea.
Methods: Among 247 patients, who were enrolled during 2003–07, 185 patients with invasive H. pylori test positive received proton pump inhibitor-based triple therapy, and follow-up H. pylori testing, including histology, CLOtest, culture, and serology, were evaluated 2, 10, and 18 months after H. pylori eradication.
Results: The initial H. pylori IgG optical density (OD_450nm), 2.06, gradually decreased to 0.63 (67% reduction) at 18 months after H. pylori eradication. The seroreversion rate was 5, 10, and 45% at 2, 10, and 18 months after H. pylori eradication, respectively. The recrudescence of H. pylori was 3.49%, and the annual reinfection rate was 2.94% per year. H. pylori IgG titers abruptly increased in cases with recrudescence and reinfection, and correlated with the results of the invasive H. pylori tests.
Conclusion: The results of this study showed that H. pylori IgG serology could be used for the determination of reinfection of H. pylori, but not for the diagnosis of H. pylori eradication. The reinfection rate of H. pylori , in Korea, was found to be very low, 2.94% per year.     

Combined effect of rebamipide and ecabet sodium on Helicobacter pylori adhesion to gastric epithelial cells

Helicobacter pylori is a major etiological agent in gastroduodenal disorders. The adhesion of H. pylori to gastric epithelial cells is the initial step of H. pylori infection. Inhibition of H. pylori adhesion is thus a therapeutic target in the prevention of H. pylori infection. We have reported that rebamipide and ecabet sodium, mucoprotective antiulcer agents, independently inhibit H. pylori adhesion. However, the antiadhesion activity of each antiulcer agent was incomplete. Experiments were performed to evaluate the combined effect of rebamipide and ecabet sodium on H. pylori adhesion to gastric epithelial cells. MKN-28 and MKN-45 cells, derived from human gastric carcinomas, were used as target cells. Twelve clinical isolates of H. pylori were used in this study. We evaluated the effects of rebamipide and ecabet sodium, individually and in combination, on H. pylori adhesion to target cells quantitatively using our previously established enzyme-linked immunosorbent assay. Rebamipide and ecabet sodium each partially inhibited H. pylori adhesion. In contrast, adhesion was almost completely inhibited by pretreating target cells and H. pylori with the combination of rebamipide and ecabet sodium. Our studies suggest that the synergistic antiadhesion activity of rebamipide and ecabet sodium is greater than that of each antiulcer agent alone.     

不同类型幽门螺杆菌对GES-1细胞Cx32、Cx43表达的影响

目的观察不同类型幽门螺杆菌（Helicobacter pylori,H.pylori）对人胃黏膜上皮细胞系GES-1细胞间隙连接蛋白（Connexin,Cx）32和43表达的影响,探讨与Cx32、Cx43表达异常有关的H.pylori菌株类型。方法将临床培养分离的不同H.pylori菌株类型包括东亚型CagA＋H.pylori、西方型CagA＋H.pylori及CagA-H.pylori与GES-1细胞共培养24 h及48 h,对照组不加H.pylori培养24 h及48 h。采用间接免疫荧光方法（IIF）及计算机图像分析技术检测GES-1细胞Cx32、Cx43表达。结果对照组24 h和48 h及加H.pylori各组24 h GES-1细胞Cx32、Cx43表达阳性率均为100%,东亚型CagA＋H.pylori组48 h Cx32、Cx43表达阳性率均低于对照组、CagA-H.pylori组和西方型CagA＋H.pylori组（P〈0.05）;对照组24 h和48 h Cx32、Cx43绿色荧光位于细胞膜,西方型CagA＋H.pylori组和东亚型CagA＋H.pylori组24 h和48 h Cx32绿色荧光大部分位于细胞膜,少部分位于细胞浆,Cx43绿色荧光大部分位于细胞浆,少部分位于细胞膜;东亚型CagA＋H.pylori组和西方型CagA＋H.pylori组24 h及48 hCx32、Cx43表达强度低于对照组和CagA-H.pylori组（P〈0.05）,且东亚型CagA＋H.pylori组较西方型CagA＋H.py-lori组减弱更明显（P〈0.05）。结论 H.pylori下调GES-1细胞Cx32、Cx43表达,以CagA＋H.pylori菌株特别是东亚型CagA＋H.pylori菌株作用更明显。     

A change of IL-2 and IL-4 production in patients with Helicobactor pylori infection

Hellcobacter pylori is the most common cause of gastroduodenal inflammation. However, the exact immune pathogenesis is not fully understood. To look for evidence of the immunological mechanism in H. pylori associated disease, we measured cytokine interleukin-2 (IL-2) and IL-4 levels produced by peripheral blood lymphocytes (PBL) and gastric biopsies in 20 subjects with or without H. pylori infection. H. pylori can stimulate IL-2 and IL-4 production from PBL in H. pylori negative as well as H. pylori positive individuals. The spontaneous IL-2 production by PBL and gastric biopsies was greater (p < 0.0025, <0.001)in H. pylori negative individuals than that in H. pylori infected patients. Increased IL-4 levels from PBL in H. pylori infected patients were found in the presence of H. pylori (p < 0.0025). An increased spontaneous production of IL-4 from gastric biopsies was also observed in H. pylori infected patients (p < 0.025). In conclusion, an enhanced type 2 cytokine production was observed in H. pylori infected patients, which may be responsible for H. pylori chronic infection.     

幽门螺杆菌外膜囊泡研究进展

幽门螺杆菌(Helicobacter pylori)被认为是引起人类胃部疾病的元凶之一。外膜囊泡(Outer Membrane Vesicles,OMVs)是由细菌外膜自发脱落而形成的囊泡状结构,其具有细菌外膜多数成分,包括外膜蛋白、多糖、脂质以及其他蛋白组分。越来越多的研究正在关注外膜囊泡在幽门螺杆菌感染、发生、发展过程中的作用。同时,研究表明幽门螺杆菌外膜囊泡作为疫苗,在防治幽门螺杆菌感染中也展现了良好的应用潜力。因此,本综述总结了目前关于幽门螺杆菌外膜囊泡组成成分的研究,并讨论了外膜囊泡在幽门螺杆菌存活和致病机制中的作用,以及外膜囊泡在幽门螺杆菌感染治疗中发挥的作用。     

L-lactic acid secreted from gastric mucosal cells enhances growth of Helicobacter pylori

BACKGROUND: Helicobacter pylori mainly inhabit the mucus layer in the gastric mucosa. However, mechanisms involving H. pylori colonization and proliferation in gastric mucosa are not well established. This study focuses on elucidating the role of gastric mucosal cells on growth of H. pylori. MATERIALS AND METHODS: H. pylori was co-cultured with the murine gastric surface mucosal cells (GSM06), and the growth of H. pylori on the cells was assessed by enumerating the colony-forming units (CFU). The H. pylori growth factor in the culture media conditioned by GSM06 cell was purified by HPLC, and the chemical structure of the growth factor was identified by analyses of (1)H- and (13)C-NMR spectra. RESULTS: A marked increase in the number of CFU of H. pylori was observed in the GSM06 cells. The enhanced H. pylori growth was also observed when indirectly incubated with GSM06 cells through semi-permeable membrane. In addition, culture media conditioned by GSM06 cell stimulated H. pylori growth approximately one thousand-fold. By bioassay-guided purification, the H. pylori growth factor was isolated from the conditioned medium of GSM06 cells and identified as L-lactic acid. The H. pylori growth-enhancing activity under microaerobic condition was well correlated with L-lactic acid concentrations in the conditioned media. CONCLUSIONS: This study demonstrates that L-lactic acid secreted by gastric mucosal cells enhances the growth of H. pylori, and this L-lactic acid-dependent growth of H. pylori may be important to the long-term colonization of H. pylori in the stomach.     

幽门螺杆菌在牙菌斑及胃粘膜分布研究

目的:探讨幽门螺杆菌(Helicobacter pylori,Hp)在牙菌斑及胃粘膜分布状况。方法:采用聚合酶链式反应(polym erase chain reaction,PCR)技术对80 例慢性胃溃疡患者的牙菌斑及胃粘膜组织进行Hp 检测。结果:48 例胃粘膜Hp 阳性的患者中12 例牙菌斑Hp 阳性;胃粘膜Hp 阴性的患者无一例牙菌斑Hp 阳性;胃Hp 阳性检出率大于牙菌斑。结论:幽门螺杆菌不仅存在于胃粘膜组织中,也存在于口腔牙周组织中。提示牙菌斑可能是Hp 的重要寄居地。     

Potential role of CagA in the inhibition of T cell reactivity in Helicobacter pylori infections

The pathogenicity of chronic gastroduodenal diseases is very often related to Helicobacter pylori infections. Most H. pylori strains carry the cagA gene encoding an immunodominant 120- to 128-kDa protein which is considered a virulence marker. The majority of CagA-positive H. pylori isolates also produce a 95-kDa protein cytotoxin (VacA) causing vacuolation and degradation of mammalian cells. In our previous study we have shown that live H. pylori bacteria and their sonicates inhibit PHA-driven proliferation of human T lymphocytes. The H. pylori CagA and VacA proteins were suspected of a paralyzing effect of H. pylori on T cell proliferation. In this report, by using isogenic H. pylori mutant strains defective in CagA and VacA proteins, we determined that CagA is responsible for the inhibition of PHA-induced proliferation of T cells.     

Role of mast cells as a trigger of inflammation in Helicobacter pylori infection.

Helicobacter pylori (H. pylori) induces severe inflammation and plays a key role in gastric mucosal diseases. In general, mast cells have been believed to play an important role in inflammation. Although mast cells were detected in the gastric mucosa, the role of mast cells in the gastric mucosal inflammation caused by H. pylori is still unclear. Therefore, we examined the effects of H. pylori water extract on the degranulation of mast cells to clarify the role of these cells in gastric mucosal inflammation induced by H. pylori. Mast cells prepared from rat abdominal cavity were incubated with H. pylori for 30 min. The protein concentrations of H. pylori water extract used in this study were 0.5-3 mg/ml. The degranulation of mast cells were monitored morphologically by phase contrast microscopy equipped with time-lapse video recording system and biochemically by measuring histamine and beta-hexosaminidase. H. pylori water extract induced the degranulation of mast cells dose-dependently. The identical experiment was performed without extracellular calcium, and no significant degranulation was found. The data indicates that the degranulation of mast cells by H. pylori water extract depend on extracellular calcium. The present results indicate that H. pylori might be involved in the gastric mucosal inflammation as a trigger of mast cell degranulation for releasing chemical mediators.     

Helicobacter pylori arginase inhibits T cell proliferation and reduces the expression of the TCR zeta-chain (CD3zeta)

Helicobacter pylori infects approximately half the human population. The outcomes of the infection range from gastritis to gastric cancer and appear to be associated with the immunity to H. pylori. Patients developing nonatrophic gastritis present a Th1 response without developing protective immunity, suggesting that this bacterium may have mechanisms to evade the immune response of the host. Several H. pylori proteins can impair macrophage and T cell function in vitro through mechanisms that are poorly understood. We tested the effect of H. pylori extracts and live H. pylori on Jurkat cells and freshly isolated human normal T lymphocytes to identify possible mechanisms by which the bacteria might impair T cell function. Jurkat cells or activated T lymphocytes cultured with an H. pylori sonicate had a reduced proliferation that was not caused by T cell apoptosis or impairment in the early T cell signaling events. Instead, both the H. pylori sonicate and live H. pylori induced a decreased expression of the CD3zeta-chain of the TCR. Coculture of live H. pylori with T cells demonstrated that the wild-type strain, but not the arginase mutant rocF(-), depleted L-arginine and caused a decrease in CD3zeta expression. Furthermore, arginase inhibitors reversed these events. These results suggest that H. pylori arginase is not only important for urea production, but may also impair T cell function during infection.     

Effect of cagA status on the sensitivity of enzyme immunoassay in diagnosing Helicobacter pylori-infected children

Background. The aims of our study were twofold. First, we sought to evaluate in symptomatic children the influence of the Helicobacter pylori genotype on gastritis, abdominal pain, and circulating anti– H. pylori IgG antibodies (anti– H. pylori IgG) or pepsinogen A (PGA) and C (PGC). Additionally, we sought to assess anti– H. pylori IgG, PGA, and PGC patterns in a large cohort (N = 921) of asymptomatic children.
Materials and Methods. In 183 symptomatic children, H. pylori infection and the presence of gastritis were evaluated by histology. In a subgroup of 20 H. pylori –positive children, the H. pylori genotype was evaluated also by polymerase chain reaction. Nine hundred and twenty-one asymptomatic children, aged 11 to 14 years, were studied by anti– H. pylori IgG, PGA, and PGC serum determination.
Results. The infection was found in 33 of 183 symptomatic children; among the 20 H. pylori –positive children for which the H. pylori genotype was available, cag A was present or absent in equal percentages. H. pylori infection was associated with more severe gastritis and higher serum levels of anti– H. pylori IgG and PGC but not with abdominal pain. In infected children, higher levels of anti– H. pylori IgG and the presence of abdominal pain were associated with infections caused by cag A-positive strains. In the cohort of 921 asymptomatic children, raised levels of anti– H. pylori IgG, PGA, and PGC were found in approximately 5% of the cases.
Conclusions. Infection with cag A-positive H. pylori strains can be associated with increased frequency of reported abdominal pain and higher circulating levels of anti– H. pylori IgG. The serological assessment of H. pylori IgG using H. pylori antigens containing significant amounts of cagA protein may, therefore, underestimate the true prevalence of infection.     

Demonstration and characterization of mutations induced by Helicobacter pylori organisms in gastric epithelial cells

BACKGROUND: Helicobacter pylori gastritis increases gastric cancer risk. Microsatellite instability-type mutations are secondary to deficient DNA mismatch repair. H. pylori gastritis is more frequent in patients with microsatellite instability-positive gastric cancers, and H. pylori organisms independently of inflammation can reduce DNA mismatch repair protein levels, raising the hypothesis that H. pylori organisms might lead to mutagenesis during infection. MATERIALS AND METHODS: Mutations were detected using a green fluorescent protein reporter vector (pEGFP-CA13). Gastric cancer AGS cells transfected with pEGFP-CA13 were cocultured with H. pylori or Escherichia coli. The numbers of green fluorescent protein (GFP)-positive cells were determined, and GFP, hMSH2, and hMLH1 protein levels were measured by Western blot. The effect of H. pylori on CpG methylation status of hMLH1 was determined by methylation-specific polymerase chain reaction. RESULTS: GFP levels and GFP-positive cell numbers in AGS cells cocultured with H. pylori significantly increased, as the levels of hMLH1 and hMSH2 dropped. H. pylori cocultures induced low-level CpG methylation of the hMLH1 promoter. Sequence analysis of cells cocultured with H. pylori showed an increased number of frameshift mutations and point mutations as compared to cells not cocultured with H. pylori (p = .03 and p = .001, respectively). CONCLUSIONS: This is the first report showing that H. pylori bacteria may lead to accumulation of genomic mutations, independently of underlying inflammation. This is associated with reduced DNA mismatch repair, and is at least in part associated with CpG methylation of the hMLH1 promoter. These data support the notion that H. pylori-induced mutations and epigenetic alterations in gastric epithelial cells during chronic gastritis may contribute to an increased risk of gastric cancer associated with H. pylori infection.