[The Application of Estrogen Receptor Aptamer in Immunohistochemical Detection of Breast Cancer].

OBJECTIVE: To synthesize and select an estrogen receptors aptamer that can be used in immunostaining of breast cancer tissues. METHODS: ER protein was purified. ER aptamer that showed a high affinity and specificity for ER was synthesized and selected and by SELEX. Ligand -receptor interactions assay was adopted to measure the affinity of the aptamer-ER complex. Both the biotinylated aptamer and the anti-ER monoclonal antibody were tested for immunohistochemical staining of ER status on 105 breast cancer samples. Agreement on the detection of ER expression was determined by Kappa statistics. RESULTS: The dissociation contant (Kd) of the biotinylated aptamer-ER complex, as calculated by a linear regression analysis, was determined to be (0.34±0.05) nmol/L ( n=3, r=0.989). The binding capacity (B max) was 769.23 fmol/(mg prot·nmol -1·L -1). The ER aptamer and the anti-ER antibody both exhibited identical specificity to ER-expressing breast cancer cells. There was a high agreement between the two methods ( n=105, Kappa value=0.943, 95% confident interval=0.879-1.006, P<0.05 for the ER positive and negtive samples; n=75, Kappa value=0.805, 95% confident interval=0.642-0.967, P<0.05 for the ER weak and moderate/strong expression samples). Both the anti-ER antibody and the ER aptamer can also recognized breast cancer cells at the same sites. There was no expression in the negative controls. There were also positive expressions in the 2 endometrial cancer tissues by using biotinylated aptamer. CONCLUSIONS: Our results indicated that the synthesized ER aptamer has a high affinity to bind ER. ER aptamer and the anti-ER antibody can both be used for immunohistochemical staining of ER status in breast cancer tissue.

Pronounced decline of serum HBsAg in chronic hepatitis B patients with long-term effective nucleos(t)ide analogs therapy.

AIMS: This study aims to investigate the kinetics of serum HBsAg levels in chronic hepatitis B patients with long-term nucleos(t)ide analogs (NAs) therapy. METHODS: This was a retrospective clinical study. Serum HBsAg in serial samples of 94 patients, who received at least 8 years of NAs therapy, were measured using Elecsys® HBsAg II Quant Assay. RESULTS: In this cohort, serum HBsAg levels reduced from 3.80 log10 IU/mL at baseline to 2.72 log10 IU/mL at year 8 (p < .001), and the percentage of patients with HBsAg <1000 IU/mL increased from 14.9% at baseline to 55.3% at year 8 (p < .001). The reduction of serum HBsAg did not differ significantly between patients stratified by baseline virological parameters and type of antiviral agents. But as compared to patients without HBeAg seroconversion, HBsAg levels were significant lower in patients with HBeAg seroconversion (3.19 vs. 2.47 log10 IU/mL at year 8, p = .001). As compared to patients with slow (0-1 log10 IU/mL) or steady HBsAg(≤0 log10 IU/mL) decline at year 1, patients with a rapid HBsAg (≥1 log10 IU/mL) decline had a significantly lower HBsAg levels from year 2 to 8. However, Cox regression analysis showed that only absolute HBsAg levels at year 1 was an independent predictor of subsequent HBsAg <1000 IU/mL at year 8 of antiviral therapy(HR 0.242, p = .004). CONCLUSION: Pronounced HBsAg declines could be achieved in patients after long-term effective therapy with NAs, and on-treatment low serum HBsAg level at year 1 might be a predictor of serum HBsAg <1000 IU/mL at year 8.

[Association of chronic obstructive pulmonary disease and lung cancer].

OBJECTIVE: To investigate the association between chronic obstructive pulmonary disease (COPD) and lung cancer. METHODS: A case-control study was undertaken, with 180 cases of lung cancer and 200 cases of controls. RESULTS: The odd of lung cancer was higher in patients with COPD, emphysema, chronic bronchitis, and pulmonary tuberculosis (P < 0.05). The odd of lung cancer increased significantly in patients with a family history of lung cancer or COPD (P < 0.05). The odd of lung cancer also increased when forced expiratory volume in one second (FEV1) < 80%. CONCLUSION: Patients with COPD or a family history of COPD have higher risk of lung cancer.

Construction of a plasmid vector for liver-specific inhibition of hepatocyte nuclear factor 4 alpha expression.

Hepatocyte nuclear factor-4alpha (HNF-4a) is an important transcription factor in the liver, and regulates a large number of genes involved in many aspects of hepatocyte functions. In this study, a liver-specific transcriptional regulatory element comprised of albumin promoter (ALBp) and alpha-fetoprotein enhancer (AFPe) was obtained and cloned into the plasmid pHNF4sh-CMV(short hairpin RNA targeting HNF4α) with original CMV promoter removed, resulting to pHNF4sh-EP for liver-specific knockdown of HNF4α expression. In an attempt to verify its characteristics, pHNF4sh-EP was transfected to L02, HepG2, and COS1 cell lines in vitro and delivered into mice in vivo. pHNF4sh-CMV and pNCsh-EP were used as controls. For in vitro, the level of HNF4α mRNA and protein was decreased in all cell lines transfected with pHNF4sh-CMV whereas HNF4α mRNA and protein decreasing was only observed in L02 and HepG2 cell lines upon transfection with pHNF4sh-EP, and this decreasing was more significant as compared with pHNF4sh-CMV transfected cells. For in vivo, the decreasing of HNF4α mRNA and protein was observed in both liver and kidney tissues upon transfection with pHNF4sh-CMV. After transfection with pHNF4sh-EP, decreasing of HNF4α mRNA and protein was only found in liver tissue and this decreasing was more significant. No obvious HNF4α mRNA and protein decreasing was detected either in vitro or in vivo after transfected with pNCsh-EP. In conclusion, pHNF4sh-EP could highly-active and liver-specific knockdown of HNF4α expression liver and it will be useful for further study of the funcitions of HNF4α in liver.

A mutation in the interferon regulatory element of HBV may influence the response of interferon treatment in chronic hepatitis B patients.

BACKGROUND: A functional interferon regulatory element (IRE) has been found in the EnhI/X promoter region of hepatitis B virus (HBV) genome. The purpose of this study is to compare the gene order of responder and non-responder to interferon therapy in patients with chronic hepatitis B (CHB), so as to evaluate the relationship between IRE mutation and the response to interferon treatment for CHB patients. RESULTS: Synthetic therapeutic effect is divided into complete response (CR), partial response (PR) and non-response (NR). Among the 62 cases included in this study, 40 cases (64.5%) were in the response group (CR and PR) and 22 (35.5%) cases were in the NR group. Wild type sequence of HBV IRE TTTCACTTTC were found in 35 cases (56.5%), and five different IRE gene sequences. included TTTtACTTTC, TTTCAtTTTC, TTTtAtTTTC, TTTtACTTTt and cTTtACcTTC, were found in 22 cases (35.5%), 1 case (1.6%), 1 case (1.6%), 2 cases (3.2%) and 1 case (1.6%) respectively. There were 41.9%cases (26/62) with forth base C→T mutation, consisted of 32.5% (13/40) cases in response group and 59.1% (13/22) cases in NR group. Among the 35 cases with IRE sequences, there were 67.5% (27/40) cases in response group and 36.4% (8/22) in NR group, and the difference in IRE sequences between two groups was statistic significantly (P = 0.027). The result suggested that there is likely relationship between the forth base mutation (C→T) of IRE region and the response of HBV to Interferon therapy, and this mutation may partially decrease the inhibition effect of interferon on HBV. CONCLUSION: The forth base C→T mutation in IRE element of HBV may partially influence the response of Interferon treatment in CHB patients.

Inhibition of hepatitis B Virus replication by hepatocyte nuclear factor 4-alpha specific short hairpin RNA.

BACKGROUND: Previous studies showed that hepatocyte nuclear factor 4α (HNF4α) may play a critical role in hepatitis B virus (HBV) replication. AIMS: This study aimed to investigate the effect of knocking down of HNF4α with RNA interference technique on HBV replication in a HBV replication mouse model. METHODS: Four HNF4α, specific short hairpin RNA (shRNA)-producing plasmids were constructed. HBV mRNA and DNA replication intermediates were analysed using Northern and Southern blot respectively. The expression of HNF4α and HBV core antigen (HBcAg) was detected using immunohistochemistry technique. RESULTS: One of the HNF4α shRNAs, HNF4α shRNA1, efficiently inhibited the expression of HNF4α in HepG2 cells and mice liver. HBV RNA transcripts and DNA replication intermediates in HNF4α shRNA1 group were decreased 67.3 and 76%, respectively, in HepG2 cells, and 68.1 and 70.6% in mice liver respectively. The expression level of HBcAg in the liver was also decreased with the inhibition of HNF4α expression. CONCLUSIONS: These results suggested that decreasing of HNF4α expression was associated with the reduced level of HBV replication in HepG2 cells and mice liver. These data indicated that HNF4α played a critical role in HBV replication in vivo, and HNF4α shRNA could inhibit HBV replication in vivo.

Pre-core/basal-core promoter and reverse transcriptase mutations in chronic HBV infected-patients.

BACKGROUND/AIMS: Some HBV mutations have been shown to have an association with liver disease. The aim of the study was to investigate the incidence of mutations in hepatitis B virus (HBV) pre-core/basal core promoter (BCP) and reverse transcriptase (RT) regions and their relationship with disease progression in chronic HBV-infected patients. METHODOLOGY: A total of 133 patients were enrolled in this study, comprising the acute-on-chronic hepatitis B liver failure (ACLF-HBV) and chronic hepatitis B (CHB) patients. The pre-core/ BCP and RT gene fragments were amplified by high-fidelity PCR. Mutations of pre-core/BCP and RT regions were examined by direct sequencing. RESULTS: There were no significant differences in age, the average level of ALT and course of disease between the ACLF-HBV and CHB groups. The HBeAg positive rate and average values of HBV-DNA loads of the ACLF-HBV patients were lower than that of CHB patients. In HBV pre-core/ BCP region, the point mutations T1753C (39.06% vs. 21.74%, p<0.01), A1762T (26.56% vs. 13.04%, p<0.05), G1764A (31.25% vs. 18.84%, p<0.01), G1896A (29.69% vs. 15.94%, p<0.05) and G1899 (23.44% vs. 10.14%, p<0.05) were significantly more frequent in the ACLFHBV than CHB patients. For combined mutations, A1762T+G1764A (23.43% vs. 11.59 %, p<0.05) and G1896A+ G1899A (21.88% vs. 13.04%, p<0.05) were significantly more frequent in ACLF-HBV than CHB patients. However, there were no significant differences in RT mutations between two groups. CONCLUSIONS: ACLFHBV patients had more frequent mutations in HBV precore/ BCP region than that of CHB patients. Some mutations in HBV pre-core/BCP region might be related to the aggravation of chronic HBV infection.

Construction of a highly-active, liver-specific transcriptional regulatory element through combination of the albumin promoter and α-fetoprotein enhancer.

In an attempt to construct a highly active, liver-specific transcriptional regulatory element, the mouse albumin promoter (ALBp) and α-fetoprotein enhancer (AFPe) were obtained. To verify its hepatic specificity and activity, the AFPe-ALBp-containing fragment was cloned into the plasmids, pVAX-S and pGL3-Luc with original promoter removed. Plasmid pVAX-AFPe-ALBp-S was then transfected into hepatic and non-hepatic cells in vitro, and delivered into mouse by intravenous injection and intramuscular injection, respectively. In addition, pGL3-AFPe-ALBp-Luc was transfected into hepatic and non-hepatic cell lines; pVAX1, pVAX1/S, and pGL3-ALBp-Luc were used as controls. The expression of hepatitis B surface antigen (HBsAg) was observed, and luciferase activity in cells was measured. For plasmid pVAX-AFPe-ALBp-S, the expression of HBsAg was observed in hepatic cell lines, but not in a non-hepatic cell line. Using pVAX-S, the expression of HBsAg was observed in both hepatic and non-hepatic cell lines. In cells expressing pGL3-AFPe-ALBp-Luc, the level of luciferase activity was significantly higher in hepatic cell lines, compared with the non-hepatic cell lines. In addition, the level of luciferase activity in cells expressing pGL3-AFPe-ALBp-Luc was significantly higher than that of pGL3-ALBp-Luc in hepatic cell lines, suggesting that AFPe could enhance target gene expression under the control of ALBp. The expression of HBsAg was detected in mouse liver, but not muscle when using pVAX-AFPe-ALBp-S. In contrast, the expression of HBsAg was detected in both mouse liver and muscle upon transfection with pVAX-S. In conclusion, the AFPe-ALBp element could be used as a tool to induce liver-specific expression of a target gene.

Histological changes in chinese chronic hepatitis B patients with ALT lower than two times upper limits of normal.

The role of ALT as a predictor of liver injury has been questioned. The aim of this study is to use liver biopsy to assess the degree of liver injury in patients with chronic hepatitis B(CHB) whose ALT < 2 x upper limit of normal (ULN). A total of 49.2% of patients in this study had significant inflammation (grade >or=2) and 36.4% had significant fibrosis (stage >or=2). The frequency of serious inflammation and fibrosis was similar in patients with different ALT levels. The level of serum HBV DNA was not significantly associated with the extent of inflammation and fibrosis. Advanced age was a significant independent predictor of histological damage and the presence of more significant inflammation and fibrosis. We conclude that many CHB patients with ALT < 2 x ULN have significant liver inflammation or fibrosis and that liver biopsy is necessary to assess liver damage and should be used to assess the need for anti-viral therapy.

TRAIL inhibits HBV replication and expression by down-regulating liver-enriched transcription factors.

BACKGROUND AND STUDY AIMS: To investigate the role of low-concentration TRAIL on HBV replication and expression. MATERIAL AND METHODS: MTT assay was performed to determine the minimum concentrations of TRAIL protein in HepG2 cell apoptosis. HepG2 cells were transfected by HBV replication plasmid pHBV4.1. After the treatment with low concentration of TRAIL, the culture supernatant was collected to detect HBsAg and HBeAg by ELISA. Proteins were extracted from the resulted cells, followed by total RNA and HBV DNA intermediate replication. Southern Blot and Northern Blot were carried out to detect HBV RNA and HBV DNA replication intermediates, respectively. RT-PCR and Western Blot were carried out to detect gene and protein expressions for HNF4α, PPARα, and RXRα, respectively. RESULTS: 50 ng/ml of TRAIL protein led to significant decline on the secretions of HBsAg and HBeAg. Expression levels of HBV RNA and HBV DNA replication intermediates were significantly decreased too. In addition, gene and protein expressions of HNF4α, PPARα and RXRα also dropped, especially for PPARα whose expressions significantly decreased. CONCLUSION: TRAIL could inhibit HBV replication and expression by downregulating the expressions of liver-enriched transcription factors HNF4α, PPARα, and RXRα.

Establishment of cell lines using a doxycycline-inducible gene expression system to regulate expression of hepatitis B virus X protein.

The hepatitis B virus (HBV) X gene plays an important role in HBV-associated pathogenesis, especially hepatocarcinogenesis. Establishment of a stable and regulable HBx expression system will allow study of the function of this gene. Here, we describe the development of a doxycycline-inducible recombinant plasmid (pBPSTR3-FlagX) with the full-length HBV X gene and all components of the tetracycline-on ("Tet-on") gene expression system. This vector exhibited dose-dependent doxycycline-dependent induction of the Flag-HBx protein in HepG2 and Hep3B cells. We also observed dose-dependent doxycycline transactivation of HBx in HepG2 cells. After transfecting HepG2 cells with the pBPSTR3-FlagX plasmid, we isolated five puromycin-resistant cell clones with stable HBx expression, two of which exhibited stable and tight control of HBx expression by doxycycline. This new system has great potential for functional studies of the HBV X gene.

Prevalence and risk factors of fatty liver disease in Chengdu, Southwest China.

BACKGROUND: Fatty liver disease (FLD) is increasingly recognized as one of the most common chronic liver diseases in China. This study aimed to investigate the prevalence and risk factors of FLD in Chengdu, Southwest China, and to provide a relevant basis for the prevention and intervention of FLD. METHODS: Altogether 9094 subjects (4721 men and 4373 women) of over 18 years old who had received a medical checkup in the West China Hospital of Sichuan University between January and December 2007 were evaluated for FLD. FLD was diagnosed by ultrasonography. Body mass index (BMI), height, body weight, blood pressure, fasting plasma glucose (FPG), triglycerides (TG), total cholesterol (TCh), alanine aminotransferase (ALT), hepatitis B surface antigen (HBsAg), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured using routine laboratory methods. RESULTS: The overall prevalence of FLD was 12.5%, which was more than 3-fold higher in males than in females (18.9% vs. 5.7%, X2=359.624, P<0.001). The prevalence increased with age in females and males of less than 50 years. The prevalence of alcoholic, suspected alcoholic, and non-alcoholic FLD was 2.6%, 3.6%, and 6.3%, respectively. Multiple logistic regression analyses showed that 10 factors (male sex, age, BMI, FPG, hypertension, TG, TCh, HDL-C, LDL-C, and ALT abnormalities) were closely related to FLD. In heavy drinkers, obesity increased the risk of FLD by 23.78-fold (95% CI, 10.22-55.33), but heavy drinking was only associated with a 2-fold (95% CI, 1.50-2.66) increased risk in obese subjects. CONCLUSIONS: The prevalence of FLD among a health-checkup population in Chengdu, Southwest China was lower than the published for other areas of China. FLD in Chengdu adults was found to be closely associated with sex, age, BMI, and other metabolic syndrome features.

[The inhibitory effects of siRNA expression vector on cell proliferation and expression of hnRNPB1 gene in lung cancer A549 cells].

OBJECTIVE: To investigate the inhibitory effects of specific siRNA on the expression of heterogeneous nuclear ribonucleoproteins (hnRNPB1) and cell proliferation in lung cancer A549 cells. METHODS: After the construction of siRNA expression vector for hnRNPB1, cultured A549 cells were tansfected with the specific siRNA vector, so RNA of A549 cells was interfered by RNA interference technique. The cells were retrieved at 1st, 4th and 6th week after transfection, cell cycles and cell apoptosis were measured with flow cytometry, the mRNAs levels of hnRNPB1, were detected by fluorescence quantity RT-PCR, the protein levels of hnRNPB1 was detected by Western blot technique. RESULTS: It was found that plasmid-derived siRNAs could inhibit hnRNPB1 mRNA and protein expression. The expressions of hnRNPB1 mRNA and protein were decreased about 46%-73%, and 77.69%-83.04% respectively. We also observed that siRNA in A549 cell suppressed the cell proliferation by increasing the amounts of G1 cells, and induced cell apoptosis. CONCLUSION: These results demonstrated the effectiveness of siRNA in lung caner cell, which indicated a possible clinical therapy method for lung cancer.

Performance of serum HBcrAg in chronic hepatitis B patients with 8-year nucleos(t)ide analogs therapy.

AIM: This study aimed to investigate long-term kinetics of serum hepatitis B core-related antigen (HBcrAg) and its correlation with serum hepatitis B surface antigen (HBsAg) in a real-world cohort of patients who had received over 8 years of nucleos(t)ide analogs(NAs) therapy. METHODS: This was a retrospective study. All patients were recruited from our previous published study, who started therapy with NAs between 2007 and 2008. Serum HBcrAg and HBsAg levels were quantitatively measured at baseline, the sixth month and each year of follow-up, using the stored serum samples. RESULTS: Among the 94 patients, serum HBcrAg presented a gradually decreasing trend from baseline to year 8, either in HBeAg-negative or HBeAg-positive patients. After 8 years of NAs treatment, 21.3% of patients achieved serum HBcrAg < 3 log 10 U/mL, and only baseline HBcrAg was an independent predictor. Additionally, good correlation of HBcrAg and HBsAg was observed at baseline, but this correlation weakened remarkably during treatment. CONCLUSION: Serum HBcrAg is decreasing gradually with the duration of antiviral therapy, and baseline HBcrAg level is an independent predictor of long-term HBcrAg below the limit of detection.

Insight into the role of TRAIL in liver diseases.

TNF-related apoptosis inducing ligand (TRAIL) is a potential antitumor protein known for its ability to selectively eliminate various types of tumor cells without exerting toxic effects in normal cells and tissues. TRAIL has recently been suggested as a potential therapeutic target in hepatocellular carcinoma (HCC) because it promotes apoptosis in cancer cells. Furthermore, studies on the role of TRAIL in liver injury have reported that TRAIL plays an essential role in viral hepatitis, fatty liver diseases, etc. However, several contradictory and confounding effects of TRAIL in these liver diseases have not been fully elucidated or placed into perspective. Hence, this review summarizes recent progress in studies on TRAIL, including its role in apoptotic signaling, potential therapeutic applications of TRAIL in HCC, hepatitis virus infection, and liver fibrosis and cirrhosis.

[RNA interference of hnRNP B1 gene in human A549 lung cells].

OBJECTIVE: To test the effect of RNA interference of hnRNP Bx gene in human lung cell line A549. METHODS: The RNAs of cell line A549 were interfered by the RNA interference technique. The hnRNP B1 mRNAs were detected by fluorescence quantity RT-PCR. The proteins of hnRNP B1 were detected by Western blot technique. The growth rate of the cells was analysed by MTT. RESULTS: The growth of the cells was suppressed significantly. The mRNA and protein expressions of hnRNP B1 were inhibited. CONCLUSION: The expression of hnRNP B1 can be inhibited by RNA interference. RNA interference could become a possible clinical therapy for lung cancers.

Establishment and primary application of a mouse model with hepatitis B virus replication.

AIM: To establish a rapid and convenient animal model with hepatitis B virus (HBV) replication. METHODS: A naked DNA solution of HBV-replication-competent plasmid was transferred to BALB/C mice via the tail vein, using a hydrodynamic in vivo transfection procedure. After injection, these mice were sacrificed on d 1, 3, 4, 5, 7 and 10. HBV DNA replication intermediates in the liver were analyzed by Southern blot hybridization. The expression of hepatitis B core antigen (HBcAg) and hepatitis B surface antigen (HBsAg) in the liver was checked by immunohistochemistry. Serum HBsAg and hepatitis B e antigen (HBeAg) was detected by enzyme-linked immunosorbent assay (ELISA). Inhibition of HBV replication was compared in HBV replication model mice treated intraperitoneally with polyinosinic-polytidylin acid (polyIC) or phosphate-buffered saline (PBS). RESULTS: After hydrodynamic in vivo transfection, HBV DNA replication intermediates in the mouse liver were detectable on d 1 and abundant on d 3 and 4, the levels were slightly decreased and remained relatively stable between d 5 and 7, and were almost undetectable on d 10. The expression patterns of HBcAg and HBsAg were similar to that of HBV replication intermediate DNA, except that they reached a peak on d 1 after injection. No obvious differences in HBV DNA replication intermediates were observed in the left, right and middle lobes of the liver. After treatment with polyIC, the level of HBV intermediate DNA in the liver was lower than that in the control mice injected with PBS. CONCLUSION: A rapid and convenient mouse model with a high level of HBV replication was developed and used to investigate the inhibitory effect of polyIC on HBV replication, which provides a useful tool for future functional studies of the HBV genome.

[The study about immune response to Balb/c mice inoculated with hepatitis B virus DNA vaccine pCI/S2S].

OBJECTIVE: To construct the hepatitis B virus (HBV) DNA vaccine pCI/S2S, and study the expression of specific antigen protein in muscle tissue section and the specific immune responses of humoral and cellular immunity in BALB/c mice being inoculated with pCI/S2S. METHODS: DNA templates were acquired from the mixed serum of patients with chronic hepatitis B, and the gene fragment S2S of HBV was cloned. The pCI vector and DNA S2S fragment were doubly cut by Kpn I and Not I, and then linked. HBV DNA vaccine pCI/S2S were intramuscularly injected into BALB/c mice, and boosted after 4 and 8 week. The expressing protein product in muscle tissue in situ and the specific cellular immune response were detected in 12 week after the first injection, the specific humoral immune responses were detected at 1, 2, 4, 6, 8, 12 week after the first injection. RESULTS: After pCI/S2S intramuscularly injected into BALB/c mice, HBsAb and preS2Ab were gradually determined with cytotoxic T lymphocyte responses induced, and HBsAg protein was expressed in situ in muscle tissue. CONCLUSION: pCI/S2S is effective HBV DNA vaccine that can induce the specific humoral and cellular immune responses.

[On the role of liver-enriched transcription factors in regulating HBV transcription and replication].

OBJECTIVE: To investigate the effects of various liver-enriched transcription factors in regulating HBV transcription and replication, and to explore their potential roles in HBV hepatotropism. METHODS: The replication-competent HBV recombinant plasmid pHBV4.1 plus different liver-enriched transcription factor (HNF1, HNF3, HNF4, HNF6, C/EBP and RXRa/PPARa) expression plasmids were co-transfected into nonhepatic cell lines (NIH3T3, HeLa, 293T, SW1353, CV-1 and COS1). The transcription levels of 3.5 kb, 2.4/2.1 kb and 0.7 kb HBV RNA were analyzed by Northern blot hybridization, and the level of HBV DNA replication intermediates was detected by Southern blot hybridization analysis. RESULTS: In the absence of co-transfected liver enriched transcription factor expression vectors, the 3.5 kb HBV RNA is not transcribed and HBV DNA replication is not detected after transfecting of NIH 3T3 cells with pHBV4.1. Expression of the liver-enriched transcription factor HNF4 or RXRalpha/PPARalpha, stimulates the transcription of 3.5 kb HBV RNA and the replication of HBV DNA. In contrast, expression of HNF1, HNF3, HNF6 and C/EBP does not stimulate the transcription of 3.5 kb HBV RNA and therefore does not activate viral replication. HNF4 and RXRalpha/PPARalpha were also shown to activate the transcription of 3.5 kb HBV RNA and viral replication in divers cell types including HeLa, 293T, SW1353, CV-1 and COS1 cells. Mutation of the proximal nucleocapsid HNF4 binding site results in a greatly decreased level of HNF4 or RXRalpha/PPARalpha dependent HBV replication. CONCLUSION: This study demonstrated that the liver-enriched transcription factors HNF4 and RXRa/PPARa can support HBV transcription and replication in nonhepatic cells, indicating that liver-specific gene transcription is one of the determinants of HBV hepatotropism.

The Serum Anti-HBs Level Among Children Who Received Routine Hepatitis B Vaccination During Infancy in Mianyang City, China: A Cross-Sectional Study.

Hepatitis B virus (HBV) prevalence has declined remarkably in children due to nationwide universal vaccination program for HBV in China. However, the persistence of immune response against HBV infection and the optimal time point when a booster vaccination should be performed remain to be elucidated. To assess the persistence and level of antibody against hepatitis B surface antigen (anti-HBs) in a representative population of age 15 and younger who received routine hepatitis B vaccination in Mianyang City, China. A cross-sectional study was conducted in 2011. One thousand five hundred twenty-six children of age 15 and younger who received three doses of 5 µg hepatitis B vaccine series during infancy but did not receive a booster vaccination later were enrolled. Of the 1,526 children, the mean age was 8.2 ± 4.1 and 739 children were male. The median anti-HBs level was 23.0 mIU/mL, and the total percentage of anti-HBs levels ≥10 mIU/mL was 60.9%. With an increase of age, median anti-HBs level, percentage of anti-HBs levels ≥10 mIU/mL, and percentage of anti-HBs levels ≥100 mIU/mL declined remarkably in the early period and reached the lowest level at the age of 3 and then remained relatively stable. The median anti-HBs level, the percentage of anti-HBs levels ≥10 mIU/mL, and the percentage of anti-HBs levels ≥100 mIU/mL in 1- and 2-year-old children were much higher than that in children aged 3-15 (p < 0.05, respectively). Immunity against HBV infection gradually decreased in early ages of children of 15 and younger who received three doses of 5 µg hepatitis B vaccine series during infancy in China. Three dosages of 10 µg hepatitis B vaccine for infants and repeated vaccination or additional booster vaccination for some children at or before age 3 should be provided to get much more powerful immunity to HBV.