2O8
GaslroenterologiaJaponica Vol. 10, No. 3. --1975~
- - O r i g i n a l Article--
HBs-Ag, HBc-Ag AND VIRUS-LIKE PARTICLES IN LIVER TISSUE Seiichi F U R U T A , Atsuo N A G A T A , K e n d o K I Y O S A W A , Toshihiro T A K A H A S H I , Yoshihiro A K A H A N E , Yuriko K O I K E , Takeshi S A H A R A , Yoshihiro I I J I M A , Kenichi F U R U K A W A , T o m o y u k i W A T A N A B E , Masako H A R A , Akihiko O M O R I a n d Masayuki O D A
Second Department of Internal Medicine, Faculty of Medicine, Shinshu University, Matsumoto
Summary Hepatitis B surface antigen (HBs-Ag) arLd hepatitis B core antigen (HBc-Ag) in hepatic tissue of 3 cases with various liver diseases were investigated by immunofluorescent method. Virus-like particles were demonstrated by electron microscopy in the nuclei of these 3 cases. The localization of HBs-Ag was restricted in cytoplasma or on the surface of hepatoeyte, while HBc-Ag was almost in the hepatocytic nuclei. However, there was unexplainable discrepancy between the distribution of HBs-Ag and that of HBc-Ag, as more the former in number and less the latter, of hepatocytes. The size of virus-like particles in nuclei was 22-27 nm in diameter. Most of them were hollow, but some of them were wholly electron dense. Their distribution was various from case to case. Discussion was made on the correlation between the presence of Hepatitis B virus and HBs-Ag or HBc-Ag.
Key Words~ hepatitis B surface antigen, hepatitis B core antigen, immunofluorescent studies, virus-like particle, electronmicroscopic studies, liver disease Introduction T h e r e have been conflicting reports concerning the localization of H B - A g in the liver tissue, as some observed it p r e d o m i n a n t l y in cytoplasma but others in nuclei of hepatocytes or in both 1-7~. I n 1971 A l m e i d a a n d her colleagues 8) reported H B - A g as two antigen-antibody systems. T h e one is a surface antigen (HBs-Ag) and a n t i b o d y (anti-HBs) a n d the other is a core antigen of D a n e particle (HBc-Ag) and antib o d y (anti-HBc), however there are a few reports demonstrating HBs-Ag a n d H B c - A g independently in a single liver tissue. 9-1~ Virus-like particles have been observed by m a n y investigators in hepatocytic nuclei of hepatitis B virus infected liver 2~ tl-20) The antigenicity of these particles have been d o c u m e n t e d identical to that of H B c - A g 21).
Barker et alY 2) observed virus-like particles of 22-28 n m in size electronmicroscopically in hepatocytic nuclei of chimpanzees, experimentally infected with H B V , a n d consequently demonstrated H B c - A g in nuclei a n d HBs-Ag on the surface of hepatocytes in the single liver tissue by immunofluorescent technique. I n this paper, three cases with various liver diseases are reported, in which HBs-Ag in cytoplasma a n d H B c - A g a n d virus-like particles in nuclei of hepatocytes are demonstrated independently.
Patients and Methods Case 1 : Y.A. T h i r t y seven year-old house wife. H e r serum was found HBs-Ag positive on routine check up for the blood donation in June, 1974. She was a s y m p t o m a t i c a n d the physical examination a n d laboratory studies
HBs-Ag, HBc-Ag and Virus-like Portides
on her first visit to our clinic revealed all negative. She underwent liver biopsy on August 2, 1974, which showed non-specific reactive hepatitis histologically (Fig. 1). H e r antigenemia persisted until the latest check up in January, 1975. Case 2: H . T . Thirty nine year-old male revealed the positive HBs-Ag on blood donation on J u n e 17, 1974. His physical examination was non-contributory except for the palpable (1/2 f.b. below the costal margin), soft and non-tender liver. Subsequent blood chemistry was within normal range on J u l y 3, 1974, although slight elevation of S G O T and SGPT to 55 and 82 respectively on the second check-up on J u l y 26, 1974. H e underwent liver biopsy on three occasions, that is July 15, September 20, and December 3, 1974. The histological findings of each biopsy were as follows; the first was non-specific reactive hepatitis, the second acute viral hepatitis and the third persistent hepatitis (Fig. 2). His antigenemia has been persistent. Case 3: Y.Y. Fifty three year-old house wife was transferred to our Dept. with the chief complaints of anasarca and generalised weakness on November 25, 1974. She had been enjoying her life until August, 1974, when she developed generalised malaise and fever up to 38~ accompanying leukocytosis of 23,300 with differential count of 66% atypical cells. She admitted to a private
Fig. 1. Liver histology of case 1. A few cell infiltration in portal area, identical to nonspecific reactive hepatitis. (H.E. stain, x 100)
209
hospital because of subequent ascites and malaise on October 12, 1974. The serum HBs-Ag was positive on her admission to the hospital. H e r general status was deteriorated since the transference to our Dept. and expired on J a n u a r y 7, 1975. The autopsy specimen of the liver revealed diffuse fibrosis without any findings of cirrhosis or malignancy (Fig. 3). Detection of HBs-Ag, anti-I-IBs and anti-HBc in serum : Detection of HBs-Ag and anti-HBc in serum were carried out by immunoadherence haemeagglutination method and that of anti-HBs by passive haemagglutination method 23). Demonstration of HBs-Ag and HBc-Ag in the liver tissue:
Fig. 2. Liver histology cf case 2. A few periportal and intraparenchymal cell infiltration and Kupffer cell mobilization are observed. (H.E. • 100)
Fig. 3. Liver histology of case 3. Periportal fibrosis with scarce cell infiltration. (H.E. • 100)
210
S. F U R U T A E T AL.
The tissue were obtained by needle biopsy or at autopsy. One part of the liver specimen was frozen at - - 7 0 ~ in dryice aceton-hexan and processed to cryostat section of 4 ~z. Anti-HBs serum of h u m a n source, which was negative for anti-HBc, was labelled with F I T C and was applied to the direct immunofluorescent studies of HBs-Ag as previously reported6h Indirect immunofluorescent technique was applied to demonstrate the HBc-Ag in the liver tissue. The primary sera with anti-HBc but no anti-HBs were obtained from patients with persistent HBsAg carriers. Specificity of the immunofluorescence for HBs-Ag was ascertained by adsorption with positive and negative HBs-Ag sera, and by blocking test of the hepatic tissue. HBc-Ag specificity was confirmed by control stainings as follows; instead of primary serum with anti-HBc, normal serum with negative HBs-Ag, anti-HBs nor anti-HBc was used in the procedures of the above described indirect immunofluorescent technique. As another control staining, the normal liver obtained from gastric ulcer patient with no history of liver disease or blood transfusion was proprocessed to the same indirect immunofluorescent technique. These control stainings revealed negative for specific fluorescence. Electron microscopic studies: The remaining part of the liver specimen was fixed with 2.5% glutar-aldehyde and 1% osmium tetroxide serially and embeded in E P O N 812. Ultrathin sections were double stained with lead citrate and uranyl acetate, subsequently underwent electron microscopic study with Hitachi 11 A EM.
Fig. 4-a. HBs-Ag in case i. Diffuse-membranous localization of HBs-Ag on the surface of hepat0cytes. (•
Fig. 4-b. ttBc-Ag in case 1. Three hepatocytic nuclei show a specific fluorescence for HBc-Ag. ( • 3oo)
Results Fig. 4 a - f show the specific immunofluorescence for HBs-Ag and HBc-Ag in hepatic tissues of 3 patients. I n case 1, HBs-Ag was observed around the membrane of hepatocytes diffusely in lobules. In case 2 and 3, granular fluorescence of HBs-Ag was distributed in hepatocytic cytoplasma. No nuclear fluorescence of HBs-Ag was observed in any cases.
Fig. 4-c. HBs-Ag in case 2.
Diffuse intracyt0plasmic localization of HBs-Ag in almost all hepatocytes. No nuclear fluorescence. (•
HBs-Ag, HBc-Ag and Virus-like Portieles
211
Fig. 4-d. HBc-Ag in case 2. Many hepatocytic nuclei show a specific fluorescence for HBc-Ag, but no cytoplasmic fluorescence is demonstrated. ( X 300)
Fig. 5. Electron microscopical findings in case 1. Viruslike particles are observed in cluster in the nucleus of a hepatocyte. ( x 100,000)
Fig. 4-e. HBs-Ag in case 3.
Fig. 6.
Fig. 4-f. HBc-Ag in case 3.
Fig. 7. Electron microscopical findings in case 3. Thickly packed virus-like particles in a hepatocytic nucleus. ( x 100,000)
Fine granular HBsAg in cytoplasma of many hepatocytes. (•
Many hepatocytic nuclei show a specific fluorescence for HBc-Ag. (x
300)
Electron microscopical findings in case 2. Virus-like particles are sparsely distributed in the nucleus of a hepatocyte. ( x 100,000)
212
S. F U R U T A E T AL.
I n contrast to HBs-Ag, HBc-Ag was quite restricted in the nucleus except for a few hepatocytes where perinuclear cytoplasmic localization of HBc-Ag was found. Hepatocytes with nuclear HBc-Ag were scarce in case 1 in spite of diffuse distribution of HBs-Ag in the liver tissue. In case 2 and 3, m a n y hepatocytic nuclei revealed HBc-Ag, although HBc-Ag fluorescence was a little lesser in n u m b e r than that of HBs-Ag. HBc-Ag in most of the nuclei showed diffuse granular distribution, but in some they were loose and speckled. T h e size of virus-like particles observed in hepatocytic nuclei under electronmicroscopy was 22-27 n m in diameter. Some of them were wholly electron dense, but most of them were hollow. No double-shelled large particles (Dane particle) were found. The distribution of these particles was various in character from case to case. In case 1 (Fig. 5), only two hepatocytes a m o n g those studied showed intranuclear particles. These particles were in cluster in one nucleus and scarcely and randomly distributed in another nucleus. In case 2 (Fig. 6), very few but scattered viruslike particles were seen in several hepatocytic nuclei. Virus-like particles in case 3 were abundantly packed in nuclei of approximately 48% of hepatocytes (Fig. 7). Only a few of similar particles were detected in peri-nuclear cytoplasma in the third case, but none in case 1 and 2. Discussion
In 1970, prior to Almeida et al. s), Nowoslowski and his colleagues 2) reported the duality of the localization of HB-Ag in hepatocytes. N a m e l y one is predominantly in the nucleus and the other in the cytoplasma. However, the precise character of the duality was not clarified at that time. They observed, in addition, virus-like particles of approximately 20 n m in diameter in nuclei of hepatocytes of the patients with carrier state and chronic active haptitis. T h e morphological appearance of the pariticles they observed was quite similar
to that we observed in our cases except for a little smaller in size than that observed here. Almeida suggested that the core particles which were released as inner component after treatment of Dane particles with Tween 80 were identical to the naked particles of 27 nm in diameter under electron microscopy in nuclei of H B V infected hepatocytes. As reported in previous paper 6~, we were not able to see any HB-Ag in hepatocytic nuclei by direct immunofluorescent method. This m a y come from that the anti-serum used for FITC-labelling had anti-HBs alone but no anti-HBc. O n the other hand anti-serum for HBc-Ag which was used for indirect immunofluorescent study of HBc-Ag in this paper contained no anti-HBs. O u r observation in the present paper confirmed the cytoplasmic localization of HBs-Ag and nuclear localization of HBc-Ag in the liver tissue and moreover the presence of viruslike particles in hepatocytic nuclei. The identity of the antigenicity of the inner component of Dane particles and HB-Ag purified from intranuclear virus-like particles has been documented by immune-electron microscopic study21 ) We believe that HB-Ag observed in the nuclei by several investigators were identical to HBc-Ag, as demonstrated here. Recently Hirshman et al. 24) isolated intranuclear particles from hepatocytes of HBV infected liver tissue and identified DNA in the particles. Furthermore, Robinson and his colleagues 25) have detected DNA-polymerase activity in the core prepared from Dane particle in the h u m a n sera. These findings suggest that H B V m a y be consisted of HBc-Ag at least in some part, if not HBcAg itself, and also suggest that H B V may be identical to D N A virus. In spite of these facts, it is a intrigueing issue to be discussed why we see the discrepancy between increased HBs-Ag with rare HBc-Ag in a single liver tissue. HBcAg positive nuclei of hepatocytes of asympto-
HBs-Ag, HBe-Ag and Virus-like Particles
matic HBs-Ag carrier are rarely observed i n spite of diffuse d i s t r i b u t i o n of HBs-Ag positive hepatocytes. O n the contrary, there are a b u n d a n t n u c l e a r HBc-Ag i n cases with chronic active hepatitis, as G e r b e r et al. reported 26~. T h e y suggested the following posibilities. H B V infection i n carrier state may result i n little virus replication i n the nucleus, whereas a b u n d a n t f o r m a t i o n of virally coded p r o t e i n i n the cytoplasma of the hepatocyte. W h i l e i n chronic hepatitis i n t r a n u c l e a r virus-like particles m a y b e c o m e d o m i n a n t with the c o n c o m i t a n t decrease of virally coded material i n hepatocytic cytoplasma. Some genetic or e n v i r o n m e n t a l factors m a y play a role for these events. Moreover, it is n o t clear w h e t h e r all of these virus-like particles c o n t a i n D N A or not. Since some particles are electron dense wholly but others are hollow i n the center. Eventually, the exact relationship b e t w e e n the presence of H B V a n d i n t r a n u c l e a r viruslike particles or synthesis of HBs-Ag in the liver tissue r e m a i n s to the future study.
Acknowledgement We are deeply grateful to Dr. M. M a y u m i for his k i n d l y co-operation for the detection of anti-HBc i n sera.
References 1) Millman, I., Zavatone, V., Gerstley, B.J.S. and Blumberg, B.S.: Australia antigen detected in the nuclei of liver ceils of patients with viral hepatitis by the fluorescent antibody technique. Nature 222: 181-184, 1969. 2) Nowoslawski, A., Brzosko, W.J., Madalinski, K. and Krawczynski, K. : Cellular localization of Australia antigen in the liver of patients with lymphoproliferative disorders. Lancet 1: 494~ 1970. 3) Nielsen, J.O. and Elling, P. : Investigations of liver biopsies for Australia antigen by immuuofluorescent technique. Clin. Exp. Immunol. 9: 699-705, 1971. 4) Edgington, T.S. and Ritt, D.J. : Intrahepatic expression of serum hepatitis virus associated antigen. J. Exp. Med. 134: 871-885, 1971. 5) Hadziyannis, S., Moussouros, A., Vissoulis, C. and Afroudakis, A. : Cytoplasmic localization of Australia antigen in the liver. Lancet i : 976-979, 1972. 6) Furuta, S., Hanaoka, S., Omori, A., Chiba, K., Tsukioka, J., Nagata, A., Takahashi, T., Kiyosawa,
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K., Akahane, Y. and Oda, M. : Australia antigen in serum and liver tissue of patients with various liver diseases. Kanzo 13: 280-287, 1972. 7) Mfiller, R., Maess, J. und Deicher, H.: Fluoreszenzserologischer Nachweis yon Australia-antlgen in der Leber. Dtsch. Med. Wschr. 98: 93-99, 1973. 8) Almeida, J.D., Bubenstein, D. and Scott, E.J.: New antigen-antibody system in Australia-antigenpositive hepatitis. Lancet 11: 1224-1227, 1971. 9) Hadziyannis, S., Moussouros, A., Giustozi, A. and Almeida, J.: Immunofluorescence study of the "core" antigen in the liver. Digestion 8: 437, 1973. 10) Shikata, T., Yoshizaki, C., Matsushita, H., Toyokawa, H. and Ishida, N. : Studies on the duality of intrahepatocytic HB-Ag by immunofluorescent antibody technique. Kanzo 15: 695, 1974. 11) Nelson, J.M., Barker, L.F. and Danovitch, S.H.: Intranuclear aggregates in the liver of a patient with serum hepatitis. Lancet 11: 773-774, 1970. 12) Huang, S.: Hepatitis-associated antigen hepatitis: An electron microscopic study of virus-like particles in liver cells. Amer. J. Pathoh 64: 483-500, 1971. 13) Dunn, A.E.G., Peter, R.L., Schweltzer, I.L. and Spears, R.L. : Virus-like particles in livers of infants with vertically transmitted hepatitis. Arch. Pathol. 94: 258-264, 1972. 14) Scotto, J., Homberg, J.C. and Caroli, J.: Electron microscopy studies of severe virus hepatitis. Cytoplasmic inclusions, virus-like particles, Australia antigen. Amer. J. Dis. Child. 123: 311-312, 1972. 15) Campion, E.C., Ludbrook, J., McLeod, J.M., Marshall, V.R., Mukherjee, T. and Wangel, A.G. : Primary hepatoma and hepatitis-assoclated antigen in a young white woman. Brit. Med. J. 4: 149-150, 1972. 16) Caramia, F., DeBac, C. and Ricci, G.: Virus-like particles within hepatocytes of Australia antigen carriers. Amer. J. Dis. Child. 123: 309-311, 1972. 17) Hirschman, S.Z., Gerber, M. and Garfinkel, E.: Purification of naked intranuclear particles from human liver infected by hepatitis B virus. Proc. Nat. Acad. Sci. USA. 71: 3345-3349, 1974. 18) Tapp, E., Jones, D.M., Anfield, C., Whittaker, J.S., Woolf, I.L. and Dymock, J.W.: Intranuclear particles in hepatocytes of HB-Ag positive blooddonors. Lancet I: 934, 1974. 19) Yamada, G., Kobayashi, T., Ohta, Y. and Kosaka, K. : I-IB-Agassociated particles in the sera and the liver of asymptomatie carriers. J. Clin. Electron Microscopy. 7: 368-369, 1974. 20) Kamimura, T., Murayama, H., Sasaki, H. and Ichida, F.: Electron microscopic studies on hepatocytes of asymptomatic carriers of hepatitis B antigen. ibid. 7: 370-371, 1974. 21) Barker, L.F., Almeida, J.D., Hoofnagle, J.It., Gerety, R.T., Jackson, D.R. and McGrath, P.P.: Hepatitis B core antigen: Immunology and electron microscopy. J. Virology. 14: 1552-1558, 1974. 22) Barker, L.F., Chisari, F.V., McGrath, P.P., Dalgard,
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D.W., Kirschstein, R.L., Almeida, J.D., Edgington, T.S., Sharp, D.G. and Peterson, M.R. : Transmission of type B viral hepatitis to chimpanzees. J. Infect. Dis. 127: 648-662, 1973. 23) Mayumi, M., Okochi, K. and Nishioka, K. : Detection of Australia antigen by means of immune adherence haemagglutination test. Vox Sang.: 20 178-181, 1971. 24) Hirschman, S.Z., Gerber, M. and Garfinkel, E.: DNA purified from naked intranuclear particles of
human liver infected with hepatitis B virus. Nature. 251 : 540-542, 1974. 25) Robinson, W.S. and Greenman, R.L. : DNA polymerase in the core of the h u m a n hepatitis B virus candidate. J. Virology. 13: 1231-1236, 1974. 26) Gerber, M.A., Hadziyannis, S., Vissoulis, C., Schffner, F., Paronetto, F. and Popper, H. : Hepatitis B antigen: Nature and distribution of cytoplasmic antigen in hepatocytes of carriers. Proc. Soc. Exp. Biol. Med. 145: 863-867, 1974.
Received April 18, 1975 Accepted May 19, 1975 Address requests for reprints to: Seiichi Furuta, M.D., The Second Department of Internal Medicine, Faculty of Medicine, Shinshu University, 3-1-1 Asahi-cho, Matsumoto, Nagano Prefecture, 390 Japan
GaslroenterologiaJaponica Vol. 10, No. 3. --1975~
- - O r i g i n a l Article--
HBs-Ag, HBc-Ag AND VIRUS-LIKE PARTICLES IN LIVER TISSUE Seiichi F U R U T A , Atsuo N A G A T A , K e n d o K I Y O S A W A , Toshihiro T A K A H A S H I , Yoshihiro A K A H A N E , Yuriko K O I K E , Takeshi S A H A R A , Yoshihiro I I J I M A , Kenichi F U R U K A W A , T o m o y u k i W A T A N A B E , Masako H A R A , Akihiko O M O R I a n d Masayuki O D A
Second Department of Internal Medicine, Faculty of Medicine, Shinshu University, Matsumoto
Summary Hepatitis B surface antigen (HBs-Ag) arLd hepatitis B core antigen (HBc-Ag) in hepatic tissue of 3 cases with various liver diseases were investigated by immunofluorescent method. Virus-like particles were demonstrated by electron microscopy in the nuclei of these 3 cases. The localization of HBs-Ag was restricted in cytoplasma or on the surface of hepatoeyte, while HBc-Ag was almost in the hepatocytic nuclei. However, there was unexplainable discrepancy between the distribution of HBs-Ag and that of HBc-Ag, as more the former in number and less the latter, of hepatocytes. The size of virus-like particles in nuclei was 22-27 nm in diameter. Most of them were hollow, but some of them were wholly electron dense. Their distribution was various from case to case. Discussion was made on the correlation between the presence of Hepatitis B virus and HBs-Ag or HBc-Ag.
Key Words~ hepatitis B surface antigen, hepatitis B core antigen, immunofluorescent studies, virus-like particle, electronmicroscopic studies, liver disease Introduction T h e r e have been conflicting reports concerning the localization of H B - A g in the liver tissue, as some observed it p r e d o m i n a n t l y in cytoplasma but others in nuclei of hepatocytes or in both 1-7~. I n 1971 A l m e i d a a n d her colleagues 8) reported H B - A g as two antigen-antibody systems. T h e one is a surface antigen (HBs-Ag) and a n t i b o d y (anti-HBs) a n d the other is a core antigen of D a n e particle (HBc-Ag) and antib o d y (anti-HBc), however there are a few reports demonstrating HBs-Ag a n d H B c - A g independently in a single liver tissue. 9-1~ Virus-like particles have been observed by m a n y investigators in hepatocytic nuclei of hepatitis B virus infected liver 2~ tl-20) The antigenicity of these particles have been d o c u m e n t e d identical to that of H B c - A g 21).
Barker et alY 2) observed virus-like particles of 22-28 n m in size electronmicroscopically in hepatocytic nuclei of chimpanzees, experimentally infected with H B V , a n d consequently demonstrated H B c - A g in nuclei a n d HBs-Ag on the surface of hepatocytes in the single liver tissue by immunofluorescent technique. I n this paper, three cases with various liver diseases are reported, in which HBs-Ag in cytoplasma a n d H B c - A g a n d virus-like particles in nuclei of hepatocytes are demonstrated independently.
Patients and Methods Case 1 : Y.A. T h i r t y seven year-old house wife. H e r serum was found HBs-Ag positive on routine check up for the blood donation in June, 1974. She was a s y m p t o m a t i c a n d the physical examination a n d laboratory studies
HBs-Ag, HBc-Ag and Virus-like Portides
on her first visit to our clinic revealed all negative. She underwent liver biopsy on August 2, 1974, which showed non-specific reactive hepatitis histologically (Fig. 1). H e r antigenemia persisted until the latest check up in January, 1975. Case 2: H . T . Thirty nine year-old male revealed the positive HBs-Ag on blood donation on J u n e 17, 1974. His physical examination was non-contributory except for the palpable (1/2 f.b. below the costal margin), soft and non-tender liver. Subsequent blood chemistry was within normal range on J u l y 3, 1974, although slight elevation of S G O T and SGPT to 55 and 82 respectively on the second check-up on J u l y 26, 1974. H e underwent liver biopsy on three occasions, that is July 15, September 20, and December 3, 1974. The histological findings of each biopsy were as follows; the first was non-specific reactive hepatitis, the second acute viral hepatitis and the third persistent hepatitis (Fig. 2). His antigenemia has been persistent. Case 3: Y.Y. Fifty three year-old house wife was transferred to our Dept. with the chief complaints of anasarca and generalised weakness on November 25, 1974. She had been enjoying her life until August, 1974, when she developed generalised malaise and fever up to 38~ accompanying leukocytosis of 23,300 with differential count of 66% atypical cells. She admitted to a private
Fig. 1. Liver histology of case 1. A few cell infiltration in portal area, identical to nonspecific reactive hepatitis. (H.E. stain, x 100)
209
hospital because of subequent ascites and malaise on October 12, 1974. The serum HBs-Ag was positive on her admission to the hospital. H e r general status was deteriorated since the transference to our Dept. and expired on J a n u a r y 7, 1975. The autopsy specimen of the liver revealed diffuse fibrosis without any findings of cirrhosis or malignancy (Fig. 3). Detection of HBs-Ag, anti-I-IBs and anti-HBc in serum : Detection of HBs-Ag and anti-HBc in serum were carried out by immunoadherence haemeagglutination method and that of anti-HBs by passive haemagglutination method 23). Demonstration of HBs-Ag and HBc-Ag in the liver tissue:
Fig. 2. Liver histology cf case 2. A few periportal and intraparenchymal cell infiltration and Kupffer cell mobilization are observed. (H.E. • 100)
Fig. 3. Liver histology of case 3. Periportal fibrosis with scarce cell infiltration. (H.E. • 100)
210
S. F U R U T A E T AL.
The tissue were obtained by needle biopsy or at autopsy. One part of the liver specimen was frozen at - - 7 0 ~ in dryice aceton-hexan and processed to cryostat section of 4 ~z. Anti-HBs serum of h u m a n source, which was negative for anti-HBc, was labelled with F I T C and was applied to the direct immunofluorescent studies of HBs-Ag as previously reported6h Indirect immunofluorescent technique was applied to demonstrate the HBc-Ag in the liver tissue. The primary sera with anti-HBc but no anti-HBs were obtained from patients with persistent HBsAg carriers. Specificity of the immunofluorescence for HBs-Ag was ascertained by adsorption with positive and negative HBs-Ag sera, and by blocking test of the hepatic tissue. HBc-Ag specificity was confirmed by control stainings as follows; instead of primary serum with anti-HBc, normal serum with negative HBs-Ag, anti-HBs nor anti-HBc was used in the procedures of the above described indirect immunofluorescent technique. As another control staining, the normal liver obtained from gastric ulcer patient with no history of liver disease or blood transfusion was proprocessed to the same indirect immunofluorescent technique. These control stainings revealed negative for specific fluorescence. Electron microscopic studies: The remaining part of the liver specimen was fixed with 2.5% glutar-aldehyde and 1% osmium tetroxide serially and embeded in E P O N 812. Ultrathin sections were double stained with lead citrate and uranyl acetate, subsequently underwent electron microscopic study with Hitachi 11 A EM.
Fig. 4-a. HBs-Ag in case i. Diffuse-membranous localization of HBs-Ag on the surface of hepat0cytes. (•
Fig. 4-b. ttBc-Ag in case 1. Three hepatocytic nuclei show a specific fluorescence for HBc-Ag. ( • 3oo)
Results Fig. 4 a - f show the specific immunofluorescence for HBs-Ag and HBc-Ag in hepatic tissues of 3 patients. I n case 1, HBs-Ag was observed around the membrane of hepatocytes diffusely in lobules. In case 2 and 3, granular fluorescence of HBs-Ag was distributed in hepatocytic cytoplasma. No nuclear fluorescence of HBs-Ag was observed in any cases.
Fig. 4-c. HBs-Ag in case 2.
Diffuse intracyt0plasmic localization of HBs-Ag in almost all hepatocytes. No nuclear fluorescence. (•
HBs-Ag, HBc-Ag and Virus-like Portieles
211
Fig. 4-d. HBc-Ag in case 2. Many hepatocytic nuclei show a specific fluorescence for HBc-Ag, but no cytoplasmic fluorescence is demonstrated. ( X 300)
Fig. 5. Electron microscopical findings in case 1. Viruslike particles are observed in cluster in the nucleus of a hepatocyte. ( x 100,000)
Fig. 4-e. HBs-Ag in case 3.
Fig. 6.
Fig. 4-f. HBc-Ag in case 3.
Fig. 7. Electron microscopical findings in case 3. Thickly packed virus-like particles in a hepatocytic nucleus. ( x 100,000)
Fine granular HBsAg in cytoplasma of many hepatocytes. (•
Many hepatocytic nuclei show a specific fluorescence for HBc-Ag. (x
300)
Electron microscopical findings in case 2. Virus-like particles are sparsely distributed in the nucleus of a hepatocyte. ( x 100,000)
212
S. F U R U T A E T AL.
I n contrast to HBs-Ag, HBc-Ag was quite restricted in the nucleus except for a few hepatocytes where perinuclear cytoplasmic localization of HBc-Ag was found. Hepatocytes with nuclear HBc-Ag were scarce in case 1 in spite of diffuse distribution of HBs-Ag in the liver tissue. In case 2 and 3, m a n y hepatocytic nuclei revealed HBc-Ag, although HBc-Ag fluorescence was a little lesser in n u m b e r than that of HBs-Ag. HBc-Ag in most of the nuclei showed diffuse granular distribution, but in some they were loose and speckled. T h e size of virus-like particles observed in hepatocytic nuclei under electronmicroscopy was 22-27 n m in diameter. Some of them were wholly electron dense, but most of them were hollow. No double-shelled large particles (Dane particle) were found. The distribution of these particles was various in character from case to case. In case 1 (Fig. 5), only two hepatocytes a m o n g those studied showed intranuclear particles. These particles were in cluster in one nucleus and scarcely and randomly distributed in another nucleus. In case 2 (Fig. 6), very few but scattered viruslike particles were seen in several hepatocytic nuclei. Virus-like particles in case 3 were abundantly packed in nuclei of approximately 48% of hepatocytes (Fig. 7). Only a few of similar particles were detected in peri-nuclear cytoplasma in the third case, but none in case 1 and 2. Discussion
In 1970, prior to Almeida et al. s), Nowoslowski and his colleagues 2) reported the duality of the localization of HB-Ag in hepatocytes. N a m e l y one is predominantly in the nucleus and the other in the cytoplasma. However, the precise character of the duality was not clarified at that time. They observed, in addition, virus-like particles of approximately 20 n m in diameter in nuclei of hepatocytes of the patients with carrier state and chronic active haptitis. T h e morphological appearance of the pariticles they observed was quite similar
to that we observed in our cases except for a little smaller in size than that observed here. Almeida suggested that the core particles which were released as inner component after treatment of Dane particles with Tween 80 were identical to the naked particles of 27 nm in diameter under electron microscopy in nuclei of H B V infected hepatocytes. As reported in previous paper 6~, we were not able to see any HB-Ag in hepatocytic nuclei by direct immunofluorescent method. This m a y come from that the anti-serum used for FITC-labelling had anti-HBs alone but no anti-HBc. O n the other hand anti-serum for HBc-Ag which was used for indirect immunofluorescent study of HBc-Ag in this paper contained no anti-HBs. O u r observation in the present paper confirmed the cytoplasmic localization of HBs-Ag and nuclear localization of HBc-Ag in the liver tissue and moreover the presence of viruslike particles in hepatocytic nuclei. The identity of the antigenicity of the inner component of Dane particles and HB-Ag purified from intranuclear virus-like particles has been documented by immune-electron microscopic study21 ) We believe that HB-Ag observed in the nuclei by several investigators were identical to HBc-Ag, as demonstrated here. Recently Hirshman et al. 24) isolated intranuclear particles from hepatocytes of HBV infected liver tissue and identified DNA in the particles. Furthermore, Robinson and his colleagues 25) have detected DNA-polymerase activity in the core prepared from Dane particle in the h u m a n sera. These findings suggest that H B V m a y be consisted of HBc-Ag at least in some part, if not HBcAg itself, and also suggest that H B V may be identical to D N A virus. In spite of these facts, it is a intrigueing issue to be discussed why we see the discrepancy between increased HBs-Ag with rare HBc-Ag in a single liver tissue. HBcAg positive nuclei of hepatocytes of asympto-
HBs-Ag, HBe-Ag and Virus-like Particles
matic HBs-Ag carrier are rarely observed i n spite of diffuse d i s t r i b u t i o n of HBs-Ag positive hepatocytes. O n the contrary, there are a b u n d a n t n u c l e a r HBc-Ag i n cases with chronic active hepatitis, as G e r b e r et al. reported 26~. T h e y suggested the following posibilities. H B V infection i n carrier state may result i n little virus replication i n the nucleus, whereas a b u n d a n t f o r m a t i o n of virally coded p r o t e i n i n the cytoplasma of the hepatocyte. W h i l e i n chronic hepatitis i n t r a n u c l e a r virus-like particles m a y b e c o m e d o m i n a n t with the c o n c o m i t a n t decrease of virally coded material i n hepatocytic cytoplasma. Some genetic or e n v i r o n m e n t a l factors m a y play a role for these events. Moreover, it is n o t clear w h e t h e r all of these virus-like particles c o n t a i n D N A or not. Since some particles are electron dense wholly but others are hollow i n the center. Eventually, the exact relationship b e t w e e n the presence of H B V a n d i n t r a n u c l e a r viruslike particles or synthesis of HBs-Ag in the liver tissue r e m a i n s to the future study.
Acknowledgement We are deeply grateful to Dr. M. M a y u m i for his k i n d l y co-operation for the detection of anti-HBc i n sera.
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K., Akahane, Y. and Oda, M. : Australia antigen in serum and liver tissue of patients with various liver diseases. Kanzo 13: 280-287, 1972. 7) Mfiller, R., Maess, J. und Deicher, H.: Fluoreszenzserologischer Nachweis yon Australia-antlgen in der Leber. Dtsch. Med. Wschr. 98: 93-99, 1973. 8) Almeida, J.D., Bubenstein, D. and Scott, E.J.: New antigen-antibody system in Australia-antigenpositive hepatitis. Lancet 11: 1224-1227, 1971. 9) Hadziyannis, S., Moussouros, A., Giustozi, A. and Almeida, J.: Immunofluorescence study of the "core" antigen in the liver. Digestion 8: 437, 1973. 10) Shikata, T., Yoshizaki, C., Matsushita, H., Toyokawa, H. and Ishida, N. : Studies on the duality of intrahepatocytic HB-Ag by immunofluorescent antibody technique. Kanzo 15: 695, 1974. 11) Nelson, J.M., Barker, L.F. and Danovitch, S.H.: Intranuclear aggregates in the liver of a patient with serum hepatitis. Lancet 11: 773-774, 1970. 12) Huang, S.: Hepatitis-associated antigen hepatitis: An electron microscopic study of virus-like particles in liver cells. Amer. J. Pathoh 64: 483-500, 1971. 13) Dunn, A.E.G., Peter, R.L., Schweltzer, I.L. and Spears, R.L. : Virus-like particles in livers of infants with vertically transmitted hepatitis. Arch. Pathol. 94: 258-264, 1972. 14) Scotto, J., Homberg, J.C. and Caroli, J.: Electron microscopy studies of severe virus hepatitis. Cytoplasmic inclusions, virus-like particles, Australia antigen. Amer. J. Dis. Child. 123: 311-312, 1972. 15) Campion, E.C., Ludbrook, J., McLeod, J.M., Marshall, V.R., Mukherjee, T. and Wangel, A.G. : Primary hepatoma and hepatitis-assoclated antigen in a young white woman. Brit. Med. J. 4: 149-150, 1972. 16) Caramia, F., DeBac, C. and Ricci, G.: Virus-like particles within hepatocytes of Australia antigen carriers. Amer. J. Dis. Child. 123: 309-311, 1972. 17) Hirschman, S.Z., Gerber, M. and Garfinkel, E.: Purification of naked intranuclear particles from human liver infected by hepatitis B virus. Proc. Nat. Acad. Sci. USA. 71: 3345-3349, 1974. 18) Tapp, E., Jones, D.M., Anfield, C., Whittaker, J.S., Woolf, I.L. and Dymock, J.W.: Intranuclear particles in hepatocytes of HB-Ag positive blooddonors. Lancet I: 934, 1974. 19) Yamada, G., Kobayashi, T., Ohta, Y. and Kosaka, K. : I-IB-Agassociated particles in the sera and the liver of asymptomatie carriers. J. Clin. Electron Microscopy. 7: 368-369, 1974. 20) Kamimura, T., Murayama, H., Sasaki, H. and Ichida, F.: Electron microscopic studies on hepatocytes of asymptomatic carriers of hepatitis B antigen. ibid. 7: 370-371, 1974. 21) Barker, L.F., Almeida, J.D., Hoofnagle, J.It., Gerety, R.T., Jackson, D.R. and McGrath, P.P.: Hepatitis B core antigen: Immunology and electron microscopy. J. Virology. 14: 1552-1558, 1974. 22) Barker, L.F., Chisari, F.V., McGrath, P.P., Dalgard,
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human liver infected with hepatitis B virus. Nature. 251 : 540-542, 1974. 25) Robinson, W.S. and Greenman, R.L. : DNA polymerase in the core of the h u m a n hepatitis B virus candidate. J. Virology. 13: 1231-1236, 1974. 26) Gerber, M.A., Hadziyannis, S., Vissoulis, C., Schffner, F., Paronetto, F. and Popper, H. : Hepatitis B antigen: Nature and distribution of cytoplasmic antigen in hepatocytes of carriers. Proc. Soc. Exp. Biol. Med. 145: 863-867, 1974.
Received April 18, 1975 Accepted May 19, 1975 Address requests for reprints to: Seiichi Furuta, M.D., The Second Department of Internal Medicine, Faculty of Medicine, Shinshu University, 3-1-1 Asahi-cho, Matsumoto, Nagano Prefecture, 390 Japan
