EPs® 7630, an extract from Pelargonium sidoides roots inhibits adherence of Helicobacter pylori to gastric epithelial cells

Helicobacter pylori specifically adheres to gastric host cells, mainly based on carbohydrate-mediated cell–cell interaction. The extract of Pelargonium sidoides roots (EPs^® 7630), a South African herbal remedy, is currently used to treat acute bronchitis. EPs^® 7630 prevents bacteria from attaching to cell membranes. Therefore, the ability of EPs^® 7630 to interfere with H. pylori growth and adhesion to gastric epithelial cells (AGS cells) was tested in vitro. EPs^® 7630 inhibited H. pylori growth and with higher potency adhesion to gastric AGS cells. EPs^® 7630 (50 and 100 μg/ml) reduced bacterial count attached to AGS cells by 77% and 91%, respectively. The results suggest that the mode of action of EPs^® 7630 is mainly related to its anti-adhesive activity.     

Proliferative activity of gastric epithelial cells in Helicobacter pylori infected children

Helicobacter pylori infection has been associated with gastric carcinogenesis. Gastric epithelial cells proliferative rate is accelerated in H. pylori infected adult patients. Our study was performed to evaluate proliferative cell activity in gastric epithelium in the course of H. pylori infection in the early stage of its natural history. Gastric antral biopsy specimens were obtained from thirteen H. pylori positive and seven negative children. To assess replication rates we used nucleolar organiser regions staining with colloidal silver nitrate technique (AgNOR). The number of AgNORs per nucleus, area of single AgNOR, and the quotient of these two parameters (AgNOR content) were analysed. The mean area of AgNOR was lower in H. pylori positive than in negative children. Conversely, both the mean number of AgNOR per nucleus and AgNOR content were higher in infected than non infected subjects. These results show accelerated proliferation of gastric antral epithelial cells in the course of H. pylori infection in children. Such alteration of cell replication occurring in an initial phase of natural history of long lasting infection provides an explanation for the association between acquisition of H. pylori infection in the first years of life and the development of gastric cancer.     

Nutrients released by gastric epithelial cells enhance Helicobacter pylori growth

BACKGROUND: Helicobacter pylori survives and proliferates in the human gastric mucosa. In this niche, H. pylori adheres to the gastric epithelial cells near the tight junctions. In vitro, H. pylori proliferated well in tissue-culture medium near gastric epithelial cells. However, in the absence of epithelial cells, growth of H. pylori could only be established in tissue-culture medium when, prior to the experiment, it was preincubated near gastric epithelial cells. Therefore, we aimed to determine whether diffusion of nutrients derived from epithelial cells was required for H. pylori growth in Dulbecco's modified Eagle's minimal essential medium (DMEM) cell culture medium. MATERIALS AND METHODS: Cell culture conditions essential for H. pylori growth in vitro were determined with gastric epithelial HM02 cells. RESULTS: Deprivation of iron in cell-culture-conditioned DMEM resulted in a growth arrest of H. pylori. However, near gastric epithelial cells, growth of H. pylori was resistant to iron deprivation. Evidently, when residing close to epithelial cells, H. pylori was able to fulfil its iron requirements, even when the DMEM was deprived of iron. Nevertheless, supplementation with iron alone did not restore H. pylori growth in DMEM, hence other nutrients were deficient as well in the absence of epithelial cells. Growth of H. pylori in DMEM was restored when hypoxanthine, L-alanine and L-proline were added to the DMEM. CONCLUSIONS: Diffusion of (precursors of) these nutrients from the gastric epithelial cells is essential for H. pylori growth in vitro. We hypothesize that in vivo, H. pylori favors colonization near the tight junctions, to gain maximal access to the nutrient(s) released by gastric epithelial cells.     

Protein changes in gastric epithelial cells RGM-1 in response to Helicobacter pylori infection

Helicobacter pylori-induced inflammation significantly increases the risk of gastric cancer. To investigate the role of H. pylori infection in gastric epithelial cell carcinogenesis, flow cytometry was used to analyze the apoptosis of gastric epithelial cells infected by H. pylori. Next, LTQ MS mass spectrometry (MS) was applied to identify protein changes in gastric epithelial cells infected with H. pylori, and then bioinformatics was adopted to analyze the cellular localization and biological function of differential proteins. LTQ MS/MS successfully identified identified 22 differential proteins successfully, including 20 host-cell proteins and two H. pylori bacterial proteins. Also, human proteins were located in all areas of cells and involved in various cell biological functions. The oncogene proteins p53, p16, and C-erbB-2 proteins in H. pylori-infected RGM-1 cells were remarkably increased from the analysis by Western blot analysis. H. pylori infection of gastric epithelial cells leads to changes in various protein components in the cell, and enhances the expression of oncogene proteins, thereby increasing the possibility of possibility of carcinogenesis of H. pylori infection.     

Effects of Helicobacter pylori on biological characteristics of gastric epithelial cells

Infection with Helicobacter pylori (H. pylori) strains is linked to an increased risk of inflammation and gastric cancer. To investigate the effects of H. pylori on biological characteristics of gastric epithelial cells SGC-7901, derived from human adenocarcinoma, morphological appearances of both the pathogen and these cells, as well as features of attachment and internalization were observed by using transmission electron microscopy (TEM). We also investigated cell junctions and invasion by TEM and Transwell Invasion Assay. Cell proliferation and apoptosis were assessed by using chromogenic methylthiazol tetrazolium bromide (MTT) dye and flow cytometry. Three types of H. pylori were observed around, attaching to, or invading tumor cells. Cellular damage was characterized by vacuolar degeneration, dilated endoplasmic reticulum (ER), and reduction of organelles. Cell junctions and cell microvilli reduced or disappeared. H. pylori inhibited cell proliferation, whereas it had no effect on apoptosis. It also promoted gastric carcinoma cell invasion. H. pylori damages cell construction, destroys cell junctions, inhibits cell proliferation, promotes cell invasive ability, and, therefore, might accelerate the malignant progress and metastasis of gastric cancer.     

Helicobacter pylori CagA transfection of gastric epithelial cells induces interleukin-8

To determine the effect of Helicobacter pylori CagA expression on interleukin-8 (IL-8) induction in AGS cells, cagA and five of its fragments from strains 147A and 147C that vary in the 3' repeat region were cloned into the eukaryotic expression plasmid pSP65SRalpha. IL-8, but not RANTES or IL-Ibeta, levels were increased in AGS cells transfected with 147A-cagA and to a greater extent with 147C-cagA, compared with negative controls. The 5' b fragment from the two strains had similar effects, but the 3' d and e fragments from 147C CagA had greater effects than those from 147A-CagA. When the Western CagA-specific sequence (WSS) of 147C-cagA was replaced with East Asian CagA-specific sequence (ESS) and cloned into pSP65SRalpha as an East/West chimera, there was no significant effect on IL-8 production. Use of specific inhibitors indicates that Src kinase activation, and the mitogen-activated protein (MAP) kinase and NF-kappaB pathways are the major intermediates for CagA effects on IL-8 induction, but the p38 MAP kinase pathway has little effect. These results indicate a direct CagA effect on IL-8 induction by gastric epithelial cells, and indicate signal pathway loci that can be targeted for amelioration.     

Helicobacter pylori infection increases cell kinetics in human gastric epithelial cells without adhering to proliferating cells

We investigated the effect of H. pylori infection on cell proliferation of gastric mucosa using immunostaining for H. pylori or Ki67. H. pylori cells attached to surface mucous cells covering luminal surface and the upper part of gastric foveolae, and up-regulated the proliferative activity of gastric epithelial cells without adhering to the proliferating epithelial cells.     

External membrane vesicles from Helicobacter pylori induce apoptosis in gastric epithelial cells

The Helicobacter pylori infection of gastric mucosa is one of the most common infectious diseases and is associated with a variety of clinical outcomes, including peptic ulcer disease and gastric cancer. Helicobacter pylori-induced damage to gastric mucosal cells is controlled by bacterial virulence factors, which include VacA and CagA. Outer membrane vesicles are constantly shed by the bacteria and can provide an additional mechanism for pathogenicity by releasing non-secretable factors which can then interact with epithelial cells. The present report shows that external membrane vesicles are able to induce apoptosis not mediated by mitochondrial pathway in gastric (AGS) epithelial cells, as demonstrated by the lack of cytochrome c release with an activation of caspase 8 and 3. Apoptosis induced by these vesicles does not require a classic VacA+ phenotype, as a negative strain with a truncated and therefore non-secretable form of this protein can also induce cell death. These results should be taken into account in future studies of H. pylori pathogenicity in strains apparently VacA-.     

幽门螺杆菌急性感染对胃上皮细胞凋亡的影响

目的 探讨幽门螺杆菌(Helicobacter pylori)急性感染对GES 1细胞凋亡的影响,揭示H. pylori引起GES 1细胞凋亡变化的分子机制。 方法 将H. pylori临床分离株SBK与胃上皮细胞GES 1按不同比例(感染复数MOI分别为50∶1和100∶1)共培养24 h,建立H. pylori急性感染模型。采用流式细胞仪分析GES 1的凋亡,通过Western Blot检测凋亡相关蛋白Bcl xL、Bcl 2、Bax、Caspase 3和NF κB p65的表达。经H. pylori感染的GES 1细胞为处理组细胞,未经H. pylori感染的GES 1细胞即为对照细胞。使用SPSS 21.0软件对结果进行统计学分析。 结果 GES 1细胞经H. pylori处理24 h后,与对照细胞相比,MOI为50∶1(t=11.040,P结论 H. pylori急性感染通过改变线粒体途径中凋亡相关蛋白Bax、Bcl 2及Bcl xL的表达促进GES 1细胞凋亡,且GES 1细胞的凋亡程度与H. pylori的感染复数有关。     

幽门螺杆菌感染与胃黏膜上皮细胞DNA甲基化的研究进展

在癌症发生表观遗传学研究中,DNA甲基化是研究最多也最深入的一种机制,异常甲基化可导致胃黏膜上皮细胞癌变.幽门螺杆菌(Helicobacter pylori,Hpylori)是胃癌的第Ⅰ类致癌原,在“慢性浅表性胃炎-慢性萎缩性胃炎-肠上皮化生-不典型增生-胃癌”的癌变模式中可能起先导作用.胃癌相关基因的异常甲基化和H.pylori的致癌机制均是目前研究的热点,本研究综述H.pylori感染与胃黏膜上皮细胞DNA异常甲基化的关系,阐述H.pylori感染介导慢性炎症诱发胃黏膜上皮细胞DNA甲基化的机制.     

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.     

CagA tyrosine phosphorylation in gastric epithelial cells caused by Helicobacter pylori in patients with gastric adenocarcinoma

Background. Tyrosine phosphorylation of Helicobacter pylori cytotoxin‐associated protein of in gastric epithelial cells is reported. The goals of this study are first to examine the occurrence of CagA tyrosine phosphorylation in H. pylori strains isolated from patients with gastric adenocarcinoma and gastritis, and second to clarify the relationship between the diversity of tyrosine phosphorylation motifs and the presence of CagA tyrosine phosphorylation. Methods. Fifty‐eight clinical isolates of H. pylori from patients with gastric adenocarcinoma (29 cases) and gastritis (29 cases) were studied for CagA tyrosine phosphorylation by Western blotting. Sequence diversity of tyrosine phosphorylation motifs was analysed among positive‐ or negative‐CagA tyrosine phosphorylation isolates. Results. Positive CagA tyrosine phosphorylation was found in 93.1% (27 of 29) of strains from gastric adenocarcinoma patients and 51.7% (15 of 29) of strains from gastritis patients (p < 0.001). Intact motifs were found in H. pylori isolates with CagA tyrosine phosphorylation. Of the 16 negative CagA tyrosine phosphorylation isolates, intact tyrosine phosphorylation motifs were found in 15 isolates. Conclusions. CagA tyrosine phosphorylation, which is significantly greater in strains from gastric adenocarcinoma patients, may play a role in gastric carcinogenesis, and could be a better marker of more virulent strains than the cag pathogenicity island in Asia, where the cag pathogenicity island is present in nearly all H. pylori strains. Sequence diversity of tyrosine phosphorylation motifs on CagA was not related to the presence of tyrosine phosphorylation. The absence of tyrosine phosphorylation motif might result in negative tyrosine phosphorylation phenotypes, but such motifs are not the sole factors associated with CagA tyrosine phosphorylation.     

Functional variability of cagA gene in Japanese isolates of Helicobacter pylori

CagA protein of Helicobacter pylori is injected into the epithelium, where CagA undergoes tyrosine phosphorylation and activates proliferation signals. However, the importance of these CagA activities for pathogenesis has yet to be resolved. The aim of this study is to analyze the genetic and functional variability of cagA gene of clinical strains in relation to gastric diseases. Thirty-six H. pylori strains were isolated from Japanese patients with various gastric diseases and examined. All 36 strains were found to contain cagA and cagE gene and to induce CagA phosphorylation upon infection. The intensity of CagA phosphorylation expressed in HeLa cells by transfection was highly correlated to the number of R1 region. The phosphorylation intensity was slightly higher in strains from chronic atrophic gastritis (CG); however, the differences were not statistically significant. These CagA proteins also activated the serum response element (SRE) reporter by 5- to 14-fold, above the level of the control. CagA proteins which lack R2 or R3 region exhibited smaller ability for SRE activation. The average of SRE activation was slightly higher in strains from cases of gastric cancer (GC; 11.4+/-1.6), MALT lymphoma (ML; 10.7+/-1.0), and chronic atrophic gastritis (CG; 11.2+/-1.6) than in those of duodenal ulcer (DU; 8.3+/-1.9) or gastric ulcer (GU; 9.0+/-1.1). In summary, most Japanese H. pylori strains contained CagA transport system and induced CagA phosphorylation, and the levels of the intensity of phosphorylation and the ability to induce SRE varied among strains. Although the association between CagA activities and disease outcome shown in this study is not very strong, variety of CagA structure, which induces variable activities, may be one of the reasons why H. pylori induces distinct diseases on host.     

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.     

Helicobacter pylori cag pathogenicity island-positive strains induce syndecan-4 expression in gastric epithelial cells

Helicobacter pylori is recognized as the main cause of gastritis and is associated with gastric carcinogenesis. Syndecan-4 represents the major source of heparan sulfate (HS) in the gastric cells. HS proteoglycans expressed on the cell surface constitute targets for H. pylori at the early stage of infection. The aim of this study was to determine whether H. pylori induction of syndecan-4 expression is affected by the virulence characteristics of the infecting strain, namely the cytotoxic-associated gene ( cag ) pathogenicity island (PAI). We observed that individuals infected with highly pathogenic H. pylori strains express syndecan-4 in the foveolar epithelium of the gastric mucosa. The association between the cag PAI status of the infecting strain and syndecan-4 expression was further demonstrated by infection of gastric epithelial cell lines with a panel of cag PAI⁺ and cag PAI⁻ H. pylori strains, showing that expression of syndecan-4 was significantly increased in response to infection with the highly pathogenic strains. Moreover, infection of gastric cells with cag A and cag E mutant strains further confirmed that syndecan-4 induction is dependent on an intact cag PAI. The present study shows that highly pathogenic H. pylori strains induce syndecan-4 expression, both in human gastric mucosa and in gastric cell lines, in a cag PAI-dependent manner.     

Mechanism of clarithromycin resistance in clinical isolates of Helicobacter pylori

Abstract The genes coding for the protease inhibitors, SSI and API-2c', have been analyzed by comparing DNA macrorestriction patterns of Streptomyces albogriseolus S-3253 and S. griseoincamatus KTo-250 with those of inhibitor-deficient mutants. The mutants were found to suffer from chromosomal deletions rather than plasmid loss which resulted in the loss of the relevant genes. Hybridization experiments indicated that the ssi homologs in S. lividans and S. coelicolor A3(2) are located near the end of the linear chromosome.     

Antigenic diversity among Portuguese clinical isolates of Helicobacter pylori

Background: The human gastroduodenal pathogen, Helicobacter pylori, is characterized by an unusual extent of genetic heterogeneity. This dictates differences in the antigenic pattern of strains resulting in heterogeneous human humoral immune responses. Here, we examined the antigenic variability among a group of 10 strains isolated from Portuguese patients differing in age, gender, and H. pylori‐associated gastric diseases. Material and Methods: Immunoassays were performed on two‐dimensional electrophoresis gels obtained for the proteome of each strain, using a commercial pool of antibodies produced in rabbit, against the whole cell lysate of an Australian H. pylori strain. Relevant proteins were identified by mass spectrometry. Results: Immunoproteomes of the Portuguese strains showed no correlation between the number of antigenic proteins or the antigenic profile, and the disease to which each strain was associated. The Heat shock protein B was the unique immunoreactive protein common to all of them. Additionally, seven proteins were found to be antigenic in at least 80% of strains: enoyl‐(acyl‐carrier‐protein) reductase (NADH); Catalase; Flagellin A; 2 isoforms of alkyl hydroperoxide reductase; succinyl‐CoA transferase subunit B; and an unidentified protein. These proteins were present in the proteome of all tested strains, suggesting that differences in their antigenicity are related to antigenic variance. Conclusions: This study showed evidence of the variability of antigenic pattern among H. pylori strains. We believe that this fact contributes to the failure of anti‐H. pylori vaccines and the low accuracy of serological tests based on a low number of proteins or antigens of only one strain.