Fourteen new geranyl phenyl ethers (1-14) along with three known compounds (15-17) were isolated from Illicium micranthum, and their structures were elucidated by comprehensive spectroscopic methods. Illimicranins A-H (1-8) were characterized as geranyl vanillin ethers, while 9 and 10 were dimethyl acetal derivatives. Illimicranins I and J (11 and 12) were rare geranyl isoeugenol ethers. Illimicranins K and L (13 and 14) represented the first example of geranyl guaiacylacetone ether and geranyl zingerone ether, respectively. Compounds 1, 2 and 15 exhibited anti-HBV (hepatitis B virus) activity against HBsAg (hepatitis B surface antigen) and HBeAg (hepatitis B e antigen) secretion, and HBV DNA replication.
Antiviral Agents/pharmacology* ; Hepatitis B Surface Antigens ; Hepatitis B e Antigens ; Illicium/chemistry* ; Phenyl Ethers

OBJECTIVE: To investigate the effect of extracts with water and alcohol from Radix Trichosanthis on the cell survival and the expression of hepatitis B surface antigen (HBsAg) and hepatitis B e-antigen (HBeAg) in HepG2.2.15 cell supernatant. METHODS: The cell survival rate of HepG2.2.15 cells was detected by MTT assay. The HBsAg and HBeAg in HepG 2.2.15 cell supernatant were evaluated by enzyme linked immunosorbent assay. RESULTS: The water and alcohol soluble extracts from Radix Trichosanthis significantly inhibited the levels of HBsAg and HBeAg in HepG2.2.15 cells in a time-and-concentration-dependent manner. However, the therapeutic index of extracts with water from Radix Trichosanthis was better than that in the alcohol group. CONCLUSION The activity of water-soluble extract from Radix Trichosanthis is stronger on anti-hepatitis B virus than that of the alcohol-soluble extract.
Antiviral Agents ; pharmacology ; Drugs, Chinese Herbal ; classification ; pharmacology ; Hep G2 Cells ; Hepatitis B Surface Antigens ; biosynthesis ; Hepatitis B e Antigens ; biosynthesis ; Hepatitis B virus ; drug effects ; Humans

<b>OBJECTIVEb>To prepare the oral solution of egg yolk hepatitis B virus (HBV)-specific transfer factor (EYHBV-TF) and evaluate its immunological activity as an immune regulator against hepatitis B.

<b>METHODSb>From hens immunized with the Hepatitis B vaccine the egg yolk was isolated to extract the specific transfer factor EYHBV-TF, and its physicochemical properties were examined. Leukocyte adhesion inhibition test (LAI) was performed to detect the immunogenic activity of EYHBV-TF. The solution of EYHBV-TF was then administered orally in normal mice, and the specific cellular immune activity induced was assayed with delayed type skin hypersensitivity test (DTH), with the non-specific immune activity assessed with immune organ index. The immune responses induced by oral EYHBV-STF solution were compared with those by EYHBV-STF injection and by different dosages (injection and oral) of porcine spleen HBV-specific transfer factor (PSHBV-STF), porcine spleen nonspecific transfer factor, and egg yolk extracts from non-immunized hens.

<b>RESULTSb>The prepared EYHBV-STF oral solution, which met the standards for biological products, could inhibit leukocyte adhesion in vitro and significantly enhance mouse foot pad swelling, demonstrating its capability of transferring antigen-specific delayed type hypersensitivity reactions to naive recipient. EYHBV-STF oral solution also significantly improved the immune organ index in mice (P<0 01) with similar effects to those caused by EYHBV-STF injections and by PSHBV-STF injection and oral solution.

<b>CONCLUSIONb>Orally administered EYHBV-STF and EYHBV-STF injection both possess hepatitis B antigen-specific cellular immune activity and can significantly enhance specific cellular immune responses.

Animals ; Chickens ; Egg Yolk ; chemistry ; Hepatitis B ; drug therapy ; Hepatitis B Antigens ; Hepatitis B virus ; drug effects ; Immunity, Cellular ; Immunization ; Mice ; Swine ; Transfer Factor ; administration & dosage ; pharmacology

In this study, the anti-HBV effects of tea polyphenols (TP) were examined. After cells were exposed to TP for 3, 6, 9 days, amounts of HBsAg, HBeAg and HBV-DNA released into the supernatant of the cultured HepG2 2.2.15 cells were detected. TP, to some extent, inhibited the secretion of HBsAg and strongly suppressed the secretion of HBeAg in a dose-dependent (P<0.01) and time-dependent manner, with 50% maximal inhibitory concentration (IC50) value being 7.34 microg/mL on the 9th day, but the time-dependence was not significant (P=0.051). Expression of HBV-DNA in the supernatant of the cell culture also was significantly decreased in a dose-dependent fashion (P<0.01). The IC50 of TP in inhibiting HBV DNA was 2.54 microg/mL. It concluded that TP possessed potential anti-HBV effects and may be used as a treatment alternative for HBV infection.
Antiviral Agents/*pharmacology ; DNA, Viral/analysis ; Dose-Response Relationship, Drug ; Flavonoids/*pharmacology ; Hep G2 Cells ; Hepatitis B Surface Antigens/analysis ; Hepatitis B e Antigens/analysis ; Hepatitis B virus/*drug effects ; Inhibitory Concentration 50 ; Phenols/*pharmacology ; Tea/*chemistry

<b>OBJECTIVEb>To evaluate the biological activity of polysaccharide of Cipangopaludina chinensis (PCC) against HBV in vitro.

<b>METHODb>HepG2 2. 2. 15 cells were taken as the in vitro experimental model. The cell toxicity was observed by MTT. PCC of different safe concentrations and positive control medicine 3TC were added into the cells. Cell control without medicine was set at the same time. Cultural supernatants were collected at 9 d. HBsAg and HBeAg in cultural supernatants were tested by ELISA. The content of HBV-DNA was detected by TaqMan probe fluorescence quantitative PCR.

<b>RESULTb>TC0 and TC50 of PCC in HepG2 2. 2. 15 cell culture were 1 g . L-1 and >10 g . L-1, respectively, suggesting low toxicity in cells. IC50 of PCC in HepG2 2. 2. 15 cells HBsAg and HBeAg were 0. 501, 0. 401 g. L-1, with SI being >19.96 and >24. 94, respectively. PCC could effectively inhibit the secretion of HBsAg and HBeAg, and have a better effect on HBeAg than on HBsAg. PCC had a significant inhibitory effect on HBV-DNA in HepG2 2. 2. 15 cells at concentrations of 0. 1, 1 g . L-1 P <0.05).

<b>CONCLUSIONb>PCC has the effect against HBV activity in vitro to some extent, with low toxicity, thereby having a good prospect for application.

Animals ; Antiviral Agents ; adverse effects ; pharmacology ; DNA, Viral ; metabolism ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; drug effects ; metabolism ; Humans ; Polysaccharides ; adverse effects ; pharmacology ; Snails ; chemistry

<b>OBJECTIVEb>To investigate the effect of synthetic non-methylated CpG-ODN combined with recombined HBsAg on the phenotype, function and the expression level of nucleic transcription factor NF-kappa B (NF-kB) and AP-1 of monocyte-derived dendritic cells (DC) in patients with chronic hepatitis B (CHB).

<b>METHODSb>Purified monocytes were isolated from the peripheral blood of CHB patients and healthy volunteers and cultured with human granulocyte-monocyte colony stimulating factor added together with interleukin-4. On the fifth day of culture, CpG-ODN, HBsAg and other reagents, such as TNF alpha and PBS, were added to the medium, and 18 hours later cells were collected for the detection of surface molecules (HLA-DR/CD86/CD1a). IL-12p70 levels in the supernatant and stimulating capacity to allogenic T lymphocytes were detected. The nucleic proteins of NF-kB and AP-1 in DC were extracted and purified for the gel shift assay.

<b>RESULTSb>Compared with those of the PBS group, the expression rates of HLA-DR of DC treated with CpG-ODN and/or HBsAg were obviously increased. Both the IL-12p70 level and the stimulating capacity of DC to allogenic T lymphocytes in mixed lymphocyte reaction increased significantly in the CpG-ODN group and in the CpG-ODN/HBsAg combination group (P less than 0.01 and 0.05, respectively). The expression rates of CD1a were raised only in the CpG-ODN/HBsAg combination group. Three kinds of immunological adjuvants, TNF alpha, CpG-ODN and CpG-ODN/HBsAg enhanced the expression of nucleic NF-kB and inhibited the expression of AP-1 in DC.

<b>CONCLUSIONb>CpG-ODN, like TNF alpha, has remarkable stimulatory effect on the impaired phenotype and function of monocyte-derived DC in patients with CHB; CpG-ODN and HBsAg have a synergetic effect in increasing the antigen presenting function. The regulating ability of CpG-ODN and TNF alpha on the expression levels of NF-kB and AP-1 might be one of the mechanisms of their immunostimulatory effects on DC of the CHB patients.

Adjuvants, Immunologic ; pharmacology ; Adult ; Cells, Cultured ; Dendritic Cells ; metabolism ; HLA-DR Antigens ; metabolism ; Hepatitis B Surface Antigens ; pharmacology ; Hepatitis B, Chronic ; metabolism ; Humans ; Male ; NF-kappa B ; metabolism ; Oligodeoxyribonucleotides ; pharmacology ; Recombinant Fusion Proteins ; Transcription Factor AP-1 ; metabolism

<b>OBJECTIVESb>To explore the effect of IL-18 on peripheral blood monocytes (PBMCs) from chronic hepatitis B (CHB) patients and HBV DNA released by HepG2.2.15 cells, which were transfected with the gene of HBV.

<b>METHODSb>PBMCs were isolated from 25 healthy persons and 25 CHB patients, which were co-cultured with HBcAg and IL-18 at different concentrations for 72 hours. The level of IFN-gamma in the culture supernatant of PBMCs was determined by ELISA. One patient' PBMCs were co-cultured for 96 hours with various concentrations of IL-18 and HepG2.2.15 cells which had been cultured for 24 hours, the supernatant was collected to detect HBV DNA level by PCR.

<b>RESULTSb>When PBMCs were stimulated by HBcAg and IL-18 at various concentrations, the levels of supernatant IFN-gamma in the CHB group were much higher than those in the normal control group (at 0.2ng/ml: t=11.7, P<0.01; at 1.0ng/ml: t=16.19, P<0.01; at 5.0ng/ml: t=20.12, P<0.01), especially when the PBMCs were stimulated by HBcAg, IL-18 and IL-12 (1313.20pg/ml+-187.76pg/ml vs. 390.75pg/ml+-43.23pg/ml, t=23.94, P<0.01). The IFN-gamma level in the patients who were stimulated by HBcAg alone was much lower than the levels in the patients who were stimulated by HBcAg and IL-18 at various concentrations, and which were lower than those in the patients stimulated by HBcAg, IL-12 and IL-18 at the same concentrations (light: t=2.2, P<0.05; moderate: t=2.97, P<0.05). The HBV DNA content in the supernatant of co-cultivation with HepG2.2.15 cells and PBMCs was much higher than that of the two kinds of cells stimulated by HBcAg and IL-18 at various concentrations or HBcAg, IL-18 and IL-12/IFN-a1b.

<b>CONCLUSIONb>IL-18 can improve the PBMCs from CHB patients to produce a great deal of IFN-gamma, so it has a good application prospect in two aspects: immunoregulatory effects and increasing the ability to kill the cells infected with virus.

Adjuvants, Immunologic ; pharmacology ; Adult ; Female ; Hepatitis B Core Antigens ; pharmacology ; Hepatitis B virus ; drug effects ; genetics ; immunology ; Hepatitis B, Chronic ; immunology ; Humans ; Interferon-gamma ; biosynthesis ; Interleukin-18 ; immunology ; pharmacology ; Leukocytes, Mononuclear ; drug effects ; immunology ; Male ; Transfection

<b>Objective:b> To explore the relationship between the status of HBsAg-positive infection of mothers and the non/low-response to hepatitis B vaccine of their infants. <b>Methods:b> A total of 225 pairs of mothers and their infants were recruited in our cohort from June 2011 to July 2013. Infants were given three doses of hepatitis B vaccine at hour 24, first month and month 6(t)h respectively and were followed up for one year after birth. HBV serological markers and HBV DNA in the peripheral blood of both mothers and infants were detected by Electro-chemiluminescence immunoassay and fluorescence quantitative Polymerase Chain Reaction. <b>Results:b> Six HBV infection models were detected in HBsAg-positive mothers, and "HBsAg (+), HBeAg (+), anti-HBc (+)" (model one) and "HBsAg (+), anti-HBe (+), anti-HBc (+)" (model two) accounted for 92.5%(208/225) of all the models. Rate of non/low-response to hepatitis B vaccine in infants born to mothers in model one was lower than those in model two, the differences are statistically significant (χ(2)=4.80, P=0.029). The rate of non/low-response to hepatitis B vaccine in infants showed a downward trend with the rising of HBeAg level in their mothers (χ(2)=4.86, P=0.028). Results from the unconditional logistic regression analysis showed that the HBeAg of the HBsAg-positive mothers was significantly correlated with the low risk of non/low-response to hepatitis B vaccine in infants (OR=0.598, 95%CI: 0.378-0.947). The positive rate of serum HBV DNA in HBsAg-positive mothers was 54.2%, while the rate of non/low-response to hepatitis B vaccine in infants born to HBV DNA positive mothers was similar to those infants born to HBV DNA negative mothers (χ(2)=0.22, P=0.640). <b>Conclusions:b> "HBsAg (+), HBeAg (+), anti-HBc (+)" and "HBsAg (+), anti-HBe(+), anti-HBc (+)" were the common models seen in HBsAg-positive mothers, and the rate of non/low-response to hepatitis B vaccine was different between the two models. HBeAg of HBsAg-positive mothers might have positive effects on the immune response to hepatitis B vaccine in infants but the mechanisms remained not clear. HBV DNA of the HBsAg-positive mothers did not seem to be correlated with the immune response to hepatitis B vaccine in infants.
Adult ; Biomarkers/blood* ; DNA, Viral/blood* ; Diagnostic Tests, Routine ; Female ; Hepatitis B/prevention & control* ; Hepatitis B Antibodies/blood* ; Hepatitis B Surface Antigens/blood* ; Hepatitis B Vaccines/pharmacology* ; Hepatitis B e Antigens/blood* ; Hepatitis B virus/isolation & purification* ; Humans ; Infant ; Infectious Disease Transmission, Vertical/prevention & control* ; Mothers ; Pregnancy ; Pregnancy Complications, Infectious/virology*

<b>OBJECTIVEb>To evaluate the inhibition effects of DNAzymes specific to Hepatitis B Virus(HBV) s gene and e gene on the expressions of Hepatitis B surface antigen(HBsAg) and Hepatitis B e antigen(HBeAg).

<b>METHODSb>DNAzymes DrzBS and DrzBC specific to HBV s gene ORF A157UG and e gene ORF A1816UG, respectively, were designed and synthesized. The inhibition effects of DrzBS or DrzBC on the expressions of HBV s and e genes were observed in 2.2.15 cells.

<b>RESULTSb>The expression of HBV s or e genes was dramatically depressed after 2.2.15 cells treated by DrzBS or DrzBC. The concentration for effective inhibition was within 0.1-2.5 micromol/L and the inhibition showed a dose dependence within that concentration range. The maximum inhibition was 94.2% and 91.8% for DrzBS and DrzBC, respectively. The inhibition was maintained for 72 hours. The efficiency of inhibiting HbsAg, HbeAg in 2.2.15 cells by DrzBS, DrzBC was higher than that by antisense oligonucleotides for the same target genes. The concentrations for effective inhibition of the DNAzymes were at least 10-fold lower compared with antisense oligonucleotides. Neither inhibition on the replication of HBV DNA nor toxicity to 2.2.15 cells was observed.

<b>CONCLUSIONb>DrzBS and DrzBC can highly block the expressions of HBV s gene and e gene in 2.2.15 HBV cell model and are proved a specific and effective anti-HBV gene therapeutic means.

DNA, Catalytic ; pharmacology ; therapeutic use ; DNA, Viral ; analysis ; Dose-Response Relationship, Drug ; Gene Expression ; drug effects ; Genetic Therapy ; Hepatitis B ; therapy ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B e Antigens ; genetics

<b>OBJECTIVEb>To study the role of tumor necrosis factor-alpha (TNFalpha) in the anti-replication effects of tetracycline (Tet) on hepatitis B virus (HBV).

<b>METHODSb>The Tet-dependent regulatory fragment (TO) was PCR amplified from the pcDNA4TM/TO vector, inserted into the pUC118 cloning vector, and verified by sequencing. The counterpart fragment in the pVITRO3 expression vector, which contains two multiple cloning sites (MCSs), was replaced with the confirmed TO to generate a pVITRO3-TO vector. The Tet repressor (TR) gene from the pcDNA6/TR regulatory vector was incorporated into one MCS of pVITRO3-TO and the TNFalpha gene was subsequently incorporated into the other MCS. The resultant vector, pVITRO3-TOTR-TNFalpha, was transiently transfected into HepG2 cells. TNFalpha expression from the vector was induced by exposure to various concentrations of Tet and measured by enzyme-linked immunosorbent assay to determine the appropriate Tet concentration for experimentation. To investigate whether Tet inhibits TNFalpha expression as a mechanism of its anti-replication activity against HBV, the HepG2.2.15 cell line stably transfected with pVITRO3-TOTR-TNFalpha was used as an HBV replication model. Levels of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) were detected by immunoassay. HBV DNA level was detected by fluorescence quantitative PCR.

<b>RESULTSb>The TNFalpha expression from the newly constructed pVITRO3-TOTR-TNFalpha vector was Tet-controllable in the eukaryotic cells examined. The optimal concentration of Tet for the experimental system was 1.0 mug/ml. HBsAg and HBeAg expression was down-regulated in the HepG2.2.15 cells stably transfected with the pVITRO3-TO-TR-TNFalpha vector. After incubation with Tet for 1, 3 and 5 days, the inhibition rate of HBsAg was 2%, 1.1% and 0, compared to 14.8%, 11.5% and 28.4% in the non-Tet control group. The corresponding inhibition rates of HBeAg were 50.0%, 26.7% and 47.9%, compared to 0.3%, 1.6% and 0.0%, in the control group. HBV DNA levels in the cells and the cell culture supernatants exposed to Tet were decreased by 70.3% and 79.9%, respectively. TNFalpha inhibited production of HBsAg mRNA.

<b>CONCLUSIONb>A Tet-dependent regulatory fragment double-expressing TNFalpha single vector system was constructed successfully, achieving controllable TNFalpha expression in both transiently transfected eukaryotic cells and stable cell lines. In this HBV cell model system, Tet-induced overexpression of human TNFalpha inhibited HBV DNA replication and reduced HBsAg and HBeAg expression. Inhibition of HBV transcription may be a key role of TNFalpha against HBV replication.

DNA, Viral ; biosynthesis ; Genetic Vectors ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; drug effects ; physiology ; Humans ; Tetracycline ; pharmacology ; Transfection ; Tumor Necrosis Factor-alpha ; genetics ; Virus Replication