AIM:To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori(H.pylori) infection.METHODS:Aiming to overcome this limitation,clones of the ...AIM:To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori(H.pylori) infection.METHODS:Aiming to overcome this limitation,clones of the heterogenic cancer-derived NCI-N87 cell line were isolated,by stably-transducing it with the human telomerase reverse-transcriptase(h TERT) catalytic subunit gene.The clones were first characterized regarding their cell growth pattern and phenotype.For that we measured the clones' adherence properties,expression of cell-cell junctions' markers(ZO-1 and E-cadherin) and ability to generate a sustained transepithelial electrical resistance.The gastric properties of the clones,concerning expression of mucins,zymogens and glycan contents,were then evaluated by haematoxylin and eosin staining,Periodic acid Schiff(PAS) and PAS/Alcian Blue-staining,immunocytochemistry and Western blot.In addition,we assessed the usefulness of the h TERT-expressing gastric cell line for H.pylori research,by performing co-culture assays and measuring the IL-8 secretion,by ELISA,upon infection with two H.pylori strains differing in virulence.RESULTS:Compared with the parental cell line,themost promising NCI-hT ERT-derived clones(CL5 and CL6) were composed of cells with homogenous phenotype,presented higher relative telomerase activities,better adhesion properties,ability to be maintained in culture for longer periods after confluency,and were more efficient in PAS-reactive mucins secretion.Both clones were shown to produce high amounts of MUC1,MUC2 and MUC13.NCI-hT ERT-CL5 mucins were shown to be decorated with blood group H type 2(BG-H),Lewis-x(Lex),Ley and Lea and,in a less extent,with BG-A antigens,but the former two antigens were not detected in the NCI-h TERT-CL6.None of the clones exhibited detectable levels of MUC6 nor sialylated Lex and Lea glycans.Entailing good gastric properties,both NCIhT ERT-clones were found to produce pepsinogen-5 and human gastric lipase.The progenitor-like phenotype of NCI-hT ERT-CL6 cells was highlighted by large nuclei and by the apical vesicular-like distribution of mucin 5AC and Pg5,supporting the accumulation of mucus-secreting and zymogens-chief mature cells functions.CONCLUSION:These traits,in addition to resistance to microaerobic conditions and good responsiveness to H.pylori co-culture,in a strain virulence-dependent manner,make the NCI-hT ERT-CL6 a promising model for future in vitro studies.展开更多
The gram-negative bacterium Helicobacter pylori(H. pylori) infects the stomachs of approximately half of the world's population. Although infection induces an immune response that contributes to chronic gastric in...The gram-negative bacterium Helicobacter pylori(H. pylori) infects the stomachs of approximately half of the world's population. Although infection induces an immune response that contributes to chronic gastric inflammation, the response is not sufficient to eliminate the bacterium. H. pylori infection causes peptic ulcers, gastric cancer and mucosa-associated lymphoid tissue lymphoma. Disease outcome is linked to the severity of the host inflammatory response. Gastric epithelial cells represent the first line of innate immune defence against H. pylori, and respond to infection by initiating numerous cell signalling cascades, resulting in cytokine induction and the subsequent recruitment of inflamma-tory cells to the gastric mucosa. Pathogen recognition receptors of the toll-like receptor(TLR) family mediate many of these cell signalling events. This review dis-cusses recent findings on the role of various TLRs in the recognition of H. pylori in distinct cell types, describes the TLRs responsible for the recognition of individual H. pylori components and outlines the influence of innate immune activation on the subsequent development of the adaptive immune response. The mechanistic iden-tification of host mediators of H. pylori-induced patho-genesis has the potential to reveal drug targets and opportunities for therapeutic intervention or prevention of H. pylori-associated disease by means of vaccines or immunomodulatory therapy.展开更多
基金Supported by Grants from the Fundao para a Ciência e a Tecnologia(FCT,Portugal),No.PPCDT/SAL-IMI/57297/2004 and No.PTDC/BIM-MEC/1051/2012The Swedish Cancer foundation+2 种基金The Swedish Research Council,No.K2010-79X-21372-01-3Forska utan djurfrsk,Animal Free ResearchResearch fellowship 2011 from the Sociedade Portuguesa de Gastrenterologia(Portugal)
文摘AIM:To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori(H.pylori) infection.METHODS:Aiming to overcome this limitation,clones of the heterogenic cancer-derived NCI-N87 cell line were isolated,by stably-transducing it with the human telomerase reverse-transcriptase(h TERT) catalytic subunit gene.The clones were first characterized regarding their cell growth pattern and phenotype.For that we measured the clones' adherence properties,expression of cell-cell junctions' markers(ZO-1 and E-cadherin) and ability to generate a sustained transepithelial electrical resistance.The gastric properties of the clones,concerning expression of mucins,zymogens and glycan contents,were then evaluated by haematoxylin and eosin staining,Periodic acid Schiff(PAS) and PAS/Alcian Blue-staining,immunocytochemistry and Western blot.In addition,we assessed the usefulness of the h TERT-expressing gastric cell line for H.pylori research,by performing co-culture assays and measuring the IL-8 secretion,by ELISA,upon infection with two H.pylori strains differing in virulence.RESULTS:Compared with the parental cell line,themost promising NCI-hT ERT-derived clones(CL5 and CL6) were composed of cells with homogenous phenotype,presented higher relative telomerase activities,better adhesion properties,ability to be maintained in culture for longer periods after confluency,and were more efficient in PAS-reactive mucins secretion.Both clones were shown to produce high amounts of MUC1,MUC2 and MUC13.NCI-hT ERT-CL5 mucins were shown to be decorated with blood group H type 2(BG-H),Lewis-x(Lex),Ley and Lea and,in a less extent,with BG-A antigens,but the former two antigens were not detected in the NCI-h TERT-CL6.None of the clones exhibited detectable levels of MUC6 nor sialylated Lex and Lea glycans.Entailing good gastric properties,both NCIhT ERT-clones were found to produce pepsinogen-5 and human gastric lipase.The progenitor-like phenotype of NCI-hT ERT-CL6 cells was highlighted by large nuclei and by the apical vesicular-like distribution of mucin 5AC and Pg5,supporting the accumulation of mucus-secreting and zymogens-chief mature cells functions.CONCLUSION:These traits,in addition to resistance to microaerobic conditions and good responsiveness to H.pylori co-culture,in a strain virulence-dependent manner,make the NCI-hT ERT-CL6 a promising model for future in vitro studies.
文摘The gram-negative bacterium Helicobacter pylori(H. pylori) infects the stomachs of approximately half of the world's population. Although infection induces an immune response that contributes to chronic gastric inflammation, the response is not sufficient to eliminate the bacterium. H. pylori infection causes peptic ulcers, gastric cancer and mucosa-associated lymphoid tissue lymphoma. Disease outcome is linked to the severity of the host inflammatory response. Gastric epithelial cells represent the first line of innate immune defence against H. pylori, and respond to infection by initiating numerous cell signalling cascades, resulting in cytokine induction and the subsequent recruitment of inflamma-tory cells to the gastric mucosa. Pathogen recognition receptors of the toll-like receptor(TLR) family mediate many of these cell signalling events. This review dis-cusses recent findings on the role of various TLRs in the recognition of H. pylori in distinct cell types, describes the TLRs responsible for the recognition of individual H. pylori components and outlines the influence of innate immune activation on the subsequent development of the adaptive immune response. The mechanistic iden-tification of host mediators of H. pylori-induced patho-genesis has the potential to reveal drug targets and opportunities for therapeutic intervention or prevention of H. pylori-associated disease by means of vaccines or immunomodulatory therapy.
