GASTROENTEROLOGY
1992;102:956-962
High-Level Expression of Hepatitis B Viral Antigens in Fibrosing Cholestatic Hepatitis JOHNSON Y. N. LAU, VINCENT G. BAIN, SUSAN JOHN G. O’GRADY, ALFRED0 ALBERTI, GRAEME
E. DAVIES, J. M. ALEXANDER,
and ROGER WILLIAMS Institute of Liver Studies, King’s College Hospital School of Medicine and Dentistry, London, England; and Clinica Medica II, Padova, Italy
The expression of hepatitis B viral antigens was quantified in liver tissue from four transplant recipients with fibrosing cholestatic hepatitis (FCH) and compared with five other transplant recipients who did not develop this syndrome and 30 patients with chronic hepatitis B virus (HBV) infection. As measured by radioimmunoassays, the liver tissue from patients with FCH had significantly greater amounts of both hepatitis B surface antigen (HBsAg) and nucleocapsid antigens than to transplant patients without this syndrome (P < 0.01) or patients with chronic HBV infection (P< 0.001). Intrahepatic expression of pre-S,/pre-S, in FCH was also extensive with a distribution parallel to that of HBsAg. High-level expression of intrahepatic HBsAg and hepatitis B core antigen in the explanted liver was associated with subsequent development of FCH in the liver graft, suggesting that viral/host factors may also be important. This pattern of intrahepatic hepatitis viral antigen expression, by analogy with Chisari’s transgenic mice model and Roingeard’s HBV-transfected HepG2 cell model, may be the cause of direct hepatocytopathic injury in this condition.
C
hronic hepatitis B virus (HBV) infection is potentially one of the commonest indications for liver transplantation worldwide, but recurrence of HBV infection in the grafted liver is associated with increased morbidity and mortality after transplantation.lm3 The histological implications of HBV reinfection of the liver graft are variable, including minor histological changes, chronic persistent/active hepatitis, and a unique histological pattern of fibrosing cholestatic hepatitis (FCH) that occurred in 24.1% of the hepatitis B surface antigen (HBsAg)-seropositive transplant patients who survived for more than 2 months.3v4 Clinically, FCH is characterized by rapidly progressive liver failure and a high mortality within 4-6 weeks of clinical onset.3 Histologically, it is characterized by a pattern of extensive serpiginous
periportal fibrosis, canalicular and cellular cholestasis, and prominent HBV antigen expression in hepatocytes but a mild mixed inflammatory cell infiltrate.3*4 The clinical pattern of acute liver failure and the relatively mild inflammatory cell infiltrate suggest that the liver cell damage in this pattern of graft reinfection is not immune mediated but might instead be cytopathic. In the present study, we have elucidated the extent of intrahepatic HBV antigen expression in transplant patients with FCH to determine its possible role in pathogenesis. The extent of HBV antigen expression in the explanted liver was also correlated with the subsequent clinical course to determine whether viral or host factors are also involved. Patients and Methods Of the 39 patients seropositive for HBsAg receiving transplants within the Cambridge/King’s College Hospital series,3 histological specimens were available in 27 patients surviving longer than .Z months. Twenty-three patients had recurrence of HBV infection as shown by the reappearance of serum HBsAg (Table 1). Seven patients developed clinically and histologically proven FCH; four other patients also ran a clinical course typical of FCH but without histological confirmation.3’4 In the majority of patients, maintenance immunoglobulin therapy was not given.3 Serum HBsAg was studied in all 23 transplant patients with HBV recurrence and serum pre-S, in 17 patients. Intrahepatic HBV antigens were quantified in four patients with FCH (5-8 months after liver transplantation) and five transplant patients with HBV recurrence but no evidence of FCH (3-10 months after transplantation: histological diagnosis: minor changes, 1; lobular hepatitis, 2; chronic active hepatitis, 1; large duct obstruction, 1). Hepatocytes were also isolated from two hepatectomy specimens for determination of the amount of intracellular soluble protein for comparative purposes. Intrahepatic expression of HBsAg and pre-S antigens were studied in 32 specimens 0 1992 by the American
Gastroenterological 0016-5065/92/$3.00
Association
HBV ANTIGEN EXPRESSION IN FCH
March 1992
957
HEPES-buffered RPM1 1640 with 0.2% ethylenediaminetetraacetic acid (EDTA; pH 7.4), and hepatocytes were released into suspension by gently scraping the biopsy specimens with two 21-G needles in a Petri dish. After orthotopic The resulting cell suspension was washed three times in Chronic liver transplant RPM1 1640 by centrifugation at 50g at 4°C for 5 minutes. HBV infection (HBV recurrence] Cells were counted and 200,000 cells were resuspended in 23 30 No. 3 mL of RPM1 1640. Evaluation by phase-contrast micros26:4 19:4 Sex (male:female) copy showed that the isolated hepatocytes retained an in35(23-59) 37(23-61) Median age [yr (range)] tact plasma membrane and should have retained the HBV Serology antigens.7 In addition, the residual fibrosis tissue con1:25,600 1:12,800 Median HBsAg titer tained minimal or no attached hepatocytes. (1:32-214,800) (1:64-214,800) (range) The cell suspensions were sonicated over ice at 10 am16 19 HBeAg positive (n) 21 13 HBV DNA positive (n) plitude microns 10 seconds per spell for three spells to re7 0 Anti-HDV positive (n) lease the intracellular viral antigens7 Sonication accordHistology ing to this method did not denature the HBsAg and Normal graft (n) nucleocapsid antigense7 The cell sonicates were then Lobular hepatitis (n) stored at -20°C until tested in batches using commercially Minor changes, chronic available radioimmunoassays for HBsAg and nucleocapsid 5 carriage (n) antigen (Abbott Diagnostics). The HBsAg results were Chronic persistent quantified by constructing a standard curve using dilu11 hepatitis (n) Chronic active tions of a standard positive control sample containing 20 a hepatitis (n) ng/mL HBsAg; the nucleocapsid antigen results were ex6 Cirrhosis (n) pressed with reference to a standard taken as 1 U/mL. Acute cellular Mixing HBV antigens with cell sonicate from a hepatoblasrejection (n) toma cell line HepG2 (ECACC, Porton Down, England) did Vanishing bile duct not interfere with the radioimmunoassay. The proportion syndrome (n) of cells containing HBV antigen in these preparations was Fibrosing cholestatic also determined by immunocytochemistry as described hepatitis (n) below. Bile duct obstruction (n) The amount of soluble protein in the hepatocytes was also determined in two hepatectomy specimens for comparative purposes. Thirty million hepatocytes were prepared and sonicated in RPM1 1640 and 1 mmol/L phenylfrom the 23 transplant patients with HBV recurrence. methysulforyl fluoride (Sigma) as a protease inhibitor. The Thirty consecutive patients with chronic HBV infection samples were then ultracentrifuged (270,OOOgat 4°C for 30 who underwent liver biopsy for diagnostic purposes in minutes), and the amount of soluble proteins in the super1989-1990 were also studied for comparison (Table 1). natant was determined using a commercial protein assay kit (Pierce, Cheshire, England). Serology
Table 1. Clinical, Serological, and Histological Data of Patients Studied at the Time of the Latest Liver Biopsy
Serum was tested for HBsAg, hepatitis B e antigen (HBeAg), and anti-HBe by radioimmunoassay (Abbott Diagnostics, Maidenhead, England). HBsAg was quantified by serial dilution using reverse passive hemagglutination (Wellcome Diagnostics, Dartford, England). Antibody to hepatitis D virus was studied by enzyme immunoassay (Abbott Diagnostics), and serum HBV DNA was assayed using a quantitative dot-blot technique.5 Serum HBV preS, antigen was measured by a sandwich enzyme-linked immunosorbent assay using two monoclonal antibodies to pre-S, with different specificities with peroxidase labeling and orthophenylene diamine as the substrate. lntrahepatic
HEW Antigen
Quantity
Intrahepatic HBV antigens were quantified in nine transplant patients with HBV recurrence (four from patients with FCH; five from patients with no feature of FCH) and 30 patients with chronic HBV infection. Hepatocytes were isolated by the method described by Trevisan et a1.6 The liver biopsy specimens were immediately immersed
in
Detection of HBV Antigens in Liver Sections and Hepatocytes in the Cytospins The intrahepatic expression of the pre-S antigens (pre-S,/pre-S,) in the posttransplant liver biopsy or hepatectomy specimens was assessed together with HBsAg in serial paraffin liver sections by standard indirect immunohistochemistry using respective monoclonal antibodies (HBsAg: D2H5, 1:20 dilution; pre-S, : MA18/7, 1:lOO dilution; pre-S,: 5535, 1:25 dilution), alkaline phosphatase as the enzyme label and naphthol and Fast Red salt (Sigma Chemical Co., St. Louis, MO) as the substrate.7” The expression of HBsAg and HBcAg in the explanted livers was also assessed in similar fashion using respective monoclonal (HBsAg; D2H5) and polyclonal antibodies (HBcAg; I:200 dilution; Dako, Bucks, England) to determine the relationship between intrahepatic HBV antigen expression before transplantation and subsequent development of FCH. The expression of HBV antigen was scored independently by two of us (J.Y.N.L., S.E.D.) on a O-4+ scale corre-
958 LAU ET AL.
GASTROENTEROLOGYVol. loz,No.3
spondingto positivity in 0%, l%-5%, 5%-30%, 30%-60%, and >60% of hepatocytes examined. The proportion of HBV antigen-containing cells in the hepatocyte suspension was determined by immunocytochemistry. Cell smears were prepared using a cytospin (Shandon, England) to obtain 20,000 cells per slide previously coated with 0.1% poly-l-lysine (Sigma) to enhance cell adhesion, and slides were stored at -20°C before staining. After thawing, cells were fixed in ice-cold acetone for 2 minutes and air dried. The HBV antigens were then sought by standard immunocytochemistry using alkaline phosphatase as the enzyme label and Fast Red salt as the substrate.7’8 Statistics Results are expressed as medians and ranges and were analyzed using Wilcoxon’s rank-sum test, Spearman’s rank correlation test, or Fisher’s Exact Test. Results Serum HBsAg and Pre-S, Levels There was no significant difference in the median titer of serum HBsAg between transplanted patients with FCH (1:51, ZOO;range, 1:3, ZOO-160,000; n = 7), transplant patients with no evidence of FCH (1:25,600; range, 1:8-214,800; n = 16; anti-HDV+, 7), and patients with chronic HBV infection (1:12,800; range, 1:64-214,800; n = 30). In patients with chronic HBV infection, those with nonspecific reactive changes or chronic persistent hepatitis had higher serum HBsAg titers (median, 1:25,600; range, 1:256214,800; n = 16) than patients with chronic active hepatitis and cirrhosis (median, 1:3200; range, 1:6425,600; n = 14). Patients with FCH had serum titers of HBsAg comparable to those of the HBV carrier patients with nonspecific reactive changes or chronic persistent hepatitis. Serum pre-S, (range, O-25.2 U/mL) correlated well with serum HBsAg (R, = 0.6831; n = 16; P = 0.004) in transplant patients with HBV recurrence. All patients with FCH were seropositive for both serum pre-S, and HBV DNA (n = 7). Among patients without evidence of FCH, 6 of 10 tested were seropositive for pre-S, and 14 of 16 were seropositive for HBV DNA (P = NS). Intrahepatic
HBV Antigen Expression
In FCH, intrahepatic expression of HBsAg, pre-S,, and pre-S, was 4+ (i.e., positive in virtually all cells) with a diffuse cytoplasmic distribution. Cells with prominent surface antigen expression also showed a variable degree of ballooning with groundglass transformation, bile pigmentation, and clearing of the cytoplasm. Intrahepatic expression of the nu-
cleocapsid antigens was also extensive, both nuclear and cytoplasmic, and has been detailed previously.4 In the liver specimens from transplant patients without evidence of FCH, the median intrahepatic expression of HBsAg was 2+ (range, l+-4+; n = 16; HDV+, 7). Biopsy specimens from three patients showed membraneous surface antigen in addition to cytoplasmic expression (histology: chronic active hepatitis, 2; lobular hepatitis, 1; all negative for HDV). The expression of hepatitis B core antigen (HBcAg) was mainly nuclear l+ (range, O-3+; n = 16; HDV+, 7) with six patients also showing cytoplasmic HBcAg l-l- (range, O-2+). In the biopsy specimens from patients with chronic HBV infection, the median intrahepatic expression of HBsAg was 3+ (range, O-4+; n = 30), and that of HBcAg was 2+ in patients seropositive for HBeAg (range, O-3+; n = 19; cytoplasmic HBcAg present in three patients). Patients with FCH had a significantly higher proportion of hepatocytes expressing both HBsAg and HBcAg than transplant patients without FCH and patients with chronic HBV infection (P < 0.01 for both HBsAg and HBcAg). Intrahepatic
HBVAntigen
Quantity
The amount of intrahepatic HBsAg in patients with FCH, as measured by radioimmunoassay of 200,000 sonicated hepatocytes, was 191.1 ng (range, 164.4-312.2 ng; n = 4), compared with 4.5 ng (range, 0.2-28.1 ng; n = 5; HDV+, 2; P < 0.01) in transplant patients with no features of FCH and 44.8 ng (range, 0.2-107.9 ng; n = 30; P < 0.001) in patients with chronic HBV infection. To determine the average quantity of intracellular HBsAg per hepatocyte, we divided the amount of HBV antigens by the estimated number of positive hepatocytes as determined by immunocytochemical staining of the hepatocyte cytospins. The amount of HBsAg per hepatocyte was also higher in patients with FCH 0.98 pg/cell (range, 0.95-1.56 pg/cell; n = 4) than in patients with no evidence of FCH [0.22 pg/cell (range, 0.05-0.70 pg/cell; n = 5; HDV+, 2; P < O.Ol)] and patients with chronic HBV infection [0.64 pg/mL (range, 0.05-1.02 pg/cell; n = 30; P < O.OOl)] (Figure ?A). In relation to the amount of hepatocytesoluble protein, HBsAg constitutes between 0.5% and 1.0% of the soluble protein per hepatocyte in patients with FCH. The median quantity of intrahepatic nucleocapsid antigen was also significantly higher in patients with FCH [lo.2 U/200,000 hepatocytes (range, 4.2-22.4 U; n = 4)] than in transplant patients with no evidence of FCH [0.2 U (range, 0.0-0.5 U; n = 5; HDV+, 2; P < O.Ol)] or patients with chronic HBV infection seropo-
HBV ANTIGEN
March 1992
,
EXPRESSION
P
1992;102:956-962
High-Level Expression of Hepatitis B Viral Antigens in Fibrosing Cholestatic Hepatitis JOHNSON Y. N. LAU, VINCENT G. BAIN, SUSAN JOHN G. O’GRADY, ALFRED0 ALBERTI, GRAEME
E. DAVIES, J. M. ALEXANDER,
and ROGER WILLIAMS Institute of Liver Studies, King’s College Hospital School of Medicine and Dentistry, London, England; and Clinica Medica II, Padova, Italy
The expression of hepatitis B viral antigens was quantified in liver tissue from four transplant recipients with fibrosing cholestatic hepatitis (FCH) and compared with five other transplant recipients who did not develop this syndrome and 30 patients with chronic hepatitis B virus (HBV) infection. As measured by radioimmunoassays, the liver tissue from patients with FCH had significantly greater amounts of both hepatitis B surface antigen (HBsAg) and nucleocapsid antigens than to transplant patients without this syndrome (P < 0.01) or patients with chronic HBV infection (P< 0.001). Intrahepatic expression of pre-S,/pre-S, in FCH was also extensive with a distribution parallel to that of HBsAg. High-level expression of intrahepatic HBsAg and hepatitis B core antigen in the explanted liver was associated with subsequent development of FCH in the liver graft, suggesting that viral/host factors may also be important. This pattern of intrahepatic hepatitis viral antigen expression, by analogy with Chisari’s transgenic mice model and Roingeard’s HBV-transfected HepG2 cell model, may be the cause of direct hepatocytopathic injury in this condition.
C
hronic hepatitis B virus (HBV) infection is potentially one of the commonest indications for liver transplantation worldwide, but recurrence of HBV infection in the grafted liver is associated with increased morbidity and mortality after transplantation.lm3 The histological implications of HBV reinfection of the liver graft are variable, including minor histological changes, chronic persistent/active hepatitis, and a unique histological pattern of fibrosing cholestatic hepatitis (FCH) that occurred in 24.1% of the hepatitis B surface antigen (HBsAg)-seropositive transplant patients who survived for more than 2 months.3v4 Clinically, FCH is characterized by rapidly progressive liver failure and a high mortality within 4-6 weeks of clinical onset.3 Histologically, it is characterized by a pattern of extensive serpiginous
periportal fibrosis, canalicular and cellular cholestasis, and prominent HBV antigen expression in hepatocytes but a mild mixed inflammatory cell infiltrate.3*4 The clinical pattern of acute liver failure and the relatively mild inflammatory cell infiltrate suggest that the liver cell damage in this pattern of graft reinfection is not immune mediated but might instead be cytopathic. In the present study, we have elucidated the extent of intrahepatic HBV antigen expression in transplant patients with FCH to determine its possible role in pathogenesis. The extent of HBV antigen expression in the explanted liver was also correlated with the subsequent clinical course to determine whether viral or host factors are also involved. Patients and Methods Of the 39 patients seropositive for HBsAg receiving transplants within the Cambridge/King’s College Hospital series,3 histological specimens were available in 27 patients surviving longer than .Z months. Twenty-three patients had recurrence of HBV infection as shown by the reappearance of serum HBsAg (Table 1). Seven patients developed clinically and histologically proven FCH; four other patients also ran a clinical course typical of FCH but without histological confirmation.3’4 In the majority of patients, maintenance immunoglobulin therapy was not given.3 Serum HBsAg was studied in all 23 transplant patients with HBV recurrence and serum pre-S, in 17 patients. Intrahepatic HBV antigens were quantified in four patients with FCH (5-8 months after liver transplantation) and five transplant patients with HBV recurrence but no evidence of FCH (3-10 months after transplantation: histological diagnosis: minor changes, 1; lobular hepatitis, 2; chronic active hepatitis, 1; large duct obstruction, 1). Hepatocytes were also isolated from two hepatectomy specimens for determination of the amount of intracellular soluble protein for comparative purposes. Intrahepatic expression of HBsAg and pre-S antigens were studied in 32 specimens 0 1992 by the American
Gastroenterological 0016-5065/92/$3.00
Association
HBV ANTIGEN EXPRESSION IN FCH
March 1992
957
HEPES-buffered RPM1 1640 with 0.2% ethylenediaminetetraacetic acid (EDTA; pH 7.4), and hepatocytes were released into suspension by gently scraping the biopsy specimens with two 21-G needles in a Petri dish. After orthotopic The resulting cell suspension was washed three times in Chronic liver transplant RPM1 1640 by centrifugation at 50g at 4°C for 5 minutes. HBV infection (HBV recurrence] Cells were counted and 200,000 cells were resuspended in 23 30 No. 3 mL of RPM1 1640. Evaluation by phase-contrast micros26:4 19:4 Sex (male:female) copy showed that the isolated hepatocytes retained an in35(23-59) 37(23-61) Median age [yr (range)] tact plasma membrane and should have retained the HBV Serology antigens.7 In addition, the residual fibrosis tissue con1:25,600 1:12,800 Median HBsAg titer tained minimal or no attached hepatocytes. (1:32-214,800) (1:64-214,800) (range) The cell suspensions were sonicated over ice at 10 am16 19 HBeAg positive (n) 21 13 HBV DNA positive (n) plitude microns 10 seconds per spell for three spells to re7 0 Anti-HDV positive (n) lease the intracellular viral antigens7 Sonication accordHistology ing to this method did not denature the HBsAg and Normal graft (n) nucleocapsid antigense7 The cell sonicates were then Lobular hepatitis (n) stored at -20°C until tested in batches using commercially Minor changes, chronic available radioimmunoassays for HBsAg and nucleocapsid 5 carriage (n) antigen (Abbott Diagnostics). The HBsAg results were Chronic persistent quantified by constructing a standard curve using dilu11 hepatitis (n) Chronic active tions of a standard positive control sample containing 20 a hepatitis (n) ng/mL HBsAg; the nucleocapsid antigen results were ex6 Cirrhosis (n) pressed with reference to a standard taken as 1 U/mL. Acute cellular Mixing HBV antigens with cell sonicate from a hepatoblasrejection (n) toma cell line HepG2 (ECACC, Porton Down, England) did Vanishing bile duct not interfere with the radioimmunoassay. The proportion syndrome (n) of cells containing HBV antigen in these preparations was Fibrosing cholestatic also determined by immunocytochemistry as described hepatitis (n) below. Bile duct obstruction (n) The amount of soluble protein in the hepatocytes was also determined in two hepatectomy specimens for comparative purposes. Thirty million hepatocytes were prepared and sonicated in RPM1 1640 and 1 mmol/L phenylfrom the 23 transplant patients with HBV recurrence. methysulforyl fluoride (Sigma) as a protease inhibitor. The Thirty consecutive patients with chronic HBV infection samples were then ultracentrifuged (270,OOOgat 4°C for 30 who underwent liver biopsy for diagnostic purposes in minutes), and the amount of soluble proteins in the super1989-1990 were also studied for comparison (Table 1). natant was determined using a commercial protein assay kit (Pierce, Cheshire, England). Serology
Table 1. Clinical, Serological, and Histological Data of Patients Studied at the Time of the Latest Liver Biopsy
Serum was tested for HBsAg, hepatitis B e antigen (HBeAg), and anti-HBe by radioimmunoassay (Abbott Diagnostics, Maidenhead, England). HBsAg was quantified by serial dilution using reverse passive hemagglutination (Wellcome Diagnostics, Dartford, England). Antibody to hepatitis D virus was studied by enzyme immunoassay (Abbott Diagnostics), and serum HBV DNA was assayed using a quantitative dot-blot technique.5 Serum HBV preS, antigen was measured by a sandwich enzyme-linked immunosorbent assay using two monoclonal antibodies to pre-S, with different specificities with peroxidase labeling and orthophenylene diamine as the substrate. lntrahepatic
HEW Antigen
Quantity
Intrahepatic HBV antigens were quantified in nine transplant patients with HBV recurrence (four from patients with FCH; five from patients with no feature of FCH) and 30 patients with chronic HBV infection. Hepatocytes were isolated by the method described by Trevisan et a1.6 The liver biopsy specimens were immediately immersed
in
Detection of HBV Antigens in Liver Sections and Hepatocytes in the Cytospins The intrahepatic expression of the pre-S antigens (pre-S,/pre-S,) in the posttransplant liver biopsy or hepatectomy specimens was assessed together with HBsAg in serial paraffin liver sections by standard indirect immunohistochemistry using respective monoclonal antibodies (HBsAg: D2H5, 1:20 dilution; pre-S, : MA18/7, 1:lOO dilution; pre-S,: 5535, 1:25 dilution), alkaline phosphatase as the enzyme label and naphthol and Fast Red salt (Sigma Chemical Co., St. Louis, MO) as the substrate.7” The expression of HBsAg and HBcAg in the explanted livers was also assessed in similar fashion using respective monoclonal (HBsAg; D2H5) and polyclonal antibodies (HBcAg; I:200 dilution; Dako, Bucks, England) to determine the relationship between intrahepatic HBV antigen expression before transplantation and subsequent development of FCH. The expression of HBV antigen was scored independently by two of us (J.Y.N.L., S.E.D.) on a O-4+ scale corre-
958 LAU ET AL.
GASTROENTEROLOGYVol. loz,No.3
spondingto positivity in 0%, l%-5%, 5%-30%, 30%-60%, and >60% of hepatocytes examined. The proportion of HBV antigen-containing cells in the hepatocyte suspension was determined by immunocytochemistry. Cell smears were prepared using a cytospin (Shandon, England) to obtain 20,000 cells per slide previously coated with 0.1% poly-l-lysine (Sigma) to enhance cell adhesion, and slides were stored at -20°C before staining. After thawing, cells were fixed in ice-cold acetone for 2 minutes and air dried. The HBV antigens were then sought by standard immunocytochemistry using alkaline phosphatase as the enzyme label and Fast Red salt as the substrate.7’8 Statistics Results are expressed as medians and ranges and were analyzed using Wilcoxon’s rank-sum test, Spearman’s rank correlation test, or Fisher’s Exact Test. Results Serum HBsAg and Pre-S, Levels There was no significant difference in the median titer of serum HBsAg between transplanted patients with FCH (1:51, ZOO;range, 1:3, ZOO-160,000; n = 7), transplant patients with no evidence of FCH (1:25,600; range, 1:8-214,800; n = 16; anti-HDV+, 7), and patients with chronic HBV infection (1:12,800; range, 1:64-214,800; n = 30). In patients with chronic HBV infection, those with nonspecific reactive changes or chronic persistent hepatitis had higher serum HBsAg titers (median, 1:25,600; range, 1:256214,800; n = 16) than patients with chronic active hepatitis and cirrhosis (median, 1:3200; range, 1:6425,600; n = 14). Patients with FCH had serum titers of HBsAg comparable to those of the HBV carrier patients with nonspecific reactive changes or chronic persistent hepatitis. Serum pre-S, (range, O-25.2 U/mL) correlated well with serum HBsAg (R, = 0.6831; n = 16; P = 0.004) in transplant patients with HBV recurrence. All patients with FCH were seropositive for both serum pre-S, and HBV DNA (n = 7). Among patients without evidence of FCH, 6 of 10 tested were seropositive for pre-S, and 14 of 16 were seropositive for HBV DNA (P = NS). Intrahepatic
HBV Antigen Expression
In FCH, intrahepatic expression of HBsAg, pre-S,, and pre-S, was 4+ (i.e., positive in virtually all cells) with a diffuse cytoplasmic distribution. Cells with prominent surface antigen expression also showed a variable degree of ballooning with groundglass transformation, bile pigmentation, and clearing of the cytoplasm. Intrahepatic expression of the nu-
cleocapsid antigens was also extensive, both nuclear and cytoplasmic, and has been detailed previously.4 In the liver specimens from transplant patients without evidence of FCH, the median intrahepatic expression of HBsAg was 2+ (range, l+-4+; n = 16; HDV+, 7). Biopsy specimens from three patients showed membraneous surface antigen in addition to cytoplasmic expression (histology: chronic active hepatitis, 2; lobular hepatitis, 1; all negative for HDV). The expression of hepatitis B core antigen (HBcAg) was mainly nuclear l+ (range, O-3+; n = 16; HDV+, 7) with six patients also showing cytoplasmic HBcAg l-l- (range, O-2+). In the biopsy specimens from patients with chronic HBV infection, the median intrahepatic expression of HBsAg was 3+ (range, O-4+; n = 30), and that of HBcAg was 2+ in patients seropositive for HBeAg (range, O-3+; n = 19; cytoplasmic HBcAg present in three patients). Patients with FCH had a significantly higher proportion of hepatocytes expressing both HBsAg and HBcAg than transplant patients without FCH and patients with chronic HBV infection (P < 0.01 for both HBsAg and HBcAg). Intrahepatic
HBVAntigen
Quantity
The amount of intrahepatic HBsAg in patients with FCH, as measured by radioimmunoassay of 200,000 sonicated hepatocytes, was 191.1 ng (range, 164.4-312.2 ng; n = 4), compared with 4.5 ng (range, 0.2-28.1 ng; n = 5; HDV+, 2; P < 0.01) in transplant patients with no features of FCH and 44.8 ng (range, 0.2-107.9 ng; n = 30; P < 0.001) in patients with chronic HBV infection. To determine the average quantity of intracellular HBsAg per hepatocyte, we divided the amount of HBV antigens by the estimated number of positive hepatocytes as determined by immunocytochemical staining of the hepatocyte cytospins. The amount of HBsAg per hepatocyte was also higher in patients with FCH 0.98 pg/cell (range, 0.95-1.56 pg/cell; n = 4) than in patients with no evidence of FCH [0.22 pg/cell (range, 0.05-0.70 pg/cell; n = 5; HDV+, 2; P < O.Ol)] and patients with chronic HBV infection [0.64 pg/mL (range, 0.05-1.02 pg/cell; n = 30; P < O.OOl)] (Figure ?A). In relation to the amount of hepatocytesoluble protein, HBsAg constitutes between 0.5% and 1.0% of the soluble protein per hepatocyte in patients with FCH. The median quantity of intrahepatic nucleocapsid antigen was also significantly higher in patients with FCH [lo.2 U/200,000 hepatocytes (range, 4.2-22.4 U; n = 4)] than in transplant patients with no evidence of FCH [0.2 U (range, 0.0-0.5 U; n = 5; HDV+, 2; P < O.Ol)] or patients with chronic HBV infection seropo-
HBV ANTIGEN
March 1992
,
EXPRESSION
P
