Copies Of Hepatitis Research Articles

Sorafenib after resection improves the outcome of BCLC stage C hepatocellular carcinoma.

To evaluate whether sorafenib use after resection impacts tumor relapse and survival in Barcelona Clinic Liver Cancer (BCLC) stage C hepatocellular carcinoma (HCC). This retrospective study enrolled 36 male BCLC stage C HCC patients with portal vein thrombus and Child-Pugh class A liver function. Twenty-four patients received only surgical resection (SR), and 12 patients received oral sorafenib within 30 d after surgery. The primary outcomes were time to progression (TTP) (the time from surgical resection until HCC recurrence or extrahepatic metastases) and overall survival (OS). The secondary outcome was the rate of postoperative recurrence or metastasis. TTP and OS were analyzed using Kaplan Meier curves. There were no significant differences between the two groups in the serum levels of alpha-fetoprotein, copies of hepatitis B virus-DNA, preoperative laboratory results, degree of hepatic fibrosis, types of portal vein tumor thrombus, number of satellite lesions, tumor diameter, pathological results, volume of blood loss, volume of blood transfusion, or surgery time (all P > 0.05). Patients in the SR + sorafenib group had a significantly longer TTP (29 mo vs 22 mo, P = 0.041) and a significantly longer median OS (37 mo vs 30 mo, P = 0.01) compared to patients in the SR group. The SR group had 18 cases (75%) of recurrence/metastasis while the SR + sorafenib group had six cases (50%) of recurrence/metastasis. A total of 19 patients died after surgery (five in the SR + sorafenib group and 14 in the SR group). The most common sorafenib-related adverse events were skin reactions, diarrhea, and hypertension, all of which were resolved with treatment. Sorafenib after SR was well-tolerated. Patients who received sorafenib after SR had better outcomes compared to patients who received only SR.

Neferine isolated from Nelumbo nucifera enhances anti-cancer activities in Hep3B cells: Molecular mechanisms of cell cycle arrest, ER stress induced apoptosis and anti-angiogenic response

Hepatocellular carcinoma (HCC) is one of the most aggressive malignant diseases and is highly resistant to conventional chemotherapy. Neferine, a major bisbenzylisoquinoline alkaloid derived from the embryos of Nelumbo nucifera, has been reported a few physiological activities. However, the mechanisms of anticancer effects are not well understood and its detailed activities on Hep3B cells have not been determined. Our results suggest that neferine exhibited cytotoxicity against HCC Hep3B cells, but not against HCC Sk-Hep1 and THLE-3, a normal human liver cell line. In addition, consistent with the induction of G1/S phase cell population in flow cytometry, downregulation of c-Myc, cyclin D1, D3, CDK4, E2F-1, as well as dephosphorlyation of cdc2 by western blot analysis, as evidenced by the appearance of cell cycle arrest, were observed in Hep3B cells treated with neferine. Our results demonstrated neferine induced ER stress and apoptosis, acting through multiple signaling cascades by the activation of Bim, Bid, Bax, Bak, Puma, caspases-3, -6, -7, -8 and PARP, and the protein expression levels of Bip, calnexin, PDI, calpain-2 and caspase-12 were also upregulated dramatically by neferine treatment. Overexpression of GFP-LC3B by neferine resulted in a diffuse cytosolic GFP fluorescence and the strong fluorescent spots, representing autophagosomes. The significant reduction of the migration in Hep3B cells and the capillary tube-like formation of HUVECs by neferine were also determined. These observations reveal that the therapeutic potential of neferine in treating HCC Hep3B cells, containing copies of hepatitis B virus (HBV) genomes.

Transient occult hepatitis B virus infection in a blood donor with high viremia

Screening of blood donors for viral nucleic acids has recently been introduced in several countries. With the use of transcription-mediated amplification, a blood donor was detected who had 90,000 copies of hepatitis B virus (HBV) DNA/mL but no hepatitis B surface antigen (HBsAg) or antibody to hepatitis B core antigen (anti-HBc). One month later, anti-HBc and hepatitis B surface antibody (anti-HBs) appeared; HBV DNA disappeared after 2 months. This study asked why HBsAg was undetectable in this rare case of transient occult HBV infection. The HBV DNA in the first sample was cloned and sequenced to identify mutations. The physical nature of the virus was examined by polyethylene glycol precipitation, DNase digestion, density gradient centrifugation, and immunoprecipitation. Several mutations were found all over the genome, but the HBs antigen loop was unchanged. A stop mutation in the precore region led to loss of hepatitis B e antigen (HBeAg) expression. No HBV DNA–containing immune complexes were present. The plasma did not contain nonencapsidated HBV DNA that could explain the absence of HBsAg. The virus was immune precipitated by antibodies against HBsAg or preS1 antigen. The ratio of HBV to HBsAg subviral particles was estimated to be 1 in less than 20 whereas in overt cases the ratio is 1 in more than 1000. The acute resolving occult HBV infection was caused by an HBeAg-negative variant, which otherwise was almost normal. The negative HBsAg result was probably due to an unusually low production of surplus HBsAg. The absence of the viral immunomodulator HBeAg and the early appearance of anti-HBs suggested a rapid noncytolytic HBsAg-specific T-cell response leading to low expression of HBsAg.

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.

High level virion production and surface antigen expression with 1.5 copies of hepatitis B viral genome

The present study aimed to construct a 1.5X hepatitis B virus (HBV) replication system in vitro that could generate high level of HBV viruses. This system would help compare the replication capacity among the virus strains associated with high and low risk of hepatocellular carcinoma (HCC). Four strains of HBV were isolated from two HCC patients and two HBV carriers. After molecular cloning, four corresponding constructs named as HBV-1.5Xs were generated. Each of them has one and a half copies of HBV 3.2 kb genome, a 5′-end redundant sequence of 1.1 kb to nt715 and a 3′-end redundant sequence of 500 bp to nt2325 that situated after the poly (A) sequence. The HepG2 cells were transfected with the HBV-1.5Xs, and the levels of HBsAg, HBeAg and viral DNA were then detected in both the supernatant and the cells. After 24 h and 48 h of transfection, a high OD value of HBsAg of 3.5 was observed in the supernatant and also in some of the diluted cell lysate samples. The HBeAg level was relatively low in all strain samples of HBV. The log 10 values of viral loads were also determined with the cell lysate having a higher value (10–11 per ml) than that of the supernatant (6–7 per ml). The results showed that the novel HBV-1.5X system was capable to generate high level of HBV in a consistent manner. However, no significant difference was found among the replication capacities among these strains in vitro. The HBV-1.5X system may be a useful platform that assists the establishment of stable cell lines and transgenic mice for the investigation of viral pathogenesis, particularly for the various strains of HBV.

Treatment of chronic hepatitis C in Europe.

The lifetime cumulative risk of developing cirrhosis and hepatocellular carcinoma is the rationale for treating patients with chronic hepatitis C with antivirals. The standard treatment is combination therapy with interferon-alfa and ribavirin. In patients with high transaminases and histologic signs of chronic hepatitis, 6- to 12-month therapy with 3 mega units (MU) interferon-alfa thrice weekly, combined with ribavirin, yielded up to 30% sustained responders, and this was increased to 50% with pegylated interferon combined with ribavirin. Favorable predictors of response to the former treatment were genotype 2 or 3, less than 2 million copies of hepatitis C virus (HCV), no portal fibrosis at biopsy, age less than 40 years, and female sex. The same was true for the latter treatment; however, with body weight less than 82 kg replacing female sex. A 98% cure of community-acquired acute hepatitis C was achieved with early treatment with daily doses of 5 MU interferon, compared with a calculated 30% HCV-RNA clearance in untreated patients. More cost-effective strategies for ceasing treatment, based upon early clearance of HCV, are under investigation, with cutoff equal to or more than a 2 log decrease in serum HCV-RNA at week 12. This approach has 100% negative predictive value and 80% positive predictive value. Treatment can also be optimized by combination retreatment of relapsers and nonresponders to monotherapy, which yielded sustained responses of 50% and 25%, respectively. There are difficult-to-treat patients who have high viremia, genotype 1 and 4, or coinfection with HIV or HBV, or carry an organ graft, and those who did not respond to combination therapy. Extended treatment of the latter patients with pegylated interferon might slow down the progression of fibrosis.

Failure to detect hepatitis C virus genome in human secretions with the polymerase chain reaction.

Although hepatitis C infection has been clearly demonstrated to be transmitted through blood products or blood contamination, most cases of sporadic hepatitis C infection are unassociated with parenteral risk factors, and it is unclear how infection might be acquired by nonparenteral means. One potential mode of nonparenteral transmission is through body secretions. We used a highly sensitive and specific polymerase chain reaction assay to determine whether hepatitis C viral genomic RNA could be detected in secretions obtained from nineteen individuals with chronic hepatitis C virus infection. Although hepatitis C genomic RNA was found in all 19 sera, hepatitis C virus RNA was not detected in any samples of saliva, semen, urine, stool or vaginal secretions from these patients. Viral titers in serum ranged from 10(2) to 10(7) polymerase chain reaction units/ml. The sensitivity of our polymerase chain reaction assay indicates that, if hepatitis C virus were in secretions, it would be present in amounts less than 1 to 4 polymerase chain reaction units/ml. This contrasts with hepatitis B virus infection, in which serum titers frequently are in excess of 10(9) copies of hepatitis B genomes/ml. Body secretions have been found to contain up to 10(6) copies of hepatitis B genomes/ml. Our findings support seroepidemiological studies indicating that nonparenteral transmission of hepatitis C through secretions is uncommon and probably much less efficient than hepatitis B virus infection.

HBV production in transgenic mice

We produced transgenic mice by microinjecting a partially duplicated copies of hepatitis B virus (HBV) gene into fertilized eggs of C57BL/6 mice. One mouse was a high producer of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in the serum. All offspring carrying HBV DNA were positive for both antigens in the serum. The HBV DNA was expressed in liver- and kidney-specific manner. The normal process of HBV replication, including the packaging of the pregenome 3.5-kb RNA into a nucleocapsid, the reverse-transcription of the complete minus strand DNA, and the release of Dane particles into the serum before the completion of synthesis of plus strand, occurred in the liver of these transgenic mice. These results suggest that the species specificity of HBV infection is not due to the inability to replicate in nonnatural host but to the lack of receptors or factors needed for virus adsorption and internalization. The founder mouse is now 19 months of age but shows no clinical or pathological change, suggesting that HBV itself is not cytopathic.

A yeast system for stable expression of hepatitis B surface antigen

We have constructed a yeast strain that has integrated into its chromosomal ribosomal RNA gene site two copies of Hepatitis B virus surface (HBS) antigen gene under the control of the yeast ( Saccharomyces cerevisiae) glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and terminator. The level of expression of HBS gene was low in the strain, but upon chemical and physical mutageneses, in combination with an immunological screening procedure, a mutant clone which expressed HBS protein at a high level was obtained. This mutant strain produces HBS antigen stably under non-selective conditions.

Transgenic mice containing hepatitis B virus sequences are more susceptible to carcinogen-induced hepatocarcinogenesis

Transgenic mice containing one copy of hepatitis B virus (HBV) genome without the core gene and expressing hepatitis B surface antigen (HBsAg) were crossed with C3H/He mice. The F1 hybrids (approximately 50% HBV positive and approximately 50% HBV negative) were treated with a single dose of diethylnitrosamine (NDEA) or p-dimethylaminoazobenzene (DAB) given at 7 days of age, or were untreated. Mice were kept under observation without further treatments until 30 weeks old and then killed. Stereological analysis of liver nodules and estimations of their size distribution demonstrated a significative enhancing effect of HBV transgene in both NDEA- and DAB-induced hepatocarcinogenesis in male mice. Hepatocellular adenomas and carcinomas were also more frequent in NDEA-treated HBV-positive than HBV-negative male mice. Female mice showed a lower tumorigenic response than males without significant differences between groups of HBV-positive and HBV-negative mice. It is proposed that the presence of the transgene enhanced carcinogen-induced hepatocarcinogenesis.

Hybrid hepatitis B virus-host transcripts in a human hepatoma cell.

The human PLC/PRF/5 hepatoma cell line (the Alexander cell) contains at least seven copies of hepatitis B virus (HBV) DNA integrated in its genome; but it selectively expresses the HBV surface antigen (HBsAg) gene and perhaps low levels of the core gene. We have prepared a cDNA library from PLC/PRF/5 cell poly(A)+ RNA and isolated clones containing HBV sequences. Hybridization experiments show that the great majority of HBV-specific RNAs in this cell line contain HBsAg coding sequences and are presumably derived from the HBsAg gene. Primer extension experiments show that these HBsAg mRNAs are, however, derived from multiple initiation sites in the HBsAg gene and involve two promoters: one at the 5' end of the gene that can produce a protein of 45 kDa, and one located in the pre-S region that can produce two proteins of 31 kDa and the mature HBsAg, 25 kDa, respectively. The HBV RNAs are hybrid RNA species that contain HBV sequences at their 5' ends and host DNA sequences at the 3' ends. The great majority of these hybrid RNAs are transcribed from two closely related yet distinct HBV integrants. The viral-host sequences of these two related hybrid RNAs suggest that the related HBV sequences were generated from a parental fragment via duplication, translocation, and mutagenesis. These processes may play a role in HBV-related oncogenesis.