T-Cell response to woodchuck hepatitis virus (WHV) antigens during acute self-limited WHV infection and convalescence and after viral challenge

The infection of woodchucks with woodchuck hepatitis virus (WHV) provides an experimental model to study early immune responses during hepadnavirus infection that cannot be tested in patients. The T-cell response of experimentally WHV-infected woodchucks to WHsAg, rWHcAg, and WHcAg peptides was monitored by observing 5-bromo-2'-deoxyuridine and [2-3H]adenine incorporation. The first T-cell responses were directed against WHsAg 3 weeks after infection; these were followed by responses to rWHcAg including the immunodominant T-cell epitope of WHcAg (amino acids 97 to 110). Maximal proliferative responses were detected when the animals seroconvered to anti-WHs and anti-WHc (week 6). A decrease in the T-cell response to viral antigens coincided with clearance of viral DNA. Polyclonal rWHcAg-specific T-cell lines were established 6, 12, 18, and 24 weeks postinfection, and their responses to WHcAg peptides were assessed. Five to seven peptides including the immunodominant epitope were recognized throughout the observation period (6 months). At 12 months after infection, T-cell responses to antigens and peptides were not detected. Reactivation of T-cell responses to viral antigens and peptides occurred within 7 days after challenge of animals with WHV. These results demonstrate that a fast and vigorous T-cell response to WHsAg, rWHcAg, and amino acids 97 to 110 of the WHcAg occurs within 3 weeks after WHV infection. The peak of this response was associated with viral clearance and may be crucial for recovery from infection. One year after infection, no proliferation of T cells in response to antigens was observed; however, the WHV-specific T-cell response was reactivated after challenge of woodchucks with WHV and may be responsible for protection against WHV reinfection.     

Efficacy of the carbocyclic 2'-deoxyguanosine nucleoside BMS-200475 in the woodchuck model of hepatitis B virus infection

Daily oral treatment with the cyclopentyl 2'-deoxyguanosine nucleoside BMS-200475 at doses ranging from 0.02 to 0.5 mg/kg of body weight for 1 to 3 months effectively reduced the level of woodchuck hepatitis virus (WHV) viremia in chronically infected woodchucks as measured by reductions in serum WHV DNA levels and endogenous hepadnaviral polymerase activity. Within 4 weeks of daily therapy with 0.5 or 0.1 mg of BMS-200475 per kg, endogenous viral polymerase levels in serum were reduced about 1,000-fold compared to pretreatment levels. Serum WHV DNA levels determined by a dot blot hybridization technique were comparably decreased in these treated animals. In the 3-month study, the sera of animals that had undetectable levels of WHV DNA by the dot blot technique were further analyzed by a highly sensitive semiquantitative PCR assay. The results indicate that BMS-200475 therapy reduced mean WHV titers by 10(7)- to 10(8)-fold, down to levels as low as 10(2) to 10(3) virions/ml of serum. Southern blot hybridization analysis of liver biopsy samples taken from animals during and after BMS-200475 treatment showed remarkable reductions in the levels of WHV DNA replicative intermediates and in the levels of covalently closed circular viral DNA. WHV viremia in BMS-200475-treated WHV carriers eventually returned to pretreatment levels after therapy was stopped. These results indicate that BMS-200475 should be evaluated in clinical trials for the therapy of chronic human hepatitis B virus infections.     

Preliminary clinical study of the distribution of HPMA copolymers bearing doxorubicin and galactosamine

Galactose-targeted delivery of macromolecules and drug conjugates to asialoglycoprotein receptor (ASGPR) positive cells has been widely documented in animals, although targeting in humans has never been demonstrated. In this study we report the pharmacokinetics and imaging determined in the first patient enrolled in a phase I clinical study of the poly[N-(2-hydroxypropyl)methacrylamide] copolymer bearing doxorubicin and galactosamine, known as PK2. Gradient high performance liquid chromatography (HPLC) evaluation of plasma and urine has been combined with 123I-based imaging to show biphasic clearance of the drug from the plasma (half-lives of 78+/-1 and 990+/-15), and approximately 30% delivery of the drug to the hepatic region, as determined by planar whole body imaging at 24 h. This patient has a multifocal hepatoma, and single photon emission computed tomography (SPECT) analysis showed a ratio of tumour tissue to normal liver uptake of approximately 1:3, at 24 h. On the basis of this patient, effective hepatic targeting can be achieved following an intravenous dose of 20 mg/m2 doxorubicin as PK2, however the therapeutic usefulness of this targeted drug has yet to be established.     

A benchmark of cell survival models using survival curves for human cells after completion of repair of potentially lethal damage

In this study, we report the synthesis and the evaluation as MRI contrast agent of arabinogalactan/pyrrolidinoxyl radicals (PCA) covalent adduct (SLAG:Spin Labelled ArabinoGalactan). Arabinogalactan was used as targeting device, as it is recognized by the asialoglycoprotein receptor specific to the hepatocytes. The higher relaxivity R1 in water of SLAG, compared with small hydrophilic nitroxyl radicals, was explained by the molecular dynamics study using EPR spectroscopy that showed some immobilization of the radical into the polysaccharide. A binding study on isolated hepatocytes revealed that SLAG still recognizes the asialoglycoprotein receptor. MR imaging was performed using spin-echo T1 weighted images on mice to compare the contrast effect obtained with SLAG and PCA after IV injection (1 mmol/kg free radical). The percent signal enhancement observed in the liver 5 min after IV injection was 40 +/- 3% and 13 +/- 5% for SLAG and PCA, respectively. The signal was also dramatically increased in the renal cortex. This latter effect as well as the prolonged duration of the contrast (+/- 3 h), indicates at least a partial nonselective biodistribution; the high concentration needed to obtain a contrast effect could account for the saturation of the asialoglycoprotein receptor and hence for the apparent nonselective biodistribution.     

Metabolic labeling of woodchuck hepatitis B virus X protein in naturally infected hepatocytes reveals a bimodal half-life and association with the nuclear framework

In order to identify potential sites of hepadnavirus X protein action, we have investigated the subcellular distribution and the stability of woodchuck hepatitis virus (WHV) X protein (WHx) in primary hepatocytes isolated from woodchucks with persistent WHV infection. In vivo cell labeling and cell fractionation studies showed that the majority of WHx is a soluble cytoplasmic protein while a minor part of newly synthesized WHx is associated with a nuclear framework fraction (20%) and with cytoskeletal components (5 to 10%). Pulse-chase experiments revealed that cytoplasmic WHx has a short half-life and decays with bimodal kinetics (approximately 20 min and 3 h). The rates of association and turnover of nucleus-associated WHx suggest that compartmentalization may be responsible for the bimodal turnover observed in the cytoplasm.     

Absence of mutations in the p53 tumor suppressor gene in woodchuck hepatocellular carcinomas associated with hepadnavirus infection and intake of aflatoxin B1

Infection with hepadnaviruses and exposure to aflatoxin B1 (AFB1) are considered major risk factors in the development of hepatocellular carcinoma (HCC) in humans and in animals. A high rate of mutations in the p53 tumor suppressor gene in hepatocellular carcinomas of predominantly hepatitis B virus (HBV) carrier patients has been recently related to dietary aflatoxin. Another member of the hepadnavirus family, the woodchuck hepatitis virus (WHV), infects woodchucks in a manner similar to that of HBV in humans. Therefore, it was of particular interest to determine whether the p53 gene in woodchuck HCCs associated with hepadnavirus infection and with exposure to AFB1 is affected in the same manner as in human HCCs. By direct PCR-sequencing, we analyzed exons 4-9 of the p53 gene in 13 HCCs from 12 woodchucks (two uninfected, ten WHV carriers). Six WHV carrier and two uninfected woodchucks were treated with AFB1. None of the analyzed HCC samples exhibited mutations, either in p53 gene exons 4-9, or in splicing donor-acceptor sites. The present data are consistent with our previous study that indicated a low rate of p53 mutations in HCCs of AFB1-treated ground squirrels, either infected or not infected with ground squirrel hepatitis virus, and in WHV carrier woodchucks not exposed to AFB1. Overall, our findings indicate that in woodchucks and in ground squirrels exposure to aflatoxin may affect the development of p53 mutations less than in humans.     

Anti-prolactin (PRL) autoantibodies cause asymptomatic hyperprolactinemia: bioassay and clearance studies of PRL-immunoglobulin G complex

The causes of hyperprolactinemia are varied, but some cases are classified as "idiopathic" because of unknown causes. We examined whether anti-prolactin (PRL) autoantibodies can cause hyperprolactinemia, especially the asymptomatic type. Serum PRL in four women with anti-PRL autoantibodies and five control patients with prolactinoma was characterized by a sensitive enzyme immunoassay, Nb2-bioassay, gel chromatography, affinity chromatography for immunoglobulin G (IgG), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, and clearance studies using anesthetized rats. In four women with anti-PRL autoantibodies, serum immunoreactive PRL concentrations were elevated (326 +/- 216 micrograms/L, normal < 30 micrograms/L), and PRL (84 +/- 5.5%) mostly consisted of the large molecular form in which a significant amount of 23 kDa PRL (60.6 +/- 14.7%) was noncovalently bound to IgG. Although three of the four women lacked clinical symptoms of hyperprolactinemia such as amenorrhea and galactorrhea, the IgG-bound PRL was fully bioactive in vitro. It was cleared more slowly from circulation than free PRL. The data suggest that PRL forms a complex with IgG, and this probably results in delayed clearance of PRL and leads to hyperprolactinemia in women with anti-PRL autoantibodies.     

Strategy to design peptide inhibitors: structure of a complex of proteinase K with a designed octapeptide inhibitor N-Ac-Pro-Ala-Pro-Phe-DAla-Ala-Ala-Ala-NH2 at 2.5 A resolution

To assess the relationship between hepatitis C virus infection and Fas antigen expression on hepatocytes, we examined changes in hepatic Fas antigen expression in the presence or absence of active hepatitis C virus infection. Twenty patients with chronic hepatitis C infection were treated with interferon and underwent pre- and posttreatment liver biopsies. Patients were classified according to the absence (group A; n = 9) or the presence (group B; n = 11) of hepatitis C virus RNA (HCV-RNA) in the liver after interferon therapy. An immunohistochemical assay showed Fas antigen staining in hepatocytes membranes and cytoplasm with expression concentrated mainly in periportal areas. The percentage of Fas-positive cells in the liver before treatment was not different between group A (39.5+/-19.1%) and group B (32.5+/-15.6%). Hepatic Fas expression was reduced significantly after treatment (24.3+/-10.6%) compared with the pretreatment values in group A (p < 0.05) but not in group B (25.9+/-16.9%). There was no significant difference between the two groups in the degree of histologic improvement. These results suggest that hepatic Fas expression is associated with persistent infection of hepatitis C virus.     

Covalently closed circular viral DNA formed from two types of linear DNA in woodchuck hepatitis virus-infected liver

We found that livers from woodchucks chronically infected with woodchuck hepatitis virus (WHV) contained covalently closed circular DNA (cccDNA) molecules with deletions and insertions indicative of their formation from linear viral DNA by nonhomologous recombination, as we previously described for the duck hepatitis B virus (W. Yang and J. Summers, J. Virol. 69:4029-4036, 1995). However, evidence for two different types of linear precursors was obtained by analysis of the recombination joints in WHV cccDNA. Type 1 linear precursors possessed the structural properties that correspond to those of in situ-primed linear DNA molecules, which constitute between 7 and 20% of all viral DNA replicative intermediates synthesized in the liver. Type 2 linear precursors are hypothetical species of linear DNAs with a terminal duplication of the cohesive-end region, between DR1 and DR2. This type of linear DNA has not been previously described and was not detected among the DNA species present in nucleocapsids. A fraction of cccDNAs formed from both type 1 and type 2 linear DNAs are predicted to be functional for further DNA synthesis, and some evidence for the formation of two or more generations of cccDNA from linear DNA was observed.     

High degree of conservation in the hepatitis B virus core gene during the immune tolerant phase in perinatally acquired chronic hepatitis B virus infection

BACKGROUND: Mutations in the hepatitis B virus genome have been implicated in the persistence of hepatitis B virus infection and the pathogenesis of hepatitis B virus related liver disease. In view of the heterogeneity in published sequences, data from cross-sectional studies of unrelated subjects cannot differentiate true mutations from infections with variant sequences. AIMS/METHODS: We compared the hepatitis B virus core gene sequences of 42 HBsAg positive subjects from 11 Chinese families with those of the index patients (maternal carriers) to determine the frequency and rate of true hepatitis B virus core gene mutations in patients with chronic hepatitis B virus infection. RESULTS: Completely identical nucleotide sequences were present in all the family members and index patients in two families, suggesting that the hepatitis B virus core gene can be conserved for more than 20 years. The high degree of sequence conservation in these families is related to the young age of the subjects (mean 19.2+/-8.9 years), the fact that they were all HBeAg positive and that 75% of them had persistently normal aminotransferase levels. Longitudinal studies confirmed that mutations were rare in those who remained HBeAg positive with normal aminotransferase levels (immune tolerant phase), but significantly more common in HBeAg positive subjects who had elevated aminotransferase levels and in those who cleared HBeAg (immune clearance phase), the rates of nucleotide and amino acid changes were respectively: 0.28+/-0.12 vs 1.30+/-0.26/10(3) nt position/yr and 0.04+/-0.01 vs 0.18+/-0.5/10(2) codon/yr. CONCLUSIONS: Identical nucleotide differences could be found in the sequences of all the subjects in some families. These differences were more likely to be due to intra-familial transmission of stable variants. Sequence analysis based on comparisons with published sequences would have led to over-reporting of mutations. The hepatitis B virus core gene can remain highly conserved for more than two decades during the immune tolerant phase of perinatally acquired chronic hepatitis B virus infection. However, significant changes can occur within 2-3 years during the immune clearance phase.     

Role of liver endothelial and Kupffer cells in clearance of human C1q in rats

In the present study the contribution of rat liver endothelial cells (EC) and Kupffer cells (KC) in the clearance of human (hu) C1q in rats was investigated. In untreated rats and rats depleted from KC the clearance kinetics and the tissue distribution of hu C1q were measured. In untreated rats, the clearance of hu C1q occurred in a monophasic manner with a half-life of 66 +/- 26.7 min. The clearance of hu C1q in KC-depleted rats was delayed significantly (p < 0.001) and occurred with a half-life of 217 +/- 78.8 min. Fifteen min after injection, 11 +/- 3.5% of hu C1q was found in the liver of untreated rats and 8 +/- 1.4% was found in the liver of KC-depleted rats. The percentage non-trichloroacetic acid precipitable activity in the circulation, as a measure for degradation of C1q, reached a level of 11.6 +/- 5.6% at 240 min in untreated rats compared with 4.6 +/- 5.8% in KC-depleted rats. Double immunofluorescence staining 5 min after administration of C1q in untreated rats, revealed that C1q was associated with KC and EC in the liver. Fifteen minutes after i.v. injection of hu C1q, there was an uptake of C1q in the hepatocytes. In KC-depleted rats, 5 min after administration of hu C1q, C1q was bound to the EC. Fifteen minutes after injection, C1q was also found in the hepatocytes. Electron microscopical studies revealed that C1q binds to EC, and that it is internalized in the hepatocytes and KC. The clearance of hu C1q in untreated rats was inhibited by preadministration of high concentrations of bovine C1q. These data show that rats depleted from KC are able to bind, internalize and degrade C1q, and that EC may play a role in the handling of C1q and C1q bound to immune complexes.     

Role of surgical interventions in the treatment of patients with trophoblastic tumor resistant to chemotherapy

A novel strategy for anti-viral intervention of hepatitis B virus (HBV) through the disruption of the proper folding and transport of the hepadnavirus glycoproteins is described. Laboratory reared woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated with N-nonyl-deoxynojirimycin (N-nonyl-DNJ), an inhibitor of the endoplasmic reticulum (ER) alpha-glucosidases. The woodchucks experienced significant dose dependent decreases in enveloped WHV, resulting in undetectable amounts in some cases. The reduction in viremia correlated with the levels of hyperglucosylated glycan in the serum of treated animals. This correlation supports the mechanism of action associated with the drug and highlights the extreme sensitivity of the virus to this type of glycan inhibitor. At N-nonyl-DNJ concentrations that prevented WHV secretion, the glycosylation of most serum glycoproteins appeared unaffected, suggesting great selectivity for this class of therapeutics. Indeed, this may account for the low toxicity of the compound over the treatment period. We provide the first evidence that glucosidase inhibitors can be used in vivo to alter specific steps in the N-linked glycosylation pathway and that this inhibition has anti-viral effects.     

Increased technetium-99m-GSA uptake per hepatocyte in rats with administration of dimethylnitrosamine or hepatocyte growth factor

Technetium-99m-diethylenetriaminepentaacetic acid-galactosyl-human serum albumin (GSA) is a new scintigraphic agent that binds specifically to asialoglycoprotein receptors on hepatocytes, and can be used to evaluate hepatic function. Asialoglycoprotein receptor is a hepatocellular membrane receptor responsible for the endocytosis of asialoglycoproteins, and the function of this receptor is affected in various disease states. The aim of this study was to investigate GSA uptake per hepatocyte in the convalescent stage from hepatic damage. METHODS: We used rats with dimethylnitrosamine (DMN)-induced hepatic injury and rats with recombinant human hepatocyte growth factor (rhHGF) stimulation. Plasma clearance of GSA and the number of hepatocytes in whole liver were calculated. RESULTS: In the DMN-treated rats, the total number of hepatocytes and GSA plasma clearance were reduced significantly at 3 wk after the final administration of DMN. However, calculated GSA uptake per individual hepatocyte was significantly greater by 53.2% than in the normal controls. The area of hepatic nucleus was also significantly greater than in the normal controls. In the rhHGF-treated rats, an increase in the total number of hepatocytes was not demonstrated on the final day of rhHGF administration (Day 4). However, calculated GSA uptake per hepatocyte was significantly greater (59%) than in the controls. CONCLUSION: Augmented GSA uptake per hepatocyte during the convalescent stage after hepatic injury suggests a cellular compensation to the decreased number of hepatocyte. This mechanism may be caused by the secretion of some hepatotropic factors such as HGF.     

Hepatitis B virus and hepatocellular carcinoma

Hepatitis B is a very important public health problem. Epidemiologic studies have shown a relationship between the hepatitis B virus (HBV) chronic carrier state and the development of hepatocellular carcinoma. HBV belongs to the Hepadna viruses family which includes the woodchuck hepatitis virus (WHV), Woodchucks infected with WHV represent a good experimental model to study the viral oncogenesis. In 85% of the studied cases, WHV acts by insertional mutagenesis in a gene of the myc family, mostly the N-myc2 gene. Expression of the myc genes is increased, suggesting the role of the viral enhancer. Study of transgenic mice have shown the liver specificity of the WHV action. In man, the liver oncogenesis is not explained. Studies are in progress to detect inactivation of tumor suppressor genes.     

Stimulation of asialoglycoprotein uptake by recombinant human hepatocyte growth factor in normal and damaged rat liver

BACKGROUND: Hepatocyte growth factor (HGF) is a strong mitogen of hepatocytes. However, little is known about the effect of HGF on the asialoglycoprotein receptors (ASGPR) of hepatocytes. The aim of this study was to identify alterations in binding of ligand to ASGPR by recombinant human HGF (rhHGF) infusion. METHODS: RhHGF was administered to rats with either normal or dimethylnitrosamine (DMN)-damaged livers. Technetium-99m-diethylenetriaminepentaacetic acid-galactosyl-human serum albumin (GSA) blood clearance was used to measure ASGPR activity. RESULTS: In normal and damaged rats, liver weight, hepatocyte nuclear size, and number of hepatocytes (cells/mm2) were not altered by rhHGF, but GSA blood clearance after rhHGF infusion was significantly increased over the preinfusion rate. CONCLUSIONS: Independent of proliferation of hepatocytes, rhHGF stimulates a hepatocytic function of the receptor-mediated uptake of ASGP.     

Multilocus quantitative trait analysis using the multipoint identity-by-descent method

A hepatocyte-directed vector has been developed; it includes several key features thought to favor in vivo gene delivery to the liver: electrostatically neutral particles which avoid nonspecific binding to other cells, to the extracellular matrix, and to complement proteins; asialoglycoprotein receptor-mediated endocytosis which may address the complexes to the perinuclear region; and polyethylenimine (PEI)-mediated endosome buffering and swelling as an escape mechanism to the cytoplasm. This system is based on a 5% galactose-bearing polyethylenimine (PEI-gal) polymer which is condensed with plasmid DNA to neutrality. Murine (BNL CL.2) and human (HepG2) hepatocyte-derived cell lines were transfected 10(4)-10(5)-fold more efficiently than murine fibroblasts (3T3), whether transfection was assessed globally (luciferase expression from the cell extract) or following histochemical staining (beta-galactosidase). Under these conditions, over 50% of the hepatocytes were selectively transfected in the presence of 10% serum. Transfection was suppressed by removal of the targeting galactose residues, by their replacement with glucose, or by the addition of excess asialofetuin. Thus, results from comparative and competitive experiments indicate the asialoglycoprotein receptor is involved in transfection of hepatocytes with neutral PEI-gal/DNA complexes.     

Structure and glycosylation patterns of surface proteins from woodchuck hepatitis virus

Woodchucks chronically infected with woodchuck hepatitis virus (WHV) are a valuable model for human hepatitis B virus (HBV) in studies of pathogenesis, immunity, and antiviral therapy. For this reason, substantial efforts to characterize both the similarities and the differences between HBV and WHV are being made. The structure of the WHV surface proteins (WHs proteins) has not yet been adequately elucidated. The bands that would be expected for glycosylated and nonglycosylated small (S) WHs protein are found by sodium dodecyl sulfate gel electrophoresis of purified WHs protein, but the bands corresponding to the middle (M) and large (L) WHs proteins of HBV are not seen at the expected sizes, even though the sequences of the WHV and HBV surface protein genes are 60% homologous. By amino-terminal sequencing we have identified two bands at 41 and 45 kDa as the MWHs proteins, 8 kDa larger than expected. We have also confirmed that two bands at 24 and 27 kDa are SWHs proteins. A protein of 49 kDa was blocked at the N terminus, which using immunoblotting with an antiserum against WHV pre-S1 (positions 126 to 146) was identified, together with a part of the 45-kDa protein, as glycosylated and nonglycosylated LWHs protein of the expected size. Sialidase and O-glycosidase digestion showed that the larger size of MWHs protein results from the presence of O glycoside groups which are probably in the pre-S2 domain of MWHs protein. Since the pre-S2 domains of HBV and WHV have similar numbers of potential O glycosylation sites, it appears to be likely that the glycosyltransferases act differently on the viral proteins in woodchucks and humans.     

Dopamine restores lung ability to clear edema in rats exposed to hyperoxia

Exposure to hyperoxia causes lung injury, decreases active sodium transport and lung edema clearance in rats. Dopamine (DA) increases lung edema clearance by stimulating vectorial Na+ flux and Na, K-ATPase function in rat alveolar epithelium. This study was designed to test whether DA (10(-)5 M) would increase lung edema clearance in rats exposed to 100% O2 for 64 h. Active Na+ transport and lung edema clearance decreased by approximately 44% in rats exposed to acute hyperoxia (p < 0.001). DA increased lung edema clearance in room air breathing rats (from 0.50 +/- 0.02 to 0.75 +/- 0.06 ml/h) and in rats exposed to 100% O2 (from 0.28 +/- 0.03 to 0. 67 +/- 0.03 ml/h). Disruption of cell microtubular transport system by colchicine blocked the stimulatory effect of DA on active Na+ transport in control and hyperoxic rats, whereas the isomer beta-lumicolchicine, which does not affect cell microtubular transport, did not inhibit the stimulatory effects of dopamine. The Na,K-ATPase alpha1-subunit protein abundance increased in the basolateral membranes of alveolar type II (ATII) cells incubated with 10(-)5 M DA for 15 min, probably by recruiting Na+ pumps from intracellular pools. Colchicine, but not beta-lumicolchicine, prevented the recruitment of alpha1 subunits to the plasma membrane by DA. Accordingly, DA restored lung ability to clear edema in hyperoxic-injured rat lungs. Conceivably, dopamine induces recruitment of Na+ pumps from intracellular pools to the plasma membrane of alveolar epithelial cells and thus increases lung edema clearance.