Richeng Mao

Impact of nucleos(t)ide analogues on the estimated glomerular filtration rate in patients with chronic hepatitis B: a prospective cohort study in China.

Chronic hepatitis B therapy with nucleos(t)ide analogues, particularly tenofovir or adefovir, may affect renal function. To date, there has not been a head‐to‐head controlled study to assess estimated glomerular filtration rate (eGFR) fluctuations in nucleos(t)ide‐treated CHB patients. We aimed to evaluate the long‐term effects of nucleos(t)ide on eGFR in Chinese patients with chronic hepatitis B. This prospective cohort study included 275 patients. Patient subgroups included those treated with lamivudine (n = 50), adefovir (n = 60), telbivudine (n = 68) and entecavir (n = 61); untreated patients (n = 36) served as control. After an average follow‐up duration of 23 months, eGFR calculated by Cockcroft–Gault and Modification of Diet in Renal Disease formulas increased by 18.35 mL/min and 19.34 mL/min (P < 0.0001) in the telbivudine group, respectively, and decreased by 10.95 mL/min and 12.17 mL/min (P = 0.0001) in the adefovir group, respectively. Even if renal function was normal or mildly impaired at baseline, eGFR increased significantly more in the telbivudine group than in the other groups (P < 0.001). More patients in the adefovir group (23%) had a ≥20% decrease in eGFR than the other groups (P < 0.0001). More patients in the telbivudine group (31%) had a ≥20% increase in eGFR than the other groups (P < 0.0001). In conclusion, prolonged telbivudine therapy resulted in improved eGFR, while adefovir therapy was associated with decreased eGFR. Lamivudine and entecavir therapy did not significantly influence eGFR.

Transcription of Hepatitis B Virus Covalently Closed Circular DNA Is Regulated by CpG Methylation during Chronic Infection

The persistence of hepatitis B virus (HBV) infection is maintained by the nuclear viral covalently closed circular DNA (cccDNA), which serves as transcription template for viral mRNAs. Previous studies suggested that cccDNA contains methylation-prone CpG islands, and that the minichromosome structure of cccDNA is epigenetically regulated by DNA methylation. However, the regulatory effect of each CpG island methylation on cccDNA activity remains elusive. In the present study, we analyzed the distribution of CpG methylation within cccDNA in patient samples and investigated the impact of CpG island methylation on cccDNA-driven virus replication. Our study revealed the following observations: 1) Bisulfite sequencing of cccDNA from chronic hepatitis B patients indicated that CpG island I was seldom methylated, 2) CpG island II methylation was correlated to the low level of serum HBV DNA in patients, and in vitro methylation studies confirmed that CpG island II methylation markedly reduced cccDNA transcription and subsequent viral core DNA replication, 3) CpG island III methylation was associated with low serum HBsAg titers, and 4) Furthermore, we found that HBV genotype, HBeAg positivity, and patient age and liver fibrosis stage were also relevant to cccDNA CpG methylation status. Therefore, we clearly demonstrated that the status of cccDNA methylation is connected to the biological behavior of HBV. Taken together, our study provides a complete profile of CpG island methylation within HBV cccDNA and new insights for the function of CpG methylation in regulating HBV cccDNA transcription.

Comparative Analysis of CpG Islands among HBV Genotypes

DNA methylation is being increasingly recognized to play a role in regulation of hepatitis B virus (HBV) gene expression. The aim of this study was to compare the CpG island distribution among different HBV genotypes. We analyzed 176 full-length HBV genomic sequences obtained from the GenBank database, belonging to genotypes A through J, to identify the CpG islands in the HBV genomes. Our results showed that while 79 out of 176 sequences contained three conventional CpG islands (I–III) as previously described, 83 HBV sequences harbored only two of the three known islands. Novel CpG islands were identified in the remaining 14 HBV isolates and named as CpG island IV, V, and VI. Among the eight known HBV genotypes and two putative genotypes, while HBV genomes containing three CpG islands were predominant in genotypes A, B, D, E, and I; genotypes C, F, G, and H tended to contain only two CpG islands (II and III). In conclusion, the CpG islands, which are potential targets for DNA methylation mediated by the host functions, differ among HBV genotypes, and these genotype-specific differences in CpG island distribution could provide new insights into the understanding of epigenetic regulation of HBV gene expression and hepatitis B disease outcome.

Prevalence of basal core promoter and precore mutations in Chinese chronic hepatitis B patients and correlation with serum HBeAG titers

The A1762T and G1764A mutations in the basal core promoter (BCP) region and the G1896A mutation in the precore (PC) region of hepatitis B virus (HBV) genome are found commonly in HBeAg‐negative patients. Experiments in vitro suggest that BCP and PC mutation reduce and abolish HBeAg expression, respectively. In the present study, the prevalence of the BCP and PC mutations were determined in 207 patients with HBeAg positive chronic hepatitis B from China and correlated with the titers of serum HBeAg. None of the patients received antiviral therapy. The HBV genotype was determined by direct sequencing of the HBsAg gene. The BCP and PC mutations were detected by the polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) and confirmed by DNA sequencing. The HBeAg titer was measured by the microparticle enzyme immunoassay. Fifty‐one of the 207 patients (24.6%) were infected with genotype B and the remainder with genotype C. The BCP mutations were detected in 103 patients (50%) while the PC mutation was present in 43 (20.8%). Thirteen patients (6.3%) harbored both BCP and PC mutations. No significant difference in the titers of HBeAg was found between patients infected with the two HBV genotypes, but the presence of either the BCP or PC mutation was associated with reduced HBeAg titer (P < 0.05). The presence of both the BCP and PC mutations was accompanied by even lower HBeAg titer (P < 0.05). These findings confirm that in patients with HBeAg, the BCP and PC mutations reduced the expression of HBeAg. J. Med. Virol. 81:807–814, 2009.

Metformin inhibits hepatitis B virus protein production and replication in human hepatoma cells

Hepatitis B virus surface antigen (HBsAg) plays an important role in maintaining the tolerance and may interfere with host innate and adaptive immune responses; therefore, novel therapeutic strategies to reduce HBsAg loads in patients infected with hepatitis B virus (HBV) are emerging as an attractive but challenging issue. Metformin could regulate hepatic metabolism while the latter interacts with HBV infection. We hypothesized that metformin could affect HBsAg expression and HBV replication and may work synergistically when combined with current antivirals. In our study, a notably inhibitory effect on HBsAg production, as well as a moderate inhibition in HBV replication and HBeAg expression was observed following metformin treatment. The 50% effective concentration (EC50) for extracellular HBsAg and intracellular HBsAg in HBV‐producing HepG2.2.15 cells was 2.85 mm and 2.75 mm, respectively, with a similarly selective index of about 18. When administered in combination, metformin enhanced the inhibitory effects of interferon‐α2b on HBsAg expression and HBV replication and provided a complimentary role in HBsAg expression for lamivudine (LMV). This novel action of metformin derives partially from its inhibition on multiple HBV cis‐acting elements. By the virtues of preferably hepatocyte distribution and safety profile, collectively, our results suggest that metformin would be potentially clinically helpful as an HBsAg production inhibitor.

Serum microRNA-124 is a novel biomarker for liver necroinflammation in patients with chronic hepatitis B virus infection

Patients with chronic hepatitis B virus (HBV) infection and normal or mildly increased transaminases may have sustained significant liver damage, as verified by liver biopsy. However, no suitable noninvasive method exists for identifying liver necroinflammation in such patients. We aimed to investigate the power of microRNA‐124 as a novel biomarker for liver necroinflammation. A total of 131 recruited patients with chronic HBV infection underwent liver biopsy for grading of necroinflammation (G) and staging of fibrosis (S). Thirty healthy individuals were included as controls (HCs). Serum microRNA‐124 and microRNA‐122 levels were measured using qRT‐PCR. Forty‐five patients from the study population receiving entecavir therapy were monitored for changes in serum microRNA‐124 levels in association with improved liver histology. The capacity of serum microRNA‐124 levels in discriminating the grade of liver necroinflammation was compared with alanine aminotransferase (ALT) with liver biopsy validation. Serum microRNA‐124 levels were significantly higher in patients with chronic HBV infection than in HCs (P < 0.0001). Patients with considerable liver necroinflammation (G ≥ 2) had significantly higher serum miRNA‐124 levels than those without or with mild necroinflammation (P < 0.0001). After 48 weeks of antiviral therapy, serum microRNA‐124 levels considerably declined in 45 patients (P < 0.0001), which were associated with histological improvement. In patients with normal ALT and a serum HBV DNA load >104 copies/mL, receiver operating characteristic (ROC) curve of serum microRNA‐124 levels yielded an area under ROC curve (AUC) of 0.840, with 58.3% sensitivity and 91.7% specificity in discriminating between moderate‐to‐severe liver necroinflammation (G ≥ 2).

Impact of Nucleos(t)ide Analogue Combination Therapy on the Estimated Glomerular Filtration Rate in Patients With Chronic Hepatitis B

AbstractMonotherapy with telbivudine or adefovir can affect estimated the glomerular filtration rate (eGFR). However, only a few studies have assessed changes in eGFR in patients who have chronic hepatitis B (CHB) and are receiving nucleos(t)ide analogue (NA) combination therapy. In our study, we aimed to evaluate the effects of long-term NA combination therapy on eGFR in Chinese CHB patients.This retrospective study included 195 CHB patients. Patient subgroups included those treated with lamivudine plus adefovir (n = 73), telbivudine plus adefovir (n = 51), and entecavir plus adefovir (n = 35); untreated patients (n = 36) served as a control group.After an average follow-up duration of 24 months with combination therapy, analysis of changes in eGFR from baseline values, calculated by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) and Modification of Diet in Renal Disease (MDRD) formulas, showed decrease by11.08 and 18.34 mL/min (P < .001), respectively, in the lamivudine plus adefovir group; decrease by 3.73 and 10.04 mL/min (P = .012), respectively, in the entecavir plus adefovir group; and increase by 0.91 and 2.12 mL/min (P = .46), respectively, in the telbivudine plus adefovir group. The eGFR in the telbivudine plus adefovir group was similar to that for the untreated group. The eGFR decreases due to adefovir therapy could be rescued by adding telbivudine, and the eGFR increase due to telbivudine could be compromised by adding adefovir.Adefovir in combination with lamivudine or entecavir therapy was significantly associated with decreased eGFR, but telbivudine could rescue the eGFR decrease that results from adefovir treatment.

Detection of lamivudine- or adefovir-resistant hepatitis B virus mutations by a liquid array

A novel polymerase chain reaction (PCR)-Luminex assay was developed for rapid, accurate, and high-throughput detection of the most important hepatitis B virus (HBV) variants, including those with reverse transcriptase (RT) domain L180M, M204I/V, A181T/V/S, I233V and N236T mutations associated with resistance to lamivudine (LAM) or adefovir (ADV). Using mixtures of mutant and wild-type HBV, this method was sufficiently sensitive for detecting 10(3)HBV ml(-1) and could detect minor mutants when they comprised 5% of the total viral population. Comparison of the PCR-Luminex assay with INNO-LiPA for detecting clinical LAM- or ADV-resistant chronic hepatitis B virus infection in 64 patients confirmed the following: the 2 methods were 97.9% (48 of 49) and 93.3% (14 of 15) concordant for detecting LAM- or ADV-resistance mutations, respectively. The agreement with direct sequencing was 70.3% (45 of 64). The PCR-Luminex assay or multi-analyte suspension array can detect simultaneously and efficiently minor populations HBV mutants early during infection in many clinical samples. It is a simple, cost-effective method for resistance surveillance or selecting appropriate antiviral agents and initiating timely rescue treatment before the development drug-resistance related virus or biochemical breakthrough.

A comparison of telbivudine and entecavir in the treatment of hepatitis B e antigen-positive patients: a prospective cohort study in China

There are few studies directly comparing the efficacy and safety of telbivudine and entecavir. The present prospective cohort study aimed to evaluate the long-term efficacy and safety of these compounds in 196 hepatitis B e antigen (HBeAg)-positive patients with chronic hepatitis B for a median follow-up period of 172 weeks; 97 were treated with telbivudine and 99 were treated with entecavir. Patients showing suboptimal responses could also take adefovir at 24-48 weeks and all patients with viral breakthrough were started on adefovir. The 240-week cumulative proportions of patients showing undetectable hepatitis B DNA levels and serum alanine transaminase (ALT) normalization were similar in the two study groups. Viral breakthrough developed in 14% of the telbivudine group and in 2% of the entecavir group (p 0.002). Interestingly, the cumulative proportions of patients treated with entecavir and telbivudine showing HBeAg seroconversion were 12% versus 21% at 48 weeks (p 0.041), 15% versus 38% at 96 weeks (p 0.001), 24% versus 50% at 144 weeks (p 0.001), 33% versus 53% at 192 weeks (p 0.004) and 36% versus 53% at 240 weeks (p 0.005), respectively. Patients treated with telbivudine were therefore significantly more likely to show HBeAg seroconversion than those receiving entecavir and similar results were observed in study sub-groups matched for age, serum ALT, and HBV DNA levels. A safety analysis identified no differences between grade 3/4 creatine kinase elevations in the study groups and only telbivudine was associated with improved kidney function.

Preserved Function of Circulating Invariant Natural Killer T Cells in Patients With Chronic Hepatitis B Virus Infection

AbstractTo date, the role of invariant natural killer T (iNKT) cells in chronic hepatitis B virus (HBV) infection is not fully understood. In previous reports, iNKT cells were identified by indirect methods. However, discrepancies regarding the prevalence and function of iNKT cells during HBV infection were observed.In this study, we have devised a direct, highly specific CD1d tetramer-based methodology to test whether patients with HBV infection have associated iNKT-cell defects. In our study, a total of 93 chronic HBV-infected patients and 30 healthy individuals (as control) were enrolled. The prevalence of iNKT cells, their cytokine producing capacity, and in vitro expansion were determined by flow cytometric analysis with CD1d tetramer staining.Our observation demonstrated that there was no significant difference in circulating CD1d-tetramer positive iNKT cell numbers between HBV-infected patients and healthy controls. The capacity of iNKT cells to produce IFN-&ggr; or IL-4 as well as their in vitro expansion was also comparable between these 2 groups. However, among chronic HBV-infected patients, a decrease in iNKT cell-number was observed in chronic hepatitis B (CHB) and cirrhosis patients in comparison to that in immune tolerant (IT) patients.These results indicated that patients with chronic HBV infection may have normal prevalence and preserved function of circulating iNKT cells. And antiviral therapy with nucleot(s)ide analogue does not alter the frequency and function of circulating iNKT cells in chronic Hepatitis B patients.