Up-regulated Reg proteins induced by Interleukin-22 treatment ameliorate acute liver injury in rat model.

BACKGROUND: The regenerating gene (Reg), encoding lectin-related protein, was originally isolated from a rat regenerating pancreatic islets. Interleukin-22 (IL-22), a recently identified cytokine, is produced by Th 17 cells and natural killer cells. Both of them have been shown to play an important role in controlling tissue repair. But, it is unclear whether the IL-22/Reg axis is involved in liver regeneration and the improvement of liver function in a rat model of acute liver injury. AIMS: We investigated the expression levels of Reg proteins after IL-22 stimulation in a rat model of acute liver injury, and estimated the effects of Reg proteins ameliorating acute liver injury. METHODS: Western blot was used to measure the expressions of Reg I, Reg III, Reg IV proteins after treatment with recombinant lentivirus IL-22. At the same time, the expression levels of TB, ALT, AST, endotoxin (ETM), superoxide dismutase (SOD), malondialdehyde (MDA) were detected by related reagents. RESULTS: In a rat model of acute liver injury, the expression levels of Reg I, Reg III, Reg IV proteins were increased after treatment with IL-22 recombinant lentivirus compared with treatment with lentivirus-empty vector, especially, Reg IV protein expression. Meanwhile, treatment with IL-22 recombinant lentivirus reduced serum levels of TB, ALT, AST, ETM, and decreased MAD levels in rat liver tissues, but increased SOD levels in rat liver tissues. CONCLUSION: IL-22 stimulation enhanced the expressions of Reg proteins in liver cell, especially, Reg IV protein, and ameliorated liver injury in a rat model of acute liver injury. Reg protein, especially Reg IV protein, might act as a biological mediator of immune cell-derived IL-22 in the recovering mechanism of liver injury.

Phenotypic assay of a hepatitis B virus strain carrying an rtS246T variant using a new strategy.

Phenotypic assays of hepatitis B virus (HBV) play an important role in research related to the problem of drug resistance that emerges during long-term nucleot(s)ide therapy in patients with chronic hepatitis B. Most of the phenotypic assay systems that are available currently rely on the transfection of recombinant replication-competent HBV DNA into hepatoma cell lines. Cloning clinical HBV isolates using conventional digestion-and-ligation techniques to generate replication-competent recombinants can be very difficult because of the sequence heterogeneity and unique structure of the HBV genome. In this study, a new strategy for constructing an HBV 1.1× recombinant was developed. The core of this strategy is the "fragment substitution reaction" (FSR). FSR allows PCR fragments to be cloned without digestion or ligation, providing a new tool for cloning fragments or genomes amplified from serum HBV DNA, and therefore making the assay of HBV phenotypes more convenient. Using this strategy, a phenotypic assay was performed on an HBV strain carrying an rtS246T variant isolated from a patient with chronic hepatitis B that was only responsive partially to entecavir therapy. The results indicated that this strain is sensitive to entecavir in vitro.

Comparison of a novel real-time PCR assay with sequence analysis, reverse hybridization, and multiplex PCR for hepatitis B virus type B and C genotyping.

We compared a novel real-time genotyping and quantitative PCR (GQ-PCR) assay, direct sequence analysis, reverse hybridization, and multiplex PCR for genotyping hepatitis B virus (HBV) in 127 HBV-infected patients. We found that GQ-PCR had the highest concordance with sequence analysis and the highest detection rate for mixed genotype detecting.

Simultaneous genotyping and quantification of hepatitis B virus for genotypes B and C by real-time PCR assay.

Hepatitis B virus (HBV) is an important cause of human chronic liver diseases and is a major public health problem. Viral load and HBV genotype play critical roles in determining clinical outcomes and response to antiviral treatment in hepatitis B patients. Viral genotype detection and quantification assays are currently in use with different levels of effectiveness. In this study, the performance of a real-time genotyping and quantitative PCR (GQ-PCR)-based assay was evaluated. Through the use of genotype-specific primers and probes, this assay provides simultaneous identification and quantification of genotypes B and C in a single reaction. Our GQ-PCR correctly identified all predefined genotypes B and C, and no cross-reaction between genotypes B and C were observed. The GQ-PCR identified more cases of HBV infections with mixed genotypes B and C than direct sequencing did. Samples from 127 HBV-infected Chinese patients were genotyped with GQ-PCR, revealing 56.7% HBV as genotype B, 13.4% as genotype C, and 29.8% as mixed genotypes B and C. This assay provides a reliable, efficient, and cost-effective means for quantification of the B and C genotypes of HBV in single or mixed infections. This assay is suitable for sequential monitoring of viral load levels and for determining the relationship between the genotype viral load and stage of disease in Asians.

[Dissection of mechanism for the adefovir dipivoxil resistance in chronic hepatitis B patients].

OBJECTIVE: To explore the mechanism for adefovir dipivoxil (ADV) resistance occurred in chronic hepatitis B patients of a series of phase III clinical trails. METHODS: 30 resistant HBV strains were selected out from 177 cases of ADV treated chronic hepatitis B patients. HBV polymerase RT region were amplified by nested PCR and analyzed with the standard nucleotide sequence of HBV strains deposited in GeneBank. RESULTS: 21 out of 30 HBV strains were primary resistant strains, among them 5 HBV strains (23.8%, 5/21) had the polymorphism site of rtN118H. While the other 9 HBV strains showed secondary resistance, variations in conservative region C (rtM207V) and other non-conservative regions were found. The classic mutation sites such as rtN236T and rtA181V/T were not found. CONCLUSIONS: Polymorphism site of rtN118H might be responsible for HBV primary resistance to ADV therapy. rtM207V variation in HBV RT C domain and other variation sites might play a role in HBV secondary resistance to ADV treatment, and natural resistant quasispecies may be the basis for the ADV quick resistance. These conclusions await further confirmation by phenotype test.

A new strategy for constructing in vitro replication-competent 1.3 copies of hepatitis B virus genome.

In the absence of a robust infectable cell culture system, assays related to replication of clinical HBV isolates are based on the transfection of replication-competent HBV DNA into hepatoma cell lines that are able to replicate and secrete HBV virions. Current methods for constructing HBV 1.1 genomes work well for drug susceptibility assays, but are not very suitable for research on HBV replication capacity or regulation since a heterogeneous promoter is required to drive pgRNA transcription. A new strategy for constructing HBV 1.3 genomes that contain HBV intrinsic promoter necessary for pgRNA transcription is reported in this paper. Using this strategy, three HBV 1.3 genomes from isolates of three patients were constructed. When the three HBV 1.3 genomes were transfected into the HepG2 cell line, replicative intermediates were detectable by Southern blotting with digoxigenin-labeled DNA probe in two of the three constructs. Using overlap extension PCR and avoiding as much as possible the digestion-and-ligation process, this strategy could be applied to constructing longer-than-genome units for most genotypes of HBV strains.

Dynamics of hepatitis B virus resistance to entecavir in a nucleoside/nucleotide-naïve patient.

The genotypic evolution of HBV quasi-species was analyzed in a nucleoside/nucleotide-naïve patient who developed resistance to entecavir. The lamivudine resistant quasi-species (rtM204V+/-rtL180M), absent at baseline, were emerged as early as 48 weeks after entecavir administration. Entecavir-resistant quasi-species (rtM204V+/-rtL180M plus S202G) were found after week 112 and gradually became the predominant mutations afterwards. The lamivudine- and entecavir-resistant mutations emerged closely in combination with the rtV207L, rtA222T, rtP237T or rtI163V substitutions. Our results indicated that the lamivudine-resistant mutations were developed first and may serve as a prequisite for subsequent entecavir-resistant mutations in this nucleoside/nucleotide-naïve patient.

Hepatitis B virus genotype-associated variability in antiviral response to adefovir dipivoxil therapy in Chinese Han population.

It is well known that different genotypes of hepatitis B virus (HBV) have a different sensitivity to interferon-alpha or lamivudine (nucleoside analogue) antiviral therapy. However, for adefovir dipivoxil (ADV, a nucleotide analogue), the antiviral response of the different genotypes remains to be clarified. In order to evaluate the response of HBV genotypes to ADV therapy and to identify factors that might affect initial virological response, we performed a retrospective analysis on patients with chronic hepatitis B (CHB) in Chinese Han population. The study included 183 patients, who had been tested positive for hepatitis B e antigen (HBeAg) and had been treated with ADV (10 mg/day) for 48 weeks. The numbers of patients infected with HBV genotype B and genotype C were 98 and 75 cases, respectively, and the remaining 10 patients were mixture infection of genotypes B plus C or genotypes B plus D. The mean HBV-DNA reduction and HBV-DNA seroclearance of genotypes B and C at 48 weeks were 3.6 log(10) and 3.1 log(10) copies/ml (p < 0.05) and 41.8% and 34.6% (p < 0.05), respectively. There were no statistically significant differences between genotypes B and C in terms of HBeAg loss, anti-HBe seroconversion and normalization of serum alanine aminotransferase (ALT). Multivariate analysis showed that young age, low pretreatment HBV-DNA and/or elevated ALT level might be independent predictive factors associated with initial virological response. Thus, in Han CHB patients who are HBeAg-positive, HBV genotype B shows a better virological response to ADV therapy than does genotype C.

[Comparison of antiviral responses to adefovir dipivoxil therapy of genotype B and genotype C HBV infected patients].

OBJECTIVE: To investigate the role of HBV genotypes on their response to adefovir dipivoxil (ADV) antiviral therapy. METHODS: HBV genotypes from 177 HBeAg-positive chronic hepatitis B (CHB) patients were identified and the patients were treated with ADV 10 mg per day for 48 weeks. The clinical data in terms of serum HBV DNA seroclearance, mean HBV DNA reduction (log value), HBeAg loss, anti-HBe seroconversion and serum ALT of those patients were analyzed against their HBV genotypes. RESULTS: Genotype B and genotype C were found in 102 and 65 cases, respectively. The mean HBV DNA reduction in patients with genotype B and genotype C at their treatment times of 12, 24 and 48 weeks was 2.2 log10copies/ml, 2.1 log10copies/ml (P more than 0.05), 2.7 log10copies/ml, 2.4 log10copies/ml (P more than 0.05) and 3.6 log10copies/ml, 3.1 log10copies/ml (P less than 0.05), respectively. At the end of the therapy (48 weeks), 43 (42.2%) patients with genotype B HBV infection and 22 (33.8%) patients with genotype C HBV infection had achieved HBV DNA seroclearance (P less than 0.05). CONCLUSIONS: Our results suggest that genotype B HBV has a better virological response to ADV therapy in HBeAg-positive chronic hepatitis B patients than that of genotype C. Longer terms of ADV treatment are needed to confirm this conclusion.

[Genotyping 238 HBV strains using type-specific primer PCR combined with type-specific nucleotide analysis].

OBJECTIVE: To establish a set of suitable and reliable methods for HBV genotyping and to study the distribution of HBV genotypes. METHODS: Type-specific nucleotides were searched through alignment of S genes (more than 1000 sequences) listed in GenBank. Then, type-specific primers were designed and type-specific primer PCR was used to genotype the 238 HBV strains. S genes of the untyped strains were further amplified and sequenced to find out their genotypes with type-specific nucleotide analysis. RESULTS: All the 238 HBV strains were genotyped. 159 (66.8%) cases were genotype B, 69 (28.9%) were genotype C, 6 (2.5%) were mixtures of genotypes B and C and 4 (1.6%) were mixtures of genotypes B and D. No genotypes of A, E, F, G, and H were found. CONCLUSION: Genotypes B and C are the most common types for HBV strains. Mixtures of genotypes B and C or genotypes B and D coinfection rarely existed. There is no relationship between the gender of the patients and HBV genotypes (X2 = 0.794, P more than 0.05).