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Gastroenterologia Japonica Vol.I0, No. 3. --1975----Original A r t i c l e

HBc A N D HBs A N T I G E N IN H E P A T O C Y T E S Toshio S H I K A T A * , Chiho Y O S H I Z A K I * , Keikoh O K A Z A K I * , Teruko U Z A W A * , Hiroshi M_ATSUSHITA**, Shuji T O Y O K A W A * * * , Takashi T A K A H A S H I * * * * and Atsushi G O T O H * * * * *

*Department of Pathology, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, ** The Institute of Public Health, Shiroganedai, Minato-ku, Tokyo 108, ***Mishuku Hospital, Meguro-ku, Tokyo 153, * * * *Kita zato Institute, Shiba-shirogane, Minato-ku, Tokyo 108, *****Jichi Medical College, Minami-kawachi-cho, Tochigi 329-04 Sumxnary The relationship between the electron microscopical structures and the immunofluorescent appearance of HB antigen found in the hepatocytes of patients with HB antigenemia was carefully studied, with the purpose to clarify the nature of those structures. The nuclei of hepatocytes, containing numerous virus-like particles, showed positive immunofluoreseence by antiserum against the core of Dane particles. The areas in which filamentous structures were observed in the cisternae of endoplasmic reticulums and hyaloplasma, corresponded to orcein or aldehyde-fuchsin positive substances in the light microscope, whereas direct relationship between filamentous structures and HBs antigen was still obscure. Accordingly, it may be concluded that virus-like particles in the nucleus correspond to core and filamentous structures in the cytoplasma correlate with coat protein of hepatitis B virus. Key Words:

HB antigen, HBsAg, HBcAg, hepatitis B. Introduction

Hepatitis B antigen (HBAg) in the liver tissue has duality not only immunologically but also biochemically, since some antisera against HBAg only react with substance in the cytoplasm of the hepatocytes and others stain nuclei on direct and indirect immunofluorescent examination 1). Antisera, obtained from animals which were immunized with HBAg positive h u m a n sera, usually react with cytoplasmic HBAg and sera of patients with I-IB antigenemia react with nuclei 2). Furthermore, new staining methods using dyes which were developed by present author, stain cytoplasmic HBAg and do not stain nuclear I-IBAg3). Those procedures probably react with disulfide bond or tryptophan. Therefore, nuclear HBAg contains neither so m u c h disufide bonds nor tryptophan. O n the other

hand, m a n y electron microscopical studies disclosed two different patterns of the appearance of HBAg4, 5). T h a t is, spherical virus-like particles, 200 to 250 A in diameter, are mainly observed in the nuclei. Second pattern is proliferation of the smooth endoplasmic reticulums and filamentous structures in them. Those structures gather in one part of cytoplasma and form cytoplasmic inclusion body. In advanced stage of cytoplasmic inclusion body, filamentous structures do not restrict in the cisternae of endoplasmic reticulums 6). Nuclear virus-like particles have been thought as an inner core of Dane particle and called as HBcAg (hepatitis B core antigen). Substance in the cytoplasm might be correlated with small particle, tubular structure and coat of Dane particle, which are tbund in the

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7". S H I K A TA E T AL.

serum (HBsAg, hepatitis surface antigen). In this study, immunofluorescent findings and electron microscopical one were compared carefully in the biopsied liver tissue, for the purpose to clarify the nature of the electron microscopical structures. Materials and Methods

Biopsy materials from 9 cases of asymptomatic HBAg carriers and 16 cases of chronic persistent and active hepatitis with HB antigenemia were used as materials. Biopsy specimens were divided into three parts. One part was fixed with formalin for the routine histological examination, including orcein and aldehyde-fuchsin stain 3). The second part was fixed in 2.5% glutaraldehyde for electron microscopical as well as immunoelectron microscopieal observation (findings obtained by immunoelectron microscopical methods will be reported in an other paper). For the conventional electronmicroscopical observation, specimens were postfixed in 1% osmium tetroxide and embedded in Epon 812. One thick sections were stained by aldehydefuchsin. HBsAg were stained faintly purple and those HBsAg containing areas were chosen for the electron microscopical observation. T h e third part was freezed in N-hexan, cooled in - - 7 0 ~ by C O 2 and acetone. Those specimens were cut with cryostat and examined by the fluorescent antibody technique. For the detection of HBsAg, anti HBs rabbit serum conjugated with F I T C or rhodamine was used. Rabbits were immunized b y mixture of adr and adw subtypes and antisera were absorbed with normal h u m a n serum and conjugated with F I T C or rhodamine. Indirect immunofluorescent methods were applied for HBcAg according to the method discribed by Brozosco et al)). Namely, HBsAg positive sera were dilututed at a ratio of 1:10 and used as p r i m a r y antisera. F I T C labeled antihuman IgG, commercially distributed from Hoechst, was used as the second serum. Direct immunofluorescent method was also applied for the detection of HBcAg. Rabbits were immunized by Dane

particles, which were gathered from sera by the gradient centrifugation and the rate zonal centrifugation. Obtained antiserum was absorbed with small particles of HBAg and normal h u m a n serum and then conjugated with F I T C . The antiserum was tested for specificity by the electrosyneresis with stripped core of Dane particles. Double stain for HBcAg and HBsAg was also performed. Results

A.

HBsAg in the cytoplasm Specific immunofluorescence for HBsAg was observed in 11 a m o n g 16 cases with symptomatic chronic hepatitis and 8 cases of 9 asymptomatic carrier. Histological examination of asymptomatic carrier disclosed 4 cases of chronic persistent hepatitis. Remaining 5 cases did not show any particular changes at least H E stain except for ground glass hepatocytes 7~. The a m o u n t of HBsAg varied case to case. Single cells containing HBsAg scattered in the liver cell cord. Those cells were rather few in chronic active hepatitis. I n same cases, cell m e m b r a n e of hepatocytes showed positive reaction in almost all areas of submitted biopsied material (Fig. 1). Orcein stain and aldehyde-fuchsin stain were less sensitive for the detection of HBsAg in the hepatocytes at least in biopsy materials. Positive reaction was observed in 8 cases of chronic hepatitis and 5 cases of asymptornatic carrier. However, aggregated HBsAg was clearly demonstrated in the cytoplasma (Fig. 2). Some of the hepatocytes, containing HBsAg, were surrounded by small lymphocytes. Cytoplasmic inclusion bodies were recognized in the 1 ~z thin section from Epon embedded material by aldehyde-fuchsin stain. Electron microscopical examination of those areas showed two different patterns which were shifted each other. I n one of them, proliferation of smooth endoplasmic reticulums and filamentous structures in them were observed as reported by several authors 4,5,8~ (Figs. 5, 6). Second features were thought to be in more advanced stage, and filamentous structures were observed not only

HBe and HBs Antigen in Hepatocytes

Fig. 1. HBsAg in a case with chronic hepatitis. Specific immunofluorescence is observed in the cytoplasm of one hepatocyte and cell membrane of other cells. (x600)

Fig. 3. HBcAg in the nuclei of hepatocytes of cases with HBAg carrier. Specific immunofluorescence is found only in the nuclei. Direct method using anti HBc rabbit serum. (X 600)

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]Fig. 2. HBsAg stained by orcein method in case with HBAg carrier. Brown color in the cytoplasm is HBsAg. Paraffin section. Orcein stain. ( • 300)

Fig. 4. Double stain of HBcAg and HBsAg. Green color in the nuclei demonstrats HBcAg and orange color in the cytoplasm is HBsAg.

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Figs. 5 a n d 6.

T. S H I K A T A E T AL.

Electron mlcrograph of HBsAg in the cisternae of proliferated endoplasmic reticulums. (Fig. 5 x 35,000 and Fig. 6 x 80,000)

HBe and HBs Antigen in Hepatocytes

Fig. 7.

Fig. 8.

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Filamentous structures in the hyaloplasm, which were observed in a cytoplasmic inclusion body. (•

HBcAg in the nuclei of hepatocytes, lmmunofluorescent method using anti ttBc Ag rabbit serum. In some of the nuclei, specific immunofluorescence is seen just beneath nuclear membrane. ( x 1,200)

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Figs. 9 a n d 10.

T. S H I K A T A E T AL.

Electron micrograph of the nuclei of hepatocytes. Same case with Fig. 8. Nemerous round virus-like particles exist in the nuclei. (Fig. 9 x40,000 and Fig. 10 x84,000)

HBe and HBs Antigen in Hepatoeytes

Fig. 11.

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Coexistence of virus-like particles in the nucleus and filamentous structures of cytoplasma within one hepatocytes. ( x 82~000)

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T. S H I K A T A E T AL.

in the cisternae of endoplasmic reticulums, but also in the hyaloplasma, as reported in the previous report 6~ (Fig. 7). In this case, granular structure was also observed electron microscopically in the cytoplasmic inclusion bodies. B. HBcAg in the nucleus. Specific immunofluorescence of HBcAg was found in 2 cases out of 9 asymptomatic carriers and one case among 16 cases of symptomatic chronic hepatitis (Fig. 3). Amount of HBsAg in those 3 cases was rather a b u n d a n t compared to the cases without HBcAg. HBcAg containing hepatocytes were more numerous compared to the HBsAg containing hepatocytes in each case. HBcAg containing cells and HBs Ag containing cells seemed not to be related each other at least under immunofluorescent microscope (Fig. 4). Specific immunofluorescence in the nuclei showed a fine granular appearance and the nuclear m e m b r a n e only showed positive reaction in some nuclei (Fig. 8). In some few areas, specific immunofluorescence was also observed in the cytoplasm. Twelve HBsAg positive sera which were examined by indirect immunofluorescent method, all contained anti HBcAg, though staining titer was different serum to serum. Findings obtained by F I T C labeled rabbit anti core of Dane particle in direct method was almost same with that found out by the indirect method. An electronmicroscopical observation of those biopsy materials was done, and round virus-like particles in the nuclei, measured about 250 A in diameter, were observed in the cases with HBcAg, which was demonstrated by immunofluorescent method (Fig. 9, 10). In other cases, round virus-like particles were very few and often found only in the cytoplasma around nucleus and the cell surface. Both structures, such as virus-like particles in the nucleus and filamentous structures in the cytoplasma, were observed electron-microscopically in one hepatocyte, though by the immunofluorescent method HBcAg and HBs Ag were scarcely found within one hepatocyte

(Fig. 11).

Discussion Dane particles in the serum have been thought as real hepatitis B virus and small particles and tubular structures have been explained as excess coat protein. Corresponding ultrastructual findings of HBAg containing hepatocytes and relationship between serum particles and structures in the hepat0cytes are still controversial. The duality of HBAg in the liver tissue reported by Brozosko l~ might correspond to different structures in the nucleus and the cytoplasm. Virus-like particles in the nucleus were confirmed as the core of Dane particles, because of the specific immunofluorescence observed in such nuclei by means of antiserum against to the core of Dane particle. Those nuclei containing HBcAg did not show any positive reaction for orcein or aldehydefuchsin stain, suggesting that HBcAg does not contain so much disulfide bonds nor tryptophan. Filamentous structures in the cytoplasma might be correlated to HBsAg, because they were poind in aldehyde-fuchsin positive areas by the electron microscopical observation. Filamentous structures were found in those areas. Gather et al. 4~ reported on the correspondence of those filamentous structures and HBsAg using the immunoelectron microscopical method. However, our preliminary examination using the samemethod did not show such results. Positive reaction was only found in the wall of smooth endoplasmic reticulum containing filamentous structures. Furthermore, the size and form of those filamentous structures are quitedifferent from those of HBsAg in the serum. Therefore, relationship between filamentous structures in the cytoplasma and those of HBsAg is still controversial. Concerning early stage of HBsAg production, it was reported in previous reports using the immunoelectron microscopical method 6>. As a summary of those observations, HBsAg m a y be produced by the ribosomes of hepatocytes under influence of viral genom, and

HBe andHBs Antigenin Hepatocytes released i n t o cisternae of e n d o p l a s m i c reticulums. Those H B s A g is a m o r p h o u s . An a g g r e g a t e of H B s A g forms c y t o p l a s m i c inclusion b o d y in w h i c h p r o l i f e r a t i o n of s m o o t h endoplasmic r e t i c u l u m s a n d filamentous structures is observed, w h e r e a s the e x a c t r e l a t i o n ship b e t w e e n the filamentous structure a n d HBsAg is still obscure. Concerning H B c A g , n u m e r o u s virus-like particles in the nuclei were o n l y f o u n d in the cases w i t h specific fluorescence o f H B c A g . Those virus-like particles m i g h t be the core of D a n e particles, since t h e y were stained b y antiserum a g a i n s t the core o f D a n e particles. References

1) Brozosko, W.J., Madalinski, K., Krawczynski, K , Nowoslawski, A. : Duality of hepatitis B antigen and its antibody. I. Immunofluorescence studies. J. Infect. Dis., 127: 424--428, 1973. 2) Barker, L.F., Chisari, F.V., McGrath, P.P. : Transmission of type B viral hepatitis to chimpanzees. J. Inf, Dis. 127: 648-662, 1973.

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3) Shikata, T., Uzawa, T., Ynshiwara, N., Akatsuka, T. and Yamazakl, S. : Staining methods of Australia antigen in paraffin section --Detection of cytoplasmic inclusion bodies--. Japan. J. Exp. Med., 44: 25-36, 1974. 4) Gerber, M.A., Hadziyannis, S., V[ssoulis, C., Schaffner, F., Paronetto, F. and Popper, H. : Electron microscopy and immunoelectron microscopy of cytoplasmic hepatitis B antigen in hepatocytes. Amer. J. Path., 75: 489-502, 1974. 5) Huang, S.N., Groh, V., Beaudoin, J.G., Dauphinee, W.D., Guttmann, R.D., Morehouse, D.D., Aronoff, A. and Gault, H.: A study of the relationship of virus-like particles and Australia antigen in liver. Human Path., 5: 209-222, 1974. 6) Shikata, T., Okazaki, K., Kako, M. and Uzawa, T.: Electron microscopical study of cytoplasmic inclusion body in hepatitis B. Japan. J. Exp. Med., 44: 519530, 1974. 7) Hadziyannis, S., Gerber, M.A., Vissoulis, C. and Popper, H.: Cytoplasmic hepatitis B antigen in "ground-glass" hepatocytes of carriers. Arch. Path., 96: 327-330, 1973. 8) Stein, O., Fainaru, M. and Stein, Y.: Virus-like particles in the cytoplasm of the liver of Australia antigen carriers. Amer. J. Dis. Child., 123 : 313-314, 1972.

Received March 27, 1975 Accepted May 19, 1975 Address requests for reprints to: Dr. Toshio Shikata, M.D., Department of Pathology, Faculty of Medicine, University of Tokyo, 3-1 Hongo 7 chome, Bunkyo-ku, Tokyo 113 Japan

HBc and HBs antigen in hepatocytes.

The relationship between the electron microscopical structures and the immunofluorescent appearance of HB antigen found in the hepatocytes of patients...
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