Gastroenterologia Japonica Copyright ~ 1977 by The Japanese Society of Gastroenterology
Vol. 12, No. 6 Printed in Japan
--Original Artlcle--
SIGNIFICANCE OF e-ANTIGEN/ANTI-e, WITH SPECIAL REFERENCE TO HBc-ANTIGEN IN THE LIVER
CLINICAL
Seiichi F U R U T A , M.D., Kendo K I Y O S A W A , M.D., Atsuo N A G A T A , M.D., Yuriko K O I K E , M.D., Takeshi SAHARA, M.D., Kenichi F U R U K A W A , M.D., Yoshihiro I I J I M A , M.D., Shinkichi Y A M A M U R A , M.D., Hironao K O M A T S U , M.D., Kenziro K A W A H A R A , M.D., Masazumi M I U R A , M.D., Yukio G I B O , M.D., Ken S O D E Y A M A , M.D., Masayuki ODA, M.D., Fumio T S U D A , M.D., Yoshihiro A K A H A N E , M.D. and Makoto M A Y U M I , M.D.
Department of Internal Medicine, Faculty of Medicine, Shinshu University, Matsumoto, Japan and Hepatitis Division, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
S~mmary e-antigert and anti-e were assayed in sera of asymptomatic HBs-Ag carriers and of patients with liver diseases. Thirteen out of 34 (38.2%) asymptomatic carriers were positive for e-antigen, which was in sharp contrast to the reports from USA and Europe. e-antigen was detected to a greater extent in patients with chronic active hepatitis, reversely anti-e in patients with chronic persistent hepatitis. However, eantigen was found rarely in patients with cirrhosis and never in 23 cases with hepatoma positive for HBs-Ag. HBc-Ag in the liver was detected in 4 out of 8 e-antigen positive asymptomatic carriers and in 4 out of 5 patients with chronic liver diseases with e-antigen respectively, and moreover in 3 out of 14 anti-e positive cases, so that the presence of anti-e did not necessarily mean the negativity of HBc-Ag in the liver. AntiHBc titer, however, was lower in anti-e positive sera than in e-antigen positive ones. This may implicate the decreased rephcation of HBV in cases with anti-e. These results emphasize that the investigation of e-antigen/anti-e is mandatory for the evaluation of the prognosis of asymptomatic carriers and of patients with chronic hepatitis.
Key Words: e-antigenand anti-e, asymptomaticHBs-Ag carrier,chronichepatitis, HBc-Ag in the liver, anti-HBc titer. Introduction
HBc-Ag, the core antigen of Dane particle
e-antigen/anti-e system described by Magnius et al l) in 1972 was found exclusively in association with H B V infection. Yet the prevalence of e-antigen in HBs-Ag carriers is different from reports to reports 2>12). Dane particle which is a candidate for H B V is rarely, if any, observed in anti-e positive serum 13)'16). This is assumed to be linked to the paucity or absence of the infectivity of anti-e positive serum, although its precise mechanism remains for debate. Several investigators have reported that
which is identical in antigenicity to the viruslike particles in hepatocytic nuclei, was unable to be detected in the liver of anti-e positive patientn, 17), whereas HBs-Ag is positive in the serum with anti-e except for a few cases. Thus the presence of anti-e does not imply the termination of H B V infection, yet the activity of its replication in the liver appears to be decreased. The presence of e-antigen in serum has been suggested to be closely related to the chronicity and activity of hepatitis 2"6)15,IS, 19)
December 1977
461
e-Antigen]Anti-e and HBc-Ag in the Liver
In order to clarify the clinical implication of e-antigen/anti-e in asymptomatic HBs-Ag carriers and in patients with liver diseases, we have tested for e-antigen/anti-e in sera of HBs-Ag positive individuals with a special reference to HBc-Ag in the liver.
Materials and Methods Subjects studied: One hundred and fiftyseven HBs-Ag positive cases were enrolled in this studies as shown in T a b l e 1. The diagnosis of all of the patients with various liver diseases was confirmed histologically by liver biopsy or autopsy. Asymptomatic carrier was designated on individuals who had no past history of liver disease or blood transfusion, nor abnormal chemical data as liver function tests for more than 3 months and whose liver showed histologically normal or only minimal changes on the first occasion of liver biopsy. Methods: HBs-Ag was assayed by R - P H A method 20), anti-HBs by P H A method, eantigen/anti-e was tested by the double immunodiffusion technique of Ouchterlony using 0.90/0 agarose in Tris-HC1 buffer (0.01 M, p H 7.6) in which 0.1 M NaC1 and Dextran T-240 were contained 13~. Sera were concentrated to about one-third of its original volume with Lyphogel before test. Wells, 3 m m in diameter, were punched out 3 m m spart from edge to edge. In order to compare the titer of anti-HBc antibody in e-antigen positive serum with that in anti-e positive one, the determination of anti-HBc titer was
performed by I A H A method 2r), blindly in respects to the presence or absence of eantigen/anti-e, using the same reagents throughout this studies. Test-serum was stored at --20~ until use. HBc-Ag in the liver was investigated by indirect immunofluorescence method on 4 ix cryostat sections of liver biopsy or autopsy specimens immediately frozen at --70~ The specificity of the positive fluorescence for HBc-Ag was evaluated previously 22).
Results 1. Prevalence of e-antigen/antive: T a b l e 1 shows the prevalence of e-antigen and anti-e in asymptomatic carriers and patients with various liver diseases, e-antigen was detected most frequently in asymptomatic carriers (13/34, 38.2%) and next in patients with chronic active hepatitis (12/36, 33.3%). None of 23 patients with hepatoma and only 2 out of 32 cases with cirrhosis of the liver had e-antigen. In contrast, anti-e was detected in 5 cases with cirrhosis and hepatoma respectively, and most frequently in asymptomarie carriers and in patients with chronic persistent hepatitis. 2. e-antigen/anti-e and HBc-Ag in the liver: T a b l e 2 shows the relationship between the presence of e-antigen/anti-e in serum and the detection of HBc-Ag in the liver. In this studies, e-antigen/anti-e was assayed on the serum obtained on the day of liver biopsy or autopsy. HBc-Ag was
Table 1. Prevalence of e-antigen and anti-e in various liver diseases Liver Diseases Asymptomatic carrier Acute hepatitis Chronic persistent hepatitis Chronic active hepatitis Cirrhosis Itepatoma
No. 34 8 16 36 35 23
e-antigen -tNo. % 13 38.2 2 25.0 2 12.5 12 33.3 2 5.7 0 0
anti-e -tNo. % 13 38.2 2 25.0" 5 31.3 2 5.6 5 14.3 5 21.4
neitherdetectable No. % 8 23.6 4 50.0 9 56.2 22 61.1 28 80.0 18 78.3
462
S. F U R U T A
Table 2.
Relationship between e-antigen/anti-e in serum and HBc-Ag in the liver
HBs-Ag positive cases Asymptomatic HBs-Ag carrier (22) Chronic liver diseases (48)
e-antigen/anti-e e-antigen + anti-e -4neither detectable e-antigen 4anti-e + neither detectable
HBs-A~(2n) >6 >6 anti- HBs HBe-A~ -
7 >6
anti-HBe
+
+
7
>6
20
~
I0
........... o----o_ ~
No. 8 9 5 5 5 38
HBc-Ag in the liver (IF) Positive rate + % 4 4 50.0 2 7 22.2 2 3 4-0~0 4 1 80.0 1 4 20.0 23 15 60.5
7
+
Biopsy 50[ 1 40 30 ~_
Vol. 12, No. 6
E T AL.
+
Biopsy 2
~. . . . . . . . . . . . . . . . 0-
. ~ S-60T
22~
S-GPT
Jun. Jul. Au~. Sep. Oct. Nov. Dec. Jan. 1976 Fig. 1 Clinical course of an asymptomatic HBs-Ag carrier with anti-e whose liver showed positive HBc-Ag (Case : Y.T. 21 y.o.) 1975
positive in 4 o u t o f 8 a s y m p t o m a t i c carriers a n d in 4 out o f 5 p a t i e n t s with chronic liver diseases positive for e-antigen. T h e presence o f anti-e did not necessarily exclude the positive H B c - A g in the liver. As shown in the table, H B c - A g was detected in the livers of 3 out of 14 a n t i - e positive cases. F u r t h e r more, H B c - A g was d e m o n s t r a t e d in 23 o u t o f 38 patients w i t h c h r o n i c liver diseases w h o h a d n e i t h e r e - a n t i g e n n o r anti-e. F i g . 1 illustrates the clinical course o f a n anti-e positive c a r r i e r w h o h a d been q u i t e well a n d was n o t e d H B s - A g positive on b l o o d d o n a t i o n . His s e r u m t r a n s a m i n a s e level has r e m a i n e d in n o r m a l r a n g e a n d his liver histology on two occasions of liver b i o p s y showed almost n o r m a l except for several h e p a t o c y t e s w h i c h h a d ground-glass a p p e a r i n g cytoplasma.
As
shown
in
F i g , 2,
several
Fig. 2. Positive immunofluorescence specific for HBc-Ag was seen in several hepatocytic nuclei of the liver of the case illustrated in Fig. 1. (on the second occasion of the liver biopsy)
h e p a t o c y t i c nuclei of the liver r e v e a l e d a positive i m m u n o f l u o r e s c e n c e specific for H B c Ag. 3. e - a n t i g e n / a n t i - e a n d the titer o f antiHBc a n t i b o d y : A n t i - H B c titer, w h i c h is believed to be a favorable i n d i c a t o r o f a n active r e p l i c a t i o n o f H B V , was tested to see w h e t h e r the p a u c i t y of H B c - A g in the liver o f anti-e positive cases were a b l e to reflect on the a n t i - H B c titer in serum. As shown in F i g . 3, the g e o m e t r i c m e a n of the a n t i - H B c titer was lower in sera with anti-e (213.6) t h a n in e-antigen positive ones (214.9). T h o u g h this difference was statistically significant ( p < 0 . 0 5 ) , a n t i - H B c titer in sera with a n t i - e was high e n o u g h at not less t h a n 2 l~ to suggest the persistent s t i m u l a t i o n b y H B c - A g 23).
December 1977
e-Antigen/Anti-e and HBc-Ag in the Liver
9
Vol. 12, No. 6 Printed in Japan
--Original Artlcle--
SIGNIFICANCE OF e-ANTIGEN/ANTI-e, WITH SPECIAL REFERENCE TO HBc-ANTIGEN IN THE LIVER
CLINICAL
Seiichi F U R U T A , M.D., Kendo K I Y O S A W A , M.D., Atsuo N A G A T A , M.D., Yuriko K O I K E , M.D., Takeshi SAHARA, M.D., Kenichi F U R U K A W A , M.D., Yoshihiro I I J I M A , M.D., Shinkichi Y A M A M U R A , M.D., Hironao K O M A T S U , M.D., Kenziro K A W A H A R A , M.D., Masazumi M I U R A , M.D., Yukio G I B O , M.D., Ken S O D E Y A M A , M.D., Masayuki ODA, M.D., Fumio T S U D A , M.D., Yoshihiro A K A H A N E , M.D. and Makoto M A Y U M I , M.D.
Department of Internal Medicine, Faculty of Medicine, Shinshu University, Matsumoto, Japan and Hepatitis Division, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
S~mmary e-antigert and anti-e were assayed in sera of asymptomatic HBs-Ag carriers and of patients with liver diseases. Thirteen out of 34 (38.2%) asymptomatic carriers were positive for e-antigen, which was in sharp contrast to the reports from USA and Europe. e-antigen was detected to a greater extent in patients with chronic active hepatitis, reversely anti-e in patients with chronic persistent hepatitis. However, eantigen was found rarely in patients with cirrhosis and never in 23 cases with hepatoma positive for HBs-Ag. HBc-Ag in the liver was detected in 4 out of 8 e-antigen positive asymptomatic carriers and in 4 out of 5 patients with chronic liver diseases with e-antigen respectively, and moreover in 3 out of 14 anti-e positive cases, so that the presence of anti-e did not necessarily mean the negativity of HBc-Ag in the liver. AntiHBc titer, however, was lower in anti-e positive sera than in e-antigen positive ones. This may implicate the decreased rephcation of HBV in cases with anti-e. These results emphasize that the investigation of e-antigen/anti-e is mandatory for the evaluation of the prognosis of asymptomatic carriers and of patients with chronic hepatitis.
Key Words: e-antigenand anti-e, asymptomaticHBs-Ag carrier,chronichepatitis, HBc-Ag in the liver, anti-HBc titer. Introduction
HBc-Ag, the core antigen of Dane particle
e-antigen/anti-e system described by Magnius et al l) in 1972 was found exclusively in association with H B V infection. Yet the prevalence of e-antigen in HBs-Ag carriers is different from reports to reports 2>12). Dane particle which is a candidate for H B V is rarely, if any, observed in anti-e positive serum 13)'16). This is assumed to be linked to the paucity or absence of the infectivity of anti-e positive serum, although its precise mechanism remains for debate. Several investigators have reported that
which is identical in antigenicity to the viruslike particles in hepatocytic nuclei, was unable to be detected in the liver of anti-e positive patientn, 17), whereas HBs-Ag is positive in the serum with anti-e except for a few cases. Thus the presence of anti-e does not imply the termination of H B V infection, yet the activity of its replication in the liver appears to be decreased. The presence of e-antigen in serum has been suggested to be closely related to the chronicity and activity of hepatitis 2"6)15,IS, 19)
December 1977
461
e-Antigen]Anti-e and HBc-Ag in the Liver
In order to clarify the clinical implication of e-antigen/anti-e in asymptomatic HBs-Ag carriers and in patients with liver diseases, we have tested for e-antigen/anti-e in sera of HBs-Ag positive individuals with a special reference to HBc-Ag in the liver.
Materials and Methods Subjects studied: One hundred and fiftyseven HBs-Ag positive cases were enrolled in this studies as shown in T a b l e 1. The diagnosis of all of the patients with various liver diseases was confirmed histologically by liver biopsy or autopsy. Asymptomatic carrier was designated on individuals who had no past history of liver disease or blood transfusion, nor abnormal chemical data as liver function tests for more than 3 months and whose liver showed histologically normal or only minimal changes on the first occasion of liver biopsy. Methods: HBs-Ag was assayed by R - P H A method 20), anti-HBs by P H A method, eantigen/anti-e was tested by the double immunodiffusion technique of Ouchterlony using 0.90/0 agarose in Tris-HC1 buffer (0.01 M, p H 7.6) in which 0.1 M NaC1 and Dextran T-240 were contained 13~. Sera were concentrated to about one-third of its original volume with Lyphogel before test. Wells, 3 m m in diameter, were punched out 3 m m spart from edge to edge. In order to compare the titer of anti-HBc antibody in e-antigen positive serum with that in anti-e positive one, the determination of anti-HBc titer was
performed by I A H A method 2r), blindly in respects to the presence or absence of eantigen/anti-e, using the same reagents throughout this studies. Test-serum was stored at --20~ until use. HBc-Ag in the liver was investigated by indirect immunofluorescence method on 4 ix cryostat sections of liver biopsy or autopsy specimens immediately frozen at --70~ The specificity of the positive fluorescence for HBc-Ag was evaluated previously 22).
Results 1. Prevalence of e-antigen/antive: T a b l e 1 shows the prevalence of e-antigen and anti-e in asymptomatic carriers and patients with various liver diseases, e-antigen was detected most frequently in asymptomatic carriers (13/34, 38.2%) and next in patients with chronic active hepatitis (12/36, 33.3%). None of 23 patients with hepatoma and only 2 out of 32 cases with cirrhosis of the liver had e-antigen. In contrast, anti-e was detected in 5 cases with cirrhosis and hepatoma respectively, and most frequently in asymptomarie carriers and in patients with chronic persistent hepatitis. 2. e-antigen/anti-e and HBc-Ag in the liver: T a b l e 2 shows the relationship between the presence of e-antigen/anti-e in serum and the detection of HBc-Ag in the liver. In this studies, e-antigen/anti-e was assayed on the serum obtained on the day of liver biopsy or autopsy. HBc-Ag was
Table 1. Prevalence of e-antigen and anti-e in various liver diseases Liver Diseases Asymptomatic carrier Acute hepatitis Chronic persistent hepatitis Chronic active hepatitis Cirrhosis Itepatoma
No. 34 8 16 36 35 23
e-antigen -tNo. % 13 38.2 2 25.0 2 12.5 12 33.3 2 5.7 0 0
anti-e -tNo. % 13 38.2 2 25.0" 5 31.3 2 5.6 5 14.3 5 21.4
neitherdetectable No. % 8 23.6 4 50.0 9 56.2 22 61.1 28 80.0 18 78.3
462
S. F U R U T A
Table 2.
Relationship between e-antigen/anti-e in serum and HBc-Ag in the liver
HBs-Ag positive cases Asymptomatic HBs-Ag carrier (22) Chronic liver diseases (48)
e-antigen/anti-e e-antigen + anti-e -4neither detectable e-antigen 4anti-e + neither detectable
HBs-A~(2n) >6 >6 anti- HBs HBe-A~ -
7 >6
anti-HBe
+
+
7
>6
20
~
I0
........... o----o_ ~
No. 8 9 5 5 5 38
HBc-Ag in the liver (IF) Positive rate + % 4 4 50.0 2 7 22.2 2 3 4-0~0 4 1 80.0 1 4 20.0 23 15 60.5
7
+
Biopsy 50[ 1 40 30 ~_
Vol. 12, No. 6
E T AL.
+
Biopsy 2
~. . . . . . . . . . . . . . . . 0-
. ~ S-60T
22~
S-GPT
Jun. Jul. Au~. Sep. Oct. Nov. Dec. Jan. 1976 Fig. 1 Clinical course of an asymptomatic HBs-Ag carrier with anti-e whose liver showed positive HBc-Ag (Case : Y.T. 21 y.o.) 1975
positive in 4 o u t o f 8 a s y m p t o m a t i c carriers a n d in 4 out o f 5 p a t i e n t s with chronic liver diseases positive for e-antigen. T h e presence o f anti-e did not necessarily exclude the positive H B c - A g in the liver. As shown in the table, H B c - A g was detected in the livers of 3 out of 14 a n t i - e positive cases. F u r t h e r more, H B c - A g was d e m o n s t r a t e d in 23 o u t o f 38 patients w i t h c h r o n i c liver diseases w h o h a d n e i t h e r e - a n t i g e n n o r anti-e. F i g . 1 illustrates the clinical course o f a n anti-e positive c a r r i e r w h o h a d been q u i t e well a n d was n o t e d H B s - A g positive on b l o o d d o n a t i o n . His s e r u m t r a n s a m i n a s e level has r e m a i n e d in n o r m a l r a n g e a n d his liver histology on two occasions of liver b i o p s y showed almost n o r m a l except for several h e p a t o c y t e s w h i c h h a d ground-glass a p p e a r i n g cytoplasma.
As
shown
in
F i g , 2,
several
Fig. 2. Positive immunofluorescence specific for HBc-Ag was seen in several hepatocytic nuclei of the liver of the case illustrated in Fig. 1. (on the second occasion of the liver biopsy)
h e p a t o c y t i c nuclei of the liver r e v e a l e d a positive i m m u n o f l u o r e s c e n c e specific for H B c Ag. 3. e - a n t i g e n / a n t i - e a n d the titer o f antiHBc a n t i b o d y : A n t i - H B c titer, w h i c h is believed to be a favorable i n d i c a t o r o f a n active r e p l i c a t i o n o f H B V , was tested to see w h e t h e r the p a u c i t y of H B c - A g in the liver o f anti-e positive cases were a b l e to reflect on the a n t i - H B c titer in serum. As shown in F i g . 3, the g e o m e t r i c m e a n of the a n t i - H B c titer was lower in sera with anti-e (213.6) t h a n in e-antigen positive ones (214.9). T h o u g h this difference was statistically significant ( p < 0 . 0 5 ) , a n t i - H B c titer in sera with a n t i - e was high e n o u g h at not less t h a n 2 l~ to suggest the persistent s t i m u l a t i o n b y H B c - A g 23).
December 1977
e-Antigen/Anti-e and HBc-Ag in the Liver
9
