Klin. Wschr. 53, 1069- 1074 (1975) - © by Springer-Verlag 1975

The Diagnostic Significance of Intrahepatocellular HepatitisB-Surface-Antigen (HB Ag), Hepatitis-B-Core-Antigen (HBcAg) and IgG for the Classification of Inflammatory Liver Diseases* (Studies on HB~Ag-positive and -negative patients) W. Arnold, K.H. Meyer zum Btischenfelde, G. Hess, and J. Knolle II. Med. Univ.-Klinik der Johannes Gutenberg-Universitfit Mainz (Direktor: Prof. Dr. reed. P. Sch61merich)

Die diagnostische Bedeutung yon intrahepatocelluliirem Hepatitis-B-surface-Antigen (HBsAg), Hepatitis-Bcore-Antigen (HB~Ag) und IgG fiir die Klassifizierung yon entziindliehen Lebererkrankungen. ( Untersuchungen an HB~Ag-positiven und-negativen Patienten.) Zusammenfassung. Wir untersuchten mit der direkten Immunfluorescenztechnik die Leberbiopsien yon Patienten mit entz/indlichen Lebererkrankungen auf intracellulfires HB~Ag, HB~Ag und IgG. Die Untersuchungen haben fotgende Ergebnisse erbracht: 1. Klinisch gesunde HB~Ag-Trfiger hatten in den meisten FS.11en HB~Ag im Cytoptasma, jedoch niemals HB~Ag in den Leberzellkernen. 2. Patienten mit HBsAg-positiver akuter Hepatitis hatten in der Fr/ihphase HB~Ag und/oder HB~Ag in den Hepatocyten. Unsere Befunde sprechen bei normalem Verlauf ffir eine komplette Immunelimination der HB-Komponenten zu verschiedenen Stadien der Erkrankung mit v611iger Wiederhersteltung. 3. Kontrollpunktate 1 Jahr nach Beginn einer ttB~Ag-positiven Hepatitis zeigten bei v611ig gesunden Patienten kein HB~Ag oder HBcAg. Bei zwei Patienten ohne Immunelimination von HB~Ag entwickelte sich innerhalb eines Jahres eine chronisch aktive Hepatitis. Diese Patienten hatten HB~Ag in den Leberzellkernen. 4. Patienten mit HB,Ag-positiver CAH und Zeichen entzfindlicher Aktivitfit hatten HB~Ag in den Zellkernen und in geringem Prozentsatz HB~Ag im Cytoplasma. HB~Ag-negative FS.11e zeigten niemals HB-Komponenten im Gewebe. 5. In keinem Fall einer HB~Ag-positiven und -negativen CAH in Remission lieB sich HB,Ag und HBrAg in Hepatocyten nachweisen. 6. In den meisten F/illen yon HB~Ag-positiver AH und CAtt, die HB~Ag-positiv waren, fand sich in den gleichen Zellkernen IgG. Die strenge Koinzidenz yon HB~Ag und IgG 1/iBt an das Vorliegen yon anti-HB~ in den Zellkernen denken. Die Bedeutung dieses Antik6rpers in den Zellkernen fiir den weiteren Verlauf der Erkrankung ist unbekannt. Schliisselw6rter : Hepatitis-B-core-Antigen, Hepatitis-B-surfaceAntigen, akute Hepatitis, chronisch aktive Hepatitis, gesunde ttB~Ag-Trfiger, Leberbiopsie, fluorescierende Antik6rpertechnik. Summary. Liver biopsies of patients with inflammatory liver diseases and clinically healthy ItB~Ag-carriers were examined for * This work was supported by the Deutsche Forschungsgemeinschaft, °Sonderforschungsbereich' 107 and Ar t06/1, Me 244/8 and 9, Mainz.

presence of intracellular HB~Ag, HBcAg and IgG by direct immunofluorescence. The studies revealed the following results : 1. In most cases healthy HBsAg-carriers had HBsAg in the cytoplasm, but they did never show HBcAg in the nuclei of hepatocytes. 2. In the early phase some patients with HB~Ag-positive acute hepatitis had HBcAg and/or HB,Ag in their hepatocytes. In a normal course with complete recovery the immunoelimination may clear either phenomenon at variable stages of the disease. 3. Cases one year after complete recovery of acute virus Bhepatitis had no HB-components in their liver tissue. 2 cases without immunoelimination of HB~Ag developed chronic active hepatitis within one year and had HB~Ag in their liver cell nuclei. 4. Patients with HBsAg-positive CAH and highly inflammatory activity had HBcAg in the nuclei and a low percentage of cells with HBsAg in the cytoplasm of hepatocytes. HB~g-negative cases with CAH never had HB-components in their tissue. 5. Patients with HBsAg-positive and -negative CAH in complete remission never had HB~Ag and HBsAg in their hepatocytes. 6. Most cases with HB~Ag-positive acute hepatitis and chronic active hepatitis positive for HB~Ag had also IgG in the same liver cell nuclei. The coincidence of this finding gives strong evidence for the presence ofanti-HB,.in these liver cell nuclei. The importance of this finding for the course of the disease is unknown. Key words: Hepatitis-B-core-antigen, hepatitis-B-surfaceantigen, acute hepatitis, chronic active hepatitis, healthy HB~Agcarriers, liver biopsy, fluorescent antibody technique.

Introduction Using autoimmune markers and HBsAg in the serum it is possible to distinguish two main groups of chronic inflammatory liver diseases. The first group predominantly consists of female patients who have autoimmune phenomena and frequently HL-A 8. The second group is HBsAg-positive in the serum, negative for autoimmune markers and has no higher incidence of HL-A 8 as compared to control groups (Doniach, 1974; Meyer zum Bfischenfelde et al., 1975). The latter group of chronic active hepatitis frequently develops directly out of a virus B-hepatitis with persistance of HBsAg (Nielsen etal., t971 and own unpublished data).

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W. Arnold etal. : Intrahepatocellular Hepatitis-B-Antigens

T h e d e m o n s t r a t i o n o f H B ~ A g a n d H B ~ A g in liver biopsies with the fluorescent antibody technique and/ or by electronmicroscopy allowed the further characterization of HB~Ag-positive courses of liver diseases ( A l m e i d a e t al., 1969; E d g i n g t o n a n d R i t t , 1970; N o w o s l a w s k i e t a l . , 1970; A k e y a m a e t a l . , 1972; B r z o s k o e t a l . , 1972; H u a n g e t a l . , 1974; G u d a t e t a l . , 1975). The pathogenetic importance of these phenomena for t h e c o u r s e o f t h e d i s e a s e s h o w e v e r is still u n known.

We therefore examined defined groups of patients for the occurrence of HB~Ag, HB~Ag and IgG in their hepatocytes.

2. Methods 2. t. Serum examination

Blood for biochemical and serological tests was drawn at the time of biopsy and examined on the same day. The presence of HB~Ag was tested by immunodiffusion, counter-immunoelectrophoresis, complement-fixation and by radioimmunoassay. Anti-HB~ was tested by counter-immunoelectrophoresis and radioimmunoassay (b). 2.2. Liver biopsies

Liver tissue was obtained by percutaneous biopsy or by laparoscopy. All biopsy specimens were examined by conventional histology (a) and by direct immunofluorescent technique. 2.3. Preparation o f tissue material

Patients and Methods 1. Patients 1.1. Healthy HB~Ag carriers, n = 29

Atl patients had no evidence of inflammatory liver disease by biochemical tests and in their liver histology.

2.3.1. Cryostat sections. 7 gm cryostat sections were obtained after rapid freezing of the biopsy material in liquid nitrogen. After air-drying the sections were used either unfixed or fixed in acetone for 1 min. 2.3.2. Isolated hepatocytes. The mechanical isolation of hepatocytes was performed in 1 ml EMBA (Eagle's medium/Serva plus 2.5% bovine serum albmnine/Behringwerke, Marburg) at 37°C according to the method described by Hopf etal. (1974). Liven" cells were smeared on slides and air-dried before incubation with FITC-labetled antiserum.

1.2. Acute hepatitis ( A H ) , n=41

This group consisted of 36 patients with HB~Ag-positive hepatitis and 5 patients with HB~Ag-negative hepatitis. In all cases SGPTvalues were higher than 300 mU/ml.

1.3. One year after recovery o f HB~Ag-positive acute hepatitis, n : 19

None of the 19 patients hat any signs of inflammatory liver diseases on biochemical testing and on histologic examination of their liver biopsy specimens. Anti-HBs could be demonstrated by radioimmunoassay in the serum of each patient.

1.4. Chronic active hepatitis ( CAH), n = 15

All patients had evidence of a highly active inflammatory liver disease by biochemical testing and in their biopsies (elevated values for SGOT, SGPT; according to morphological definition typical findings of a chronic aggressive hepatitis). 11 patients were tlB~Agpositive and two of these had developed CAH out of a biopsy-proven acute virus-B-hepatitis within one year. 4 patients were HB,Ag-negative in the serum. 7 of the HB~Ag-positive and all patients with HB~Ag-negative CAH received immunosuppressive therapy (azathioprine 2 mg/kg/day and prednisone 10 mg/day).

1.5. C A H in complete remission (either spontaneously or under immunosuppression), n =5

2.4. hnmunofluorescent studies

Intracellular localization of HBsAg, HBcAg and IgG in tissue sections or smears of hepatocytes was investigated by a direct immunofluorescent technique. For these studies the following FITC-conjugates were used: 2.4.1. Human anti-HbcFITC-conjugate, mol F/P-ratio 3.2, dilution 1:20 (original protein content 5.0 mg/ml), CF-titer 1:800 and a self-prepared human anti-HBcAg-FITC-conjugate, mol F/P-ratio 2.2 with the same dilution as above (purification according to Arnold and v. Mayersbach, 1972). The first conjugate was kindly provided by Dr. R.H. Purcell, NIH, Bethesda/Maryland and served as a reference for the further investigations. 2.4.2. Human anti-HBs-FITC-conjugate, mol F/P-ratio 2.8, protein content 5.0 mg/ml; titer in the radioimmunoassay 1:4096 (b). 2.4.3. Self-prepared rabbit-anti-human-lgG-, -anti-IgM-, -antiIgA-, and -anfi-flic-FITC-conjugates. Mol F/P-ratio between 2.2 and 2.8, protein content 5.0 mg/ml. Purification as described above. 2.4.4. Incubations were carried out for 30 min at 37°C in a humid chamber. The preparations were washed three times in PBS and mounted in glycerine-PBS. 2.4.5. Specifity controls: All studies were performed by direct immunofluorescent technique. Incubations with normal human or rabbit-IgG-FITC and blocking by preincubation with the unlabelled antisera therefore were used as specifity controls, 2.4.6. Consecutive tissue sections were examined in the following order: anti-HB~-FITC, anti-HB~-FITC and anti-IgG-FITC.

2 of the 5 cases were HBsAg-positive in the serum.

3. Results 1.6. 8 patients with metabolic liver diseases served as controls

4 patients had fatty liver, the other 4 alcoholic liver cirrhosis. All were negative for HBsAg in their serum.

T h e results a r e s u m m a r i z e d in T a b l e 1: H B s A g c o u l d b e d e t e c t e d in all b i o p s i e s o f H B ~ A g - p o s i t i v e

W. Arnold et at. : Intrahepatocetlular Hepatitis-B-Antigens

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Table 1. Summary of the results obtained for HB~Agin the serum, HB~Ag, HB,Ag and IgG in hepatocytes of patients with inflammatory and metabolic liver diseases Diagnosis

N

HB~Ag serum

Inflammatory activity

HB~Ag (tissue)

HBcAg (tissue)

IgG (tissue, isol. hepatoc.)

nucleus

cytoplasma

nucleus

cytoplasma

nucleus

cytoplasma

Healthy HB~Ag-carriers

29

+

0

0

21

0

0

0

0

AH- (HBsAg-neg.)

5

0

+

0

0

0

0

0

0

AH- (HBsAg-pos.)

36

+

+

0

11

6

0

5

0

AH- (UB,Ag-pos.) 1 year after onset of disease

19

0

0

0

0

0

0

0

0

CAH HB~Agpos. CAH HB~Agneg.

11 ~ 4

+ 0

++ ++

0 0

6 0

10 0

0 0

10 0

0 0

HB~Ag pos. and neg. CAH (inactive)

5

0/+

0

0

0

0

0

0

0

Metabolic liver disease

8

0

0

0

0

0

0

0

0

2 cases out of HB~Agpositive acute hepatitis one year after onset of the disease.

patients at a variable percentage. On the other hand, HBcAg in liver cell nuclei could only be demonstrated in HB,Ag-positive acute hepatitis (AH) and in HB~Agpositive chronic active hepatitis (CAH) with signs of inflammatory activity.

5 of the 6 HBcAg-positive patients had also IgG in the same liver cell nuclei (Fig. 1 b, 2 b, c). Investigations for intracellular IgM and IgA with the fluorescent antibody technique yielded negative results. In the 5 patients with HB~Ag-negative acute hepatitis no detectable HB~Ag, HBcAg and/or IgG were observed in their hepatocytes.

3.1. Healthy HB~Ag-carriers Heatthv HB~Ag-carriers had HB~Ag in the cytoplasm of liver cells in 21 out of 29 cases. The percentage of positive cells varied considerably between 0% and 70%. The HB~Ag-positive hepatocytes sometimes were arranged in clusters but more often as single cells. In 8 cases we were unable to demonstrate HBsAg in the investigated biopsy material. HBcAg and intracellular IgG could not be demonstrated in the nuclei or cytoplasm of liver cells in these patients.

3.2. Acute hepatitis In the early phase of HB~Ag-positive acute hepatitis we found cytoplasmic HB~Ag in I I out of 36 cases. The percentage of HB~Ag-positive cells varied between 20% and 50%. HB~Ag could never be detected in nuclei of hepatocytes. 6 out of the 36 patients had intranuclear HBcAg in their biopsy specimens (Fig. 1a). HBdkg could never be demonstrated in the cytoplasm of cryostat sections smears of isolated hepatocytes.

3.3. Recovery after acute virus B-hepatitis HBea,g, HBcAg and IgG could not be detected in the biopsy specimens of 19 patients who had recovered completely from a HB~Ag-positive acute hepatitis. All 19 patients were negative for HBsAg but showed anti HBs in their serum. Chronic active hepatitis developed within one year after acute virus B-hepatitis in two cases with HBsAgpersistance (Table t). In either case biopsies were examined during acute hepatitis and one year later: In either case HBcAg and IgG in liver cell nuclei were found on both instances.

3.4. Chronic active hepatitis 6 out of 11 cases with HBsAg-positive CAH had cytoplasmic HBsAg and 10 of the 11 patients showed intranuclear HBcAg. All these cases also had IgG in the same cell nuclei. The percentage of HBcAg and

(a)

(b)

Fig. 1 a and b. Immunofluorescent demonstration of HB~Ag and IgG in nuclei of hepatocytes from a patient with HB~Ag-positive chronic active hepatitis. Unfixed cryostat section, (a) incubation with human anti-HBc-FITC. Magnification x 250. (b) incubation with rabbit-anti-human-TgG, magnification × 250

/b)

(a)

(c)

Fig. 2. (a) Demonstration of intranuclear ~]~BcAg in isolated hepatocytes from a patient with HBsAg-positive chronic active hepatitis. Magnification × 540. (b, c) Immunofluoreseent demonstration of intranuclear IgG in isolated hepatocytes. Magnification x 400

W. Arnold et al. : IntrahepatocelIular Hepatitis-B-Antigens

IgG positive nuclei was more than 50% in every case. In 1 patient however who had 90% of his nuclei positive for HB~Ag, only 50% of the nuclei showed a positive staining with anti-IgG. The coincidence of HB~Ag and HB~Ag in the same tissue specimen could only be detected in two cases. The 4 patients with HB~Ag-negative CAH had no detectable HB~Ag, HBcAg or IgG in their hepatocytes. None of the patients with HB~Ag-positive CAH and IgG in their liver cell nuclei had antinuclear antibodies (ANA) in their serum. In two patients with HB~Ag-negative CAH and ANA in the serum no IgG was present in the nuclei of hepatocytes. 3.5. Chronic active hepatitis in complete remission

HB~Ag, HB~Ag and/or IgG in the hepatocytes could not be seen in HB~Ag-positive and -negative cases with CAH in complete remission and in patients with metabolic liver diseases.

4. Discussion

4.1. Expression of HB~Ag and HB~Ag in hepatocytes

The expression of HB~Ag and HBcAg in liver tissue was already described in previous reports (Brzosko et al., 1973; Hadziyannis et al., 1973a, b; Ten Kate et al., 1974; Gudat et al., 1975). These findings are corroborated by our results. The examination of biochemically and histologically defined groups of diseases enable us however to add some further knowledge on the expression of HBcAg and HB~Ag in hepatocytes: HB~Ag in the cytoplasm of hepatocytes frequently occurs in clinically healthy HB~Ag-carriers. The lack of cytoplasmic ItB~Ag in one biopsy specimen however does not exclude its presence in other parts of the liver.This assumption is supported by the scattered occurrence of HB~Ag in the liver. Healthy HB~Ag-carriers have no HBcAg. Asymptomatic HB~Ag-carriers however with HB~Ag in the nuclei and ttB~Ag in the cytoplasm of hepatocytes had chronic active or chronic persistent hepatitis on histological examination and abnormalities in their biochemical tests. Therefore these cases were excluded out of the group of the healthy HB~Ag-carriers in this study. Our findings in healthy HB~Ag-carriers confirm the data reported by Stein et al. (1971, 1972) and Gerber et aL (1974a), but are in contrast to others (Caramia et aL, 1972).

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Recently Gudat et al. (1975) were able to demonstrate HB~Ag in one out of 3 cases of acute HB~Agpositive hepatitis but did not detect HB~Ag in liver tissue. On the basis of these findings they classified liver diseases with this type of expression of HBcomponents in liver tissue as "elimination type". Our cases of acute HB~Ag-positive hepatitis however had demonstable cytoplasmic HB~Ag and intranuclear HBcAg in frequencies of 31% and 17%, respectively. The dissimilar findings may be explained by a different timing of the histological examinations. It is remarkable that our cases, whether or not HBAg-components were present in hepatocytes, showed an uneventful course of virus B-hepatitis with complete recovery. Our findings support the contention that both components may be present in all cases of HB~Ag-positive acute hepatitis in the very early phase of the disease. The immunoelimination may clear either phenomenon to variable time intervals during the course of the disease. The functional status of the immune system on the humoral (rise of anti-HB~, Hoofnagle et al., 1973) and cellular level (Gerber et al., 1974b; Meyer zum Bfischenfelde et al., 1975) may be responsible for these differences. Our findings in the 19 patients one year after recovery from acute virus B-hepatitis support this conclusion. In contrast, two patients without immunoelimination of HB~Ag developed chronic active hepatitis within one year. The latter findings and the results in HB~Ag-positive chronic active hepatitis are in agreement with those reported by Gudat et al. (1975). In the present study no differences were found for the hepatocellular expression of HBcAg, HB~Ag and intranuclear IgG whether or not the patients were treated by immunosuppressive therapy. Our findings suggest that the expression of HBAg-components in liver tissue depends on the inflammatory' activity of the liver disease only. In HB~Ag-negative chronic active hepatitis HBAgcomponents were never detected in hepatocytes. These findings support the existence of at least two different subgroups of CAH (Doniach, 1974; Meyer zum Bfischenfelde et al., 1975).

4.2. HB~Ng and IgG in liver ceil nuclei

It has been pointed out that IgG might occur in nuclei of patients with HB~Ag-positive liver diseases (Nielsen and Elling, 1971; Hadziyannis et al., 1973 a, b; Ten Kate et al., 1974). The direct technique of immunofluorescence permitted us to demonstrate HBdX~g and IgG in the same hepatocellular nuclei in

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W.Arnold etaL: Intrahepatoceltular Hepatitis-3-Antigens

acute virus B-hepatitis and HBsAg-positive CAH. On the other hand, nuclei without HBcAg were never positive for IgG. The coincidental occurrence of ttBcAg and IgG in the same cell nuclei is a strong evidence that IgG represents anti-HBc. Until now the question remains unresolved when and how this antibody is attached to HB~Ag in the nuclei of hepatocytes. Furthermore, the importance of these findings for the disease itself and for its outcome has to be determined. We are grateful to Miss E. Frerick for excellent technical assistance. We are indepted to Prof. Dr. O. Klinge, Institute for Pathology, University Wfirzburg for the histological examination of the biopsies (a), to Dr. W. Gerlich, Hygiene-Institut, University of G6ttingen for the determination of HB,Ag and anti-HBs by radioimmunoassay (b), to Dr. H. Bitz, Red Cross Blood Center, Bad Kreuznach for the determination of HB~Ag by complement-fixation and for kindly providing anti-HBs-serum (c), and to Dr. R.It. Purcell, National Institute of Health Bethesda/Maryland for the human-anti-HB~FITC-conjugate which served as a reference for our studies (d).

References Akeyama, T., Kamada, T., Koizumi, T., Abe, H. : The localization of the Australia-antigen in the liver by immunofluorescence. J. clin. Path. 25, 1071 (1972) Almeida, J.D., Zuckerman, A.J., Taylor, P.E.: Immune electron microscopy of the Australia-SH (serum hepatitis) antigen. Microbios 1, 117 (1969) Arnold, W., Mayersbach, H. v. : Changes in the solubility of immunoglobulines after fluorescent labelling and its influence on immunofluorescent techniques. J. Histochem. Cytochem. 12, 975 (1972) Brzosko, W.J., Madalinski, K., Krawczynski, K., Nowoslawski, A. : Duality of hepatitis B antigen and its antibody. I. Immunofluorescence studies. J. Infect. Dis. 127, 424 (1973) Caramia, F., De Bac, C., Ricci, G. : Virus-like particles within hepatocytes of Australia-antigen carriers. Am. J. Dis. Child. 123, 309 (1972) Doniach, D. : Liver autoimmunity. Current Titles in Immunology, Transplantation and Allergy 2, 1 (1974) Edgington, T.S., Ritt, D.J. : Intrahepatic expression of serum hepatitis virus associated antigen. J. exp. Med. 134, 871 (1971) Gerber, M.A., Hadziyannis, S., Vissoulis, C., Schaffner, F., Raronetto, E., Popper, H. : Hepatitis B antigen: Nature and distribu-

tion of cytoplasmatic antigen in hepatocytes of carriers. Proc. Soc. exp. med. and biol. 145, 863 (1974) Gerber, MJ., Phuangsab, D., Sudtn, M.S., Vittal, B.V., Dourdourekas, D., Steigmann, F., Clowdns, B.F. : Cell-mediated immune response to hepatitis B-antigen in patients with liver diseases. Digestive Diseases 19, 637 (1974) Gudat, F., Bianchi, L., Sonnabend, W., Thiel, G., Aenishaenshin, W., Stalder, G.A. : Pattern of core and surface expression in liver tissue reflects state of specific immune response in hepatitis B. Lab. Invest. 32, 1 (1975) Hadziyannis, S., Gerber, M.A., Vissoulis, C., Popper, H. : Cytoplasmatic hepatitis B antigen in "ground glass" hepatocytes of carriers. Arch. Path. 96, 327 (1973) Hadziyannis, S., Moussouros, A., Giustozi, A., Almeida, J. : Immunofluorescence of the "core" antigen in the liver (Abstr.). Digestion 8, 66 (1973) Hoofnagte, J.G., Gerety, R.J., Barker, L.F. : Antibody to hepatitis B-virus core in man. Lancet II, 869 (1973) Hunag, S.N., Groh, V., Beaudoin, J.G., Dauphinee, W.D., Guttmann, R.D., Morehouse, D.D., Aronoff, A., Gault, H. : A study of the relationship of virus-like particles and Australia antigen in liver. Human Pathology 5, 209 (1974) Meyer zum Btischenfelde, K.H., Alberti, A., Arnold, W., Freudenberg, J. : Organ-specifity and diagnostic value of cell-mediated immunity against a liver specific membrane protein-studies in hepatic and non-hepatic diseases. Klin. Wschr. 53, 1061 (1975) Nowoslawski, A., Brzosko, W.J., Madalinski, K., Krawzynski, K. : Cellular localisation of Australia-antigen in the liver of patients with lymphoproliferative disorders. Lancet I, 494 (1970) Nielsen, J.O., Dietrichson, O., Elling, P., Christoffersen, P. : Incidence and meaning of persistence of Australia-antigen in patients with acute viral hepatitis : development of chronic hepatitis. New Engl. J. Med. 285, 1157 (1971) Nielsen, J.O., Elling, P. : Investigations of liver biopsies for Australia-antigen by immunofluorescent technique. Clin. exp. hnmunol. 9, 699 (1971) Stein, O., Fainaru, M., Stein, Y. : Virus like particles in cytoplasm of livers of Au-antigen carriers. Lancet II, 90 (1971) Stein, O., Feinaru, M., Stein, Y. : Virus-like particles in the cytoplasm of the livers of Australia antigen carriers. Am. J. Dis. Child. 123, 313 (1972) Ten Kate, F.J.W., Feltkamp-Vroom, T., Helder, A.W., Roos, C.M., Van Blankenstein, M. : Demonstration of HB-antigens in liver tissue (Abstr.). Digestion 10, 305 (1974) Dr. W. Arnold II. Medizinische Klinik der Universit/it D-6500 Mainz Langenbeckstr. 1 Federal Republic of Germany

The diagnostic significance of intrahepatocellular hepatitis-B-surface-antigen (HBsAg), hepatitis-B-core-antigen (HBcAg) and IgG for the classification of inflammatory liver diseases. (Studies on HBsAg-positive and -negative patients).

Liver biopsies of patients with inflammatory liver diseases and clinically healthy HBsAg-carriers were examined for presence of intracellular HBsAg, H...
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