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Ehrlichia chaffeensis is an obligately intracellular bacterium that resides and multiplies within cytoplasmic vacuoles of phagocytes. The Ehrlichia-containing vacuole (ECV) does not fuse with lysosomes, an essential condition for Ehrlichia to survive inside phagocytes, but the mechanism of inhibiting the fusion of the phagosome with lysosomes is not clear. Understanding the ECV molecular composition may decipher the mechanism by which Ehrlichia inhibits phagosome-lysosome fusion. In this study, we obtained highly purified ECVs from E. chaffeensis-infected DH82 cells by sucrose density gradient centrifugation and analyzed their composition by mass spectrometry-based proteomics. The ECV composition was compared with that of phagolysosomes containing latex beads. Lysosomal proteins such as cathepsin D, cathepsin S, and lysosomal acid phosphatase were not detected in E. chaffeensis phagosome preparations. Some small GTPases, involved in membrane dynamics and phagocytic trafficking, were detected in ECVs. A notable finding was that Rab7, a late endosomal marker, was consistently detected in E. chaffeensis phagosomes by mass spectrometry. Confocal microscopy confirmed that E. chaffeensis phagosomes contained Rab7 and were acidified at approximately pH 5.2, suggesting that the E. chaffeensis vacuole was an acidified late endosomal compartment. Our results also demonstrated by mass spectrometry and immunofluorescence analysis that Ehrlichia morulae were not associated with the autophagic pathway. Ehrlichia chaffeensis did not inhibit phagosomes containing latex beads from fusing with lysosomes in infected cells. We concluded that the E. chaffeensis vacuole was a late endosome and E. chaffeensis might inhibit phagosome-lysosome fusion by modifying its vacuolar membrane composition, rather than by regulating the expression of host genes involved in trafficking. [ABSTRACT FROM AUTHOR]

Seven cytoplasmic male - sterile and six - branched male restorer lines of the improved oil sunflower were hybridized with 7 x 6 incomplete diallel, 42 F1 hybrids were further used to estimate their general combining ability (GCA)-specific combining ability (SCA) and total combining ability (TCA) effects on main economic traits of oil Sunflower. The components of variance for combining ability revealed that mean squares were highly significant for all of the cross combinations, showed that the preponderance of additive gene action played the leading role in leaves per plant, kernel oil content and kernel rate, and indicated the preponderance of non - additive gene action was predominant in plant height, head diameter and growth duration, while seed yield per plant and 1 000 - seed weight were attributed to additive gene action and non - additive gene action variance. GCA effects of the parents revealed that among the CMS LQ194A and LQ252A and among the restorers Y109, 9706R, F15 - 1R were found to be promising parents. Based on significant SCA and TCA effects in components analysis, seven hybrids LQ218 x9706R, LQ28 xY109, LQ28 x9706R, LQ178 x Y109, LQ218 xF15 - 1 R, LQ28 x F15 - 1 R and LQ28 x Y112 were identified as distinct for comprehensive traits and heredity characteristics. The CMS played a main role in the formation of the F0 traits of the head diameter and 1 000 - seed weight. The result of correlation analysis indicated a significant negative correlation between 1 000 - seed weight and kernel rate and a highly significant positive correlation between 1 000 - seed weight and seed yield per plant. The phenotypic correlations and genotypic correlations were consistent. [ABSTRACT FROM AUTHOR]

Background & Aims: Immunodominance is an important feature of antiviral, antitumor, and antibacterial cellular immune responses, but it is not well demonstrated in the immune responses against Helicobacter pylori. Antigen-specific CD4+ T cells protect mice against infection with H pylori. We investigated the immunodominant CD4+ T-cell response to neuraminyllactose-binding hemagglutinin (HpaA), which is a conserved, H pylori–specific colonization factor that is being investigated as an antigen for vaccination strategies. Methods: HpaA-specific CD4+ T cells were expanded with autologous peripheral blood mononuclear cells that had been incubated with recombinant HpaA and characterized using overlapping synthetic peptides. We compared the percentage of CD4+ T cells with specificity for HpaA88–100, restricted to HLA-DRB1*1501, among 59 H pylori–infected subjects with different gastric diseases. Results: We identified and characterized several immunodominant CD4+ T-cell epitopes derived from HpaA. The immunodominant CD4+ T-cell responses specific to HpaA88–100 were observed in most H pylori–infected individuals who expressed HLA-DRB1*1501 and were significantly more abundant in patients with less severe diseases (P < .05). Conclusions: The HLA-DRB1*1501–restricted immunodominant CD4+ T-cell response to HpaA88–100 is associated with reduced risk of severe gastric diseases. Further study of these and other immunodominant CD4+ T-cell responses to H pylori will provide insight into mechanisms of protective immunity and aid in vaccine design. [ABSTRACT FROM AUTHOR]

The article presents a study that looks into the method on how to acquire the data of virulence and adherence associated genes and polymorphism of Escherichia coli 0157. H7 isolates. The study used the polymerase chain reaction (PCR) assay to determine the measure of gene on 0157: H7 isolates. It reveals the relatively low virulence of 0157 isolates and its being genetically conservative.

Background & Aims: CD8+ T cells that produce interleukin (IL)-17 (Tc17 cells) promote inflammation and have been identified in tumors. We investigated their role in the pathogenesis of gastric cancer. Methods: We used flow cytometry analyses to determine levels and phenotype of Tc17 cells in blood and tumor samples from 103 patients with gastric cancer. We performed multivariate analysis to identify factors associated with overall survival using the Cox proportional hazards model. CD8+ T cells and monocytes were isolated and cocultured in an assay for induction of Tc17 cells. Tumor cells and myeloid-derived suppressor cells (MDSCs) were isolated and used in assays of Tc17 cell function. Results: Tc17 cells with distinct cytokine and functional profiles were found in gastric tumor samples from patients. The percentage of Tc17 cells increased with tumor progression and was associated with overall survival time. Tumor-activated monocytes secreted IL-6, IL-1β, and IL-23, which promoted development of Tc17 cell populations. Supernatants from cultured Tc17 cells induced production of the chemokine CXCL12 by tumor cells; this promoted CXCR4-dependent migration of MDSCs and impaired functions of anti-tumor CD8+ cytotoxic T cells via a cell contact–dependent mechanism. Conclusions: Percentages of Tc17 cells in gastric tumors are associated with survival times of patients. These cells promote chemotaxis of MDSCs, which might promote tumor progression. [ABSTRACT FROM AUTHOR]

Abstract: Enterohemorrhagic E. coli (EHEC) causes severe diseases in humans and animals via the production of Shiga toxins, and injection of effectors into epithelia using type III secretion system (TTSS). E. coli secreted protein A (EspA) forms the filamentous conduits of TTSS, which extends into the translocation pore embedded in host cell membranes and aids in the transportation of bacterial effectors. In addition, EspA is closely associated with initial bacterial adhesion and the formation of biofilms. EspA in its various forms elicits protective immune responses, although the epitope responsible has not to be identified. Here we report the presence of a linear, immunogenic, conserved and partially protective epitope E07 (100Lys-120Val) on EspA, which is recognized by the novel monoclonal antibody 1H10. This antibody blocks EHEC-induced actin polymerization and confers protection in mice. These findings provide a better understanding of EspA-induced immune responses and could lead to epitope-based vaccines and antibody-based therapies. [ABSTRACT FROM AUTHOR]

Abstract: CD4+ T cells play important roles in protection against Helicobacter pylori (H. pylori) infection. In order to better understand the immune responses of H. pylori infection and improve immune interventions against this pathogen, we identified the Th epitopes in UreB of H. pylori, an excellent vaccine candidate antigen. By using the RANKPEP prediction algorithm, we have identified and characterized three Th epitopes within the UreB antigen, which can be recognized by CD4+ T cells from BALB/c (H-2d) mice. They were U546–561, U229–244, and U237–251. These epitopes have important value for studying the immune response of H. pylori infection and for designing effective vaccine against H. pylori. [Copyright &y& Elsevier]

Helicobacter pylori evade immune responses and achieve persistent colonization in the stomach. However, the mechanism by which H. pylori infections persist is not clear. In this study, we showed that MIR30B is upregulated during H. pylori infection of an AGS cell line and human gastric tissues. Upregulation of MIR30B benefited bacterial replication by compromising the process of autophagy during the H. pylori infection. As a potential mechanistic explanation for this observation, we demonstrate that MIR30B directly targets ATG12 and BECN1, which are important proteins involved in autophagy. These results suggest that compromise of autophagy by MIR30B allows intracellular H. pylori to evade autophagic clearance, thereby contributing to the persistence of H. pylori infections.

MicroRNAs (miRNAs) have emerged as important gene regulators and are recognized as oncogenes or tumor suppressor genes in carcinogenesis. Gastric cancer is one of the most common malignant diseases worldwide. Our previous studies have revealed that miR-146a is upregulated in gastric epithelial cells infected with Helicobacter pylori (H. pylori) and in mucosal tissues from H. pylori-positive patients. However, the role of miR-146a in gastric cancer is largely unknown. In the current study, we showed that miR-146a was upregulated in 20 gastric cancer tissues compared with matched non-tumor adjacent tissues by quantitative RT-PCR. Furthermore, ectopic expression of miR-146a could improve cell proliferation in vitro by using Cell Counting kit 8 (CCK-8). We also found that miR-146a inhibited apoptosis of gastric cancer cells by flow cytometry (FCM) and Caspase-Glo® 3/7 assay. Using target prediction algorithms, luciferase reporter assay and Western blot assay, SMAD family member 4 (SMAD4) was identified as a target gene of miR-146a in gastric cancer. Moreover, an inverse correlation was observed between the expression of SMAD4 mRNA and miR-146a in gastric cancer tissues (R=-0.731, P=0.039, Pearson's correlation). Taken together, our results provide important evidence that miR-146a can directly target SMAD4, and suggest that miR-146a may play a role in the development of gastric cancer by modulating cell proliferation and apoptosis. miR-146a could serve as a potential biomarker and therapeutic target against gastric cancer.

Aim: To prepare hybridoma cell lines were obtained by fusing Sp2/0 with spleen cells from BALB/c mice immunized with killed enterohemorrhagic E.coli O157:H7 EspA (EHEC O157:H7 EspA)., Methods: The subclass isotype and the specificity of monoclonal antibodies (mAb) were determined and identified by ELISA, Western blot and immune fluorescence staining., Results: Isotype of 3 mAb was IgG1kappa, IgG1lambda, and IgG2kappa, respectively, and the affinity constant were 3.0x10(9), 2.8x10(9), 1.9x10(9). As demonstrated by Western blot, these 3 mAb specifically reacted with EspA protein and EHEC O157:H7. Useing immune fluorescence staining, EHEC O157:H7 could adhere to the membrace of Hela cell., Conclusion: Three hybridoma cell lines can stably secrete anti-EspA mAb with high-titer and high-specific have been established. It can be used to deeply study EHEC O157:H7 pathopoiesis mechanism.

Objective: To express a multi-copy specific epitope recombinant protein of herpes simplex virus type 1 (HSV-1) with immuno-reactivity., Methods: Multi-copy genes with a specific epitope of HSV-1-glycoprotein G 112-127 were constructed by DNA recombination and cloned in E. coli JM109 pGEM-5Zf. The positive recombinants were determined by SDS-PAGE and Western blotting., Results: The recombinants with 4, 8, 16 and 32 copies of gG 112-127 were obtained. The 8-copy recombinant was expressed by 17.5%, mainly as inclusion body. And it reacted with antiserum HSV-1, but not with antiserum HSV-2., Conclusion: The HSV-1-gG112-127 recombinant could be used to distinguish HSV-1 and HSV-2 in ELISA.

Aim: To assess the variability of adhesin gene hpaA between different Helicobacter pylori (H pylori) strains with PCR-restriction fragment length polymorphism (RFLP)., Methods: Twelve different H pylori strains were chosen to amplify the 710-bp segments of gene hpaA. These strains were NCTC11637, SS1; Chongqing clinical isolates CCS9801, CCS9802, CCS9803, CCS9806, CCS9809, CCS9810, CCS9813, which were gained from patients of gastritis; Mongolia gerbil adapted H pylori strains (abbreviation MG), which were gained from the following steps: gastric mucosal specimens of Mongolia gerbils infected by clinical isolate CCS9803 were cultured and detected, the positive H pylori strains were named as the first generation of Mongolia gerbil adapted H pylori strains (abbreviation MG1) and then were subcultured with healthy Mongolia gerbil to generate MG2, in turn to gain the ninth generation (abbreviation MG9). All hpaA segments, obtained from 12 different H pylori strains, were digested by HhaI and HaeIII individually and analyzed by agarose gel electrophoresis., Results: In all 12 strains, the 710-bp PCR products were successfully amplified and products were cloned to pMD18-T vector respectively, then the recombinant plasmids were digested simultaneously with NcoI and XhoI to recover the small fragments. The objective fragments from 12 different H pylori strains digested with Hae III could be seen as 4 types of bands and 5 types with Hha I. According to the hpaA RFLP patterns, the 12 H pylori strains could be divided into 5 groups: group I, NCTC11637 and SS1; group II, CCS9809, which RFLP type digested with HaeIII was the same as strains of group I, but HhaI RFLP showed difference compared with the other groups; group III, CCS9810; group IV, CCS9803; group V: CCS9801, CCS9802, CCS9806, CCS9813, MG1, MG3 and MG9. The sequence data of 12 hpaA segments were analyzed by DNAsis software and it was observed that: (1) The homologies of base pair and amino acid sequence between strains NCTC11637, SS1, CCS9809 were 99.6% and 98.9%, respectively; (2) The homology of base pair and amino acid sequence between CCS9803 and CCS9810 was 97.7% and 99.1%; (3) That of the rest strains, CCS9801, CCS9802, CCS9806, CCS9813, MG1, MG3, MG9 reached 99.4% and 98.4%; (4) The base pair homologies between all hpaA fragments of different sources were higher than 94.6%, therefore the correspondence of deduced amino acid sequence was higher than 96.8% between each other., Conclusion: The gene hpaA from different H pylori strains revealed variation, and this might provide an effective method for molecular epidemiological survey of H pylori.

Objective: To study the improvement of CTL effect by CTL epitopes which are modified by KDEL and recognized by CD8+ T lymphocytes to herpes simplex virus type 2., Methods: Observations were made on the specific immune response induced by the CD8+ CTL epitopes(SSIEFARL, S1), the CD8+ CTL epitopes modified by KDEL(SSIEFARL-KDEL, S1-KDEL), the tandem four copies CD8+ CTL epitopes[(SSIEFARL)4, S4] and the tandem four copies CD8+ CTL epitopes modified by KDEL[(SSIEFARL)4-KDEL, S4-KDEL], 25 male C57BL/6 mouse were randomly divided into 5 group, 5 mice per group, respectively immunized with istonic Na chloride, S1, S1-KDEL, S4 and S4-KDEL. Lymphocyte proliferation was detected by 3H-TdR and the CTL effect induced by CTL epitopes in vivo was detected by 51Cr., Results: In the 3H-TdR test, compared with the control, the group S1 and group S1-KDEL, the cpm values of Group S4 and Group S4-KDEL were markedly higher (P < 0.05) and the cpm value of Group S4-KDEL was significantly higher than Group S4 (P < 0.05), but the cpm values of Group S1 and Group S1-KDEL were not significantly different from the control (P > 0.05), nor was that of Group S1 from Group S1-KDEL (P > 0.05). In the experiment in which the EL4 cells sensitized by S1 were attacked as target cells, the CTL activities induced in Group S4 and Group S4-KDEL were markedly higher (P < 0.05) compared with the control, Group S1 and Group S1-KDEL, and that induced in Group S4-KDEL was significantly higher than Group S4 (P < 0.05), but the CTL activity induced in Group S1 and Group S1-KDEL were not significantly different from the control (P > 0.05), nor was that induced in Group S1 from Group S1-KDEL (P > 0.05). In the experiment in which the EL4 cells were attacked as target cells, the kill rate was below 10% in every group, not significantly different from the control., Conclusion: The tandem four copies CD8+ CTL epitopes, modified by KDEL and recognized by CD8+ T lymphocytes to herpes simplex virus type 2, can improve the CTL effect.

Objective: To perform genetic recombination of the urease B subunit (UreB) of Helicobacter pylori (Hp) and examine the biological properties of the recombinant protein., Methods: The gene fragment encoding Hp UreB was isolated clinically from Chinese subjects by means of PCR, and cloned subsequently into an expression vector pET-11C-UreB for the non-fusion protein expression in E.coli BL21 (DE3) strain., Results: The expression of recombinant UreB was achieved in E.coli BL21 with a relative molecular weight of approximately 62,000 at the expression ratio of 26%, and the first 15 amino acids of recombinant UreB were MKKISREYVSMYGP. The results of peptide mapping and amino acid compositional analysis were consistent with previous theoretical prediction, and enzyme-linked immunosorbent assay together with Western blotting indicated strong immunogenicity and reactivity of the recombinant protein in BalB/c mice, which were specifically recognized by polyclonal BalB/c mice anti-Hp sera or human sera infected with Hp., Conclusion: The results of this study has laid an solid immunological foundation for incorporating recombinant UreB as a subunit vaccine component against Hp.