Hepatitis B virus infection in microcarrier-attached immortalized human hepatocytes cultured in molecularporous membrane bags: a model for long-term episomal replication of HBV

Studies on the pathobiology of chronic (long-term) hepatitis B virus (HBV) infection and in vitro drug testing have been hampered by the lack of appropriate systems for culturing susceptible cells chronically infected with HBV. Most of the in vitro studies of HBV replication have been performed with HBV genome-transduced cell lines. In this system, viral production is mainly the result of chromosomal replication. In an in vitro infection system, owing to medium refreshment (which leads to the removal of infectious particles necessary for the perpetuation of infection) and to trypsinization for cell passages, it is difficult, if not impossible, to maintain chronic HBV infection, despite the use of susceptible cells. To circumvent these unfavourable factors for chronic HBV infection in vitro, we cultured microcarrier-attached immortalized human hepatocytes, infected with HBV, in molecularporous (MW 12,000-14,000) membrane (dialysis) bags for a duration of 2 months. HBV covalently-closed-circular (ccc) DNA, HBV precore/core and X mRNAs were detected in the cells cultured in this system following infection until the end of the experiment (day 58), while in classical culture conditions (monolayer), markers of HBV replication were also detected. Production of hepatitis B surface antigen (HBsAg) and HBV DNA was detected and their levels in culture medium (collected at the end of experiments from the molecularporous membrane bags) were increased 2.86- and 3.28-fold respectively. Using Southern blot analysis, HBV replicative intermediates could also be demonstrated throughout the experiments. However, integrated HBV DNA was not present. In contrast, HBV ccc DNA, HBV precore/core and X mRNAs, and replicative intermediates were not demonstrable in FTO 2B rat hepatoma cells infected in the same manner in parallel experiments. This in vitro infection system, using susceptible, immortalized human hepatocytes, therefore provides a new tool for studying the long-term effect of HBV infection, mainly involving episomal replication in hepatocytes, and for drug testing.     

Inhibitory effect of 9-(2-phosphonylmethoxyethyl)-adenine (PMEA) on human and duck hepatitis B virus infection

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) was evaluated for its inhibitory effect on hepadnavirus replication in three different cell systems, i.e., human hepatoma cell lines HepG2 2.2.15 and HB611 (transfected with human hepatitis B virus (HBV)) and primary cultures of duck hepatocytes infected with duck hepatitis B virus (DHBV). PMEA inhibited HBV release from HepG2 2.2.15 cells and HB611 cells at a 50% inhibitory concentration (IC50) of 0.7 and 1.2 microM, respectively. Intracellular viral DNA synthesis was inhibited at concentrations equivalent to those required to inhibit virus release from the cells. DHBV secretion from duck hepatocytes was inhibited by PMEA at an IC50 of 0.2 microM. HBsAg secretion was inhibited by PMEA in a concentration-dependent manner in HB611 cells and DHBV-infected duck hepatocytes but not HepG2 2.2.15 cells. The 50% cytotoxic concentration, as measured by inhibition of [3H-methyl]deoxythymidine incorporation was 150 microM for the two human hepatoma cell lines and 40 microM for the duck hepatocyte cultures. In a pilot experiment PMEA was found to reduce the amounts of DHBV DNA in the serum of Pekin ducks.     

Rat as an animal model carrying human hepatitis B virus in hepatocytes

OBJECTIVE: To establish an experimental animal model of rat carrying human hepatitis B virus in the hepatocytes using a simple and reproducible method. MATERIALS AND METHODS: Human serum rich in hepatitis B virus was injected into portal veins and caudalis veins of young male Wistar rats. One and two months after the injection, liver biopsies were done. In situ hybridization and immunohistochemical study of liver specimens were carried out. Sera were also examined for HBV DNA by polymerase chain reaction. RESULTS: All of seven rats in this experiment were HBV DNA and HBV surface antigen (HBsAg) positive in their hepatocytes. Most HBV positive hepatocytes were distributed around the central vein and scattered in the liver lobules, and HBV DNA and HBsAg were located in cytoplasm. HBsAg exists mainly as the forms of diffuses and inclusion body. No hepatocytic damage or inflammation was observed. Neither viremia nor antigenemia was detected. CONCLUSIONS: Our studies showed for the first time that natural human HBV can enter Wistar rat liver cells through intravenous injection efficiently and express for a long period. This animal model can be used in the studies of HBV molecular biology, therapeutic regimens and prophylaxis against HBV. A possible mechanism of HBV entering rat hepatocytes is also proposed.     

Taste dimensions of monosodium glutamate (MSG) in a food system: role of glutamate in young American subjects

Fibrosing cholestatic hepatitis is a histological variant of hepatitis B virus infection with a high rate of mortality. We describe a patient who acquired acute hepatitis B virus infection 8 months after renal transplantation. Clinical features of rapidly progressive liver failure, indicated by prolonged prothrombin time (57 seconds) and increased bilirubin (40.4 mg/dL) and ammonia (129 mumol/L) concentrations, were accompanied by an extremely high serum HBV DNA level (2.153 x 10(6) pg/mL). Liver biopsy specimen showed fibrosing cholestatic hepatitis with widespread balloon degeneration of hepatocytes, focal hepatocyte loss, bile stasis, periportal fibrosis, mild lymphocytic infiltration, and strongly positive immunohistochemical staining for hepatitis B surface antigen (HBsAg) and hepatitis B core antigen. Lamivudine therapy suppressed HBV DNA to < 10 pg/mL within 4 weeks, which was followed by gradual recovery of liver function from a state of hepatic precoma. Twenty-four months after the onset of hepatitis, the patient had normal prothrombin time and bilirubin, transaminase, and albumin levels. She remained HBsAg positive and hepatitis B e antigen negative. Renal allograft function was stable, with a creatinine level of 1.52 mg/dL. HBV DNA remained suppressed after 22 months of lamivudine therapy. Our experience shows that fibrosing cholestatic hepatitis and liver failure caused by HBV infection can be successfully treated with lamivudine.     

Nested polymerase chain reaction (PCR) and multiple cloned antibody capture PCR for the examination of serum hepatitis B virus DNA in negative hepatitis B surface antigen patients suffering from liver diseases

In order to find out rapidly the causes of the liver diseases suffered by patients with negative hepatitis B surface antigen (HBsAg), nested polymerase chain reaction (PCR) and multiple cloned antibody capture PCR techniques were established to examine serum hepatitis B virus (HBV) DNA. By using both techniques along with the examination of hepatitis C virus (HCV) infection, the causes of chronic liver diseases with negative HBsAg were studied. It is found that nested-PCR can increase the sensitivity of single PCR more than 1,000 fold and multiple cloned antibody capture-PCR can detect concentration of HBV DNA as low as 0.1-0.01 pg/L. HBV DNA positive patients were found in 45.5%, 30.8%, 13.3% and 100% respectively of the patients suffering from liver cirhosis with negative HBsAg (group A, 22 cases), chronic hepatitis with negative HBsAg (group B, 13 cases), normal subjects with negative HBsAg and positive hepatitis B core antibody (HBcAb, group C, 30 cases) and liver cirhosis with positive HBsAg and negative HBeAg (group D, 12 cases). HBV DNA can be also found in the serum of HBsAb positive patients and subjects supposed to be healthy, 81.8% and 53.8% of the patients were infected with HBV and/or HCV in group A and group B respectively. All these results suggest that nested-PCR and multiple cloned antibody capture-PCR are rapid and highly sensitive methods for detection of serum HBV DNA. HBV infection is an important cause of chronic liver diseases in patients with negative HBsAg. The causes of most of the HBsAg-negative chronic liver diseases are related with infection of viruses. The clinical significance of serum HBsAb in naturally infected patients should be reconsidered.     

Functional analysis of HBV genomes from patients with fulminant hepatitis

Two previous case reports suggest that hepatitis B virus (HBV) core promoter variants with a high replication competence contribute to the pathogenesis of fulminant hepatitis B (FHB). We recently found in HBV genomes from patients with FHB an accumulation of mutations within the core promoter region. Therefore, the aim of this study was to investigate the phenotype of these HBV variants. Replication competence and expression of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) of viral genomes from seven patients with FHB and one patient with fulminant recurrent hepatitis after liver transplantation were analyzed by transfection experiments in human hepatoma cells. Compared with wild-type virus, the HBV variants from the seven patients with FHB produced similar or slightly lower levels of intracellular replicative intermediates and extracellular viral particles. In contrast, the HBV genomes from the patient with fulminant recurrent hepatitis synthesized and secreted significantly more HBV DNA. All genomes tested expressed similar or even higher levels of HBeAg compared with wild-type virus, except for those from four patients with a precore stop codon mutation in the respective dominant viral populations. The level of HBsAg produced by all variant genomes was similar or reduced compared with wild-type virus. These data indicate that in some cases HBV variants with enhanced replication competence and/or a defect in HBeAg expression may contribute to the development of FHB. However, neither phenotype is an essential prerequisite; thus, an additional role of other viral or host factors in the pathogenesis of FHB is suggested.     

Fibrosing cholestatic hepatitis in a transplant recipient with hepatitis B virus precore mutant

A patient with hepatitis B virus (HBV) precore mutant (seropositive for hepatitis B surface antigen [HBsAg], anti-hepatitis B e antigen [HBeAg], and HBV DNA) who underwent orthotopic liver transplantation for end-stage liver disease is described. Sequencing of the HBV precore region of the pretransplant serum sample confirmed the presence of the precore stop-codon mutant (G-->A mutation in codon 1896) only. The patient received HBV immunoglobulin prophylaxis for 6 months but HBV recurred thereafter with a mild hepatitic flare, and he remained seropositive for HBsAg, anti-HBe, and HBV DNA. The initial hepatitic illness resolved in 3 months. The patient remained well for another 16 months before presenting with fibrosing cholestatic hepatitis (FCH). During his entire initial hepatitic flare, quiescent period, and final FCH phase, he remained seropositive for HBsAg, anti-HBe, and HBV DNA. Moreover, sequencing of the serum HBV DNA in final FCH phase showed the presence of the identical HBV precore mutant. Immunohistochemical staining showed extensive expression of HBsAg/pre-S1, pre-S2, and hepatitis B core antigen, but HBeAg was scarcely detectable. This case illustrates that (1) recurrence of HBV precore mutant infection can occur in liver; (2) it can give rise to FCH; and (3) hepatic accumulation of HBeAg is not essential for the development of FCH.     

Response of patients with dual hepatitis B virus and C virus infection to interferon therapy

Patients with dual infection with hepatitis B virus (HBV) and delta virus (HDV) responded poorly to interferon (IFN) therapy. Little is known about the effect of IFN therapy in patients with HBV and hepatitis C virus (HCV) dual infection. The patients in two randomized controlled trials with chronic HBV infection were retrospectively assayed for HCV markers. The HBV responses to IFN therapy in patients with and without HCV markers were compared. An open trial was conducted in 4 patients who had lost their serum HBV surface antigen (HBsAg) but had continuing HCV viremia and hepatitis. Of the 15 patients seropositive for HCV marker(s), only 1 (6.7%) responded with seroclearance of HBV DNA and HBV e antigen, as compared with 46 (28%) of 164 HCV-negative patients (p = 0.058). Icteric hepatitis developed in 1 patient on emergence of serum HCV RNA in association with seroclearance of HBV DNA. In contrast, good response was demonstrated in 3 of the 4 patients who had lost serum HBsAg before therapy. The results suggest that IFN therapy is not only of limited value in patients with dual infection with HBV and HCV but also has a potential risk of severe hepatitis if the clearance of one virus removes its suppressive effect on and facilitates the emergence of the other. However, patients with continuing HCV hepatitis after termination of the chronic HBsAg carrier state responded well to IFN therapy.     

Changes in ceftazidime concentration in the CSF following overdose in acute kidney failure]

The diagnosis of liver diseases induced by hepatitis B virus (HBV) is supported by the detection of HBV surface antigen (HBsAg) in serum. The present study aimed to investigate the presence of HBV deoxyribonucleic acid (DNA) in patients with liver cirrhosis using a polymerase chain reaction (PCR) based on primers derived from the pre-S1 and pre-core regions. HBsAg was detected in 10 of 48 patients (21%), total anti-hepatitis B core antigen (HBc) antibodies in 54%, anti-hepatitis B e antigen (HBeAg) in 14.6%, anti-HBc immunoglobulin M in 8%, and anti-HBs in 26%; none had detectable HBeAg. HBV DNA was detected in 73% of the cirrhotic patients. All cirrhotic patients with HBsAg also had HBV DNA; HBV DNA was detected in 64.5% of those without HBsAg. We conclude that the clearance of HBsAg does not necessarily indicate termination of viraemia in patients with liver cirrhosis and the detection of HBV DNA using a PCR based on primers from the pre-S1 and pre-core regions should be included in the diagnosis of HBV infection.     

Structural development in the newborn marsupial, the stripe-faced dunnart, Sminthopsis macroura

Human hepatitis B virus (HBV) infection has been closely linked to the occurrence of hepatocellular carcinoma (HCC). Hepatoma cell lines and nude mouse-passaged hepatoma tissues were used in this report to study the HBV DNA status in these cells after passage. DNA was extracted from seven hepatoma cell lines and three nude mouse passaged HCC lines. Southern blot hybridization technique was performed with either cloned HBV whole genome or subgenomic DNA fragments as probes to analyze the presence of HBV DNA. Integration of HBV DNA fragments was detected in one mouse passaged tissue, R. Hybridization with HBV subgenomic DNA revealed that there were some DNA rearrangements of the integrated HBV DNA in R. However, the integrated HBV DNA could not be detected in the cell line derived from R after in vitro cultivation for 2 years. Both episomal form and integrated HBV DNA were detected in a cell line NTU-h3. Episomal form HBV DNA ih NTU-h3 changed after several passages. HBV DNA in NTU-h3 was unstable after in vitro cultivation. Therefore, we concluded that the presence of HBV DNA might not be essential for the maintenance of the tumorigenicity of hepatoma and the nude mouse system was more stable for maintaining HBV DNA in HCC.     

Hepatitis B virus transmission in an elementary school setting

CONTEXT: The risk of transmission of hepatitis B virus (HBV) in day care centers and schools is low. OBJECTIVE: To investigate the source of HBV transmission for an elementary school teacher with acute hepatitis B. DESIGN: Serologic survey for HBV infection among elementary school students, school staff, and household members of an HBV-infected teacher and student. SETTING: General community and elementary school. PATIENTS: Elementary school students and staff members and household members of an HBV-infected teacher. MAIN OUTCOME MEASURES: Elementary school students, school staff, and household members of an HBV-infected teacher were tested for markers of HBV infection. Samples positive for hepatitis B surface antigen (HBsAg) were tested for HBsAg subtype using monoclonal antibodies and examined for HBV DNA homology by polymerase chain reaction techniques. RESULTS: An HBV-infected student and the teacher were found to have the same HBV subtype (ayw1-2) and to have identical HBV DNA sequences. The teacher reported none of the usual risk factors for acquiring HBV infection, and none of her family members had been infected prior to her illness. The specific means of HBV transmission from student to teacher was not identified. Of 108 total children in the same grade as the HBV-infected student, 102 (94%) were tested for serologic markers of HBV infection, and none was positive. CONCLUSIONS: This investigation documented transmission from an HBV-infected student to a teacher in an elementary school setting without a reported overt percutaneous or permucosal exposure to blood or infectious body fluids. Transmission of HBV to other students or staff members in the school was not observed.     

Genital infections and their care

The pathogenesis of hepatitis B can be subdivided into three sequentially correlated events: (a) loss of virus tolerance, (b) liver cell necrosis mediated by virus specific inflammatory response, (c) non-specific death of functionally compromised hepatocytes mediated by inflammatory cytochines released by virus specific inflammatory response. The severity of liver damage depends on the occurrence of these events as well as other factors. The HBeAg defective mutant appears to be involved in the loss of virus tolerance and therefore in the pathogenesis of acute hepatitis B. In addition it is positively selected by antiviral immunoreaction, behaves as an escape mutant, and it also contributes to the pathogenesis of chronic hepatitis B. The combination of these characteristics explains the relative prevalence of this mutant over wild-type HBV in patients with severe acute hepatitis B and in chronic HBsAg carriers during anti-HBe seroconversion and/or hepatitis B exacerbations. However, the absence of HBeAg defective mutants in some cases of severe and fulminant hepatitis B as well as its detection in asymptomatic carriers of HBsAg should not be surprising. The severity of hepatitis is influenced by many other factors: the number of virus infected cells, the competence and genetic heterogeneity of the immune system, the vigor and extent of non-specific inflammatory response and the killing of hepatocytes endangered by other diseases or infected with other hepatotropic viruses.     

The prevalence of HBsAg in hospitalized children as a marker of hepatitis B virus infection]

The aim of this study was to determine the prevalence of hepatitis B surface antigen (HBsAg) in hospitalised children, as specific marker for hepatitis B virus (HBV) infection. Our study group consists of 517 children, 68 of them diagnosed with chronic hepatitis. For HBsAg determination we used an ELISA test (Labsystems); for some children we also tested by ELISA the following markers: the antibodies and anti-hepatitis C virus (HCV) antibodies. From 517 children 24.28% were HBSAg positive and 75% of children with chronic hepatitis were positive for the same marker. Almost 100% of chronic active hepatitis (CAH) patients was positive for HBSAg. CONCLUSIONS: 1. The prevalence of HBsAg was much higher as compared with the healthy population prevalence; it is a clear prove that HBV infection has an important role in chronic hepatitis appearance. 2. For all HBsAg positive patients, it is necessary to determine other markers like HBeAg-anti-HBe antibodies system as well as markers for other viral hepatitis (HDV, HCV). 3. The anti-HBV infection vaccine will reduce significantly the prevalence of HBV and HDV infections; 4. Biological molecular technique, like PCR will be necessary in our country, in the future, even the price is so high, to monitoring the IFN treatment for chronic infection as unique solution for these patients.     

Relation between laboratory test results and histological hepatitis activity in individuals positive for hepatitis B surface antigen and antibodies to hepatitis B e antigen

BACKGROUND: Hepatitis B surface antigen (HBsAg) and antibodies to hepatitis B e antigen (anti-HBe) commonly coexist, and laboratory tests are often requested to assess histological hepatitis activity. An optimum panel of tests has not been found and the usefulness of hepatitis B virus (HBV) DNA assays in this context has not been established. We assessed various blood tests to find which best predicted hepatitis activity. METHODS: Routine plasma biochemical liver tests and serum HBV DNA (hybridisation and PCR assays) were assessed prospectively in 123 patients positive for HBsAg and anti-HBe. We scored histological hepatitis activity (hepatitis activity index) and determined whether chronic active hepatitis (chronic hepatitis with portal and periportal lesions) was present. We analysed the relation between laboratory data and the hepatitis activity index or risk of chronic active hepatitis by multiple regression and multiple logistic regression, respectively. FINDINGS: The analyses provided models for predicting either the hepatitis activity index or the risk of chronic active hepatitis. Aspartate aminotransferase was the most important test in the two models. The contribution of HBV DNA and other assays, especially alanine-aminotransferase activity, were of no practical importance. INTERPRETATION: Because screening by aspartate-aminotransferase activity could not be improved by the addition of other assays or HBV DNA, patients positive for HBsAg and anti-HBe could be screened for chronic active hepatitis with a single assay and counselling of patients can be improved if proper reference values are used.     

So who was Gerdy... and how did he get his own tubercle?

To elucidate the role of apoptosis in the pathological lesion of hepatitis B virus (HBV) infection, biopsied liver tissue specimens of 38 patients with chronic hepatitis B of varying severity were investigated with in situ immunohistochemistry and TUNEL test. Apoptotic hepatocytes were found to be rare, while the nuclei of many cells were positively stained with TUNEL, suggesting 3'-OH ends generated as the DNA was impaired. Of the 17 cases with mild lesion or without piecemeal necrosis, 14 were negative or weakly positive with both Fas and TUNEL test. Of the 7 cases with piecemeal and bridging necrosis, none was strongly positive. In the 14 cases with active hepatitis and early cirrhosis, strongly positive results with Fas were found in 9 and with TUNEL in 3 respectively. It is suggested that the cytotoxic T lymphocyte (CTL)-Fas-apoptosis mechanism was involved in the hepatocyte death of hepatitis B as well. The Fas expression, DNA damage and apoptotic cells distributed mostly in the piecemeal necrosis region, and the ballooning and the necrotic hepatocytes were also clustering in this region. As both the apoptosis and necrosis are mediated by CTL, they are closely related: while transducted by different ways, they occurred independently.