Increased expression of transforming growth factor alpha after transfection of a human hepatoblastoma cell line with the hepatitis B virus.

The expression of transforming growth factor alpha (TGF-alpha) was examined in a human hepatoblastoma cell line, Hep G2, which does not contain hepatitis B virus (HBV) DNA, and in the cell line 2.2.15, which was formed by the transfection of Hep G2 cells with the complete HBV DNA, to study the possibility that HBV and TGF-alpha could function as co-factors in hepatocarcinogenesis. Northern blot hybridization of RNA extracted from these cell lines, with densitometric analysis, revealed expression of the TGF-alpha gene in the transfected cells at a level three times higher than in the nontransfected cells. Staining of the cells using a monoclonal antibody to TGF-alpha and the avidin-biotin-peroxidase immunohistochemical method revealed a much higher intensity of TGF-alpha staining in the transfected cell line. These findings show that the presence of HBV DNA appears to cause a significant up-regulation of the TGF-alpha gene. This effect on the TGF-alpha gene may be a mechanism by which HBV contributes to the etiology of hepatocellular carcinoma in some patients.     

Ultrastructural demonstration of hepatitis B virus production in a mouse model produced by hydrodynamic transfection

A mouse model of hepatitis B virus (HBV) infection produced by hydrodynamic injection of HBV DNA has been recently established. However, the ultrastructural demonstration of HBV particles in this mouse model has not as yet been reported. In our study, plasmid DNA containing wild-type HBV DNA was rapidly injected into 8-week-old female SCID mice via the tail vein. Serum levels of HBsAg were measured by ELISA kit. Intrahepatic HBV protein expression was detected by immunohistochemistry of HBcAg. Ultrastructural study of the serum samples was performed by transmission electron microscopy and immunogold electron microscopy. Serum HBsAg and intrahepatic HBcAg were detected in HBV DNA-injected mice for at least 14 days. Spherical and filamentous particles 22 nm in diameter and double-shelled Dane-like particles 42 nm in diameter were detected in the sera of mice. The ultrastructural features of these particles were identical to HBV particles observed in serum from chronic hepatitis B patients. These particles were confirmed to be HBV particles by immunogold electron microscopy. We conclude that our present HBV mouse model using hydrodynamic transfection of HBV DNA is appropriate for production of HBV virions including Dane particles. This mouse model may be useful for screening in vivo the efficacy of antiviral drugs.     

Serum hepatitis B virus DNA in acute hepatitis B

With the use of the dot blot hybridization technic, sera from 30 consecutive patients with acute hepatitis B were examined for the presence of hepatitis B virus (HBV)-DNA. In an additional five patients with acute hepatitis B, serial serum samples, beginning before the serum alanine amino transferase elevation to the clearance of hepatitis B surface antigen, also were tested for various hepatitis B virus markers, including HBV-DNA. Thirteen of the 30 patients (43%) had circulating HBV-DNA and HBeAg at the time of their first clinic visit. However, 11 additional patients with HBeAg did not have HBV-DNA in their sera. In the 13 patients with HBV-DNA, there was no correlation between the titers of HBeAg and the levels of HBV-DNA. Examination of the serial serum samples from the additional five patients showed HBV-DNA and HBeAg to be present several days before the peak of serum alanine amino transferase. In all five patients, HBV-DNA was present in the serum before the appearance of IgM anti-HBc. However, HBsAg was present in all these patients' sera at the time of HBV-DNA positivity. None of the patients in this study became chronic carriers of hepatitis B virus.     

Instability of integrated hepatitis B virus DNA with inverted repeat structure in a transgenic mouse

We established four cell lines, from the liver cells of a transgenic mouse, constructed with hepatitis B virus DNA that had an inverted repeat structure. The integrated DNA patterns of the four established cell lines were different from one another and from the original pattern. These data show that the instability of integrated hepatitis B virus DNA would also occur in somatic cells during replication, apart from meiosis, which was previously reported.     

Detection of hepatitis B virus DNA in sera from patients with chronic hepatitis B virus infection by DNA microarray method

We have developed a sensitive and quantitative assay using a DNA microarray for the detection of hepatitis B virus (HBV) DNA in serum. Fluorescently labeled target cDNA prepared from cloned HBV DNA or serum HBV DNA was hybridized to capture DNA on a slide. A linear relationship was obtained between the intensities of the array spot and the amount of the cloned or serum HBV DNA, indicating the quantitative accuracy of this assay system. In addition, there was a significant correlation between the number of molecules of serum HBV DNA determined by the DNA microarray and that determined by a branched-DNA assay (n = 21, r = 0.89). Given these results, we conclude that the DNA microarray assay system may be useful as a diagnostic technique in the clinical laboratory.     

Leucocyte hepatitis B virus DNA in acute and chronic hepatitis B virus infection

In the present study we have investigated 53 patients with a spectrum of acute and chronic hepatitis B virus (HBV) infection for the presence of leucocyte HBV-DNA with the aid of molecular techniques. HBV-DNA was detected in peripheral blood mononuclear cells of 31 of 45 (69%) of chronic HBsAg carriers and 2 of 8 (25%) patients with acute hepatitis B. Although HBV-DNA was detected more frequently in leucocytes from those HBsAg carriers seropositive for HBeAg (79%), 50% of those with anti-HBe in serum had leucocytes positive for HBV-DNA independent of the presence of serum HBV-DNA. Examination of various leucocyte subpopulations showed the presence of HBV-DNA in polymorphonuclear leucocytes as well as T- and non-T-enriched mononuclear cell fractions. The HBV-DNA identified was predominantly 3.2-kilobase (kb), while higher molecular weight sequences were rarely detected, and lower molecular weight sequences indicative of active viral replication were not observed. These data indicate that although leucocytes do not actively support viral replication, they frequently harbour 3.2-kb HBV-DNA and may act as a reservoir for infection and, more importantly, since leucocytes contaminate several body secretions, may be involved in virus transmission.     

Serum hepatitis B virus DNA in hepatitis B virus seropositive and seronegative patients with normal liver function

The presence of hepatitis type B virus (HBV) DNA in serum specimens from 926 apparently healthy people with normal liver functions was determined by polymerase chain reaction; 41.2% of people with positive results for HBV surface antigen (HBsAg) (94 of 228) and 95.2% of people with positive results for HBV e antigen (HBeAg) (60 of 63) were found to have positive results for serum HBV DNA. On the other hand, serum HBV DNA was found in 11.0% (77 of 698) of HBsAg-negative people and in 13% (69 of 530) of those who had positive results for serum antibodies directed against HBsAg. The results seem to suggest that HBV DNA can be found in a significant portion of apparently healthy people with normal liver function who are either seronegative for HBsAg or seropositive for antibodies directed against HBsAg.     

State of hepatitis B virus DNA in leucocytes of hepatitis B patients

Hepatitis B virus (HBV) DNA in leucocytes from 50 hepatitis patients with various patterns of HBV serological markers and serum HBV DNA and 13 normal controls were examined by Southern blot hybridization with 32P-labeled 3.2 Kb HBV DNA. A free form of HBV DNA was observed in leucocytes of 8 patients, 7 of whom were positive for serum HBeAg, and in 6 patients an integrated form of HBV DNA was identified. HBV DNA was not identified in leucocytes from 13 normal controls. The free form of HBV DNA in leucocytes existed as a heterogeneous smear from 2.0 to 3.2 Kb, similar to the pattern in liver and hepatocellular carcinoma cells but different from serum HBV DNA in which the 3.2 Kb band was absent. The banding pattern of the integrated form of HBV DNA in leucocytes varied among different patients. During preparation of white blood cells and purification of HBV DNA probes, it was important to remove plasma contamination and traces of pBR322, respectively. The presence of extrachromosomal DNA sequences partially homologous to pBR322 could cause false results. The presence of a free and integrated form of HBV DNA in leucocytes is important for explaining the biology of HBV, the harbouring and replication sites of extrahepatic origin, the mechanism of recurrent infection, and the rationale of the treatment of hepatitis B.     

False-positive results with hepatitis B virus DNA dot-hybridization in hepatitis B surface antigen-negative specimens.

Three serum samples derived from healthy hepatitis B surface antigen-negative subjects were found to be reactive for hepatitis B virus (HBV) DNA sequences when assayed by DNA dot-hybridization. All three results were shown to be due to the presence of sequences which reacted with residual bacterial plasmid vector sequences in the DNA probe, and no evidence of HBV markers was demonstrated in the sera. This is the first report of a false-positive result with the HBV DNA dot-hybridization assay.     

Patients with and without loss of hepatitis B virus DNA after hepatitis B e antigen seroconversion have different virological characteristics

The characteristic differences between patients with and without loss of hepatitis B virus (HBV) DNA after achieving hepatitis B e antigen seroconversion were analyzed by comparing changes in HBV DNA and HBV core-related antigen levels during a period from 3 years before to 3 years after the seroconversion. Of the 24 seroconverters, 6 (inactive replication group) showed continuous loss of HBV DNA in serum after the seroconversion and the remaining 18 did not lose HBV DNA (active replication group). The HBV DNA level was similar between the two groups, while the HBV core-related antigen level was significantly lower in the active replication group than in the inactive replication group before the seroconversion. The levels of both HBV DNA and HBV core-related antigen decreased remarkably around the time of seroconversion in the inactive replication group, while these levels did not change or decreased slightly in the active replication group. After the seroconversion, the HBV DNA level was significantly higher in the active replication group than in the inactive replication group, while the HBV core-related antigen level was similarly low between the two groups. Because the serum level of HBV core-related antigen mainly reflects that of HBe antigen, the low level of HBV core-related antigen seen after seroconversion in both groups might have contributed to the occurrence of seroconversion. The precore and core promoter mutations which cause diminished excretion of hepatitis B e antigen were significantly more frequent in the active replication group than in the inactive replication group. It was therefore considered that the seroconversion was caused mainly by a decrease in viral replication in the inactive replication group, and mainly by a decrease in HBe antigen production in the active replication group.     

乙型肝炎病毒感染者血清HBV DNA水平与HDL-C的相关性

目的分析乙型肝炎病毒（HBV）感染者血清HBV DNA和高密度脂蛋白胆固醇（HDL-C）水平,探讨血清HBV DNA拷贝数与HDL-C水平的相关性。方法收集116例HBV感染者血清,通过实时荧光定量PCR法测定血清HBV DNA拷贝数,通过选择性抑制均相测定法分析血清HDL-C值;计算HBVDNA拷贝数与HDL-C的相关系数,并对相关系数进行显著性检验。结果在乙型肝炎病毒感染者中,血清HBV DNA拷贝数对数均值为（4.18±1.77）/ml,分布范围为（1.38～7.85）/ml;血清HDL-C浓度均值为（1.30±0.29）mmol/L,分布范围为（0.66～2.01）mmol/L。血清HBVDNA拷贝数与HDL-C水平存在负相关（r=-0.5346,P=0.0023）。结论 HDL-C对乙型肝炎病毒的复制有抑制作用。     

Persistence of intrahepatic hepatitis B virus DNA integration in patients developing hepatocellular carcinoma after hepatitis B surface antigen seroclearance

Background/AimsThe role of hepatitis B virus (HBV) integration into the host genome in hepatocarcinogenesis following hepatitis B surface antigen (HBsAg) seroclearance remains unknown. Our study aimed to investigate and characterize HBV integration events in chronic hepatitis B (CHB) patients who developed hepatocellular carcinoma (HCC) after HBsAg seroclearance.MethodsUsing probe-based HBV capturing followed by next-generation sequencing technology, HBV integration was examined in 10 samples (seven tumors and three non-tumor tissues) from seven chronic carriers who developed HCC after HBsAg loss. Genomic locations and patterns of HBV integration were investigated.ResultsHBV integration was observed in six patients (85.7%) and eight (80.0%) of 10 tested samples. HBV integration breakpoints were detected in all of the non-tumor (3/3, 100%) and five of the seven (71.4%) tumor samples, with an average number of breakpoints of 4.00 and 2.43, respectively. Despite the lower total number of tumoral integration breakpoints, HBV integration sites in the tumors were more enriched within the genic area. In contrast, non-tumor tissues more often showed intergenic integration. Regarding functions of the affected genes, tumoral genes with HBV integration were mostly associated with carcinogenesis. At enrollment, patients who did not remain under regular HCC surveillance after HBsAg seroclearance had a large HCC, while those on regular surveillance had a small HCC.ConclusionsThe biological functions of HBV integration are almost comparable between HBsAg-positive and HBsAg-serocleared HCCs, with continuing pro-oncogenic effects of HBV integration. Thus, ongoing HCC surveillance and clinical management should continue even after HBsAg seroclearance in patients with CHB.     

Detection of hepatitis B virus DNA in serum and relation with the IgM class anti-HBc titers in hepatitis B virus infection

Sera from four groups of patients wtih different serologic markers of HBV infection were examined for HBV DNA using molecular hybridization technique and for IgM class anti-HBc using an ELISA based on the antibody capture principle. Results of HBV DNA assay were generally in good agreement with the presence of HBeAg. However, HBV DNA was found in 13% of anti-HBe+ sera and in one patient with anti-HBc as a sole marker. IgM anti-HBc was detected at high titers in acute hepatitis B patients and was also present during the "window-period." This marker was also found, though less frequently when other markers for HBV infectivity were absent, in chronic hepatitis B patients and healthy carriers. From these findings we conclude that the HBV DNA assay provides a reliable method of detecting the infectious agent, particularly in anti-HBe+ sera and sera with anti-HBc as a sole marker. The assay for IgM anti-HBc is useful for establishing the diagnosis of recent infection in patient with anti-HBc as a sole marker, and during acute hepatitis with very high aminotransferase values, a condition in which HBV DNA may be undetectable.     

Increased liver pathology in hepatitis C virus transgenic mice expressing the hepatitis B virus X protein

Transgenic mice expressing the full-length HCV coding sequence were crossed with mice that express the HBV X gene-encoded regulatory protein HBx (ATX mice) to test the hypothesis that HBx expression accelerates HCV-induced liver pathogenesis. At 16 months (mo) of age, hepatocellular carcinoma was identified in 21% of HCV/ATX mice, but in none of the single transgenic animals. Analysis of 8-mo animals revealed that, relative to HCV/WT mice, HCV/ATX mice had more severe steatosis, greater liver-to-body weight ratios, and a significant increase in the percentage of hepatocytes staining for proliferating cell nuclear antigen. Furthermore, primary hepatocytes from HCV, ATX, and HCV/ATX transgenic mice were more resistant to fas-mediated apoptosis than hepatocytes from nontransgenic littermates. These results indicate that HBx expression contributes to increased liver pathogenesis in HCV transgenic mice by a mechanism that involves an imbalance in hepatocyte death and regeneration within the context of severe steatosis.     

Clonal growth of Blastocystis hominis in soft agar with sodium thioglycollate

 The present report describes a method for establishment of colonies of Blastocystis hominis from single cells in soft agar. The percentage of colony-forming efficiency (% CFE = number of colonies grown / number of cells inoculated × 100) for the cultures was greatly improved by the addition of sodium thioglycollate. Five human Blastocystis isolates chosen for this study showed no apparent variation in colonial morphology. Isolated colonies were also successfully grown in liquid medium, providing a means of obtaining large numbers of B. hominis cells that had arisen from a single clone. Received: 12 January 1996 / Accepted: 14 May 1996     

Hepatitis B virus DNA in leukocytes of patients with hepatitis B virus-associated liver diseases

In order to determine the relationship between hepatitis B virus (HBV) infection of human white blood cells and different forms of HBV-associated liver diseases, we tested for HBV DNA in the sera and leukocytes of 11 healthy individuals without any serological markers of HBV infection and 91 patients with HBV infection and other gastrointestinal and urinary diseases by dot and Southern blot hybridization. HBV DNA was found in leukocytes of chronic HBV carriers, in acute and chronic hepatitis, and in patients with liver cirrhosis and hepatocellular carcinoma. Between 27 and 50% of individuals in different categories of patients examined were positive for leukocyte HBV DNA. HBV DNA was also detected in the sera of some of these patients but was absent in others. Serum HBV DNA-positive rates seemed to be highest in hepatitis B e antigen-positive asymptomatic carriers (8/10, 80%), and tended to drop to lower levels as the disease progressed to liver cirrhosis (0/8) while leukocyte HBV DNA-positive rates were highest in patients with cirrhosis (4/8, 50%). The results also show that in individuals who were serologically negative for hepatitis B surface antigen (HBsAg) and positive for antibodies to HBsAg and/or HBcAg, HBV DNA was absent in most of the sera (27/28, 96%) but it was present in leukocytes of some of these patients (7/28, 25%). In control experiments with 11 healthy individual, HBV DNA was not detected in either sera or leukocytes. In all the cases with leukocyte HBV DNA, the HBV DNA molecules were present in free forms with discrete sizes. The exceptions were a case of liver cirrhosis and a case of chronic hepatitis with possible HBV sequence integration into high molecular weight cellular DNA. Since HBV does infect human leukocytes, it may perhaps interfere with the immunological functions of the white blood cells, and thus play an important role in the pathogenesis of HBV-induced liver disease.     

Hepatitis B virus DNA patterns in the liver of children with chronic hepatitis B

The pattern of hepatitis B virus DNA (HBV-DNA) expression were studied in 2 sequential liver biopsies from 26 children (18 treated with interferon and 8 controls) with chronic hepatitis B. In the basal biopsy replicative forms of HBV-DNA were detected in all of the samples and integrated viral DNA was present in 1 case. At the end of the study, 8 children had lost serum HBV-DNA although 2 of the children were still HBeAg positive. (Six had been treated with interferon.) In all of the cases, HBV-DNA was not detectable in the final biopsy. For the rest of the patients, HBV-DNA was positive in serum and all of them had replicative forms of HBV-DNA in the second liver sample. None of the patients lost hepatitis B surface antigen (HBsAg). Peripheral blood mononuclear cells (PBMC) from these patients were studied. HBV-DNA was not found in the PBMC of the 8 children without serum HBV-DNA, and HBV-DNA was detected in the PBMC of 5/12 patients with serum HBV-DNA. In conclusion, HBV-DNA disappeared from the biopsies of children who lost circulating HBV-DNA, although some of the patients were still HBeAg positive. This result implies that the detection of HBV-DNA in liver is important in order to assess the efficacy of the antiviral therapy. On the other hand, HBsAG remained positive in all children at the end of the study although HBV-DNA was not detected in serum, liver, and PBMC by the conventional hybridization techniques.     

Antigenicity and immunogenicity of hepatitis B virus particles produced by mouse cells transfected with cloned viral DNA

Antigenicity and immunogenicity of the hepatitis B surface antigen (HBsAg) 22-nm particles produced by mouse cells transfected with HBV DNA were studied. Both the a group and the y subtype determinants were present on the particles. Injected into mice the particles induced formation of anti-HBs. An antibody titer of 400 IU/mP, 7 weeks after the primary injection was obtained in one experiment. The affinity constant of these antibodies to human HBsAg particles was about 1 × 10^8? mol/liter. The anti-HBs antibodies react specifically with both the a group and the y subtype HBV determinants. These results show that these particles have antigenic and immunogenic properties similar to those of the 22-nm particles present in human serum.     

Detection of hepatitis B virus DNA in hepatocellular carcinoma

DNA-DNA hybridization was used to examine tissue from 31 patients with hepatocellular carcinoma (HCC) in Taiwan for the presence of the hepatitis B virus genome. Twenty-four of the DNA samples had discrete high molecular weight bands when cut with HindIII and hybridized with a radiolabelled HBV DNA probe, and similar results were obtained with 25 of the samples when cut with EcoRI. Thus integrated HBV DNA was present in almost 80% of the specimens. None of the samples had a similar pattern and most contained HBV DNA integrated at multiple sites. Seventeen out of 19 HBsAg carriers and one out of two patients with anti-HBs were found to have integrated HBV DNA in the tumour tissue, providing further evidence of the strong association between HBV infection and development of hepatocellular carcinoma.