The expression profile in the mouse hepatitis B virus X (HBx)-transfected model was investigated in order to lay a foundation for further study on the implication of cytokines expression in hepatitis B virus (HBV) infection. Hydrodynamic injection method via the tail vein was used to establish the animal HBx-transfected model. By using microassay, the differential expression of gene in each group was analyzed, which was further confirmed by using real-time PCR and semi-quantitative PCR. Most of chemokine genes such as Ccl2, Ccl5, Ccl9, MIG and IP-10 were up-regulated in the HBx-transfected mouse model versus the control mice, which was coincided with the microarray results. Western blotting and immunohistochemistry were applied to detect the expression of MIG and IP-10 in the liver tissues. Simultaneously, ELISA was adopted to measure the content of IFN-γ in the liver tissues. DNA microassay revealed that the expression of 611 genes changed in HBx-transfected mice as compared with that in pCMV-tag2B-transfected mice, and most of the screened chemokines were up-regulated (including MIG and IP-10). Additionally, IFN-γ protein levels were increased by 20.7% (P<0.05) in pCMV-tag2B-HBx-transfected mice as compared with the untreated mice. IFN-γ protein levels were reduced by 53.9% (P<0.05) in pCMV-tag2B-transfected mice as compared with the untreated mice, which was consistent with the up-regulation of MIG and IP-10. It was suggested HBx transfection could induce the expression of MIG and IP-10 in the liver tissues, which might play the roles in HBV-related liver immunity and cytokines-mediated antiviral effect.
Animals ; Chemokine CXCL10 ; immunology ; Chemokine CXCL9 ; immunology ; Cytokines ; immunology ; DNA, Viral ; genetics ; Hepatitis B ; genetics ; immunology ; virology ; Hepatitis B virus ; genetics ; immunology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Trans-Activators ; genetics ; Transfection ; methods

Hepatitis B virus (HBV) infection disrupt the innate immunity response, which may play an important role in the chronic mechanism, while retinoic acid-induced gene I (RIG-I) mediated signaling pathway is one of the most important channel in the innate immunity. HBx and HBV polymerase may disrupt RIG-I mediated signaling pathway. The recent advances about HBV and RIG-I are reviewed in this article.
DEAD Box Protein 58 ; DEAD-box RNA Helicases ; metabolism ; Gene Products, pol ; metabolism ; Hepatitis B ; immunology ; Humans ; Immunity, Innate ; immunology ; Signal Transduction ; Trans-Activators ; metabolism

Exosomes are small membrane vesicles secreted from various types of cells. Tumor-derived exosomes contain MHC class I molecules and tumor-specific antigens, receiving attention as a potential cancer vaccine. For induction of efficient anti-tumor immunity, CD4+ helper T cells are required, which recognize appropriate MHC class II-peptide complexes. In this study, we have established an MHC class II molecule-expressing B16F1 murine melanoma cell line (B16F1-CIITA) by transduction of the CIITA (Class II transactivator) gene. Exosomes from B16-CII cells (CIITA-Exo) contained a high amount of MHC class II as well as a tumor antigen TRP2. When loaded on dendritic cells (DCs), CIITA-Exo induced the increased expression of MHC class II molecules and CD86 than the exosomes from the parental cells (Exo). In vitro assays using co-culture of immunized splenocytes and exosome-loaded DCs demonstrated that CIITA-Exo enhanced the splenocyte proliferation and IL-2 secretion. Consistently, compared to B16-Exo, CIITA-Exo induced the increased mRNA levels of inflammatory cytokines such as TNF-alpha, chemokine receptor CCR7 and the production of Th1-polarizing cytokine IL-12. A tumor preventive model showed that CIITA-Exo significantly inhibited tumor growth in a dose-dependent manner. Ex vivo assays using immunized mice demonstrated that CIITA-Exo induced a higher amount of Th1-polarized immune responses such as Th1-type IgG2a antibodies and IFN-gamma cytokine as well as TRP2-specific CD8+ T cells. A tumor therapeutic model delayed effects of tumor growth by CIITA-Exo. These findings indicate that CIITA-Exo are more efficient as compared to parental Exo to induce anti-tumor immune responses, suggesting a potential role of MHC class II-containing tumor exosomes as an efficient cancer vaccine.
Animals ; Cancer Vaccines/genetics/immunology ; Cell Line, Tumor ; Cell Proliferation ; Dendritic Cells/immunology ; Exosomes/genetics/*metabolism ; Gene Expression Regulation ; Gene Transfer Techniques ; Immunity, Cellular/immunology ; Immunity, Humoral/immunology ; Immunotherapy ; Lymphocyte Activation/immunology ; Melanoma, Experimental/mortality/pathology/*physiopathology ; Mice ; Mice, Inbred C57BL ; Nuclear Proteins/*genetics/*metabolism ; Survival Analysis ; T-Lymphocytes/immunology/metabolism ; Trans-Activators/*genetics/*metabolism ; Transduction, Genetic

To settle the foundation for the future research on the influence of wild and mutant (A1762T/ G1764A) HBV X gene on the progress of chronic HBV infection and hepatic tumorigenicity, wild and mutant (A1762T/G1764A) HBxAgs expression system was constructed. The wild and mutant (A1762T/ G1764A) HBV X genes were amplified with polymerase chain reaction (PCR) from HBV genome were inserted into pGEX-6P-2 and confirmed by sequencing respectively. Prokaryotic expression vectors pGEX-6P-2-hbvx(w) and pGEX-6P-2-hbvx(m) (A1762T/G1764A) were constructed and transformed to Trans1-blue; wild and mutant HBxAgs were expressed through IPTG induction respectively; after refolding of inclusion body, the wild and mutant HBxAgs were purified with GSTrap FF; and analysised by SDS-PAGE, Western blot and ELISA. SDS-PAGE analysis showed that the expression system was able to express target protein efficiently; the concentrations of purified wild HBxAg and mutant HBxAg were 4.88 mg/mL and 5.07 mg/mL respectively; Western blot analysis certified both the wild HBxAg and the mutant HBxAg could be recognized by the same monoclonal antibody against HBxAg; the two expressed fusion antigens coated in microtiter plate were able to react with the sera of HBV infected patients but not with the sera from healthy donors in ELISA. Results demonstrated that we successfully established a system for expression of hepatitis B x antigen and lay the foundation for further research on the role and molecular mechanisms of the mutant HBxAg in the progress of chronic HBV infection and hepatic tumorigenicity.
Antibodies, Neutralizing ; blood ; immunology ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; Genetic Vectors ; genetics ; Hepatitis B, Chronic ; blood ; immunology ; metabolism ; Humans ; Mutation ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; isolation & purification ; metabolism ; Trans-Activators ; genetics ; immunology ; metabolism

OBJECTIVETo evaluate the relationship between the clinical stages of nasopharyngeal carcinoma (NPC) and Epstein-Barr virus (EBV) antibodies Rta/IgG, EBNA1/IgA, VCA/IgA and EA/IgA.

METHODSSerum samples obtained from 211 untreated patients with NPC categorized by the project of 92' stage were examined for the presence of the EBV antibodies Rta/IgG and EBNA1/IgA by enzyme-linked immnunosorbent assay (ELISA) and for VCA/IgA and EA/IgA by immunoenzymatic assay. The positive rates and antibody levels in the NPC patients in different TNM stages and clinical stages were analyzed statistically.

RESULTSNo significant difference in Rta/IgG rA value was found in the NPC patients in different TNM or clinical stages (P>0.05). The EBNA1/IgA rA value was significantly lower in stage T1, N0, and clinical stage I than in the other corresponding T stages, N stages and other clinical stage (P<0.05). The antibody titers of VCA/IgA and EA/IgA differed significantly between the N stages and the clinical stages (P<0.05).

CONCLUSIONThe expression of EBV Rta/IgG is not associated with NPC stage. The expression of EBNA1/IgA is relatively low in early NPC. The antibody level of VCA/IgA and EA/IgA are significantly correlated to the degree of neck lymph node metastasis, and might be helpful to classify the clinical stages of NPC.

Adult ; Aged ; Aged, 80 and over ; Antibodies, Viral ; blood ; immunology ; Antigens, Viral ; immunology ; Capsid Proteins ; immunology ; Epstein-Barr Virus Nuclear Antigens ; immunology ; Female ; Herpesvirus 4, Human ; immunology ; Humans ; Immediate-Early Proteins ; immunology ; Immunoglobulin A ; blood ; Immunoglobulin G ; blood ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; immunology ; pathology ; virology ; Neoplasm Staging ; Trans-Activators ; immunology ; Young Adult

OBJECTIVEDifferent from primary membranous nephropathy, hepatitis B virus associated membranous nephropathy (HBV-MN) shows lower deposits of membrane attack complex (C5b-9) in glomerular subepithelium. The causes of relatively low complement activation in this disease remain unclear. The aim of this study was to investigate the influence of hepatitis B x protein (HBx) on the expression of CD59 and Crry in mouse podocytes.

METHODCultured mouse podocytes were divided into adenovirus vector hepatitis B virus X gene (Ad-HBx) transfected group (Ad-X group), blank podocytes group (B group) and adenovirus vector transfected group (Ad group). CD59 and Crry mRNA expression were assayed by semiquantitative RT-PCR. CD59 and Crry expression were tested by flow cytometry. The effect of HBx on complement activation was evaluated with MTT method. And then, the effects of P38MAPK, PI-3K and ERK1/2 pathway inhibitors (SB203580, LY294002, U0126) and DMSO on CD59 and Crry expression were respectively detected by flow cytometry.

RESULTProteins CD59 and Crry expression rates (%) in group B, Ad group and Ad-X group were 17.71 ± 3.81, 18.29 ± 3.36 and 45.7 ± 9.01; 18 ± 2.31, 21.78 ± 2.01 and 47.45 ± 9.95, respectively. Compared with group B, CD59 and Crry expression in group Ad was not significantly different (P values for both > 0.05), but CD59 and Crry protein expression in Ad-X group was significantly higher than that in groups B and Ad (P values for both < 0.005); CD59 and Crry gene expression in group Ad was not significantly different from that in group B (P values for both > 0.05). However, CD59 and Crry gene expression of Ad-X group was significantly higher than that in groups B and Ad (P values for both < 0.05). Flow cytometry detected CD59 protein expression rates (%) were 17.35 ± 1.24, 46.19 ± 9.77, 43.03 ± 6.83 and 40.04 ± 6.39 and Crry protein expression rates (%) were 18.14 ± 3.56, 31.95 ± 1.68, 31.95 ± 1.69 and 37.14 ± 3.92 after SB203580, LY294002, U0126 and DMSO were added to Ad-X group respectively. P38 pathway inhibition resulted in significantly lower CD59 and Crry expression than Ad-X group (P values for all < 0.005), but PI-3K, ERK1/2 pathway inhibitors and DMSO had no significant effect on the expression of CD59 and Crry (P values for all > 0.05). The inhibition rates of cell lysis were significantly higher in Ad-X group than in groups B and Ad at each serum dilution point (P values for all < 0.05), while groups B and Ad had no significant difference in cell viability.

CONCLUSIONHBx can up-regulate CD59 and Crry expression in podocytes through activating P38 pathway, resulting in decreased complement activation, which may facilitate latent HBV infection in podocytes and play a role in development of hepatitis B virus associated glomerulonephritis (HBV-GN).

Animals ; CD59 Antigens ; metabolism ; Cells, Cultured ; Mice ; Podocytes ; immunology ; metabolism ; Receptors, Complement ; metabolism ; Trans-Activators ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

Hepatitis B virus (HBV) is regarded as a stealth virus, invading and replicating efficiently in human liver undetected by host innate antiviral immunity. Here, we show that type I interferon (IFN) induction but not its downstream signaling is blocked by HBV replication in HepG2.2.15 cells. This effect may be partially due to HBV X protein (HBx), which impairs IFNβ promoter activation by both Sendai virus (SeV) and components implicated in signaling by viral sensors. As a deubiquitinating enzyme (DUB), HBx cleaves Lys63-linked polyubiquitin chains from many proteins except TANK-binding kinase 1 (TBK1). It binds and deconjugates retinoic acid-inducible gene I (RIG I) and TNF receptor-associated factor 3 (TRAF3), causing their dissociation from the downstream adaptor CARDIF or TBK1 kinase. In addition to RIG I and TRAF3, HBx also interacts with CARDIF, TRIF, NEMO, TBK1, inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon (IKKi) and interferon regulatory factor 3 (IRF3). Our data indicate that multiple points of signaling pathways can be targeted by HBx to negatively regulate production of type I IFN.
Animals ; B-Lymphocytes ; immunology ; metabolism ; Cell Line ; DEAD Box Protein 58 ; DEAD-box RNA Helicases ; antagonists & inhibitors ; immunology ; metabolism ; Hep G2 Cells ; Hepatitis B virus ; immunology ; metabolism ; Humans ; I-kappa B Kinase ; antagonists & inhibitors ; immunology ; metabolism ; Immune Evasion ; Immunity, Innate ; Interferon Regulatory Factor-3 ; antagonists & inhibitors ; immunology ; metabolism ; Interferon Type I ; antagonists & inhibitors ; immunology ; metabolism ; Mice ; Molecular Targeted Therapy ; Polyubiquitin ; antagonists & inhibitors ; metabolism ; Protein Binding ; immunology ; Sendai virus ; immunology ; metabolism ; Signal Transduction ; immunology ; TNF Receptor-Associated Factor 3 ; antagonists & inhibitors ; immunology ; metabolism ; Trans-Activators ; immunology ; metabolism

MHC class II expression is controlled mainly at transcriptional level by class II transactivator (CIITA), which is a non-DNA binding coactivator and serves as a master control factor for MHC class II genes expression. Here, we describe the function of a novel splice-isoform of CIITA, DC-expressed caspase inhibitory isoform of CIITA (or DC-CASPIC), and we show that the expression of DCCASPIC in DC is upregulated upon lipopolysaccharides (LPS) induction. DC-CASPIC localizes to mitochondria, and protein-protein interaction study demonstrates that DC-CASPIC interacts with caspases and inhibits its activity in DC. Consistently, DC-CASPIC suppresses caspases-induced degradation of nitric oxide synthase-2 (NOS2) and subsequently promotes the synthesis of nitric oxide (NO). NO is an essential regulatory molecule that modulates the capability of DC in stimulating T cell proliferation/activation in vitro; hence, overexpression of DC-CASPIC in DC enhances this stimulation. Collectively, our findings reveal that DC-CASPIC is a key molecule that regulates caspases activity and NO synthesis in DC.
Alternative Splicing ; Amino Acid Sequence ; Animals ; Base Sequence ; CARD Signaling Adaptor Proteins ; genetics ; metabolism ; Cell Line ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Humans ; In Vitro Techniques ; Lipopolysaccharides ; pharmacology ; Lymphocyte Activation ; Mice ; Mice, Inbred C57BL ; Mitochondria ; metabolism ; Molecular Sequence Data ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; metabolism ; Nuclear Proteins ; genetics ; metabolism ; Protein Isoforms ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; T-Lymphocytes ; immunology ; metabolism ; Trans-Activators ; genetics ; metabolism ; Up-Regulation ; drug effects

In order to obtain an attenuated vaccine candidate for enteropathogenic Escherichia coli (EPEC) O45, a ler deletion mutant of pig enteropathogenic E. coli (PEPEC) O45 was constructed by using the suicide vector pCVD442, termed as PEPEC O45(deltaler). The culture supernatant of PEPEC O45(deltaler) deletion mutant was inoculated in vero cell culture. PEPEC O45(deltaler) deletion mutant lost the toxigenicity to vero cell. Test group and control group of mice were orogstrically inoculated with the PEPEC O45(deltaler) deletion mutant and the virulent strain O45 respectively. Mice were observed daily for clinical signs and weight changes. Test group of mice inoculated with PEPEC O45(deltaler) gained weight normally and experienced no clinical signs. In contrast, control group of mice inoculated with virulent strain O45 exhibited weight loss and all died in four days. In another experiment, pregnant mice and pig were orally vaccinated by PEPEC O45(deltaler) twice at interval of 14 days respectively. Subsequently, the suckling mice and pig were orally challenged with O45 at 7 days of age respectively. The results showed that 80% of the sucking mice born by vaccinated mice and 75% of the sucking pig born by vaccinated pig were survival; 15% of the sucking mice born by non-vaccinated mice and 10% of the sucking pig born by non-vaccinated pig were survival. This study demonstrated that PEPEC O45(deltaler) deletion mutant lost the toxigenicity to vero cell and to be safety to mice and pig. Oral immunization can induce specific immune responses in mice and pig, and this mutant strain could be used as an attenuated vaccine candidate against PEPEC O45.
Animals ; Enteropathogenic Escherichia coli ; genetics ; immunology ; Escherichia coli Infections ; microbiology ; prevention & control ; Escherichia coli Proteins ; genetics ; Escherichia coli Vaccines ; biosynthesis ; genetics ; immunology ; Gene Deletion ; Mice ; Mutagenesis, Site-Directed ; Swine ; microbiology ; Swine Diseases ; microbiology ; prevention & control ; Trans-Activators ; genetics ; Vaccines, Attenuated ; biosynthesis ; genetics ; immunology

OBJECTIVETo develop a tight tetracycline-controlled HCV-C double transgenic mouse model.

METHODSBy crossbreeding of ApoE-rtTA-tTS transgenic mice with TRE-HCV-C transgenic mice, the double transgenic mice were produced in the F1 generation. The presence of HCV-C and tTS gene in the F1 generation was confirmed by PCR, followed by further identification and quantification of the transgene using Southern blot hybridization. The expression of HCV-C in the liver of the mouse model was detected immunohistochemically.

RESULTS AND CONCLUSIONTwo transgenic mice were obtained, which contained ApoE-rtTA-tTS and TRE-HCV-C genes in the genome. Five founders contained HCV-C gene as confirmed by PCR and Southern blot hybridization. The tight tetracycline-controlled system may facilitate further study of HCV-C gene expression and gene therapy of hepatic cellular carcinoma.

Animals ; Apolipoproteins E ; genetics ; Blotting, Southern ; Breeding ; Crosses, Genetic ; Female ; Gene Expression Regulation, Viral ; drug effects ; Hepacivirus ; genetics ; immunology ; Hepatitis C Antigens ; genetics ; immunology ; Male ; Mice ; Mice, Transgenic ; Polymerase Chain Reaction ; Tetracycline ; pharmacology ; Trans-Activators ; genetics ; Viral Core Proteins ; genetics