HLA-A31- and HLA-Aw68-restricted cytotoxic T cell responses to a single hepatitis B virus nucleocapsid epitope during acute viral hepatitis.

We have recently developed the technology to identify and characterize the human histocompatibility leukocyte antigen (HLA) class I-restricted, CD8+ cytotoxic T lymphocyte (CTL) response to hepatitis B virus (HBV)-encoded antigens in patients with acute viral hepatitis. CTL are expanded in vitro by stimulation with HBV-derived synthetic peptides and selected by restimulation with a panel of HLA-matched stable transfectants that express the corresponding HBV protein. We have recently reported the existence of an HLA-A2-restricted, CD8+ CTL response to an epitope located between residues 18 and 27 of the HBV nucleocapsid core antigen (HBcAg). We now report the discovery of a CTL epitope located between HBcAg residues 141 and 151 that completely overlaps a critical domain in the viral nucleocapsid protein that is essential for its nuclear localization and genome packaging functions as well as processing of the precore protein. The CTL response to this epitope is dually restricted by the HLA-A31 and HLA-Aw68 alleles, which, unexpectedly, appear to use a common binding motif based on the results of alanine substitution and competition analysis, and the binding properties of these two alleles predicted from their known primary sequence, and from the three-dimensional structure of HLA-Aw68. We have also demonstrated that the HBV-specific CTL response to this epitope is polyclonal during acute viral hepatitis, since these two restriction elements can present the HBcAg 141-151 epitope to independent CTL clones derived from a single patient; and that the CTL response is multispecific, since HLA-A2-restricted and HLA-Aw68-restricted CTL responses to HBcAg 18-27 and HBcAg 141-151, respectively, have been identified to coexist in another patient. The foregoing argue against the emergence of CTL escape mutants as a significant problem during HBV infection, especially at this locus, where mutations might be incompatible with viral replication. Finally, our data suggest an association between the HBV-specific CTL response and viral clearance, and they have implications for the design of immunotherapeutic strategies to terminate HBV infection in chronically infected patients.

Mechanisms of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis.

The molecular and cellular mechanisms responsible for cytotoxic T lymphocyte (CTL)-induced immunopathology are not well defined. Using a model in which hepatitis B surface antigen (HBsAg)-specific CTL cause an acute necroinflammatory liver disease in HBsAg transgenic mice, we demonstrate that class I-restricted disease pathogenesis is an orderly, multistep process that involves direct as well as indirect consequences of CTL activation. It begins (step 1) almost immediately as a direct antigen-specific CTL-target cell interaction that triggers the HBsAg-positive hepatocyte to undergo programmed cell death (apoptosis). It progresses (step 2) within hours to a focal inflammatory response in which antigen-nonspecific lymphocytes and neutrophils amplify the local cytopathic effect of the CTL. The most destructive pathogenetic function of the CTL, however, is to secrete interferon gamma when they encounter antigen in vivo, thereby activating the intrahepatic macrophage and inducing a delayed-type hypersensitivity response (step 3) that destroys the liver and kills the mouse. We propose that the principles illustrated in this study are generally applicable to other models of class I-restricted, CTL-induced immunopathology, and we suggest that they contribute to the immunopathogenesis of viral hepatitis during hepatitis B virus infection in humans.

Molecular basis of the diversity of hepatitis B virus core-gene products.

All hepatitis B viruses examined to date code for at least two different core-gene products which are referred to as the c- and the e-protein. In the case of the human hepatitis B virus, they are known as the HBcAg and the HBeAg. Although these proteins share most of their primary amino acid sequence, they exhibit quite distinct properties. The e-protein is located in the cytoplasm and the nucleus of infected cells and very efficiently assembles into nucleocapsids. By contrast, the e-protein does not form particles. It enters the secretory pathway and is actively secreted by the cells. Here we describe the biosynthetic pathways by which the c- and e-proteins are expressed and summarize recent data from our laboratory showing that the antigenic and biophysical properties which distinguish the HBeAg from the HBcAg are primarily due to the 10 amino acid long portion of the HBeAg leader sequence that remains attached to the HBeAg after cleavage.

Formation of thermally stable derivatives of chlordiazepoxide and its desmethyl metabolite for GLC.

A derivatization procedure is described for the GLC determination of subnanogram amounts of chlordiazepoxide and nanogram amounts of N-desmethylchlordiazepoxide. Treatment with acetic anhydride at elevated temperature eliminates the highly polar and unstable nitrone group of these compounds by rearrangement and acetylation. The mass spectrometric fragmentation pattern of the acetyl derivatives is recorded and interpreted.

Biosynthesis of the secretory core protein of duck hepatitis B virus: intracellular transport, proteolytic processing, and membrane expression of the precore protein.

The biosynthesis of the secretory core gene product of the duck hepatitis B virus (DHBe protein) was examined. Recombinant vaccinia viruses were constructed encoding either the full-length or C-terminally truncated forms of the DHBe precursor protein (precore protein) and used to express these proteins in the human hepatoma cell line HepG2. Western immunoblot analysis of core gene products isolated from cells producing the full-length precore protein revealed the presence of DHBe precursor proteins containing the strongly basic C-terminal sequence which is lacking in the mature DHBe protein. These proteins were not secreted, suggesting that C-terminal proteolytic processing of the precore protein represents an obligatory step for DHBe biosynthesis. Pulse-chase experiments showed that this cleavage reaction occurs late during DHBe synthesis. Interestingly, when mutated precore proteins were expressed which lacked the basic C-terminal domain, proteins were produced which were glycosylated but not secreted. This shows that the transient presence of this region is essential for intracellular transport of the precore protein. Cell sorter analyses revealed that production of a cell surface-expressed variant of the secretory core protein is a feature conserved between the duck and the human hepatitis B viruses. Surprisingly, the C terminus of the membrane-expressed DHBe protein was accessible from the outside, showing that the topology of this interesting protein is more complicated than expected.

The secretory core protein of human hepatitis B virus is expressed on the cell surface.

Human hepatitis B virus (HBV) causes acute and chronic liver disease, which can result in tumor formation. An as yet unexplained phenomenon is that virus elimination usually correlates with the development of antibodies directed against the HBeAg, a secretory HBV core gene product which can be detected in the serum of infected patients. Expression of HBeAg in a human hepatoma cell line by using recombinant vaccinia viruses revealed that the HBeAg is not only secreted from HBeAg-producing cells but also incorporated into the outer cell membrane. No membrane-expressed core gene product could be detected when the cytoplasmic core protein (HBcAg) was expressed. Immune sera from patients who developed anti-HBe antibodies efficiently recognized the membrane-bound HBeAg, suggesting that surface-expressed HBeAg can serve as a target for an antibody-mediated elimination of HBV-infected cells.

Fatal poisoning with a plant protective containing monocrotophos, dodine and dinocap.

The toxicological findings after fatal poisoning with a plant protective containing monocrotophos, dodine and dinocap are described and discussed. While monocrotophos could be measured in all tissues and in blood 12 microgram/g, lung 13 microgram/g, brain 13 microgram/g, kidney 11 microgram/g, liver 1.8 microgram/g), measurable amounts of dodine (detection limit approx. 3 microgram/g) and dinocap (detection limit approx. 4 microgram/g) were not detected in these materials. The gastric contents contained 52 mg of monocrotophos, 7.5 mg of dodine and 20 mg of dinocap.

A micromethod for the isolation of drugs from blood using amberlite XAD-2.

The extraction of drugs from small blood samples (1 ml or less) for subsequent quantitative determination is described. Isolation was carried out by adsorption of the drugs to Amberlite XAD-2 resin utilizing a batch procedure that enabled the simultaneous extraction of up to 200 samples in approx. 5 hours. A new desorption technique yielded extracts of high purity that could be used directly for gas chromatographic or radioimmunological determinations, even if hemolyzed or putrid blood was to be examined. The following 26 substances were quantitated after addition to postmortem blood speciments at concentrations of 1-10 microgram/ml: tilidine, diphenhydramine, dibenzepine, imipramine, chlorpromazine, amphetamine, pentazocine, phenacetin, methaqualone, meprobamate, parathion, diazepam, digoxin, beta-methyldigoxin, carbromal, glutethimide, amobarbital, pentobarbital, cyclobarbital, phenobarbital, diphenylhydantoin, carbutamide, tolbutamide, glycodiazin, tolazamide and chlorpropamide. Thereby recoveries of 60-100% could be achieved. The reproducibility of the procedure was satisfactory as demonstrated by coefficients of variation of 3.7-8%.

The quaternary structure, antigenicity, and aggregational behavior of the secretory core protein of human hepatitis B virus are determined by its signal sequence.

Human hepatitis B virus encodes a secretory core protein, referred to as the HBe protein, whose secretion is mediated by the pre-C signal sequence. Here we examined whether this sequence is important only for translocation of the HBe precursor (the precore protein) or whether it also contributes to the structural and biophysical properties of the mature HBe protein. When a truncated hepatitis B virus precore protein, lacking the basic C-terminal domain which is cleaved from the wild-type protein during its conversion into HBe, was expressed in human hepatoma cells, only a small amount of HBe-like protein was produced. This protein was slightly smaller than the wild-type HBe protein, suggesting that C-terminal cleavage of the precore protein does not occur at the suggested site. When the authentic signal sequence of the precore protein (the pre-C sequence) was replaced by the unrelated signal sequence of an influenza virus hemagglutinin, not only the full-length but also the C-terminally truncated protein was expressed and secreted with high efficiency. Western blot (immunoblot) analyses with nonreducing gels and conformation-specific monoclonal antibodies revealed that the HBe protein secreted under control of the pre-C signal sequence was a monomer with HBe antigenicity, whereas the HBe-like protein secreted under control of the hemagglutinin signal sequence was a disulfide-bridge-linked dimer with both HBe and HBc antigenicity. Electron microscopic examination of gradient-purified particulate core gene products showed that HBe protein secreted under control of the hemagglutinin signal sequence forms core particles, whereas HBe protein secreted under control of the pre-C sequence does not. Thus, the pre-C sequence not only mediates the secretion but also determines the structural and aggregational properties of the HBe protein.

Analysis of the earliest steps of hepadnavirus replication: genome repair after infectious entry into hepatocytes does not depend on viral polymerase activity.

Hepadnaviruses contain a relaxed circular DNA genome (RC-DNA) with discontinuities in both strands. Upon infectious entry into a host cell, this genome is converted into a covalently closed superhelical form (CCC-DNA), which later serves as the template for transcription. Here we examined whether the viral polymerase activity is required for this repair reaction. Primary hepatocytes prepared from embryonated duck eggs were infected with the duck hepatitis B virus. Conversion of the RC-DNA into the CCC-DNA was then analyzed by a newly developed polymerase chain reaction technique. This method allows the efficient discrimination between the two DNA forms and is sensitive enough to monitor repair of the infecting viral DNA in the absence of replication and amplification. Thus, we were able to monitor this process in the presence of a potent inhibitor of the viral polymerase, the nucleoside analog 2',3'-dideoxyguanosine. The data show that inhibition of the viral polymerase activity has no influence on genome repair, suggesting that this enzymatic function is not required for conversion of the RC-DNA into the CCC-DNA. Consequently, antiviral drugs blocking the polymerase activity cannot prevent the infectious entry of the virus into a host cell.

Suicide by an overdosage of N-propylajmalinium bitartrate.

The toxicological findings after suicidal poisoning with N-propylajmalinium bitartrate (NPAB) are presented. For isolation of NPAB the biological material was homogenized and the drug was isolated by adsorption on Amberlite XAD-2. After column chromatographic purification on Sephadex LH-20 quantitative determinations were carried out by gas chromatography of the trifluoroacetate. The identity of the material finally obtained was checked by various chromatographic and spectrometric methods. The following concentrations of NPAB were found: liver 58 microgram/g, kidney 32 microgram/g, brain 16 microgram/g, muscle less than 10 microgram/g, heart less than 5 microgram/g, blood less than 5 microgram/g, gastric contents 600 mg (total). 1200 mg of NPAB had been swallowed; thus the amount of NPAB, that had crossed into blood, was approx. 500-600 mg corresponding to a dose of 9-11 mg/kg body weight.

The hepatitis-B viruses: molecular biology and recent tissue culture systems.

In this report we summarize what is known about the molecular biology of the hepatitis-B viruses. In the first part we describe the general properties of these viruses, their structure and their replication strategy. In the second part we discuss the most recent attempts at the establishment of tissue culture systems allowing the study of the virus/host cell interactions in vitro. In addition we present experimental data from our laboratory in which we show that new synthesis of viral proteins can be studied in vitro either by biochemical analysis of already infected cells or after experimental infection.

Synthesis and encapsidation of duck hepatitis B virus reverse transcriptase do not require formation of core-polymerase fusion proteins.

The expression strategy of the duck hepatitis B virus (DHBV) P gene, which is assumed to encode the viral reverse transcriptase, was investigated by mutational analysis. This study showed that P gene expression starts in the region where the P gene overlaps the viral core gene. However, in contrast to retroviral reverse transcriptases, which are expressed via gag-pol fusion protein intermediates, the DHBV P gene product was found to be synthesized starting at a P gene ATG codon. The resulting protein can complement polymerase-negative mutants in trans and can reverse transcribe viral pregenomic RNA that does not encode an active polymerase. These findings raise the question of how reverse transcription of cellular RNAs can be avoided in infected cells.

A cysteine and a hydrophobic sequence in the noncleaved portion of the pre-C leader peptide determine the biophysical properties of the secretory core protein (HBe protein) of human hepatitis B virus.

The molecular basis of the biophysical and antigenic differences between the cellular core protein (HBc protein) and the secreted core protein (HBe protein) of human hepatitis B virus was examined. The data show that the properties which distinguish the HBe protein from the HBc protein are due mostly to the 10-amino-acid portion of the HBe leader sequence which remains attached to the HBe protein after cleavage. A cysteine located within this region determines the quaternary structure and the antigenicity of the HBe protein. If this cysteine is lacking, the HBe protein, which is predominantly a monomer with only HBe antigenicity, is expressed as a disulfide-linked homodimer showing both HBe and HBc antigenicity. However, dimerization of the HBe protein was found to be neither sufficient nor required for particle formation. In fact, aggregation of the HBe protein was found to be inhibited by the strongly hydrophobic tripeptide Trp-Leu-Trp, which is also located in the noncleaved portion of the signal sequence. If this tripeptide was converted into either Asp-Asn-Asn or Ala-Asp-Leu, the HBe protein assembled into particles, independent of the presence of the cysteine.

[Frequency of positive diazepam-screening in post-mortem examinations (author's transl)]. / Häufigkeit positiver Diazepam-Befunde bei Sektionsfällen.

The post-mortem blood specimens of 389 forensic autopsies were analyzed for diazepam. The age of the cases investigated was above 10 years and the survival time was less than 12 hours. Eighteen samples corresponding to 4.6% were found to be diazepam-positive. These 18 samples were distributed equally between men and women. The proportion of diazepam-positive samples was increased in the groups of suicide and poisoning (alcohol and opiates). The association between diazepam intake and poisoning was statistically highly significant. No correlation was found between diazepam intake and age. Alcohol was found to occur significantly more often in the group of the diazepam positive cases as compared to the diazepam negative group.

The duck hepatitis B virus pre-C region encodes a signal sequence which is essential for synthesis and secretion of processed core proteins but not for virus formation.

Analysis of the serum of duck hepatitis B virus (DHBV)-infected ducks has revealed the presence of C-terminally truncated viral core proteins (e antigens). These proteins are glycosylated and therefore were not released from infected cells by lysis but rather by active secretion, indicating that the DHBV core protein can be synthesized alternatively as a cytoplasmic or a secretory protein. Transient expression of cloned wild-type DHBV DNA and of a specifically designed viral mutant in a human hepatoma cell line (Hep-G2) showed that the DHBV core gene promoter is active in differentiated human liver cells and that synthesis and secretion of the processed core proteins are dependent on the expression of the pre-C region, a small open reading frame which precedes the core gene. In addition, these experiments showed that the mechanism of core protein processing and secretion is conserved between DHBV and the human hepatitis B virus and therefore might be important for the hepatitis B virus life cycle in general. In spite of this, intrahepatic injection of the pre-C mutant into uninfected ducks resulted in viremia without concomitant e-antigen synthesis, indicating that virus formation is independent of pre-C expression.

Uptake of duck hepatitis B virus into hepatocytes occurs by endocytosis but does not require passage of the virus through an acidic intracellular compartment.

The infectious entry pathway of duck hepatitis B virus (DHBV) was investigated with primary duck hepatocytes. Virus uptake was measured by a selective PCR technique which allows for the detection of a successful infection without the need for viral replication or gene expression. To test whether DHBV uptake occurs by endocytosis, the effects of energy depletion were analyzed. The requirement for an acidic intracellular pH was tested with the lysosomotropic agent ammonium chloride. The data show that energy depletion prevents the uptake of DHBV into primary hepatocytes whereas ammonium chloride has no effect. From these data, we conclude that DHBV is taken up by its host cells by endocytosis. However, in contrast to that of most other enveloped viruses, escape of DHBV from the endocytotic route does not depend on an acidic intracellular compartment.

Hepatitis B virus nucleic acids associated with human peripheral blood mononuclear cells do not originate from replicating virus.

There have been numerous reports suggesting that human peripheral blood mononuclear cells (PBMCs) can be productively infected with human hepatitis B virus (HBV). We therefore examined whether the PBMCs can be used to establish an in vitro infection system for HBV. Freshly purified PBMCs were incubated with HBV with or without mitogen stimulation. Successful infection was tested using a newly developed PCR method that can differentiate between the relaxed circular (RC) DNA of the virus inoculum and the covalently closed circular (CCC) DNA which is formed only after successful virus entry. This method enables virus uptake to be proven even if the infection is abortive because there is no gene expression because of the lack of liver specific gene expression factors. All attempts to detect CCC DNA after incubation of PBMCs with HBV failed. On the contrary, CCC DNA could easily be detected in infected liver or after in vitro infection of primary human hepatocytes. Because this result appeared to be contradictory to the published data, we analyzed PMBCs isolated from infected patients. We could confirm that HBV DNA and RNA are associated with these cells. However, even after restimulation with mitogens, we could only detect RC DNA. Moreover, we could also demonstrate that viral RNA is present in free virus. Apparently, a certain amount of defective particles do not reverse transcribe the packaged pregenomic RNA. In summary we found no evidence that PMBCs can be infected with HBV and conclude that all previous observations can be explained by adsorbed virus.