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Anti-HBc, HBeAg and DNApolymerase activity in healthy HBsAg carriers and patients with inflammatory liver diseases

ZusammenfassungIn der vorliegenden Arbeit wird über anti-HBc-Titer-Bestimmungen, HBeAg, DNApolymerase-Aktivität im Serum sowie über intrazellulär nachweisbares HBcAg bei gesunden HBsAg-Trägern und Patienten mit HBsAg-positiven entzündlichen Lebererkrankungen berichtet.32/44 Patienten mit akuter Virus-B-Hepatitis hatten keine anti-HBc-Titer-Erhöhung in der ersten Krankheitswoche. Bei gesunden HBsAg-Trägern schwanken die anti-HBc-Titer zwischen 1:10 und 1:32000 (mittlerer Titer 1:4000). Bei HBsAg-positiven chronisch-aktiven Hepatitiden fand sich ein mittlerer Titer zwischen 1:32000 bis 1:64000, bei HBsAg-positiver CPH ein mittlerer Titer von 1:16000. Alle Patienten mit autoimmuner CAH (HBsAg-negativ) hatten anti-HBc-Titer unter 1:10.In einer Gruppe von 71 asymptomatischen HBsAg-Trägern konnten immunhistologisch bei keinem der gesunden HBsAG-Träger HBcAg im Leberzellkern nachgewiesen werden. Im Gegensatz dazu wurde HBcAg bei 4/5 HBsAg-positiven CAH- und 6/9 CPH-Patienten gefunden.Eine Erhöhung der DNApolymerase-Aktivität wurde bei keinem der gesunden HBsAg-Träger nachgewiesen. Von den 44 HBsAg-positiven akuten Virus-B-Hepatitiden hatten nur 3 Fälle eine Erhöhung der DNApolymerase-Aktivität. Andererseits fand sich eine DNApolymerase-Aktivität bei 17/37 Patienten mit HBsAg-positiver CAH und 9/15 Fällen mit CPH.Die Untersuchungen konnten eine enge Korrelation zwischen dem Nachweis des HBeAg im Serum und der DNApolymerase-Aktivität zeigen.Die Befundmuster ermöglichen eine klare Abgrenzung der “gesunden” HBsAg-Träger von den HBsAg-Trägern mit entzündlichen Lebererkrankungen.SummaryIn this paper we report on anti-HBc-titers, HBeAg, DNApolymerase activity in the serum and intracellular HBsAg in healthy HBsAg-carriers and patients with HBsAg-positive inflammatory liver diseases.32/44 patients with acure virus-B-hepatitis were negative for anti-HBc in the first week of the disease. Anti-HBc-titers in healthy HBsAg-carriers varied between 1:10 and 1:32,000 (medium titer 1:4,000). In HBsAg-positive CAH we found a medium titer between 1:32,000 and 1:64,000, in cases with CPH of about 1:16,000. All autoimmune type CAH showed anti-HBc-titers less than 1:10.By immunofluorescence we could demonstrate in a group of 71 asymptomatic HBsAg-carriers in none of the healthy HBsAg-carriers HBcAg in the liver cell nuclei. In contrast HBcAg could only be found in 4/5 HBsAg positive CAH- and 6/9 CPH patients.No elevated DNApolymerase activity could be demonstrated in healthy HBsAg-carriers. Out of 44 patients with virus-B-hepatitis only 3 showed elevated DNApolymerase activity. On the other hand DNApolymerase elevation was demonstrable in 17/37 cases with CAH and 9/15 with CPH.The investigations showed a strong correlation between the demonstration of HBeAg in the serum and the DNApolymerase activity.The characteristic findungs enabled us to differentiate between “healthy” HBsAg-carriers and HBsAg-carriers with inflammatory liver diseases.

Inhibition of hepatitis-B-virus DNA polymerase by phosphonoformate: Studies on its mode of action

Phosphonoformate (PFA) and phosphonoacetate (PAA) were tested for their ability to inhibit the hepatitis‐B‐virus associated DNA polymerase. The HBV DNA polymerase was inhibited by 100 μM/liter PFA 50% while it was highly resistant to PAA. The inhibition of the Dane particle‐associated DNA polymerase by PFA was not competitive to substrates and not affected by changes in the magnesium concentration. PFA was active also after initation of the DNA polymerase reaction. Competition studies revealed that PFA had a higher affinity to a proposed pyrophosphate binding site than PAA or—alternatively—that both compounds bind to different sites.

The clinical relevance of the antibody to hepatitis B core antigen (anti-HBc): A review

The antibody against the core component of the Dane particle (anti-HBc) is generally detected in the sera of individuals with acute type B hepatitis and in chronic HBsAg carriers. While the serological demonstration of HbsAg with or without anti-HBc indicates continued replication of viral antigens, the co-occurrence of anti-HBs and anti-HBc is considered a marker of recent HBV replication. The demonstration of anti-HBc in the absence of HBsAg and anti-HBs is in agreement with at least four different states of HBV infection. As this pattern indicates persistent HBV infection in some cases and recovery from an acute type B hepatitis in others, current efforts focus on further characterization of this pattern, using additional test methods such as anti-HBe and anti-HBc of the IgM class.

Comparison of indicators for dane particles.

ZusammenfassungDas Dane Partikel wird als das komplette Hepatitis B Virus angesehen. Seren mit einer großen Zahl von Dane Partikeln gelten als hoch infektiös. In der vorliegenden Studie haben wir die Höhe der DNA Polymerase Aktivität — gemessen in 20fach Dane Partikel Konzentraten — mit dem HBsAg, dem HBeAg und dem anti-HBc Titer verglichen. Unsere Untersuchungen zeigen, daß der HBeAg Titer ein semiquantitatives Maß für die Zahl zirkulierender Dane Partikel darstellt. Die HBsAg Konzentration steigt ebenfalls mit der Zahl im Serum zirkulierender Dane Partikel an und stellt ebenso — wenn in HBeAg positiven Seren gemessen — ein semiquantitatives Maß für die Zahl zirkulierender Dane Partikel dar. Zwischen dem anti-HBc Titer und dem Serum Dane Partikel Gehalt besteht keine Beziehung.SummaryThe Dane particle is — because of its characteristics — believed to be the complete hepatitis-Bvirus. Sera containing high numbers of Dane particles were shown to be highly infectious. In the present study we related the HBeAg-, the HBsAg- and the anti-HBc titer to the level of DNA polymerase activity measured in 20 fold Dane particle concentrates. The data obtained indicate that the HBeAg concentration gives a semiquantitative estimate on the number of circulating Dane particles. Mean DNA polymerase activity was found to increase with HBsAg concentration and is therefore also of value-if determined in a HBeAg positive serum- for quantitation of Dane particles. The anti-HBc titer was found to be unrelated to the number of circulating Dane particles.

Inhibition of hepatitis B virus specific DNA polymerase by intercalating agents

Intercalating agents, some of them in clinical use, were tested for their ability to inhibit the hepatitis B virus specific DNA polymerase reaction. Ethidium bromide was shown to be the strongest inhibitor among the compounds tested. Compounds in clinical use inhibited the DNA polymerase test only at high concentrations. The inhibitory activity of all compounds tested was increased when the MgCl2 content in the reaction mixture was lowered. UV absorption studies presented no evidence that this effect was due to complex formation of magnesium and the individual compounds. The therapeutic significance of these findings is not certain and needs further work.

Etiology of Hepatitis B Surface Antigen (HBsAg)-Negative Chronic Hepatitis

A study was undertaken to elucidate the etiology of HBsAg-negative chronic hepatitis. Form 37 individuals with HBsAg-negative chronic hepatitis, 11 had liver membrane autoantibody (LMA) and were thus classified as autoimmune. 6 patients had anti-HBc, 1 of which was also positive for LMA. The majority of individuals with HBsAg-negative chronic hepatitis had antibodies to hepatitis A antigen (anti-HAV), in general at low titer. We conclude from our data that hepatitis A and hepatitis B virus infections are unlikely to play a significant role in inducing or maintaining HBs-Ag-negative chronic hepatitis. The etiological role of non-A non-B hepatitis agent(s) is difficult to estimate and must await the detection of appropriate markers for type non-A non-B hepatitis.

Quantitation of HBeAG and anti-HBe by RIA in sera of chronic HBsAG carriers and individuals with type B hepatitis.

SummaryThis paper gives the results for HBeAg and anti-HBe titers in chronic HBsAg carriers and patients with type B hepatitis using a “solid-phase” radioimmunoassay.In tumor and hemodialysis patients the HBeAg titers are statistically significant higher compared to the group of HBsAg positive CAH or CPH.High anti-HBe titers are a characteristic finding in “healthy” HBsAg carriers. On the other hand, there is a subgroup of HBsAg positive CAH with anti-HBe; although there are signs of an ongoing virus B replication these cases of CAH proceed sometimes to cirrhosis.ZusammenfassungIn dieser Arbeit werden die Ergebnisse der HBeAg und anti-HBe-Titer Bestimmungen mit einem „solid-phase“-Radioimmunoassay dargestellt.Bei Tumor- und Hämodialysepatienten sind die HBeAg-Titer statistisch signifikant höher als bei Patienten mit HBsAg positiver CAH oder CPH.Hohe anti-HBe Titer sind ein charakteristischer Befund für „gesunde“ HBsAg-Träger. Andererseits gibt es eine Untergruppe der HBsAg positiven CAH mit anti-HBe; obgleich bei dieser Form eine CAH keine Zeichen einer fortbestehenden Virus B-Replikation nachgewiesen werden kann, schreitet die Krankheit manchmal in Richtung auf eine Zirrhose fort.

Significance of Antibody to Hepatitis Be Antigen (Anti‐HBe) in HBsAg‐Negative Individuals

Abstract. A study was initiated to assess the significance of antibody to hepatitis B e antigen (anti‐HBe) in HBsAg‐negative individuals. Anti‐HBe was demonstrated in the majority of sera positive for anti‐HBs and anti‐HBc. All sera positive for anti‐HBs and negative for anti‐HBc and most sera positive for anti‐HBc but negative for anti‐HBs were anti‐HBe‐negative. This implies that the antibody response to HBeAg is of shorter duration than that to HBsAg and HBcAg. Anti‐HBe may help to discriminate between various states of hepatitis B virus infection found to be associated with anti‐HBc‐positive but HBsAg‐ and anti‐HBs‐negative sera. 2 individuals were anti‐HBe‐positive but HBsAg‐, anti‐HBs‐ and anti‐HBc‐negative, this finding is not understood.

Hepatitis-B Virus-associated Deoxyribonucleic Acid Polymerase: A Partial Characterization by the Use of Chemical Agents* **

SummaryThe very limited coding capacity of the HBV-DNA led us to study the nature of the HBV (Dane particle) — associated DNA polymerase. The HBV-associated DNA polymerase met in many respects the characteristics of the repair enzyme of the host: the DNA polymerase beta. It operates under high salt conditions, and exhibits similar salt effects with NaCl, KCL, and PO43−. It is insensitive to sulf-hydryl group blockers, such as p-hydroxymercuribenzoate and N-ethylmaleimide, is resistant to phosphonoacetic acid, and not inhibited by 5 mol/l urea. It requires a divalent cation (Mg2+) for activity, the Mg2+ concentration revealing optimal activity is somewhat higher than that described for most DNA polymerases beta. The HBV-associated DNA polymerase differs also from most DNA polymerases beta in its sensitivity to ddTTP and its optimal pH. The fact that DNA polymerases beta of different origin vary considerably in their response to chemical agents and that the DNA polymerase beta from human liver has not been studied allows no definite conclusion as to the nature of the Dane particle-associated DNA polymerase.ZusammenfassungDie HBV-DNA erscheint zu klein, um für alle Hepatitis-B-Virusantigene zu kodieren. Wir haben deshalb die Eigenschaften der HBV-DNA-Polymerase mit denen der zellulären DNA-Polymerasen verglichen. Die HBV-assoziierte DNA-Polymerase ähnelt in vielen der untersuchten Eigenschaften der zellulären DNA-Polymerase beta. Zur Entwicklung optimaler Aktivität benötigt die HBV-DNA-Polymerase ein zweiwertiges Kation (Mg2+) und hohe Salzkonzentrationen, sie verhält sich in der Gegenwart von Sulfhydryl-Gruppen-Blockern, Phosphonoacetat und 5 mol/l Urea wie die zelluläre DNA-Polymerase beta. Die HBV-DNA-Polymerase unterscheidet sich jedoch von der DNA-Polymerase beta in ihrem optimalem pH und der Empfindlichkeit gegenüber ddTTP. Da die DNA-Polymerase von humaner Leber bisher nicht untersucht wurde und sich die DNA-Polymerases verschiedenen Spezies zum Teil deutlich unterscheiden, erlauben die hier beschriebenen Untersuchungen keine endgültigen Rückschlüsse, ob die HBV-assoziierte DNA-Polymerase Virus- oder Wirts-kodiert ist.

Follow-Up of Anti-HBc Titers in Healthy HBsAg Carriers and Patients with Chronic Inflammatory Liver Diseases

Sera of 85 asymptomatic HBsAg carriers found among blood donors were tested for anti-HBc titers and were retested 4 years later. The results were correlated with the histological findings of first and final biopsy. None of 75 HBsAg carriers with normal or minimally changed liver tissue, 71 of them anti-HBe positive, developed chronic inflammatory liver disease. 4 HBsAg carriers eliminated HBsAg from the serum after a 1-to 3-year HBsAg-carrier state and 2 developed antibody against HBsAg in the sequel. In 65 of 75 cases we found unchanged anti-HBc titers. The geometrical mean titer (GMT) was 1:7,800 in the first and 1:7,000 in the final examination with a range of 1:400 and 1:25,600 in the group of HBsAg carriers with normal liver, and was 1:14,200 and 1:10,300, respectively, with a range of 1:800 and 1:51,200 in HBsAg carriers with minimal changes. In both groups the decrease of anti-HBc concentration within 4 years was not significant. The group of 10 HBsAg carriers with chronic hepatitis did not differ from healthy HBsAg carriers in respect to anti-HBc titers. Anti-HBc titers varied between 1:6,400 and 1:25,600, the GMT was 1:13,700 and 1:12,800, respectively. It is speculated that in healthy HBsAg carriers shedding of serologically undetectable quantities of complete and/or defective HBcAg from liver cell nuclei which contain HBcAg not detectable by immunofluorescence maintain the production of anti-HBc.