[Effects of cell-mediated immunity induced by intramuscular chitosan-pJME/ GM-CSF nano-DNA vaccine in BAlb/c mice].

This study aimed to investigate the immune adjuvant effect and mechanism induced by chitosan nanoparticles carrying pJME/GM-CSF. In this study, plasmid DNA (pJME/GM-CSF) was encapsulated in chitosan to prepare chitosan-pJME/GM-CSF nanoparticles using a complex coacervation process. Immunohistochemistry was used to detect the type of infiltrating cells at the site of intramuscular injection. The phenotype and functional changes of splenic DCs were measured by flow cytometry after different immunogens were injected intramuscularly. The killing activity of CTLs was assessed using the lactate dehydrogenase (LDH) release assay. The preparation of chitosan-pJME/GM-CSF nanoparticles matched the expected theoretical results. Our results also found that, after pJME/GM-CSF injection, the incoming cells were a mixture of macrophages, neutrophils, and immature DCs. Meanwhile, pJME/GM-CSF increased the expression of MHC class II molecules on splenic DCs, and enhanced their Ag capture and presentation functions. Cell-mediated immunity was induced by the vaccine. Furthermore, chitosan-pJME/GM-CSF nanoparticles outperformed the administration of standard pJME/GM-CSF in terms of DC recruitment, antigen processing and presentation, and vaccine enhancement. These findings reveal that chitosan could be used as delivery vector for DNA vaccine intramuscular immunizations, and enhance pJME/GM-CSF-induced cellular immune responses.

The dominant roles of ICAM-1-encoding gene in DNA vaccination against Japanese encephalitis virus are the activation of dendritic cells and enhancement of cellular immunity.

We investigated the cellular immune responses elicited by a plasmid DNA vaccine encoding prM-E protein from the Japanese encephalitis (JE) virus (JEV) with or without various forms of intercellular adhesion molecule (ICAM)-1 gene to maximize the immune responses evoked by the JE DNA vaccine. We observed that co-immunization with the construct containing murine ICAM-1 gene (pICAM-1) resulted in a significant increase in the percentage of CD4(+)T cells, high level of JEV-specific cytotoxic T lymphocyte response, and high production of T helper 1 (Th1)-type cytokines in splenic T cells. Furthermore, the co-expression of ICAM-1 and DNA immunogens was found to be more effective in generating T cell-mediated immune responses than those induced by immunization with pJME in combination with pICAM-1. Our results suggested that ICAM-1 enhanced T cell receptor signaling and activated Th1 immune responses in the JEV model system by increasing the induction of CD4(+)Th1 cell subset and activating dendritic cells.

[Analysis of the clinical features of and responsive factors on the prognosis in patients with fulminant hepatic failure].

OBJECTIVE: To judge the prognosis in the patients with fulminant hepatic failure and to provide the evidences of correct therapy. METHODS: The clinical features and the indexes which may affect the prognosis of the patients with fulminant hepatic failure were analyzed. Indexes including prothrombin time (PT), the routine biochemical analysis of liver and kidney functions, the plasma levels of glucose and ammonia, cortisol, lipases, amylase, age, gender and complications were analyzed using the software Statistical Product and Service Solutions (SPSS)15.0. The differences between the died and living patients were compared. RESULTS: The mortality of the patients was 65% and the highest was 80% for those with HBV and HEV coinfection. The age and gender had no influence on mortality (P value was 0.423 and 0.728 respectively). HBV infection was the main factor which caused fulminant hepatic failure (52%), The next was hepatitis E virus infection (39%). Among the indexes analyzed, the plasma levels of total bilirubin, usea nitrogen, creatinine, glucose, cholesterol and prothrombin time had positive correlations with the prognosis of the patients (P value was 0.005, 0.001, 0.001, 0.005, 0.010 and 0.049 respectively). The incidence rate of hepatic coma, hepatorenal syndrome, and adrenal insufficiency were higher in the died group than that in the living group (P value was 0.005, 0.012 and 0.025 respectively). But prothrombin time was the only factor which had correlation with the prognosis (P=0.035) analyzed by multivariate logistic regression analysis. The scores of MELD were higher in the died group than that in living group (t=18.236, P<0.01) and especially in the patients with hepatic coma and hepatorenal syndrome. The scores of MELD also had positive correlation with the plasma level of TNFa (r=0.585, P<0.01). CONCLUSIONS: The HBV infection was the main cause of fulminant hepatic failure and HBV and HEV coinfection had the highest mortality. The plasma levels of total bilirubin, cholesterol, glucose , prothrombin time and some complications including hepatic coma, hepatorenal syndrome, and adrenal insufficiency maybe had positive correlations with the prognosis of fulminant hepatic failure. The scores of MELD may predict the prognosis of these patients.

Intramuscular immunization with a plasmid DNA vaccine encoding prM-E protein from Japanese encephalitis virus: enhanced immunogenicity by co-administration of GM-CSF gene and genetic fusions of prM-E protein and GM-CSF.

OBJECTIVE: To investigate the immune responses elicited by pJME with or without various forms of granulocyte-macrophage colony-stimulating factor (GM-CSF) gene. METHODS: The changes of the T lymphocyte subsets and the levels of Th cell intracellular cytokines IFN-gamma and IL-4 were evaluated by flow cytometric analysis. The cytotoxic T lymphocyte kill activity was assessed by lactate dehydrogenase activity release test. An 80% plaque reduction neutralization test was performed to titrate the neutralization antibody before and after viral challenge. RESULTS: We demonstrated that simultaneous administration of pJME plus plasmid-ecoded GM-CSF (pGM-CSF) activated Th1 immune responses similar to those found by injecting pGM-CSF i.m. into mice 3 days before pJME vaccination, and enhancement of Th2 immunity predominated when the pGM-CSF was injected 3 days after pJME vaccination. Furthermore, the immunization with DNA vaccine encoding precursor membrane envelope/GM-CSF fusion protein was more effective in generating immune responses than that induced by immunization with pJME alone or in combination with pGM-CSF. CONCLUSIONS: These observations support the potential of GM-CSF DNA adjuvant for the Th1/Th2 balance and the enhancement of immune responses by showing that the timing of the administration of pGM-CSF and the application of different forms of GM-CSF gene influence the outcome of the resultant immune responses.

Selection and application of serotypical synthetic peptides derived from hepatitis C virus NS5A region.

BACKGROUND: Numerous studies have reported a relationship between hepatitis C virus (HCV) genotype and the response to interferon therapy. Despite high sensitivity and specificity, genotyping methods can be performed only on HCV RNA positive samples. Serotyping might be a rapid and cost effective method for determining HCV genotypes, especially in patients with previously undetectable HCV RNA. In this study, an enzyme linked immunosorbent assay (ELISA) method for HCV serotyping with the genotype specific, synthetic peptides derived from HCV nonstructural 5a (NS5A) region was developed. METHODS: Based on 45 sequences, representing HCV genotypes 1 - 6 from Genebank, we synthesised 305 overlapping 30-mer peptides within NS5A region at positions 2182 - 2343 of HCV. All peptides for antigenic reactivity were tested by enzyme immunoassay with 69 human sera with antiHCV positive representing genotype 1 - 6. Forty hepatitis C patient sera were serotyped using serotype specific, synthetic peptides and genotyped by sequencing analysis. RESULTS: The correspondence of amino acids in HCV NS5A region with amino acids in positions 2182 - 2343 was very low among different genotype peptides. The highly conserved sequences were residues 2182 - 2211 (R1), 2272 - 2301 (R7) and 2302 - 2331 (R9): the highly variable 2212 - 2241 (R3) and 2257 - 2286 (R6). Using 305 peptides, antigenic regions were located in R3, R7 and R9. Eighteen peptides from highly conserved region representing genotypes 1 to 6 showed broad immunoreactivity with sera containing antibody to all HCV genotypes. Immunoreactivity of the peptides from highly variable region was stronger with similar genotype sera. Twelve unique peptides showed highly, genotype specific, reactivity with types 1 and 3 sera. Type 2 genotype specific peptides had cross reaction with type 3 serum. No type 4, 5 or 6 specific peptides were selected. The serotyping results showed high agreement with sequencing analysis. CONCLUSIONS: The major antigenic regions in HCV NS5A region were at 2212 - 2241 (R3), 2272 - 2301 (R7) and 2302 - 2331 (R9). Eighteen peptides from highly conserved region show genotype independent, immunoreactivity, useful for antiHCV antibody test. Twelve peptides from highly variable region show genotype 1 and 3 dependent immunoreactivity, useful for determining HCV serotype, especially for patients with previously undetectable HCV RNA.

Relationship of hepatitis B virus infection of placental barrier and hepatitis B virus intra-uterine transmission mechanism.

AIM: To explore the mechanism of intra-uterine transmission, the HBV infection status of placental tissue and in vitro cultured placental trophoblastic cells was tested through in vivo and in vitro experiments. METHODS: A variety of methods, such as ELISA, RT-PCR, IHC staining and immunofluorescent staining were employed to test the HBV marker positive pregnant women's placenta and in vitro cultured placental trophoblastic cells. RESULTS: The HBV DNA levels in pregnant women's serum and fetal cord blood were correlated. For those cord blood samples positive for HBV DNA, their maternal blood levels of HBV DNA were at a high level. The HBsAg IHC staining positive cells could be seen in the placental tissues and the presence of HBV DNA detected. After co-incubating the trophoblastic cells and HBV DNA positive serum in vitro, the expressions of both HBsAg and HBV DNA could be detected. CONCLUSION: The mechanism of HBV intra-uterine infection may be due to that HBV breaches the placental barrier and infects the fetus.

Immunologic analysis induced by DNA vaccine encoding E protein of Beijing-1 strain derived from Japanese encephalitis virus.

OBJECTIVE: We have compared the gene expression and DNA immunization efficacy encoding prME and E proteins of a different strain (JaGAr-01) derived from Japanese encephalitis virus. This study aimed to construct a recombinant encoding E protein of the Beijing-1 strain derived from Japanese encephalitis virus and analyze the humoral, cellular and protective immunity induced by the above recombinant. METHODS: The recombinant pJBE containing E (1,500 bps) gene from the Beijing-1 strain of Japanese encephalitis virus was constructed and then transfected into the HepG2 cell line by liposome fusion. The expression of E (about 53 kD) protein in transfected cells was analyzed by Western blot using a specific anti-JEV-E antibody. BALB/c mice were vaccinated with 3 microg of pJBE by the gene-gun technique. JaGAr-01 and Beijing-1 strains (10(5) PFU/100 microl) of Japanese encephalitis virus were given to BALB/c mice by intraperitoneal injection 3 weeks after double DNA immunization with a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. An 80% plaque reduction neutralization test was performed to titrate the neutralization antibody before and after viral challenge. A lactate dehydrogenase activity release test was used to examine cytotoxic T lymphocyte activity after double DNA immunization. RESULTS: The expression of about 53 kD protein associated with pJBE was determined in transfected HepG2 cells with specific anti-JEV-E antibody. A higher level of neutralization antibodies and the cytotoxicity effect were induced with pJBE immunization using the gene-gun technique, and were similar to those induced with inactivated vaccine derive from the Beijing-1 strain of Japanese encephalitis virus. Balb/c mice immunized with pJBE survived the challenge with the different strains of Japanese encephalitis virus; however, Balb/c mice immunized with inactivated vaccine did not survive the challenge with the JaGAr-01 strain of Japanese encephalitis virus at all. CONCLUSIONS: DNA vaccine containing the E protein gene derived from Japanese encephalitis virus can provide not only better efficacy including humoral and cellular immunity, but also cross-protection against infection with homologous and heterologous Japanese encephalitis virus.

[Immunoreactivity studies on synthetic peptides deriving from immunodominant region of hepatitis C virus NS5a gene].

OBJECTIVE: To identify the location of major immunodominant antigenic region and study the relationship between the gene heterogeneity and immunoreactivity via detecting antigenic reactivity of synthetic peptides deriving from immunodominant region in different genotypes of hepatitis C virus (HCV) NS5a gene. METHODS: In total, 305 non-identical 30-mer long and overlapping by 15 aa peptides derived from HCV NS5a region from codon 2,182 to 2,343 among 45 unique published HCV sequences in GenBank corresponding to different genotype were designed and synthesized. The amino acid sequences of all peptides were compared with DNA Star software. The antigenic reactivity of those peptides was detected with indirect ELISA with both anti-HCV and anti-NS5 positive serum. RESULTS: The sequences showed highly conserved among HCV genotype in regions 2,272-2,301 and 2,302-2,331 as compared to regions 2,212-2,241 and 2,257-2,286. The peptides basing on amino acid residues among 2,212-2,241, 2,272-2,301 and 2,302-2,331 showed stronger immunoreactivity than any other peptides. Eighteen peptides derived from this region showed a broad immunoreactivity, 3 of them could react with 96% of anti-HCV positive sera. Whereas the immunoreactivity of the peptides derived from the region showing highly variable among HCV genotype was found to react more strongly with homologous-genotype sera. CONCLUSION: The major linear antigenic region was located at amino acid residues among 2,212-2,241, 2,272-2,301 and 2,302-2,331; the short synthetic peptide derived from NS5a region at position 2,212-2,241, 2,272-2,301 and 2,302-2,331 can be used for efficient detection of HCV antibody; some peptides showed genotype specific immuunoreactivity.

[Tumor necrosis factor-alpha, caspase-3 expression and hepatocyte apoptosis in fulminanting hepatic failure].

OBJECTIVE: To study the role of tumor necrosis factor-alpha (TNFalpha) and caspase-3 expression on hepatocyte apoptosis in experimental model of fulminant hepatic failure (FHF). METHODS: Mouse experimental model of FHF was induced by lipopolysaccharide (LPS) and D-galactosamine (D-GalN). Serum TNFalpha level and TNFalpha mRNA expression in liver were tested by ELISA and reverse transcriptase PCR (RT-PCR) method, respectively. The expression of caspase-3 in liver tissue was determined by in situ hybridization. Hepatocyte apoptosis was examined by DNA agarose gel electrophoresis and TUNEL method. TNFalpha, caspase-3 and hepatocyte apoptosis were observed in different stages after drug administration. In addition, we observed the changes of above variables after pretreatment with anti-TNFalpha IgG1. RESULTS: TNFalpha mRNA expression in liver increased significantly (0.91 +/- 0.75, that of normal control was 0.32 +/- 0.10) 2 to 4 hours after administration of LPS and D-GalN. The level of serum TNFalpha increased [(320.50 +/- 86.57) ng/L; that of normal control was (16.66 +/- 7.01) ng/L] and caspase-3 expressed to a slight extent, too. Typical manifestation of hepatocyte apoptosis appeared 8h after drug administration. 8 hours after drug administration the level of serum ALT and total bilirubin (TBil) remarkably increased [(560.66 +/- 60.20) U/L and (163.66 +/- 34.51) micro mol/L, respectively, that of normal control was (23.56 +/- 8.03) U/L and (14.90 +/- 4.80) micro mol/L, respectively] and the expression of caspase-3 reached the peak. 12 hours after drug administration, hepatocyte apoptosis and necrosis coexisted, and the level of serum ALT and TBil reached a peak [(668.30 +/- 78.69) U/L and (203.17 +/- 19.92) micro mol/L, respectively] whereas the expression of caspase-3 already decreased. Hepatocyte apoptosis and liver injury and the expression of caspase-3 could be blocked by antagonizing TNFalpha. CONCLUSIONS: TNFalpha played an important role in hepatocyte apoptosis and liver injury in fulminant hepatic failure. The hepatocyte apoptosis induced by TNFalpha is correlated with the activation of caspase-3. Hepatocyte apoptosis occurs earlier than necrosis.

Gene expression of NS5a strong antigenic regions of hepatitis C virus in E.coli and detection of their antigenicity.

OBJECTIVE: To study the effect of sequence variability between different genotypes of HCV on the antigenic properties of the NS5a protein of strong antigenic region at position 2212-2313 by using recombinant proteins. METHODS: Thirteen representative sequences from HCV genotypes 1 to 6 were selected to design synthetic genes encoding for this antigenic region. These genes were assembled by PCR from synthetic olionucleotides and expressed in E.coli as hybrid proteins with glutathione S-transferase. All 13 fusion proteins were purified from bacterial lysates and used to test a panel of anti-HCV positive sera (n=61) obtained from patients infected with HCV genotypes 1 through 6. RESULTS: A comparison of sequences derived from different HCV genotypes showed that the primary structure of this strong antigenic region is highly variable. Percent homology between different genotype sequences varied from 40.4% to 72.5%. All but one protein immunoreacted with 62% to 93% of serum samples. Although a variable degree of genotype specific antigenic reactivity was detected, only one protein demonstrated a noticeable preference to immunoreact with antibodies against the homologous HCV genotype. On the other hand, closely related proteins derived from the same subtype or genotype immunoreacted with significantly different efficiency with HCV antibodies. CONCLUSIONS: Different genotype HCV genes were successfully cloned, expressed and purified. Sequence variability has a profound effect on the antigenic properties of the HCV NS5a immunodominant region. This finding should be considered in the development of diagnostic tests for the efficient detection of anti-HCV activity in serum specimens.