Little is known about the interactions between Helicobacter pylori, which specializes in colonizing the mucin layer that covers the gastric mucosa, and primary gastric epithelial cells. The expression pattern of Toll-like receptors (TLRs) in primary gastric epithelial cells and cell lines was compared. Primary cells did not express TLR4, whereas all cell lines expressed a nonsignaling form of TLR4. Because other cells within the mucosa expressed TLR4, it was next investigated whether H. pylori can be recognized by TLR4--they cannot. Moreover, H. pylori infection of primary cells induced a regulated production of interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha, whereas infection of cell lines only resulted in IL-8 production. The cytokine production in all cell types was strictly cag dependent. These findings indicate that, although the epithelium is important in directing the immune response against H. pylori infections, the response is independent of TLR4.

Despite increasing knowledge on the biology of Helicobacter pylori, little is known about the expression pattern of its genome during infection. While mouse models of infection have been widely used for the screening of protective antigens, the reliability of the mouse model for gene expression analysis has not been assessed. In an attempt to address this question, we have developed a quantitative reverse transcriptase PCR (RT-PCR) that allowed the detection of minute amounts of mRNA within the gastric mucosa. The expression of four genes, 16S rRNA, ureA (encoding urease A subunit), katA (catalase), and alpA (an adhesin), was monitored during the course of a 6-month infection of mice and in biopsy samples from of 15 infected humans. We found that the selected genes were all expressed within both mouse and human infected mucosae. Moreover, the relative abundance of transcripts was the same (16S rRNA > ureA > katA > alpA), in the two models. Finally, results obtained with the mouse model suggest a negative effect of bacterial burden on the number of transcripts of each gene expressed per CFU (P < 0.05 for 16S rRNA, alpA, and katA). Overall, this study demonstrates that real-time RT-PCR is a powerful tool for the detection and quantification of H. pylori gene expression within the gastric mucosa and strongly indicates that mice experimentally infected with H. pylori provide a valuable model for the analysis of bacterial gene expression during infection.Helicobacter pylori is a spiral-shaped, microaerophilic, gramnegative microorganism which persistently colonizes the stomach mucosa of 30 to 50% of the human population (9). Since its discovery and isolation in 1983 (25, 35), H. pylori has been identified as the major causative agent of chronic active gastritis and peptic ulcer disease and contributes to the development of intestinal type gastric carcinoma and B-cell mucosaassociated lymphoid tissue lymphoma (9). Therefore, infection by H. pylori causes a major health problem. Moreover, the rapid emergence of H. pylori strains resistant to two drugs, clarythromycin and metronidazole (1), used to treat H. pylori infection and the fact that antibiotic therapy does not prevent reinfection (5) have led to extensive research on the development of a vaccine against this pathogen.Many studies of the biology of H. pylori have been carried out during the past decade in order to identify virulence factors associated with colonization of the gastric lumen and responsible for pathogenesis (33). Among these factors are urease (which allows H. pylori to survive in the very acidic conditions of the stomach) (7, 8); multiple adhesins like BabA responsible for binding to Lewis b histo blood group antigen (18) or a lipoprotein AlpA (26); catalase (16,27); the cytotoxin VacA (3); and H. pylori neutrophil-activating protein (30). In parallel to the identification of virulence factors, mouse models have been widely used to identify protective antigens for inclusion in a vaccine against H. pylori infection, such as urease (10,13,14,21), t...

The gastric pathogen Helicobacter pylori is known to activate multiple proinflammatory signaling pathways in epithelial cells. In this study, we addressed the question of whether expression of the interleukin-8 receptors IL-8RA (CXCR1) and IL-8RB (CXCR2) is upregulated in H. pylori-infected human gastric biopsy samples. Biopsy samples from patients infected with H. pylori strains harboring the cag pathogenicity island (PAI) expressed larger amounts of both receptors. In addition, IL-8RB expression was induced in the gastric epithelial cell line AGS upon infection with a clinical isolate containing the cag PAI, while a strain lacking the cag PAI did not. Our finding suggests that gastric epithelial cells express IL-8R in response to H. pylori infection.Helicobacter pylori is a gram-negative bacterium that is specialized in colonizing the human gastric mucosa, where it causes a variety of clinical outcomes ranging from asymptomatic carriage to gastritis, peptic ulcers, and cancer (12). One of the most important virulence determinants of H. pylori is the 40-kb pathogenicity island (PAI) that harbors the cag genes, which encode effector molecules and a type IV secretion system (4). The type IV secretion system is required for translocation of effector molecules, e.g., CagA, into host cells (14).Epithelial cells form tight mucosal barriers and are the first cells that encounter a microbial challenge and have evolved numerous ways to detect and respond to infectious agents (2). One of the most important effector molecules produced by the epithelium is the chemokine interleukin-8 (IL-8) (9, 11). Binding of IL-8 to two high-affinity receptors, IL-8RA (CXCR1) and IL-8RB (CXCR2), induces chemotaxis and transepithelial migration of neutrophils (8,10,13). The importance of IL-8R was shown in a urinary tract infection model in which mice deficient in the receptor were unable to clear an Escherichia coli infection (6). Because epithelial expression of IL-8R can be an effective means by which to augment the inflammatory response required for bacterial clearance, we examined whether epithelial cells express IL-8R in response to H. pylori infections. MATERIALS AND METHODSCollection of gastric biopsy samples. Clinical samples were obtained from 20 volunteers (18-to 50-year-old healthy males and females) enrolled in a clinical research trial (June 1999 to June 2000) in the Hepato-Gastroenterology Department at the Centre Hospitalier Universitaire Edouard Herriot (Lyon, France). Patients who had taken nonsteroidal anti-inflammatory drugs, proton pump inhibitors, or antibiotics during the preceding 3 months were excluded from the study. The H. pylori status of the volunteers was determined by serological Pyloriset Dry tests (Orion Diagnostica, Espoo, Finland) and enzyme-linked immunosorbent assays (ELISAs) as pre-endoscopy screening. Endoscopies were performed under local anesthesia, and two biopsy samples were obtained from adjacent areas of the gastric antrum. RNA and DNA were extracted with TRIZOL (Gibco BRL, Cergy Pontoise, Fran...

The human interleukin-4 ( IL-4) gene produces an exon 2-lacking alternative splice variant, termed IL-4delta2, and described as a naturally occurring antagonist of IL-4-driven activity. We report the isolation of an IL-4delta2 cDNA from chimpanzee ( Pan troglodytes) bone marrow samples and cynomolgus macaque ( Macaca fascicularis) activated peripheral lymph node cells. The complete IL-4 cDNA sequence from chimpanzee is also provided for the first time. The phylogenetic analysis of several known IL-4 sequences revealed a highly conserved structure of coding regions among primates, suggesting that alternative IL-4 transcript splicing may be a process shared by other simian and potentially pro-simian species as well. Extension of the study to other mammalian species led us to the assumption that generation of IL-4 splice variants may be common to primates, lagomorphs (rabbit), and rodents of the sciuridae family (woodchuck), but is unlikely to occur in mice and rats (muridae), for which IL-4 splice variants have indeed never been described. Potential implications of alternatively spliced cytokine products with possible antagonistic or competitive inhibitory function, for the choice of suitable animal models of IL-4-regulated immune processes, are discussed. This study also indicates the importance of considering alternative splicing when defining cytokine bioassays, most particularly in the present context of transcriptomics, involving the generalization of sequence-based detection methods such as quantitative reverse transcription PCR.

Pulsed-field gel electrophoresis and PCR were applied for the first time to the molecular characterization of Clostridium tetani. Among five strains tested, one (CN1339) turned out to contain a mixture of two genetically different clones and two (D11 and G761) to contain bacteria differing by the presence or absence of the 74-kb plasmid harboring the tetX gene.