Hepatitis B ; immunology ; prevention & control ; Hepatitis B Vaccines ; immunology ; Humans ; Immunization Schedule ; Vaccination ; Vaccines, Synthetic ; immunology

OBJECTIVETo investigate the association of heptitis B virus (HBV) genotypes and precore(PreC)/basal core promoter(BCP) mutation with interruption failure of HBV vaccination in mother-to-infant transmission.

METHODSA total number of 208 serum samples were collected from infants and mothers,including 16 infants who had become HBsAg-positive despite a complete and timely course of immunization and another 88 infants successfully protected from mother-to infant HBV transmission. HBV genotypes were determined by type-specific primers PCR method. PreC/BCP mutations were detected by direct sequencing of PCR products, and Clustal W 1.8 software was applied to analyzing the sequences.

RESULTSOf 16 mothers who were having vaccine failure infants, 15 (93.8%) were HBeAg positive and infected with genotype C (15/15, 100%). Among 88 mothers of having children being protected by vaccine, 51 (58.0%) were HBeAg positive, with 45.1% (23/51) of genotype C. The proportion of genotype C in HBeAg mothers of infants with vaccine failure, was significantly higher than that of mothers with vaccine protected infants (chi2 = 14.3, P = 0.003). However, the frequencies of T1762/A1764 mutations had no significant differences between genotype C HBeAg positive mothers with vaccine failure or protected infants (33.3% and 13.3%, respectively, P = 0.4). No A1896 mutation was found in these two groups.

CONCLUSIONHBV genotype C might contribute to the immune failure of HBV vaccination in mother-to-infant transmission, while PreC/BCP mutation might not have correlation with it.

Adult ; Female ; Genes, Viral ; Genotype ; Hepatitis B ; immunology ; prevention & control ; transmission ; Hepatitis B Vaccines ; immunology ; Hepatitis B virus ; genetics ; immunology ; Humans ; Infant, Newborn ; Infectious Disease Transmission, Vertical ; prevention & control ; Mutation ; Polymerase Chain Reaction ; Pregnancy ; Pregnancy Complications, Infectious ; immunology ; virology ; Promoter Regions, Genetic

OBJECTIVETo develop a gene chip for rapid detection of the "a" determinant hotpoint mutation of hepatitis B virus (HBV).

METHODSPrimers were designed in the HBV conservative region, and probes for detecting 126A, 126S, 144A, 145R, 145E, 144A+145R, and 144A+145E mutants were developed for that gene chip. PCR amplification and gene chip technology were optimized. The performance of the gene chip was evaluated by detecting the reference plasmids. Forty five samples of serum obtained from patients with chronic hepatitis B were used to compare the sensitivity of the gene chip and the direct sequencing of PCR products.

RESULTSThe oligonucleotide microarray was specific for mutant and native plasmids. The sensitivity of the gene chip was 5 x 10(3)copies/micro l with a high reproducibility. The gene chip could detect minor variants when they were more than 10% among the HBV strains. The positive rates of 126A, 126S-1, 126S-2 detected in the 45 specimens by the gene chip (46.67%, 35.56% and 24.44%, respectively) were higher than those detected by direct sequencing of PCR products (9.00%, 4.44% and 2.22%; P=0.000, P=0.000 and P=0.002, respectively). The sequencing of cloned PCR products demonstrated that the gene chip was specific for the "a" determinant hotpoint mutation detection.

CONCLUSIONHBV "a" determinant hotpoint mutations can be detected by oligonucleotide microarray with high sensitivity and specificity, providing a method for large scale screening of the mutants.

Hepatitis B ; blood ; diagnosis ; Hepatitis B virus ; genetics ; Humans ; Oligonucleotide Array Sequence Analysis ; methods ; Point Mutation

OBJECTIVETo investigate the effects of adenovirus-mediated PTEN and P27 on the invasion of PC-3 in vitro and angiogenesis, along with their synergy in the treatment of prostate cancer.

METHODSRecombinant adenovirus vectors of the human tumor suppressor genes PTEN and P27 were constructed. The replication-incompetent recombinant adenovirus was packaged and propagated in HEK293 cells. The viral titer was examined by plaque assay and the mRNA and protein expressions of PTEN and P27 in human prostate cancer cell line PC-3 infected with Ad-PTEN and Ad-P27 were determined by RT-PCR and Western blot respectively. The invasion of PC-3 cells in vitro was examined by Boyden chamber assay. MTT assay was used to testify the effect of supernatant from PC-3 infected with Ad-PTEN and Ad-P27 on the proliferation of endothelial cells ECV-304 and the CAM test was used to testify the effect of PTEN and P27 on angiogenesis. The difference between the combined therapy group and the single gene therapy group was also examined.

RESULTSThe viral titers of Ad-PTEN and Ad-P27 were 1.8 x 10(7) pfu/ml and 1.2 x 10(9) pfu/ml respectively. Adenovirus infection verified that the mRNA and protein expression of PTEN and P27 were steady in human PC-3 cells. The invasion in vitro of PC-3 cells was significantly inhibited by infection with Ad-PTEN or/and Ad-P27. CAM and MTT assays of ECV-304 confirmed that the supernatant from PC-3 cells infected with Ad-PTEN or/and Ad-P27 could inhibit the angiogenesis effectively. There was a significant difference between the combined therapy group and the single gene therapy group.

CONCLUSIONThe combined gene therapy of Ad-PTEN and Ad-P27 plays a synergistic role in inhibiting the invasiveness of PC-3 cells and angiogenesis.

Adenoviridae ; genetics ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; Humans ; Male ; Neoplasm Invasiveness ; PTEN Phosphohydrolase ; genetics ; Prostatic Neoplasms ; blood supply ; pathology ; Transfection

Objective To compare and analyze the sensitivity,specificity of 4 domestic ELISA kits for detection of hepatitis B virus (HBV) markers (HBsAg,anti-HBs,HBeAg,anti-HBe,and anti-HBc).Methods Five hundred and ninety four serum samples collected from patients with chronic hepatitis B and abnormal blood donors were detected for HBV markers and by 4 domestic ELISA kits.Samples with conflicting results by different diagnostic kits were retested.Samples with the HBsAg values close to the cut-off point were detected by Abbott HBsAg confirmation kit (Architect HBsAg confirm).Sensitivity of the kits was determined,using the national sensitivity reference panels for HBsAg,anti-HBs,HBeAg,anti-HBe and anti-HBc.Results The rates of sensitivity on 4 domestic kits for detection of HBsAg were 4 to 10 times lower,and on the 4 domestic kits for detection of anti-HBs,HBeAg,anti-HBc and anti-HBc were 4 to 16 times lower,as compared to Abbott Architect kits.In addition,the domestic HBV ELISA kits had some false positive results.The total coincidence rates of HBsAg,anti-HBs,HBeAg,anti-HBe,anti-HBc were 96.46%-98.15%,94.28%-98.15%,98.15%-99.49%,90.07%-96.30%,92.09%-96.80%,respectively.Conclusion Both sensitivity and specificity of the domestically produced HBV ELISA kits should be improved.

OBJECTIVETo study how hepatitis B virus(HBV) 'a' determinant hotpoint mutations were influecing the hepatitis B vaccine efficacy.

METHODSPrimers were designed in HBV conservative region, and the degenerate probes for detecting 16 'a' determinant hotpoint mutations were developed for gene chips. Sensitivity and specificity of the gene chips were evaluated by clone sequencing. Sera of 47 pairs of mothers and infants with immune failure and 323 mothers of children with immune protection of HB vaccine were detected by the gene chips.

RESULTSResult from clone sequencing demonstrated that the gene chips were specific for the detection of 'a' determinant hotpoint mutations. The wild type of HBV was still dominant, with the prevalence of 78.66%, and the mutation frequencies of 126A, 145R, 126S-1, 126S-2, 129H, 144A, and 129R were 11.27%, 5.76%, 5.28%, 4.56%, 1.20%, 0.72% and 0.24%, respectively. The prevalence of 126A mutation was significantly higher than that of other mutations(P < 0.01). No significant differences were found in mother-infant transmission rates of 126A, 126S-1, 126S-2 and 145R variants.

CONCLUSIONThe currently available hepatitis B vaccine could block mother-infant transmission of 126A, 126S and 145R variants. It appears that there is no need to develop a new hepatitis B vaccine against 126 and 145 variants at present, but the consistent epidemiological surveillance on HBV mutants should be carried out.

Adult ; Female ; Genotype ; Hepatitis B ; prevention & control ; transmission ; Hepatitis B Vaccines ; immunology ; Hepatitis B virus ; genetics ; immunology ; Humans ; Infant, Newborn ; Infectious Disease Transmission, Vertical ; prevention & control ; Mutation ; Oligonucleotide Array Sequence Analysis ; Pregnancy ; Pregnancy Complications, Infectious ; prevention & control ; virology

OBJECTIVETo evaluate the kinesis of cellular immunity in adults who were vaccinated with yeast recombinant hepatitis B(rHB) vaccine and the correlation between cellular and humoral immune responses induced by the vaccine.

METHODSEight adults were vaccinated with rHB vaccine according to 0, 1,2 month schedule. The peripheral blood mononuclear cells(PBMCs) were collected at the 3, 8, 21, 34 and 65 days after the first dose. The high purity of CD4+ and CD8+ T cells obtained by sorting from PBMCs were restimulated with recombinant hepatitis B surface antigens (rHBsAg) or peptides. The spot forming cell (SFC) of IFN-gamma, IL-2 and IL-4 of CD4+ and CD8+ T cells were detected by enzyme-linked immunospot (ELISPOT).

RESULTSThe characteristics of IFN-gamma, IL-2 and IL-4 of CD4+ and CD8+ T cells appeared different after immunization with rHB vaccine. IFN-gamma of CD8+ and CD4+ T cells could be detected early with stable SFC, while the IL-2 and IL-4 of CD4+ T cells appeared late but increased after the second and third dose of vaccination. The positive rate of IL-4 of CD4+ T cells were significantly correlated with the positive rate of anti-HBs, while the SFCs of IL-4 and IL-2 of CD4+ T cells were also significantly related to the titers of anti-FIBs.

CONCLUSIONIFN-gamma could be detected early after rHB vaccination in adults, and the positive rates of IL-4 and IL-2 were correlated with that of anti-HBs.

Adult ; CD4-Positive T-Lymphocytes ; CD8-Positive T-Lymphocytes ; Hepatitis B Vaccines ; administration & dosage ; immunology ; Humans ; Immunization Schedule ; Interferon-gamma ; blood ; Interleukin-2 ; blood ; Interleukin-4 ; blood ; Vaccines, Synthetic ; immunology

OBJECTIVETo evaluate the efficacy of hepatitis B viruse (HBV) vaccination and its influencing factors among children in rural area of Jiangsu province.

METHODSTwenty-five hundred and twenty-two children born after 1998 in rural area were selected as the study population using multistage cluster sampling method. HBsAg and anti-HBs were detected by enzyme linked immunoassay (ELISA) and radio-immunoassay (RIA), respectively. Anti-HBs negative children were boosted using different hepatitis B vaccines and the efficacy was compared. Factors causing HBV infection in HBsAg positive children were also investigated.

RESULTSHBsAg positive rates in 1-7 year olds were 0.28%-1.28%, and the anti-HBs positive rates decreased from 76.7% to 45.5%. The HBsAg positive rate in children not timely vaccinated was significantly higher than those with HBV vaccine injection within 24 hours after birth (1.4% vs. 0.5%, P = 0.031). More than 90% of the anti-HBs negative children had protective level of anti-HBs after boosted with HBV vaccine.

CONCLUSIONHBsAg positive rate in children born after 1998 in rural area of Jiangsu province decreased significantly, with an average of 0.8%. The reason for HBsAg carriage in children might be attributed to mother-to-infant transmission or not timely HBV vaccination.

Adult ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Hepatitis B ; epidemiology ; immunology ; prevention & control ; transmission ; Hepatitis B Surface Antigens ; blood ; Hepatitis B Vaccines ; administration & dosage ; immunology ; Humans ; Infant ; Infectious Disease Transmission, Vertical ; Pregnancy ; Rural Population

OBJECTIVETo study the immune memory in vaccinees after the completion of a full schedule hepatitis B immunization.

METHODSOne thousand and two hundred one infants born in 1987 -1989 were immunized with 3 doses of plasma derived hepatitis B vaccine, while 2484 newborn babies during 1996-1999 were injected with 3 doses of the yeast recombinant hepatitis B vaccine. All of the infants under observation were tested for HBsAg, anti-HBs and anti-HBc, in 2005. Of 959 individuals negative for anti-HBs (< 10 mIU/ml), HBsAg and anti-HBc, 228 were immunized with plasma-derived vaccine and 731 with yeast recombinant vaccine after birth. All of them were detected for anti-HBs 15 days after a booster of 10 Ipg yeast recombinant vaccine. In addition, interleukin-2 (IL-2) was detected in 11 non-responders and 22 responders after boostering, using an enzyme-linked immunospot (ELISPOT). The anti-HBs levels of 190 individuals (91 with plasma derived vaccine and 99 with yeast recombinant vaccine) who had had quantitative data on their antibody status after the primary hepatitis B vaccination, were compared with that after the boostering.

RESULTSAmong the individuals who received plasma derived vaccine 16-18 years ago, 79.82% of them showed the signs of immune memory after one booster, with a geometric mean titer (GMT)of 325.69 mIU/ml. Of the individuals who received the yeast recombinant vaccine 6-9 years ago, 95.62% showed immune memory after one booster,with its GMT of 745.18 mIU/ml. Anti-HBs levels induced by the booster were associated with that after the primary immunization. The positive rate of IL-2 was 40.91% in subjects with good immune memory. However, IL-2 was not detected in non-responders after the booster (P < 0.01).

CONCLUSIONMost of the individuals who had received a completed schedule of primary hepatitis B vaccination and seroconverted from anti-HBs positive to negative,showed the signs of having immune memory after the booster. Only a small proportion of the vaccinees had lost their immune memory during the long term follow-up period, suggesting that these individuals should receive a booster of hepatitis B vaccine in the highly endemic areas of hepatitis B. Hepatitis B virus; Immune memory; Booster immunization

Antibody Formation ; Hepatitis B ; immunology ; prevention & control ; Hepatitis B Vaccines ; administration & dosage ; immunology ; Humans ; Immunization, Secondary ; Immunologic Memory ; Infant ; Infant, Newborn ; Interleukin-2 ; blood

BACKGROUNDUrsolic acid (UA) is a ubiquitous molecule in the plant kingdom with specific anticancer effects that have been shown in vitro and in vivo. Although UA can inhibit the proliferation of liver cancer cells and induce apoptosis of many types of tumor cells, the molecular mechanism of its anti-hepatoma activity is still not well defined. The objective of this study was to investigate the inhibitory effect and mechanisms of UA on the human hepatoma cell line SMMC-7721.

METHODSAfter treatment with UA, the growth inhibition of SMMC-7721 cells was assessed by MTT assay. Cells were also evaluated by flow cytometric analysis, Wright-Giemasa staining, Hoechst 33258 staining and transmission electron microscope after they were induced by UA. DNA microarray technology was used to investigate the gene expression pattern of SMMC-7721 cells exposed to UA 40 micromol/L. The molecular mechanism of cells death was analyzed by real-time RT-PCR and Western blotting.

RESULTSThe proliferation of SMMC-7721 cells was significantly inhibited in a dose- and time-dependent manner after UA treatment. UA induced cell cycle arrest and apoptosis. The DNA microarray analysis indicated that 64 genes were found to be markedly up- or down-expressed, including GDF15, SOD2, ATF3, and fos. The result of Western blotting showed the apoptotic proteins p53 and Bax were up-regulated while the anti-apoptotic protein Bcl-2 was down-regulated. Real-time RT-PCR confirmed UA could up-regulate the mRNA expressions of GDF15, SOD2, ATF3 and down-regulate the mRAN expression of fos. Meanwhile these effects were partly blocked by pretreatment with the p53 inhibitor Pft-alpha.

CONCLUSIONActivation of the p53 pathway is involved in UA inhibition of SMMC-7721 human hepatocellular carcinoma cell growth and induction of apoptosis.

Apoptosis ; drug effects ; Blotting, Western ; Carcinoma, Hepatocellular ; drug therapy ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Flow Cytometry ; Humans ; Liver Neoplasms ; drug therapy ; metabolism ; Microscopy, Electron, Transmission ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects ; Triterpenes ; therapeutic use ; Tumor Suppressor Protein p53 ; metabolism