Primary Studies On The Pathogenesis And Immune Mechanisms Of The Neutrophil-activating Protein Of Helicobacter Pylori

Helicobacter pylori were a microaerophilic curved Gram-negative bacilli isolated from the human stomach. In 1982, Australia scientists J Robin Warren and Barry J Marshall noticed this pathogen in a significant number of gastritis positive gastric biopsies, and then proved that it is a cause of chronic gastritis and peptic ulcer. Furthermore H.pylori has also been associated with gastric lymphoma gastric adenocarcinoma and stomach mucosa related B cell lymphoma. In 1994 the world health organization classified H.pylori infection as a definite (class 1) carcinogen. In 2005, both sientists were awarded Nobel physiological and medical prize due to their outstanding work. H.pylori infection is the most common bacterial infection worldwide and infectes the stomach of more than 50% of the entire human population. In China, the average prevalence in adults is about 60%. How to prevent H.pylori infection effectively is the focus problem, which is concerned by a large number of researchers.Infection of the stomach mucosa by the gastric pathogen Helicobacter pylori is accompanied by a large infiltration of neutrophils and monocytes which are believed to contribute substantially to H. pylori-induced gastritis.H. pylori infection induces an immune response, which is not sufficient to either prevent or counteract bacterial colonization; rather, it actually contributes to chronic gastric inflammation (6). The severity of mucosal injury appears to be directly correlated with the extent of neutrophil infiltration. Two of the major H. pylori virulence factors are the vacuolating cytotoxin (VacA) and the H. pylori neutrophil-activating protein (HP-NAP). HP-NAP, an oligomeric protein of 150 kDa, was initially identified as a promoter of endothelial adhesion of neutrophils and was designated as neutrophil-activating protein because it stimulates high production of oxygen radicals from neutrophils. In addition, HP-NAP increases in monocles the synthesis of tissue factor and the secretion of type 2 plasminogen activator inhibitor. Recently it has been found that HP-NAP promoted Th1 immune responsesis participates in creating a particular cytokine environment at the site of infection by IL-12,IL6,IL8,TNF-a production. The cytokine profile produced during the immune response to H. pylori may represent an important factor capable of influencing the outcome of the infection. It has also been found that the majority of the infected patients have antibodies against this antigen, and that vaccination of mice with HP-NAP induces protection against a subsequent challenge with H. pylori. Therefore, HP-NAP was an important virulence factor and vaccine candidate antigen.On the pathogenesis mechanism study of HP-NAP, it was known that HP-NAP induced neutrophils to produce reactive oxygen intermediates (ROI). Because of ROI's ability to damage DNA directly to induce mutation, it was suggested that HP-NAP had relationship with HP-associated gastric cancer. Now we know less about the relationship between HP-NAP and gastric cancer. To clear that is very important for diagnosis and treatment of HP-associated gastric cancer. Since CXC chemokines were identified as proinflammatiry cytokines for neutrophil chemotaxis and activation, it has been suggested they play a critical role in neutrophil recruitment in H. pylori-induced inflammatory responses. Many previous reports have shown that IL-8 and GROs were elevated in H.pylori-infected gastric mucosa. It also was reported that H.pylori water-soluble surface proteins up-regulated IL-8 and GROa mRNA and protein production by neutrophils. IL-8 is a proinflammatory factor. GROa is growth-related oncogenes and has relation with cancer development. Now it is unknown whether HP-NAP up-regulates IL-8 and GROa production in H.pylori-infected gastric mucosa to induce inflammation and cancer.The current therapy, based on the use of a proton-pump inhibitor and antibiotics, is efficacious but faces problems such as patient compliance, antibiotic resistance, and possible recurrence of infection. If H. pylori was not totally eliminated the diseases will soon recur in 70%-80% patients. Another reason of reccurence believed by some researchers was that bacterium in patient's mouth can be transported into his stomach continously. Due to the inefficiency of antibiotic therapy, vaccine was considered to be the most effective way in controlling infection. The main antigen candidates of vaccine research were Urease subunit A (UreA) or B (UreB), CagA and VacA, Hot shock protein 60 (Hsp60) etc. But so far, these vaccine candidates showed several shortcomings. Ure A and Ure B could not induce overall and stable protective immune response. Cag A and Vac A were highly variable and it was known whether Cag A was essential in infection. Hsp 60 was high homogeneous compared with the human's so that it may not induce protective immune response and probably can cause inflammation. In order to provide basis for vaccine development, it is eager to find more new rational vaccine candidates and study immune protective mechanisms.In this study, we constructed a prokaryotic expression system of H. pylori napA gene and got recombinant HP-NAP. Then we present three parts to investigate the pathogenesis and immune mechanisms of HP-NAP.Chapter.1 Primary study on the relationship between HP-NAP antibodies and gastric cancer and pathogenesis mechanisms of HP-NAPIn this study, the napA gene in 20 HP clinical isolates was detected by PCR. To investigate the relations between NAP antibodies and gastric cancer, the positive rate and amount of NAP antibodies in sera of 82 patients with gastric cancer were detected by ELISA method, and they were compared with NAP antibodies production of chronic gastritis, peptic ulcer patients and health person. To explore the effects of HP-NAP on gastric epithelial cells, we used HP-NAP to stimulate SGC7901gastric epithelial cells and measured IL-8, GROa cytokines production in culture supernatant.It was found that nap gene exists in all of HP clinical isolates. The prevalence of NAP antibodies in healthy persons was 42.6%. But the prevalence in gastric cancer patients was 97.5% and it was higher than that of peptic ulcer (92.9%) and chronic gastritis (85.7%). The level of NAP antibodies in sera of gastric cancer patients was higher than that of chronic gastritis patients (P＜0.05) and the same with that of peptic ulcer patients. There was no relation between the level of NAP antibodies and the age of patients. The results showed that there were some relations between NAP antibodies and gastric cancer and NAP was probably a risk factor of gastric cancer. It was also been found that HP-NAP up-regulated slightly IL-8 production by gastric epithelial cell lines and had no effect on GROa production. It suggested that the action of HP-NAP on gastric epithelial cell was limited and HP-NAP caused inflammation or cancer by neutrophils activation.Chapter.2 Preparation and identification of monoclonal antibodies against HP-NAPIn this study, BALB/c mice were immunized with the supernatant and precipitation of cultured HP after ultrasonication and mAbs were obtained by means of hybridoma technique. Then, MAbs against HP-NAP were screened using recombinant HP-NAP. The obtained mAbs were evaluated for subtype and titer. The specificity of mAbs was identified by Western blot and immunohistochemical techniques.29 hybridoma cell lines that secreted HP specific antibodies were established and 3 strains of mAbs against HP-NAP were obtained. All of mAbs against HP-NAP were identified as immunoglobulin G1 (IgG1) and their titer in the culture supernatant and ascites was 1:16 and 1:32000 to 1:64000 respectively. The mAbs showed specific reaction with recombinant HP-NAP and HP clinical strains. It suggested that 3 strains of mAbs specially against HP-NAP have been obtained, which would be useful for the diagnosis, prognosis and vaccine research of HP.Chapter.3 Bioinformatics analysis and antigenic epitope mapping of HP-NAP First, the three- dimensional structure of HP-NAP was predicated and ORF of napA gene were analyzed. Prediction of the three-dimensional structure by Linux operation system shows that there are many continuousα-helix. It suggested that a cross-membrane domain exists in HP-NAP and this protein is an outer membrane protein. Analysis of napA gene ORF by DNAMAN shows that there are two forward ORF and one reverse ORF. It means that napA have many different transcript products. Secondly, according to amino acid sequence of HP-NAP, B cell epitopes are predicated based on hydrophilicity, antigenic index, second structure by DNAStar and EMBOSS. Four high antigenic peptides located in 4-24, 55-77, 95-103, 118-140aa are found. The average antigenic index is 1.0236. Especially the peptide located in 55-77aa has higher antigenic index about 1.15 and contains aβ-turn. Thirdly, three mAbs were produced using recombinant NAP-GST as the immunogen by hybridoma technique. Then the mAbs were used as target molecular to immunoscreen phage display random disulfide-constrained heptapeptide (Ph. D. -C7C) library. The positive phage clones were sequenced and analyzed. The results confirmed that three different mimicry B-cell epitopes as mimotope of NAP protein are XVXFXKV, LXHXPXX and XQKSHTV showing good match with NAP proteins at 14-20aa, 60-66aa and 131-137 respectively. And these mimotopes of NAP protein were located in predicated high antigenic peptides at 4-24, 55-77, 118-140 aa. Our work demonstrated that bioinformatics method is a good tool for protein analysis and the antigenic epitope can be mapped through screening the phage displayed peptide libraries with mAbs. The study of NAP mimotope provides a new approach for vaccine development of H. pylori.