Helicobacter pylori infection and CagA protein translocation in human primary gastric epithelial cell culture

BACKGROUND: Increasing evidence has shown that Helicobacter pylori CagA protein translocation into gastric epithelial cells plays an important role in the development of gastric inflammation and malignancy. Translocated CagA undergoes tyrosine phosphorylation in gastric adenocarcinoma cell line cells, and CagA involves disruption of cellular apical-junction complex in Madin-Darby canine kidney cells. METHODS: To elucidate whether these events take place in normal human gastric epithelium, we infected human primary gastric epithelial cells with H. pylori. RESULTS: Our results demonstrate that CagA protein was translocated into primary gastric epithelial cells and tyrosine phosphorylated. The translocated CagA induces cytoskeletal rearrangement and the disruption of tight junctions in primary gastric epithelial cells. CONCLUSIONS: This study provides direct evidence of the modulation of gastric epithelial cells by CagA protein translocation, and advances our understanding of the pathogenesis of H. pylori infection.     

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.     

Helicobacter pylori infection up-regulates gland mucous cell-type mucins in gastric pyloric mucosa

Background. Two types of mucous cell are present in gastric mucosa: surface mucous cells (SMCs) and gland mucous cells (GMCs), which consist of cardiac gland cells, mucous neck cells, and pyloric gland cells. We have previously reported that the patterns of glycosylation of SMC mucins are reversibly altered by Helicobacter pylori infection. In this study, we evaluated the effects of H. pylori infection on the expression of GMC mucins in pyloric gland cells. Methods. Gastric biopsy specimens from the antrums of 30 H. pylori‐infected patients before and after eradication of H. pylori and 10 normal uninfected volunteers were examined by immunostaining for MUC6 (a core protein of GMC mucins), α1,4‐N‐acetyl‐glucosaminyl transferase (α4GnT) (the glycosyltransferase which forms GlcNAcα1‐4Galβ‐R), and GlcNAcα1‐4Galβ‐R (a GMC mucin‐specific glycan). Results. MUC6, α4GnT, and HIK1083‐reactive glycan were expressed in the cytoplasm, supranuclear region, and secretory granules in pyloric gland cells, respectively. The immunoreactivity of MUC6 and α4GnT, but not of GlcNAcα1‐4Galβ‐R, in the pyloric gland increased in H. pylori‐associated gastritis, and after the eradication of H. pylori, the increased expression of MUC6 and α4GnT in the gastric mucosa of H. pylori‐infected patients decreased to almost normal levels. This up‐regulation was correlated with the degree of inflammation. Conclusions. In addition to the synthesis of GMC mucins increasing reversibly, their metabolism or release may also increase reversibly in H. pylori‐associated gastritis. The up‐regulation of the expression of gastric GMC mucins may be involved in defense against H. pylori infection in the gastric surface mucous gel layer and on the gastric mucosa.     

幽门螺杆菌感染新型原代人胃上皮细胞模型的建立

目的 培养三维(3D)人胃黏膜上皮类器官,转为二维(2D)原代胃黏膜细胞培养,并建成人2D原代胃上皮细胞的幽门螺杆菌感染模型。 方法 (1)从正常人胃上皮组织中分离胃腺,在含有多种生长调节和凋亡抑制等混合因子的培养基中,依附于基质胶而培养成3D类器官;(2)利用免疫荧光技术鉴定胃上皮类器官的相关分子标记;(3)研究正常原代胃上皮细胞被幽门螺杆菌感染后的形态学变化,利用免疫印迹技术鉴定幽门螺杆菌感染相关蛋白的表达水平。 结果 成功培养出可长期传代的人胃上皮3D类器官,具有典型的人胃黏膜上皮分子标记。而且3D类器官转为2D平面培养的原代胃上皮细胞,可作为幽门螺杆菌的体外原代细胞感染模型。 结论 3D胃上皮类器官,可作为2D原代胃上皮细胞的持久来源,为研究幽门螺杆菌感染人体胃上皮的分子机制带来个体化的新模型。     

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。     

Epithelial cell kinetics of the gastric mucosa during Helicobacter pylori infection

Helicobacter pylori is an important pathogen in major gastroduodenal diseases, including inflammation with ulceration and gastric malignancies. Alterations in H. pylori associated cell turnover in gastric epithelial cells are examined in relation to inflammatory activity, bacteria load and cytokines which may improve knowledge concerning the outcome of gastric diseases caused by H. pylori. Antral biopsies from 42 dyspeptic patients including 27 H. pylori-positive and 15 H. pylori-negative patients were tested for apoptotic activity by the TUNEL assay, and immuno-histochemically for p53 and the proliferative marker Ki-67. H. pylori infection, bacteria load and inflammatory activity were associated with increased cell turnover as judged by enhanced activities of TUNEL, p53 and Ki-67. Only p53 was significantly correlated to IFN-gamma, IL-8 and IL-10. The H. pylori-positive state was furthermore accompanied by varying degrees of altered distribution pattern of the markers studied, with occasional presence of apoptosis in the deeper pit zones, upward extension of Ki-67 and to a lesser degree of p53. Given a similar pattern of change in proliferation and apoptosis in some neoplastic lesions in other parts of the gastrointestinal tract, such studies in cell turnover may provide insights valuable in the investigations of potential precursors of gastric malignancies.     

The effect of Helicobacter pylori infection on levels of DNA damage in gastric epithelial cells

Background. Helicobacter pylori infection leads to an increased risk of developing gastric cancer. The mechanism through which this occurs is not known. We aimed to determine the effect of H. pylori and gastritis on levels of DNA damage in gastric epithelial cells. Methods. Epithelial cells were isolated from antral biopsies from 111 patients. DNA damage was determined using single cell gel electrophoresis and the proportion of cells with damage calculated before and 6 weeks after eradication of H. pylori. Cell suspensions generated by sequential digestions of the same biopsies were assayed to determine the effect of cell position within the gastric pit on DNA damage. Results. DNA damage was significantly higher in normal gastric mucosa than in H. pylori gastritis [median (interquartile range) 65% (58.5–75.8), n = 18 and 21% (11.9–29.8), n = 65, respectively, p < .001]. Intermediate levels were found in reactive gastritis [55.5% (41.3–71.7), n = 13] and H. pylori negative chronic gastritis [50.5% (36.3–60.0), n = 15]. DNA damage rose 6 weeks after successful eradication of H. pylori[to 39.5% (26.3–51.0), p = .007] but was still lower than in normal mucosa. Chronic inflammation was the most important histological factor that determined DNA damage. DNA damage fell with increasing digestion times (r = –.92 and –.88 for normal mucosa and H. pylori gastritis, respectively). Conclusions. Lower levels of DNA damage in cells isolated from H. pylori infected gastric biopsies may be a reflection of increased cell turnover in H. pylori gastritis. The investigation of mature gastric epithelial cells for DNA damage is unlikely to elucidate the mechanisms underlying gastric carcinogenesis.     

pH-dependent binding of Helicobacter pylori to pig gastric mucins

A microtiter-based assay was developed to study the binding of Helicobacter pylori to pig gastric mucins purified by density-gradient centrifugation in CsCl/4 M guanidinium chloride. Binding of H. pylori was observed over the 'mucin' band as well as with 'low-density' components in the gradients, and binding to the latter was more pronounced when incubations were performed at 37 degrees C as compared to 20 degrees C. At a lower pH, binding of H. pylori (strain SVA 40) to the 'high-density' mucins from pig antrum was increased but binding to the 'low-density' ones was decreased. Binding of the P466 strain (Le(b)-specific) was mainly associated with the 'mucin' band, whereas the MO19 strain reacted preferentially with the 'low-density' components. In summary, H. pylori may bind to gastric mucins and the binding is influenced by temperature, pH and the repertoire of bacterial adhesins.     

A novel in vitro infection model of Helicobacter pylori using mucin-producing murine gastric surface mucous cells

BACKGROUND: Helicobacter pylori is found within the gastric surface mucous gel layer and in the epithelial surface. Gastric cancer cells have been used in experimental H. pylori infection in vitro, although cancer cells have some abnormalities in cellular properties. The aim of this study was to develop an in vitro H. pylori infection model using normal gastric surface cells that produce gastric mucin. MATERIALS AND METHODS: Normal murine gastric surface mucous cells (GSM06) were cultured by the liquid interface method using a serum-free medium and a collagen gel containing a fibroblast cell line (L929) and infected with H. pylori. Infection by H. pylori was assessed by enumerating the colony-forming units (CFU) of H. pylori adhered to GSM06 cells and by transmission electron microscopy. The production of mucin was determined by a lectin binding assay, sugar analysis, and MUC5AC gene expression. RESULTS: GSM06 cells cultured under these conditions produced mucin containing N-acetylgalactosamine and MUC5AC as the core protein. Significantly higher numbers of H. pylori adhered to GSM06 cells under mucin-producing conditions than under nonproducing conditions. Microscopic observation showed a filamentous structure resembling a type IV secretion system apparatus formed between the surface of GSM06 cells and H. pylori. CONCLUSIONS: This study demonstrates a novel in vitro H. pylori infection model using mucin-producing murine GSM06 cells for early stages of infection.     

Helicobacter pylori-downregulated tumor necrosis factor receptor-associated protein 1 mediates apoptosis of human gastric epithelial cells

Heat shock proteins (HSPs) are crucial proteins in maintaining the homeostasis of human gastric epithelial cells. Tumor necrosis factor receptor-associated protein 1 (TRAP1), a member of the HSP90 family, has been shown to be involved in various crucial physiological processes, particularly against apoptosis. However, the regulation and function of TRAP1 in Helicobacter pylori infection is still unknown. Here, we found that TRAP1 expression was downregulated on human gastric epithelial cells during H. pylori infection by real-time polymerase chain reaction (PCR) and western blot analysis. Through virulence factors mutant H. pylori strains infection and inhibitors screening, we found that H. pylori vacuolating cytotoxin A ( vacA), but not cytotoxin-associated gene A ( cagA) protein, induced human gastric epithelial cells to downregulate TRAP1 via P38MAPK pathway by real-time PCR and western blot analysis. Furthermore, downregulation of TRAP1 with lentivirus carrying TRAP1 short hairpin RNA constructs impairs mitochondrial function, and increases apoptosis of gastric epithelial cells. The results indicate that H. pylori vacA downregulated TRAP1 is involved in the regulation of gastric epithelial cell apoptosis.     

Immune and proliferative cellular responses to Helicobacter pylori infection in the gastric mucosa of Mexican children

BACKGROUND: Helicobacter pylori infection occurs mostly during childhood, but few studies on this age group have addressed the innate immune and the proliferative response to this infection. Mexico has a high H. pylori prevalence in children, but a low risk of gastric cancer. The aim of this work was to study the cellular responses of the gastric mucosa to this infection in Mexican children. METHODS: Antral and corpus gastric biopsies were obtained from 44 H. pylori-infected children (mean age 12 +/- 3.2 years) and 44 uninfected children (mean age 10 +/- 3 years). Mucosal cellular responses were studied by immunohistochemistry, using anti-Ki67 antibodies for proliferation studies, antihuman tryptase for mast cells, and antihuman CD68 for macrophages. T and B lymphocytes were stained with a commercial integrated system. The intensity of cellular responses was estimated histologically using the software KS300. RESULTS: Epithelium proliferation and infiltration of macrophages and T and B lymphocytes were significantly higher in H. pylori-infected than in uninfected children. A balanced increase of CD4, CD8, and CD20 lymphocytes was observed in infected children. However, activated mast cells were decreased, and infiltration of neutrophil and mononuclear cells was low. Epithelial proliferation was associated with polymorphonuclear infiltration but not with infiltration of macrophages or lymphocytes. Inflammation and proliferation was higher in CagA (+)-infected children. CONCLUSIONS: Mexican children respond to H. pylori infection with a low inflammatory response, a balanced increase of T and B lymphocytes, and a high regenerative activity.     

Strain-dependent proliferation in response to human gastric mucin and adhesion properties of Helicobacter pylori are not affected by co-isolated Lactobacillus sp

Background: Helicobacter pylori colonize the mucus layer that covers the gastric epithelium and can cause gastritis, ulcers, and gastric cancer. Recently, Lactobacillus sp. have also been found to reside in this niche permanently. This study compares adhesive properties and proliferation of co‐isolated lactobacilli and H. pylori in the presence of mucins and investigates possibilities for lactobacilli‐mediated inhibition of H. pylori. Materials and methods: Binding and proliferation of four H. pylori and four Lactobacillus strains, simultaneously isolated after residing in the stomachs of four patients for >4 years, to human gastric mucins were investigated using microtiter‐based methods. Results: The H. pylori strains co‐isolated with lactobacilli exhibited the same mucin binding properties as demonstrated for H. pylori strains previously. In contrast, no binding to mucins was detected with the Lactobacillus strains. Proliferation of mucin‐binding H. pylori strains was stimulated by the presence of mucins, whereas proliferation of non‐binding H. pylori and Lactobacillus strains was unaffected. Associative cultures of co‐isolated H. pylori and Lactobacillus strains showed no inhibition of H. pylori proliferation because of the presence of whole bacteria or supernatant of lactobacilli. Conclusions: The presence of lactobacilli in the stomach did not select for different mucin binding properties of H. pylori, and Lactobacillus sp. did neither compete for binding sites nor inhibit the growth of co‐isolated H. pylori. The effects of human gastric mucins on H. pylori proliferation vary between strains, and the host–bacteria interaction in the mucus niche thus depends on both the H. pylori strain and the microenvironment provided by the host mucins.     

Clinicopathological analysis of early-stage gastric cancers detected after successful eradication of Helicobacter pylori

Background and Aims: The results of a randomized controlled study and meta‐analysis study have recently proved that Helicobacter pylori eradication has a preventive effect against the development of metachronous and primary gastric cancer. However, gastric cancer is sometimes detected after successful eradication. There is a lack of study about gastric cancers in eradicated patients. To clarify the characteristics of gastric cancers detected after H. pylori eradication, we analyzed the clinicopathological features of these cancers. Methods: The subjects were 18 early‐stage gastric cancer specimens resected from 17 patients who had received successful eradication of H. pylori from February 1995 to March 2009. The control group consisted of 36 specimens from noneradicated patients with persistent H. pylori infection who were matched with the subjects in age, sex, and depth of invasion. Clinicopathological features and mucin phenotypes of gastric cancer were clinically and immunohistologically evaluated. Results: The average diameter of gastric cancer was smaller and Ki‐67 index was lower in the eradication group. The morphological distribution of depression types was significantly lower in the control group. Immunohistochemical phenotyping revealed that 72.2% of the lesions in the eradicated group were complete gastric type or gastric predominant mixed type, whereas the percentages of gastric type and intestinal type in the control group were similar. Conclusion: Our findings indicate that the clinicopathological characteristics of gastric cancers detected after H. pylori eradication are different from those of gastric cancers in patients with persistent H. pylori infection. H. pylori eradication may suppress intestinalization during the development of gastric cancer.     

ceRNA network construction and comparison of gastric cancer with or without Helicobacter pylori infection

Gastric cancer (GC) is a lethal disease, and among its variety of etiological factors, Helicobacter pylori (H. pylori) infection is the strongest risk factor. However, the genetic and molecular mechanisms underlying H. pylori-related GC need further elucidation. We investigated the competing endogenous RNA (ceRNA) network differences between H. pylori (+) and H. pylori (−) GC. The long noncoding RNA (lncRNA), microRNA (miRNA), and messenger RNA (mRNA) expression data from 32 adjacent noncancerous samples and 18 H. pylori (+) and 141 H. pylori (−) stomach adenocarcinoma samples were downloaded from the TCGA database. After construction of lncRNA–miRNA–mRNA ceRNA networks of H. pylori (+) and H. pylori (−) GC, Panther and Kobas databases were used to analyze the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Finally, survival analysis was used to discover the key genes. In H. pylori (+) GC, we identified a total of 1,419 lncRNAs, 82 miRNAs, and 2,501 mRNAs with differentially expressed profiles. In H. pylori (−) GC, 2,225 lncRNAs, 130 miRNAs, and 3,146 mRNAs were differentially expressed. Furthermore, three unique pathways (cytokine–cytokine receptor interaction, HIF-1 signaling pathway, and Wnt signaling pathway) were enriched in H. pylori (+) GC. According to the overall survival analysis, three lncRNAs (AP002478.1, LINC00111, and LINC00313) and two mRNAs (MYB and COL1A1) functioned as prognostic biomarkers for patients with H. pylori (+) GC. In conclusion, our study has identified the differences in ceRNA regulatory networks between H. pylori (+) and H. pylori (−) GC and provides a rich candidate reservoir for future studies.     

Genome-wide DNA methylation profiles in noncancerous gastric mucosae with regard to Helicobacter pylori infection and the presence of gastric cancer

Background and Aims: To determine genome‐wide DNA methylation profiles induced by Helicobacter pylori (H. pylori) infection and to identify methylation markers in H. pylori‐induced gastric carcinogenesis. Methods: Gastric mucosae obtained from controls (n = 20) and patients with gastric cancer (n = 28) were included. A wide panel of CpG sites in cancer‐related genes (1505 CpG sites in 807 genes) was analyzed using Illumina bead array technology. Validation of the results of Illumina bead array technique was performed using methylation‐specific PCR method for four genes (MOS, DCC, CRK, and PTPN6). Results: The Illumina bead array showed that a total of 359 CpG sites (269 genes) were identified as differentially methylated by H. pylori infection (p < .0001). The correlation between methylation‐specific PCR and bead array analysis was significant (p < .0001, Spearman coefficient = 0.5054). Methylation profiles in noncancerous gastric mucosae of the patients with gastric cancer showed quite distinct patterns according to the presence or absence of the current H. pylori infection; however, 10 CpG sites were identified to be hypermethylated and three hypomethylated in association with the presence of gastric cancer regardless of H. pylori infection (p < .01). Conclusions: Genome‐wide methylation profiles showed a number of genes differentially methylated by H. pylori infection. Methylation profiles in noncancerous gastric mucosae from the patients with gastric cancer can be affected by H. pylori‐induced gastritis. Differentially methylated CpG sites in this study needs to be validated in a larger population using quantitative methylation‐specific PCR method.