Population pharmacokinetics, safety and dosing optimization of voriconazole in patients with liver dysfunction: A prospective observational study.

AIMS: Voriconazole is a broad-spectrum antifungal agent for the treatment of invasive fungal infections. There is limited information about the pharmacokinetics and appropriate dosage of voriconazole in patients with liver dysfunction. This study aimed to explore the relationship between voriconazole trough concentration (Ctrough ) and toxicity, identify the factors significantly associated with voriconazole pharmacokinetic parameters and propose an optimised voriconazole dosing regimen for patients with liver dysfunction. METHODS: The study prospectively enrolled 51 patients with 272 voriconazole concentrations. Receiver operating characteristic curves were used to explore the relationship between voriconazole Ctrough and toxicity. The pharmacokinetic data was analysed with nonlinear mixed-effects method. Dosing simulations stratified by total bilirubin (TBIL, TBIL-1: TBIL < 51 µmol/L; TBIL-2: 51 µmol/L ≤ TBIL < 171 µmol/L; TBIL-3: TBIL ≥ 171 µmol/L) were performed. RESULTS: Receiver operating characteristic curve analysis revealed that voriconazole Ctrough of ≤ 5.1 mg/L were associated with significantly lower the incidence of adverse events. A 1-compartment pharmacokinetic model with first-order absorption and elimination was used to describe the data. Population pharmacokinetic parameters of clearance, volume of distribution and oral bioavailability were 0.88 L/h, 148.8 L and 88.4%, respectively. Voriconazole clearance was significantly associated with TBIL and platelet count. The volume of distribution increased with body weight. Patients with TBIL-1 could be treated with a loading dose of 400 mg every 12 hours (q12h) for first day, followed by a maintenance dose of 100 mg q12h administered orally or intravenously. TBIL-2 and TBIL-3 patients could be treated with a loading dose of 200 mg q12h and maintenance doses of 50 mg q12h or 100 mg once daily and 50 mg once daily orally or intravenously, respectively. CONCLUSIONS: Lower doses and longer dosing intervals should be considered for patients with liver dysfunction. TBIL-based dosing regimens provide a practical strategy for achieving voriconazole therapeutic range and therefore maximizing treatment outcomes.

Betaine-assisted recombinase polymerase assay for rapid hepatitis B virus detection.

Hepatitis B virus (HBV) is a worldwide epidemic pathogen that causes hepatitis B. On-site screening the HBV infection is of critical importance for preventing and diagnosing HBV infection. In this paper, a simple, visual, and rapid method for on-site detection of HBV-DNA has been developed. This method is based on betaine-assisted recombinase polymerase assay and followed with naked-eye detection via lateral flow assay (BRPA-LF). Result show that nonspecific amplification is prone to occur in recombinase polymerase amplification (RPA) if the assay was performed with serum sample without purification. This problem has been addressed by adding 0.8 M of betaine to the RPA reactions. It was demonstrated that BRPA-LF can detect 1,000 copies of HBV-DNA in 50 µL mixture, and achieved 90% sensitivity and 100% specificity for serum sample detection. These results demonstrated that BRPA-LF can resist serum interference and has great potential for on-site screening of HBV infection.

Novel HBV mutations and their value in predicting efficacy of conventional interferon.

BACKGROUND: Accumulating studies assessing the impacts of hot spot mutations on conventional interferon (IFN) efficacy come to discrepant conclusions; studies regarding the mutations in S and RT regions are also unclear. The present study aimed to evaluate the impacts of HBV mutations on the efficacy of conventional IFN. METHODS: A total of 126 patients who received conventional IFN treatment for 48 weeks were enrolled. Biochemical and serological parameters were routinely tested. The sequences of HBV from 78 serum samples were amplified by nested-PCR; mutations were identified with sequence scanner V1.0 after ABI 3730xl direct sequencing, HBV genotypes were determined according to RT gene sequences utilizing NCBI Genotyping Tool which was based on phylogenetic analysis. RESULTS: The baseline DNA levels of virological response (VR) group were significantly lower than those of no VR group [7.13+/-0.76 vs 7.69+/-0.56 lg (copies/mL), P=0.001]. The baseline ALT levels were significantly higher in the HBeAg clearance group (204.72+/-88.65 vs 162.80+/-85.81 IU/L, P<0.05) and HBeAg seroconversion group (204.89+/-95.68 vs 166.75+/-84.43 IU/L, P<0.05). Females and lower BMI levels (20.01+/-2.33 vs 21.65+/-3.66 kg/m2, P<0.05) were prone to acquired biochemical response (BR). PC-W28STOP (ntG1896A) was significantly higher in the combined response (CR) group than that in the no CR group (91.7% vs 39.7%, P=0.001). Multivariate logistic regression analysis showed that baseline DNA, PC-P159T (ntC2288A), BCP-N118T (ntA1726C) and BCP-L134L (ntA1775C/G/T) influenced VR independently. PC-G182C (ntG2357T), PC-S64A/T (ntT2003G/A) and BMI were independent influence factors for HBeAg clearance, HBeAg seroconversion and BR, respectively. The new predicting model concluded that baseline DNA and new mutations for VR were established successfully, and ROC analysis showed that AUC was 0.842 (P<0.001) with a sensitivity of 0.652 and a specificity of 0.933. CONCLUSIONS: PC-P159T (ntC2288A), BCP-N118T (ntA1726C), BCP-L134L (ntA1775C/G/T), PC-G182C (ntG2357T) and PC-S64A/T (ntT2003G/A) were novel identified mutations that impacted IFN therapeutic efficacy. These novel mutations could serve as important predictors before conventional IFN treatment.

Hepatitis B virus mutation may play a role in hepatocellular carcinoma recurrence: A systematic review and meta-regression analysis.

BACKGROUND AND AIMS: A number of studies have confirmed that antiviral therapy with nucleotide analogs (NAs) can improve the prognosis of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) after curative therapy. However, what factors affected the prognosis of HBV-HCC after removal of the primary tumor and inhibition of HBV replication? A meta-regression analysis was conducted to explore the prognostic factor for this subgroup of patients. METHODS: MEDLINE, EMBASE, Web of Science, and Cochrane library were searched from January 1995 to February 2014 for clinical trials evaluating the effect of NAs on the prognosis of HBV-HCC after curative therapy. Data were extracted for host, viral, and intervention information. Single-arm meta-analysis was performed to assess overall survival (OS) rates and HCC recurrence. Meta-regression analysis was carried out to explore risk factors for 1-year OS rate and HCC recurrence for HBV-HCC patients after curative therapy and antiviral therapy. RESULTS: Fourteen observational studies with 1284 patients met the inclusion criteria. Influential factors for prognosis of HCC were mainly baseline HBeAg positivity, cirrhotic stage, advanced Tumor-Node-Metastasis (TNM) stage, macrovascular invasion, and antiviral agent type. The 1-year OS rate decreased by more than four times (coefficient -4.45, P<0.001) and the 1-year HCC recurrence increased by more than one time (coefficient 1.20, P=0.003) when lamivudine was chosen for HCC after curative therapy, relative to entecavir for HCC. CONCLUSIONS: HBV mutation may play a role in HCC recurrence. Entecavir or tenofovir, a high genetic barrier to resistance, should be recommended for HBV-HCC patients.

Results of a phase III clinical trial with an HBsAg-HBIG immunogenic complex therapeutic vaccine for chronic hepatitis B patients: experiences and findings.

BACKGROUND & AIMS: Even though various experimental therapeutic approaches for chronic hepatitis B infection have been reported, few of them have been verified by clinical trials. We have developed an antigen-antibody (HBsAg-HBIG) immunogenic complex therapeutic vaccine candidate with alum as adjuvant (YIC), aimed at breaking immune tolerance to HBV by modulating viral antigen processing and presentation. A double-blind, placebo-controlled, phase II B clinical trial of YIC has been reported previously, and herein we present the results of the phase III clinical trial of 450 patients. METHODS: Twelve doses of either YIC or alum alone as placebo were administered randomly to 450 CHB patients and they were followed for 24weeks after the completion of immunization. The primary end point was HBeAg seroconversion, and the secondary end points were decrease in viral load, improvement of liver function, and histology. RESULTS: In contrast to the previous phase II B trial using six doses of YIC and alum as placebo, six more injections of YIC or alum resulted in a decrease of the HBeAg seroconversion rate from 21.8% to 14.0% in the YIC group, but an increase from 9% to 21.9% in the alum group. Decrease in serum HBV DNA and normalization of liver function were similar in both groups (p>0.05). CONCLUSIONS: Overstimulation with YIC did not increase but decreased its efficacy due to immune fatigue in hosts. An appropriate immunization protocol should be explored and is crucial for therapeutic vaccination. Multiple injections of alum alone could have stimulated potent inflammatory and innate immune responses contributing to its therapeutic efficacy, and needs further investigation.

[Demethylation pattern of PD-1 gene in promoter region is associated with the PD-1 expression on peripheral blood mononuclear cells in chronic hepatitis B patients].

OBJECTIVE: To observe the expression variations and influencing factors of programmed death one (PD-1) and DNA demethylation of PD-1 promoter on peripheral blood mononuclear cells (PBMCs) in chronic hepatitis B (CHB) patients and further investigate the relationship between the demethylation pattern of PD-1 gene in promoter region and the PD-1 expression on PBMC in CHB patients. METHODS: A total of 162 subjects, including 144 CHB patients and 18 healthy blood donors, were enrolled. The expression of PD-1 on PBMCs was detected by flow cytometry. And the serum HBV markers, HBV DNA load and liver function were also measured. DNA of PBMCs was treated with sodium bisulfite; the PD-1 promoter fragments were amplified by polymerase chain reaction (PCR) and then transformed into Escherichia coli. Positive clones were selected for sequencing and the methylation status of fragments of PD-1 promoter was examined. RESULTS: With the PD-1 expression in normal controls (10.8% ± 4.4%) as a baseline level, the expression of PD-1 in CHB patients significantly increased. In CHB patients, the serum expression of PD-1 in PBMCs from patients with positive HBeAg (27.1% ± 18.4%) was much higher than that from those with negative HBeAg (19.6% ± 15.6%). And the expression level of PD-1 was not correlated with serum HBV DNA load and serum level of alanine aminotransferase. The results of bisulfite genomic sequencing showed that demethylation probability of some CG points in PD-1 promoter region (-601, -553, -538, -483, -463, -317 bp) were significantly correlated with PD-1 expression level (P < 0.05). CONCLUSION: The demethylation pattern of PD-1 gene in promoter region is associated with the PD-1 expression on PBMC in CHB patients.

[Dynamic changes in PD-1, TLR3, and TLR4 surface expression on peripheral blood mononuclear cells in chronic hepatitis C patients undergoing peg-IFNalpha-2a plus ribavirin combination therapy].

OBJECTIVE: To investigate the dynamic changes in expression of programmed death (PD)-1, Toll-like receptor (TLR)3, and TLR4 on the surface of peripheral blood mononuclear cells (PBMCs) in patients with chronic hepatitis C (CHC) that occur in response to pegylated-interferon alpha-2a (peg-IFNalpha-2a) plus ribavirin (RBV) combination therapy, and to analyze the relation to achievement of sustained virological response (SVR). METHODS Twenty-three CHC patients and 10 healthy controls were enrolled in the study. All CHC patients underwent 48 weeks of combination therapy with peg-IFNalpha-2a (180 microg, subcutaneous injection, once weekly) plus RBV (15 microg/kg, oral, once daily). Total PBMCs were isolated from both groups (CHC patients at treatment week 0, 12, 24, and 48 and post-treatment week 24; controls at enrollment) and subjected to flow cytometric analysis of PD-1, TLR3, and TLR4 surface expression. In addition, serum levels of alanine aminotransferase (ALT) and hepatitis C virus (HCV) RNA levels were analyzed by enzymatic assay and the AmpliPrep/COBAS (Roche) nucleic acid amplification test, respectively. SVR was defined as undetectable levels of HCV RNA at post-treatment week 24. Intergroup differences were assessed by one-way ANOVA. RESULTS: The expression ratios of PD-1, TLR4 and PD-1: TLR4 on PBMCs were significantly higher in CHC patients before therapy than in the healthy controls (45.20 +/- 7.12% vs. 16.82 +/- 4.13%, 58.45 +/- 15.13% vs. 21.09 +/- 2.89%, and 35.54 +/- 7.69% vs. 14.12 +/- 2.89%; all P < 0.05). In contrast, the expression ratios of TLR3 and PD-1:TLR3 were slightly, but not significantly, higher in CHC patients before therapy than in the healthy controls (P > 0.05). During the course of peg-IFNalpha-2a plus RBV combination therapy, the expression ratios of PD-1 and TLR4 on PBMCs showed a decreasing trend, while TLR3 expression showed an increasing trend. Furthermore, CHB patients who achieved SVR at post-treatment week 24 had a significantly different expression ratio of PD-1 and TLR3 than those who did not achieve SVR (P < 0.05). CONCLUSION: Surface expression of PD-1, TLR4, and PD-1:TLR4 is up-regulated in the total PBMCs of CHC patients. Peg-IFNalpha-2a plus RBV treatment-induced suppression of HCV replication results in a significant reduction in PD-1 and TLR4 expression on the surface of PBMCs, but a remarkably elevated level of TLR3 expression. The dynamic change in PD-1 and TLR3 expression on PBMCs that occurs during antiviral therapy may be related to achievement of SVR.

[Hepatitis C virus strain JFH1 down-regulates expression of growth arrest and DNA damage-inducible gene 45a in human hepatoma Huh7.5.1 cells].

OBJECTIVE: To investigate the effect of hepatitis C virus (HCV) strain JFH1 on expression of the human gene, growth arrest and DNA damage-inducible gene 45 alpha (GADD45a), in infected hepatoma cells. METHODS: HCV JFH1 RNA-containing supernatants were used to infect the human hepatoma cell line, Huh7.5.1; infection was confirmed by Western blot detection of the HCV-encoded non-structural 5A (NS5A) protein and core protein. Infection-induced changes in GADD45a mRNA and protein expressions were measured by real time PCR using SYBR Green and Western blotting, respectively. Significance of differences between the levels detected in JFH1-infected or uninfected Huh7.5.1 cells was analyzed by single factor analysis of variance testing. RESULTS: The HCV infection system was successfully established, as evidenced by expression of NS5A protein and core protein. The GADD45a mRNA and protein levels were significantly down-regulated in JFH1-infected Huh7.5.1 cells, by 0.57+/-0.09 and 0.28+/-0.03, respectively, as compared to levels in uninfected Huh7.5.1 cells (F values were 75.407 and 560.04, respectively; P less than 0.01). CONCLUSION: HCV inhibits the mRNA transcription and protein expression of host GADD45a, which may contribute to the pathogenesis of hepatocellular carcinoma caused by HCV infection.

[The therapeutic effect of Anluohuaxian capsule combined with adefovir dipivoxil on patients with chronic hepatitis B and influence on hepatic histology].

UNLABELLED: To observe the efficacy of adefovir dipivoxil(ADV) in combination with Anluohuaxian capsule in the treatment of chronic hepatitis B (CHB) patients. METHODS: 72 cases with CHB were randomly divided into two groups. 36 cases of treatment group were given ADV combined with Anluohuaxian capsule for 48 weeks. 36 cases of control group were given ADV. The levels of serum ALT, AST, Alb, TBil, HA, LN, CIV, HBV DNA and hepatic tissue were compared before and after being treated. RESULTS: After 48 weeks treatment,the liver function, serum fibrosis index and histology of treatment group and control group all have improved. After treatment, the two groups in the levels of ALT(t=0.746, P=0.342), AST (t=0.369, P=0.713), TBil (t=0.146, P=0.684), Alb(t=0.148, P=0.883), liver tissue inflammation mobility scoring (t=1.666, P=0.100) and HBV DNA negative rate (x2=0.141, P=0.708) were no evident difference.The level of HA, LN, CIV were significantly lower in treatment group(101.58+/-30.11, 147.89+/-41.72, 38.75+/-9.50) compared with control group(182.25+/-117.59, 181.50+/-56.96, 74.92+/-31.14) (P less than 0.05). After the treatment, the liver tissue fibrosis scoring was significantly lower in treatment group (10.61+/-2.37) compared with before the treatment (12.28+/-3.16) (P less than 0.05).There was no difference found between after the treatment (11.36+/-2.93) and before the treatment (12.17+/-3.01) in control group (P more than 0.05). CONCLUSIONS: The results show that the treatment with ADV in combination with Anluohuaxian capsule can play promoting antifibrotic effect and significant improved liver histology of chronic hepatitis B patients.

The oncogenic role of NS5A of hepatitis C virus is mediated by up-regulation of survivin gene expression in the hepatocellular cell through p53 and NF-κB pathways.

Approx. 4% of patients experiencing chronic infection of human HCV (hepatitis C virus) ultimately develop HCC (hepatocellular carcinoma). The NS5A (non-structural protein 5A) encoded by HCV has been reported to have an oncogenic role during HCV infection, but the precise mechanism remains largely unclear. The aim of this study is to investigate the signal transduction pathways that mediate the role of NS5A in hepatocarcinogenesis. HepG2 cells were transfected with a plasmid expressing HCV NS5A protein. Subsequently, cell proliferation was analysed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and cell counting, apoptosis was analysed by Hoechst 33342 staining, and the gene expression profile was identified by microarray and subsequently validated by RT-PCR (reverse transcription-PCR). The protein levels of survivin, p53, NOS2A (nitric oxide synthase 2A), cyclin D1 and NF-κB (nuclear factor κB) were monitored by Western blotting. Our results showed that transfection of HCV NS5A expression plasmid significantly down-regulated the expression of nine genes and up-regulated the expression of ten genes among the 104 genes detectable by the microarray associated with signalling transduction. The increased expression of survivin mRNA and protein, down-regulated p53 protein levels and increased NOS2A, cyclin D1 and NF-κB protein levels were further identified. Our results suggested that HCV NS5A protein can enhance survivin transcription by increasing p53 degradation and stimulating NOS2A expression as well as NF-κB relocation to the nucleus. The functions of survivin in anti-apoptosis and regulation of cell division might mediate the role of NS5A in HCV-induced HCC.

[HCV NS5A protein down-regulates hepcidin gene expression and increases hepatic intracellular iron storage].

OBJECTIVE: To investigate whether the nonstructural protein 5A (NS5A) encoded by the hepatitis C virus RNA genome affects the expression of hepcidin gene. METHODS: HCV NS5A expression plasmid (pCN5A) and pRc/CMV were transfected into QSG7701 cells individually, RT-PCR was employed to detect the HCV NS5A and hepcidin mRNA transcription. Western blot was used for detection of HCV NS5A and hepcidin proteins. Iron was stained to evaluate the intracellular iron level. RESULTS: HCV NS5A plasmid was successfully transfected into QSG7701 cells, which was evidenced by HCV NS5A mRNA and protein from the transfected cells. The hepcidin mRNA relative quantification in untransfected cells, pRc/CMV transfected cells and pCNS5A transfected cells were 0.711+/-0.049, 0.718+/-0.052 and 0.264+/-0.030 respectively. The transcription of hepcidin mRNA decreased remarkably in the cells transfected with pCNS5A plasmid as compared to the untransfected cells and pRc/CMV transfected cells (P less than 0.01). The level of hepcidin protein expression was found also significantly lower in the pCN5A plasmid transfected cells as compared to the untransfected cells and pRc/CMV transfected cells. The intracellular iron staining was remarkably higher in the pcNS5A transfected cells than untransfected or pRc/CMV transfected cells. CONCLUSIONS: HCV NS5A inhibits the transcription of hepcidin mRNA and expression of hepcidin protein, inducing hepatic intracellular iron storage.

Cellular context- and protein level-dependent interaction of pluripotency factor OCT4A with multiple octamer motifs of the same target gene.

AIMS: To compare how OCT4A proteins interact with and regulate multiple OCT4A-octamer motifs (OMs) in different regions of the FOS gene expressed in somatic cancer cells versus pluripotent stem cells. MATERIALS AND METHODS: Two FOS reporter gene systems harboring predicted OMs or their mutational counterparts were introduced into HeLa and NCCIT cells with varying OCT4A protein levels. The transcription of dsGFP reflecting FOS expression was quantitated by RT-qPCR, the OCT4A-OMs binding and the correlation between OCT4A and FOS transcription was determined by ChIP-PCR and RNA-Seq, respectively. KEY FINDINGS: In NCCIT cells, abundant OCT4A proteins bound to and inhibited OM1 and OM2 at the promoter of the FOS gene. RA-induced OCT4A down-regulation transiently increased FOS transcription. In contrast, in HeLa cells that contain much lower levels of endogenous OCT4A proteins, OCT4A primarily bound to and activate OM1 thereby promoting FOS transcription. OCT4A KO significantly reduced FOS expression. Ectopically introduced OCT4A, at its leaked or induced expression level, promoted FOS transcription by binding to OM2/OM3 or OM1/OM3, respectively. Thus, the interaction of OCT4A proteins with different OMs is cellular context- and protein level-dependent, and such complicated OCT4A binding mode can only be reflected by a dsGFP-based reporter harboring the full-length FOS gene but not by that merely having the FOS promoter. SIGNIFICANCE: Our findings unravel an additional layer of regulatory mechanisms that account for the cellular context- and dose-related versatile functions of OCT4A protein, and further underscore the importance of precise modulation of OCT4A in the regenerative medicine and anticancer therapies.

Short-term entecavir therapy of chronic severe hepatitis B.

BACKGROUND: Chronic severe hepatitis B patients often have limited survival. This investigation aimed to evaluate the short-term effects of nucleoside analog therapy on chronic severe hepatitis B. METHODS: We retrospectively, randomly collected the data of 129 chronic severe hepatitis B patients: 55 were treated with entecavir, and the remaining 74 were not treated with nucleoside analogues. RESULTS: No significant difference in short-term survival rate was found between the group treated with entecavir and that treated without nucleoside analogues. Although entecavir greatly reduced HBV replication in different periods of therapy (P<0.001), the model for end-stage liver disease (MELD) score and liver function (alanine aminotransferase, albumin, bilirubin, prothrombin time) showed no significant change. No significant differences were found in MELD scores and liver function in patients with different HBV DNA levels (< or =10(4) copies/ml, >10(4) to <10(6) copies/ml, > or =10(6) copies/ml). Nor correlation was observed between HBV DNA levels and MELD scores in different periods of therapy (P>0.05). The HBV DNA levels of patients who survived for over 3 months or less than 3 months were not significantly different either. However, the MELD score and parameters of liver function (albumin, bilirubin, prothrombin time) were different between the two groups (P<0.05). CONCLUSION: These results suggest that short-term suppression of HBV replication may not slow down the progression of liver failure in patients with chronic severe hepatitis B.

Cationic liposome-mediated transfection of CD40 ligand gene inhibits hepatic tumor growth of hepatocellular carcinoma in mice.

OBJECTIVE: To evaluate the efficacy of cationic liposome-mediated CD40 ligand (CD40L) gene therapy for hepatocellular carcinoma. METHODS: 1x10(6) of parental H22 cells or H22 cells transfected with the expression vector containing murine CD40L cDNA encoding the entire coding region (pcDNA3.1(+)-mCD40L) were inoculated subcutaneously into the left flanks of syngenic BALB/C mice. The tumor-bearing mice (tumor nodules 10 mm in maximal diameter) received the treatment of the intratumoral injection of pcDNA3.1(+)-mCD40L/Transfectam, pcDNA3.1(+), or phosphate-buffered saline (PBS), or no treatment. The mice were monitored for tumor growth weekly. We examined mCD40L messenger ribonucleic acid (mRNA) expression by reverse transcription polymerase chain reaction (RT-PCR) and the histologic changes in tumors at two weeks after intratumoral injection using immunohistochemical staining of tumor tissues. RESULTS: All mice inoculated with parental H22 cells developed a tumor subcutaneously, and the tumor size increased progressively within three weeks. However, the mice receiving H22-CD40L cells exhibited complete regression of the tumor two weeks after tumor cell inoculation. The tumor-bearing animals with the treatment of pcDNA3.1(+) or PBS, or without treatment had progressive tumor growth, while those mice treated with pcDNA3.1(+)-mCD40L exhibited a significant inhibition of tumor growth. RT-PCR analysis showed that 783-bp fragments corresponding to the mCD40L mRNA were amplified only from pcDNA3.1(+)-mCD40L treated tumors. The tumor samples from pcDNA3.1(+)-mCD40L-treated mice showed significant lymphocyte infiltration, apoptotic bodies, and confluent necrosis in the tumor tissues. CONCLUSION: The tumorigenicity of CD40L-expressing cells was abrogated when the cells were implanted subcutaneously. In vivo gene therapy of established liver tumor nodules in mice by the intratumoral injection of pcDNA3.1(+)-mCD40L led to significant tumor inhibition. There was mCD40L mRNA expression in the tissues from pcDNA3.1(+)-mCD40L-treated tumors. The intratumoral injection of pcDNA3.1(+)-mCD40L induced a strong inflammatory, mainly lymphocytic infiltration of the tumor, and increased the necrotic rate of the neoplastic cells.

[Effects of different mutated sites in vpr gene of HIV on apoptosis of host cells: experiment with HeLa cells].

OBJECTIVE: To investigate the effects of different mutated sites in the vpr gene of HIV on the apoptosis of host cells, and the possible mechanism thereof. METHODS: Fourteen HIV-1 vpr fragments were obtained from HIV-infected persons. Eukaryotic expression vector pcDNA3.1 (+) plasmid was extracted, the PCR purified product was double-cut by HindIII and BamH, and the cut products were ligated to vectors, thus establishing the JM109 competent cells. Sequencing was used to confirm the reconstruction of pcDNA-vpr eukaryotic expression vectors that were then transfected into HeLa cells. Blank vectors were transfected as control group. Cells were harvested after 24 hours and underwent Hoechst 33258 staining and observed under fluorescence microscope. Annexin-FITC-PI staining and flow cytometry were used to observe the percentage of apoptosis. The caspase-3 activity was detected by enzyme labeling instrument. RESULTS: The apoptotic rates shown by Hoechst and annexin--FITC-PI staining methods, and caspase-3 activity levels of the HeLa cells transfected with the gene fragments with mutated sites 70, 85, 86, and 94 cells were all lower than the cells transfected with the gene fragments without these mutated sites. The apoptosis causing ability levels of the No 1-7 recombinant plasmids (all of the Vpr AE subtype) were all lower than those of the No 8-14 plasmids (of Vpr B, AB, C, and C/BC subtypes). CONCLUSION: The apoptosis causing ability of the HIV with the vpr sequence with mutated sites 70, 85, 86, 94 is significantly lower than those without these sites. AE subtype induces lower apoptotic behavior in the hoist cells, and decreased activation of the caspase-3 pathway may be one of the mechanisms.

Response to immune complex vaccine in chronic hepatitis B patients is associated with lower baseline level of serum IgG galactosylation.

The composition of glycan in immunoglobulin G (IgG) has shown to affect various diseases and can be regulated by drugs and preventive vaccination. A hepatitis B surface antigen (HBsAg)-hepatitis B immunoglobulin (HBIG) immune complex (YIC) therapeutic vaccine for chronic hepatitis B (CHB) patients has undergone clinical trials. To explore for markers of CHB, which could be associated with responsiveness to YIC therapeutic vaccine, serum IgG glycosylation in CHB patients was analyzed.Kinetic changes of serum galactosylated IgG in 53 hepatitis Be antigen (HBeAg)-positive CHB patients treated with YIC were monitored by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) analysis. Whole blood cytokines were assayed by cytokine binding assay kits. All samples were back assayed before treatment, during therapy and follow-up for 6 months from a previous completed clinical trial.During YIC treatment, 26 patients with lower IgG galactosylation level at baseline [galactosylation level (Gal-ratio) = -0.29, 0.18 (mean, SD)] showed sustained increase of serum galactosylated IgG, and responded to YIC treatment by HBeAg seroconversion. While those who did not respond to YIC treatment [Gal-ratio = -0.40, 0.15 (mean, SD)] failed to show similar changes. Furthermore, this kinetic increase of galactosylated IgG correlated with marked up-regulated IL-2 level, confirming that effective cellular immune responses have participated in responsiveness.For HBeAg-positive CHB patients lower serum IgG galactosylation level may serve as an indicator for selecting a suitable subpopulation of candidates for YIC therapeutic vaccination.

Daily decrease of post-operative alpha-fetoprotein by 9% discriminates prognosis of HCC: A multicenter retrospective study.

BACKGROUND: Mixed evidence challenges preoperative alpha-fetoprotein (AFP) as an independent prognostic factor for patients with hepatocellular carcinoma (HCC) after hepatectomy. RESULTS: Daily post-operative decrease of AFP by 9% as compared to the preoperative level (A09) were selected as the Cut-off. The Kaplan-Meier curve showed that A09 was significantly different for OS (P=0.043) and RFS (P=0.03). A decrease in risk by 54% was observed for OS and 32% for RFS in the at-risk population (A09>9%). A better concordance was observed after adding A09 into TNM and BCLC staging systems. Moreover, a consistent concordance was observed in the internal (FDZS5:0.63; FDZS3:0.608) and external (FDZS5:0.85; FDZS3:0.762) validation cohorts, suggesting its prognostic value in HCC population with elevated AFP. CONCLUSIONS: Decrease in perioperative serum AFP rather than preoperative AFP is an independent prognostic factor for HCC patients after hepatectomy. Cut-off A09 significantly discriminates overall and recurrence-free survival and could be interpret into TNM and BCLC staging systems to improve the stratification power for HCC patients with elevated AFP. METHODS: Kaplan-Meier curve depicted the differences of overall survival (OS) and recurrence-free survival (RFS). Nomogram and concordance were employed to evaluate the superiority of the current staging system.

Hepatitis B virus-persistent infection and innate immunity defect: Cell-related or virus-related?

The outcomes of hepatitis B virus (HBV) infection are closely related to the age at which infection was acquired. Infection acquired in adult life tends to be self-limited, in contrast to perinatal acquirement, for which chronic persistence of the HBV is a general outcome. Innate immunity plays an indispensable role in early virus infection, facilitating virus clearance. However, it has been reported that HBV is under-recognized and poorly eliminated by the innate immune system in the early stages of infection, possibly explaining the long-lasting persistence of viremia afterwards. Furthermore, due to the existence of covalently closed circular DNA, chronic HBV clearance is very difficult, even when patients are given interferon-α and nucleotide/nucleoside analogs for antiviral therapy. The mechanism by which HBV evades innate immune recognition and establishes persistent infection remains a subject of debate. Besides, some researchers are becoming more interested in how to eradicate chronic HBV infection by restoring or boosting innate immunity. This review aimed to summarize the current knowledge on how intrahepatocyte signaling pathways and innate immune cells act after the onset of HBV infection and how these actions are related to the persistence of HBV. We anticipate the insights presented herein to be helpful for future development of novel immune therapeutic strategies to fight HBV infection.

CpAMs induce assembly of HBV capsids with altered electrophoresis mobility: Implications for mechanism of inhibiting pgRNA packaging.

Native agarose gel electrophoresis-based particle gel assay has been commonly used for examination of hepatitis B virus (HBV) capsid assembly and pregenomic RNA encapsidation in HBV replicating cells. Interestingly, treatment of cells with several chemotypes of HBV core protein allosteric modulators (CpAMs) induced the assembly of both empty and DNA-containing capsids with faster electrophoresis mobility. In an effort to determine the physical basis of CpAM-induced capsid mobility shift, we found that the surface charge, but not the size, of capsids is the primary determinant of electrophoresis mobility. Specifically, through alanine scanning mutagenesis analysis of twenty-seven charged amino acids in core protein assembly domain and hinge region, we showed that except for K7 and E8, substitution of glutamine acid (E) or aspartic acid (D) on the surface of capsids reduced their mobility, but substitution of lysine (K) or arginine (R) on the surface of capsids increased their mobility in variable degrees. However, alanine substitution of the charged amino acids that are not exposed on the surface of capsid did not apparently alter capsid mobility. Hence, CpAM-induced electrophoresis mobility shift of capsids may reflect the global alteration of capsid structure that changes the exposure and/or ionization of charged amino acid side chains of core protein. Our findings imply that CpAM inhibition of pgRNA encapsidation is possibly due to the assembly of structurally altered nucleocapsids. Practically, capsid electrophoresis mobility shift is a diagnostic marker of compounds that target core protein assembly and predicts sensitivity of HBV strains to specific CpAMs.

Hepatitis C virus non-structural 5A abrogates signal transducer and activator of transcription-1 nuclear translocation induced by IFN-alpha through dephosphorylation.

AIM: To study the effect of Hepatitis C virus non-structural 5A (HCV NS5A) on IFNalpha induced signal transducer and activator of transcription-1 (STAT1) phosphorylation and nuclear translocation. METHODS: Expression of STAT1 Tyr701 phosphorylation at different time points was confirmed by Western blot, and the time point when p-STAT1 expressed most, was taken as the IFN induction time for further studies. Immunocytochemistry was used to confirm the successful transient transfection of NS5A expression plasmid. Immunofluorescence was performed to observe if there was any difference in IFNalpha-induced STAT1 phosphorylation and nuclear translocation between HCV NS5A-expressed and non-HCV NS5A-expressed cells. Western blot was used to compare the phosphorylated STAT1 protein of the cells. RESULTS: Expression of HCV NS5A was found in the cytoplasm of P(CNS5A-transfected ) Huh7 cells, but not in the PRC/CMV transfected or non-transfected cells. STAT1 Tyr701 phosphorylation was found strongest in 30 min of IFN induction. STAT1 phosphorylation and nuclear import were much less in the presence of HCV NS5A protein in contrast to P( RC/CMV-transfected ) and non-transfected cells under fluorescent microscopy, which was further confirmed by Western blot. CONCLUSION: HCV NS5A expression plasmid is successfully transfected into Huh7 cells and HCV NS5A protein is expressed in the cytoplasm of the cells. IFN-alpha is able to induce STAT1 phosphorylation and nuclear translocation, and this effect is inhibited by HCV NS5A protein, which might be another possible resistance mechanism to interferon alpha therapy.