P2Y6 contributes to ovalbumin-induced allergic asthma by enhancing mast cell function in mice.

Extracelluar nucleotides have been identified as regulatory factors in asthmatic pathogenesis by activating purinergic receptors. This research aimed to investigate the function of the purinergic receptor P2Y6 in mediating airway inflammation in allergic asthma. Wild-type (WT) and P2Y6-deficient mice were stimulated with ovalbumin (OVA) to construct asthmatic mouse models. Overexpression of P2Y6 and uridine 5'-diphosphate (UDP)-releasing were demonstrated in lung tissues in ovalbumin-induced asthmatic mice. The release of the cytokine IL-4, mast cell invasion, and the airway remodeling phenotypes were more severe following the application of UDP in asthmatic mice. However, P2Y6 deficiency reduced these asthmatic pathogeneticsymptoms markedly in a mouse model. In vitro, we found that P2Y6 in purified mast cells enhanced the functions of mast cells in the inflammatory response in the asthmatic process by triggering their capability for migration, cytokine secretion and granule release. Moreover, P2Y6 stimulated the function of mast cells through activation of the AKT signaling pathway. Our data provides evidence that P2Y6 contributes to allergic airway inflammation and remodeling by enhancing the functions of mast cells in ovalbumin-induced asthmatic mice.

Gpr97 Is Dispensable for Inflammation in OVA-Induced Asthmatic Mice.

BACKGROUND: Asthma is a complex inflammatory disorder involving the activation and invasion of various immune cells. GPR97 is highly expressed in some immunocytes, including mast cells and eosinophils, which play critical roles in asthma development. However, the role of Gpr97 in regulating airway inflammation in asthma has rarely been reported. In this study, we investigated the potential role of Gpr97 in the development of allergic asthma in mice. METHODS: Relevant airway asthmatic mouse models were constructed with both wild-type and Gpr97-/- mice sensitized to 250 µg ovalbumin (OVA). The levels of interleukin IL-4, IL-6 and IFN-γ, which are involved in OVA-induced asthma, in the bronchoalveolar lavage fluid (BALF) and the IgE level in the serum were examined by enzyme-linked immunosorbent assay (ELISA). The invasion of mast cells and eosinophils into lung tissues was assessed by immunohistochemical and eosinophil peroxidase activity assays, respectively. Goblet cell hyperplasia and mucus production were morphologically evaluated with periodic acid-Schiff (PAS) staining. RESULTS: In our study, no obvious alteration in the inflammatory response or airway remodeling was found in the Gpr97-deficient mice with OVA-induced asthma. Neither the secretion of cytokines, including IL-4, IL-6 and IFN-γ, nor inflammatory cell recruitment was altered in the Gpr97-deficient mice. Moreover, Gpr97 deficiency did not affect airway remodeling or mucus production in the asthma mouse model. CONCLUSION: Our findings imply that Gpr97 might not be required for the development of airway inflammation in OVA-induced allergic asthma in mice.

[Cloning and expression of the extracellular region of human LIGHT gene in E.coli.].

AIM: To construct a recombinant prokaryotic expression vector containing the extracellular region of human LIGHT gene and perform the express in E.coli. METHODS: Total RNA was extracted from human immature bone marrow-derived dendritic cells. The extracellular region of human LIGHT gene was amplified by RT-PCR and cloned into pET32a(+) vector, the recombinant plasmid was identified by restriction endonuclease digestion analysis and DNA sequencing. After the recombinant plasmid was transformed into E.coli BL21 and induced with IPTG, the expressed protein was analyzed by SDS-PAGE and Western blot. RESULTS: A 543 bp of the extracellular region of human LIGHT gene was obtained and the sequence was confirmed correct by DNA sequencing. SDS-PAGE and Western blot analysis showed that a protein with molecular weight of 41 000 was expressed in E.coli BL21. CONCLUSION: The extracellular region of LIGHT gene is cloned successfully and expressed in E.coli BL21 and the elementary expression conditions were obtained, which lays a basis on the further functional research of LIGHT.

B7-DC-silenced dendritic cells induce stronger anti-HBV immunity in transgenic mice.

Hepatitis B virus (HBV) is a noncytopathic DNA virus and is the pathogen of acute and chronic hepatitis. Interferon and nucleotide analogues such as lamivudine and adefovir are the current treatment strategies of HBV infection; however, it is still a serious disease. Therefore, the development of new therapeutic options against HBV is needed. In the present study, we have investigated whether the vectors carrying short hairpin RNA (shRNA) targeting the murine B7-DC gene could silence the expression of B7-DC and analyzed the function of gene-modified dendritic cells (DCs) by mixed lymphocyte reaction. The results demonstrated that two shRNA vectors efficiently suppressed the expression of B7-DC. The MLR assay showed that shRNA-B7-DC-transfected DCs induced markedly higher allogeneic lymphocyte proliferation than transfected DCs with the vector plasmid pAS and untreated DCs at all dilutions. The most efficient shRNA plasmid vector against B7-DC was then used to silence the expression of B7-DC on DCs, the gene-modified DCs were pulsed with HBV-specific peptides, and HBV transgenic mice were immunized. After three rounds of immunization, the splenocytes were stimulated in vitro and tested for cytotoxicitic T lymphocyte activity, while the sera were used to detect the level of HBsAg and HBV DNA. The data demonstrated that blockade of B7-DC on DCs augmented the cytolytic activity induced by immunization with peptide-pulsed DCs and significantly reduced the concentration of serum HBsAg and HBV DNA, suggesting that silencing of B7-DC is of potential value in DC-based therapy of HBV infection.

[Construction and expression of prokaryotic vector encoding extracellular region of murine B7-DC].

AIM: To construct a prokaryotic expression vector for the extracellular domain of murine B7-DC(B7-DC(ECD)) gene, and to express the gene in E.coli BL21. METHODS: The total RNA was extracted from murine immature bone marrow-derived dendritic cells and the extracellular fragment of B7-DC cDNA was amplified by RT-PCR. The recombinant plasmid pET32a(+)-B7-DC(ECD) was constructed by cloning the extracellular fragment of B7-DC cDNA into the prokaryotic expression vector pET32a(+). After the recombinant plasmid was identified by restriction endonuclease digestion analysis and DNA sequencing, pET32a(+)-B7-DC(ECD) was transformed into E.coli BL21 through IPTG induction to express the target protein, and the protein was analyzed by SDS-PAGE and Western blot. RESULTS: A 582 bp of extracellular fragment B7-DC cDNA was obtained and the sequence was confirmed right by DNA sequencing. SDS-PAGE and Western blot analysis showed that a protein with molecular weight of 41 000 was expressed in E.coli BL21. CONCLUSION: The extracellular fragment of B7-DC is successfully cloned into pET32a (+) and expressed in E.coli BL21, which lays a foundation for the further functional research of B7-DC.

Therapeutic potential of dendritic cell-based immunization against HBV in transgenic mice.

Hepatitis B virus (HBV) transgenic mice that express HBV envelope proteins represent a model of chronic HBV infection suitable for the development of therapeutic immunization strategies. To address immunologically therapeutic effects induced by peptide-pulsed DCs, HBV transgenic mice were immunized with peptide-pulsed DCs, and the mice were killed after three times of immunization and the splenocytes were stimulated in vitro and detected by IFN-gamma ELISPOT and cytotoxic T lymphocyte (CTL) activity. The data demonstrated that HBV-specific CD8+ T cell response could be induced and CD8+ T cells had specific CTL activity. Furthermore, ELISA and fluorescent quantitative PCR were performed to detect the level of serum HBsAg and HBV DNA and the results demonstrated that HBV-specific peptide-pulsed DCs could significantly reduce the concentration of serum HBsAg and HBV DNA. The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured and no significant differences were observed between the different groups, which indicated that no hepatocellular injury occurred. Taken together, the data strongly demonstrated that CD8+ T cell responses and antiviral immunity were elicited in HBV transgenic mice, suggesting that peptide-pulsed DCs could elicit an effective antiviral immunity.

[Selection of recombinant fowlpox virus coexpressing HIV-1 gag-gp120 and IL-6].

OBJECTIVE: To construct the recombinant fowlpox virus (rFPV) coexpressing HIV-1 gag-gp120 and hIL-6. METHODS: The recombinant expressing plasmid pUTA-GE-IL6 was successfully constructed by inserting gag-gp120 gene and hIL-6 gene into the downstream of the combined promoter ATI-p7.5 and p7.5 tandem promoter respectively. After transfecting the plasmid into chicken embryonic fibroblast (CEF) cells preinfected with FPV 282E4 strain and selecting the recombinant virus under the pressure of BUdR. The recombinant virus was analyzed by nucleic acid probe hybridization and immunoblotting. In addition, the formation of virus-like particle and the expression of interested proteins in the recombinant virus-infected p815 cells were observed, and the immunogenicity of the recombinant virus was also analyzed. RESULTS: There was colorable dot for the positive recombinant virus, immunoblotting analysis showed that the recombinant virus could expressed both gag-gp120 and IL-6. Virus-like particles (VLP) were formed in virus-infected cells, and the interested proteins could be expressed in mammalian cells infected by the recombinant virus. The immunity index from the immunized mice showed that the recombinant virus had good immunogenicity. CONCLUSION: The recombinant fowlpox virus coexpressing gag-gp120 and IL-6 was successfully constructed, which may provide basis for the preparation of live vector genetic engineering vaccine and macromolecule particle vaccine against HIV-1.

[Immune response to HIV-2 gp105 DNA vaccine in mice].

AIM: To explore the immune response of mice vaccinated with HIV-2 gp105 nucleic acid vaccine. METHODS: pVAX1-gp105 recombinant DNA vaccine was constructed by inserting HIV-2 gp105 cDNA into an eukaryotic expression vector pVAX1. BALB/c mice were immunized with pVAX1-gp105, pVAX1 and PBS, respectively via intramuscular injection. Anti-HIV-2 antibody was detected by ELISA. The percentages of CD4(+) and CD8(+) T cell subsets from immunized mice were analyzed by flow cytometry and the specific killing activity of splenic CTLs was measured by lactate dehydrogenase (LDH) release assay. RESULTS: The titer of specific antibody, the numbers of CD4(+) and CD8(+) T cells and cytotoxic activity of specific CTLs in pVAX1-gp105 vaccination group were obviously higher than those in control group (P<0.01, P<0.05 and P<0.01, respectively). CONCLUSION: pVAX1-gp105 nucleic acid vaccine can elicit specific cellular and humoral immunities in mice.

[Study on the immunogenicity of HIV-1 gag vaccine].

AIM: To detect the immunogenicity of HIV-1 gag DNA vaccine. METHODS: The serum antibody titers of the immunized mice were detected by ELISA. The numbers of spleen's CD4(+) and CD8(+) T cells were analyzed by the fluorescent antibody staining and the cytotoxic activity was determined by lactate dehydrogenase (LDH) release assay. RESULTS: The titers of the serum antibodies and the numbers and cytotoxic activity of spleen's T lymphocyte subsets had significant differences (P<0.05 and P<0.01, respectively) between pVAXGAG and pVAX1 vaccinated groups. CONCLUSION: HIV-1 DNA vaccine plasmid pVAXGAG could elicit both the specific humoral and cellular immunities in BALB/c mice.

[The immune response in mice inoculated with HIV-1 gag-gp120 chimeric gene DNA vaccine and IL-18 plasmid].

AIM: To explore the immune response in mice inoculated with HIV-1 chimeric gene gag-gp120 DNA vaccine and IL-18 plasmid. METHODS: BALB/c mice were injected i.m with eukaryotic expression plasmid pVAXIL18 containing IL-18 gene and DNA vaccine plasmid pVAXGE containing HIV-1 gag-gp120 chimeric gene. The specific killing activities of spleen CTL and the titers of serum antibodies of the immunized mice were detected. RESULTS: The specific killing activities of spleen CTL and the titers of serum antibodies in the co-inoculation group were significantly higher than those in the single inoculation group(P<0.05), the vector control group (P<0.01) and PBS control group (P<0.01). CONCLUSION: The specific cellular and humoral immunity in mice can be induced by co-inoculating HIV-1 gag-gp120 chimeric gene DNA vaccine and IL-18 plasmid indicating that IL-18 augments immune response as immunoadjuvant.

[Construction of recombinant fowlpox virus coexpressing gp120 of Chinese HIV-1 strain and IL-18 and its immunogenicity in mice].

To screening out Chinese vaccine candidate against HIV-1, Chinese vaccine strain 282E4 of fowlpox virus was used as the vector to construct the recombinant fowlpox virus (rFPV) coexpressing gp120 of Chinese HIV-1 strain and IL-18, and the recombinant virus was indentified by PCR and Western blot. The specific DNA fragment could be amplified by PCR from the genome of rFPV. Western blot analysis showed that gp120 and IL-18 could be expressed not only in chicken embryo fibroblast (CEF) cells infected by rFPV, but also in mammalian cells infected by rFPV. After the recombinant fowlpox virus was inoculated into BALB/c mice, the spleen specific CTL activities and serum antibodies in the immunized mice were detected, which demonstrated that the rFPV had good immunogenicity and could induce BALB/c mice to produce specific humoral and cellular immunity. IL-18 palyed the role of immunoadjuvant. The study lays the basis on the preparation of genetic engineering live vector vaccine against HIV-1.

[Construction and Identification of DNA vaccine containing chimeric gene gag-gp120 of HIV-1].

AIM: To construct DNA vaccine expression plasmid containing the chimeric gene gag-gp120 of HIV-1. METHODS: The recombinant eukaryotic expression vector pVAXGE was constructed via inserting the chimeric gene gag-gp120 into the vector pVAX1. Hela cells had been transfected by recombinant plasmid via liposome. After 72 h, the transfected cells was detected by RT-PCR and analyzed by Dot-ELISA. RESULTS: The transcript products of target gene could be amplified from the cells transfected by recombinant plasmid. Dot-ELISA detection showed that the target gene was expressed in Hela cells. CONCLUSION: The DNA vaccine plasmid expressing chimeric gene gag-gp120 was successfully constructed, which lays the foundation for preparing DNA vaccine against HIV-1.