(Cin. exp. Immunol. (1979) 37, 15-24.
Hepatitis B core antigen immune complexes in the liver of hepatitis B patients M. B. RAY,* V. J. DESMET,* A. F. BRADBURNEt J. DESMYTER,t J. FEVERY+ & J. DE GROOTEt *Laboratory of Histochemistry and Cytochemistry, tViral Diseases Laboratory and +Division of Hepatology,
Academisch Ziekenhuis Sint Rafail, University of Leuven, Belgium
(Accepted for publication 20 December 1978) SUMMARY
Single liver biopsies from 102 clinically diagnosed hepatitis patients were examined by immunofluorescence for the presence of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg), complement and immunoglobulin deposition, and for their capacity to fix human complement in vitro. Of the sixty-five HBsAg positive livers, fifty-three were histologically diagnosed as chronic hepatitis, three as acute hepatitis, five as acute hepatitis with signs of transition to chronicity, and four as 'near normal liver'. In the group with chronic hepatitis, HBcAg was observed in thirty-nine livers, all of which also had HBsAg. Thirty-five of these thirty-nine cases also had the ability to fix complement in vitro in the hepatocyte nuclei and/or cytoplasm. Of these thirty-five cases, twenty-nine were positive for immunoglobulin deposition on the nuclei. All of these cases had antibody to HBcAg in the blood, but only five had anti-HBs. The frequency of in vitro complement fixation and immunoglobulin deposition was higher in active forms of the disease, such as chronic aggressive hepatitis and active cirrhosis, than in non-active disease such as chronic persistent hepatitis and mild cirrhosis. By the application of double fluorescent staining techniques, complement fixation was observed in some HBcAg-positive nuclei. In the 'near normal liver' cases there was no intrahepatic accumulation of HBcAg, and despite the presence of anti-HBc in the blood, in vitro complement fixation and immunoglobulin deposition were both absent. The group of three HBsAg positive 'acute hepatitis with signs of transition to chronicity' cases behaved similarly to those with chronic aggressive hepatitis and had circulating anti-HBc, in vitro complement fixation and immunoglobulin deposition in the hepatocytes. None had circulating anti-HBs. In the group with HBs-positive acute hepatitis, anti-HBc in the blood was the only other evidence of hepatitis B virus infection. INTRODUCTION Hepatitis B surface (HBsAg) and hepatitis B core (HBcAg) antigens are two morphologically and immunologically distinct components of the hepatitis B virus (HBV) (Almeida, Rubinstein & Stott, 1971; Barker et al., 1974). The intrahepatic distribution patterns of these antigens are distinct in both acute and chronic hepatitis B (Ray et al., 1976a) but the origin of these patterns of HBV antigen expression and their functional roles in the histological alterations seen in hepatitis B infection remain obscure. Early studies supported the view that HBsAg immune complexes may mediate liver cell damage in hepatitis (Nowoslawski et al., 1972). Several workers have reported the presence of bound IgG with anti-HBc Correspondence: Dr M. B. Ray, Laboratorium voor Histochemie en Cytochemie, Departement Medische Navorsing, Academisch Ziekenhuis Sint Rafael, B-3000 Leuven, Belgium. 0099-9104/79/0070-0015$02.00 (© 1979 Blackwell Scientific Publications
15
16
AM. B. Ray et al.
specificity in HBcAg-positive complexes (Arnold et al., 1975; Gerber, Sarno & Vernace, 1976; Rizzetto et al., 1976; Ten Kate et al., 1974). Such immune complexes were observed in 9000 of HBsAg chronic aggressive hepatitis (CAH) cases and in 17% of cases with acute hepatitis B, (Arnold et al., 1975). Gerber et al. (1976) reported the presence of HBcAg, IgG and in vitro complement fixation (VCF) in CAH but not in acute hepatitis (AH). Ten Kate et al. (1974) demonstrated nuclear IgG in some livers with intranuclear core particles. The present study was designed to investigate further the various intrahepatic localization patterns of HBV components, in vitro complement fixation and immunoglobulins in over 100 patients. These results are correlated with the histological diagnosis and with circulating antibodies to HBs and HBc antigens. MATERIALS AND METHODS 102 patients with clinically diagnosed hepatitis were each subjected once to needle biopsy. Some patients with diagnosed CAH and active cirrhosis received standard therapy with prednisolone alone or in combination with azothioprine. Histological diagnosis was used as the sole classification criterion of the hepatitis patients (see Review by an International Group, 1971, 1977). Acute hepatitis with signs of a possible transition to chronicity (AHTC) is a histological entity combining lobular features of acute hepatitis with periportal piece-meal necrosis as described previously (Review by an International Group, 1971; 1977). Biopsies with minimal inflammatory changes but with numerous 'ground glass' hepatocytes were categorized as 'near-normal liver'. Immunofluorescence of HBsAg and HBcAg. The procedures used for the immunofluorescent demonstration of these antigens have been described previously (Ray et al., 1976a), as have the technical aspects for the demonstration of HBsAg in frozen liver biopsies (Ray, Van Damme & Desmet, 1974). Both the serology and the fluorescent examinations were done under 'double-blind' conditions. In vitro complement fixation (VCF). The technique described by Burkholder (1961) was used, employing fresh human serum of blood group AB and negative for anti-nuclear factor as the source of complement (C3). This serum was incubated with the 4p thick unfixed cryostat sections of biopsy material. Donors of serum used as a C3 source had previously been checked to make sure that all serology for HBV infection was negative. Incubation was for 45 min at 370C, after which the sections were washed and then stained with fluoresceine isothiocyanate (FITC) conjugate rabbit anti-human C3 (Hyland) for 30 min at 370C. As a complement control, the fresh human serum was inactivated by heating at 560C for 1 hr before use. A second control omitted the source of C3 and serial sections were stained directly with the FITC-anti-C3. Immunoglobulin detection. Rabbit antisera directed against human IgG, IgA and IgM were purchased from Organon Teknika, Antwerp. These antisera were used to detect immunoglobulins in biopsy sections in indirect immunofluorescence, using FITC-conjugated goat anti-rabbit globulins (Hyland) as the second layer. Occasionally, immunoglobulins were tested for directly by using FITC-conjugated rabbit anti-human IgG, IgA and IgM (Nordic). The specificity of all these anti-human immunoglobulin antisera was controlled by staining sections of lymph node biopsies from serologically proven monoclonal myelomas and lymphomas. Double staining with contrast colour fluorochromes. Double staining was used to reveal the concomitant presence of HBV antigen and VCF activity. Using both FITC and rhodamine (TRITC) conjugated antisera, serial sections of biopsies were stained to these two schedules: (1) HBsAg and HBcAg: rabbit anti-HBs + goat anti-rabbit globulin/TRITC + human anti-HBc/FITC; (2) HBcAg and VCF: C3 as normal human serum + goat anti-human C3/TRITC + human anti-HBc/ FITC. All TRITC-conjugated antisera were purchased from Nordic, Antwerp. After completing each staining schedule, slides were washed in buffer and mounted as usual. The specimens were examined in a Leitz Ortholux microscope with an epi-illumination arrangement using an Xenon XBO 75 lamp, and equipped with excitation filters for alternative examination at fluorescein and rhodamine excitation wavelengths. Serology. Serum samples from each patient were collected within a week of liver biopsy and tested for HBsAg by radioimmunoassay (Ausria II, Abbott). All of the 102 patients in this study were tested for HBsAg and eighty-eight serum samples were available for further studies on anti-HBs and anti-H1Bc. Anti-HBs was measured by either passive haemagglutination (Electro-nucleonics) or radioimmunoassay (Ausab, Abbott). Anti-HBc was titrated by a solid-phase radioimmunoassay developed in our laboratories (Desmyter & Bradburne, 1975).
RESULTS Demonstration of HBsAg and HBcAg The intrahepatic distribution patterns of HBsAg alone, and in combination with HBcAg in the various histological groups found in biopsies from the 102 patients were similar to those reported previously (Ray et al., 1976a, b; Ray & Desmet, 1976c). HBsAg was observed in both the cytoplasm and membranes of the liver cells. HBcAg was localized mostly in the nuclei of hepatocytes but was, on rare
17 Hepatitis B core antigen immune complex occasions, to be found in the cytoplasm and periphery of the hepatocytes. The distribution patterns and mutual proportions of HBsAg and HBcAg were disease-related. Demonstration of in vitro complement fixation Complement fixation was detected in the nucleus and cytoplasm of the hepatocytes as bright, granular fluorescence. In most hepatocytes the intra- or extranuclear staining patterns were mutually exclusive, but sometimes staining throughout the cell was observed (Fig. 1). On occasions Kupffer cells were also stained positively. The in vitro complement fixation was only detected if fresh human serum was used as the source of complement; heat-inactivation or the omission of C3 abolished any reaction.
FIG. 1. In vitro complement fixation: liver biopsy from a patient with untreated chronic aggressive hepatitis. Positivity is present both in the nuclei and cytoplasm of the hepatocytes. Cytoplasmic complement fixation is coarsely granular and is observed in the periphery of the hepatocytes. (Magnification x 400.)
Localization of immunoglobulin In the majority of cases, immunoglobulin classes were only detected individually. IgG was predominant, being observed exclusively in the hepatocyte nuclei of 900 , of the biopsies positive for immunoglobulins. IgM was detected only twice (accompanying IgG) and IgA only once. The fluorescence varied in intensity and was mostly granular in appearance. Staining of the nuclei was far more common than in the cytoplasm; any in the latter was only very faint. Liver sinusoids were strongly stained, especially for IgG, and this hindered the detection of IgG in the cell membranes. However, membrane-bound IgG was sometimes definitely located. The revelation of HB V antigens by double staining procedures Double staining of positive specimens for both HBsAg and HBcAg produced different patterns of combination. Some liver cells contained nuclear HBcAg alone; others were positive for HBsAg only, either cytoplasmic or membrane-associated. However, particularly if the category of membrane-only HBsAg localization is included a substantial proportion of liver cells expressed both antigens. When using the double staining schedule for both HBcAg and VCF, the majority of cells were found to be positive for both parameters together (Fig. 2 & 3). B
18
M. B. Ray et al.
FIG. 2. HBcAg fluorescence: liver biopsy from a patient with untreated chronic aggressive hepatitis, double stained first with normal human serum followed by goat anti-human C3/TRITC and then with antiHBc/FITC. HBcAg specific green fluorescence of variable intensity is limited to the nuclei of the hepatocytes. (Magnification x 386.)
Incidence of circulating antibodies to HBV antigens Table 1 shows the frequencies of anti-HBs and anti-HBc in the sera of all the patients studied. In HBsAg sero-positive chronic hepatitis, 100% of the sera examined had circulating anti-HBc; six (150%) of them had anti-HBs as well. In twenty-nine HBsAg-negative chronic hepatitis cases, 24% were positive for anti-HBc alone and another 8% also had anti-HBs. Yet another 1200 carried anti-HBs alone. Two patients (with active cirrhosis) had anti-HBc in the serum, but although the liver was positive for HBsAg by fluorescence, the serum was negative by radioimmunoassay. In the twelve cases of acute hepatitis, half were HBsAg-positive and five (83%) had circulating antiHBc with HBsAg. In contrast only one of the five sera studied from HBsAg- negative cases had antiHBc. Out of all the patients with HBsAg, anti-HBs detected only when anti-HBc was present. These cases were confined to the group with chronic hepatitis and the anti-HBs was only detectable by radioimmunoassay. In the AHTC group, the only patient without HBsAg was negative for anti-HBs and anti-HBc as well; all four HBsAg-positive cases examined had anti-HBc, but no anti-HBs. All the HBsAg carriers ('near normal liver') had anti-HBc and no anti-HBs in the blood.
Relationship between membrane-localized HBsAg and nuclear HBcAg In chronic hepatitis and in AHTC there is a direct correlation between the membrane-localized HBsAg and the nuclear HBcAg (Ray et al., 1976a). This finding was confirmed by the present results.
Relationship between HBcAg in the liver cells and in vitro complement fixation Table 2 lists in detail the correlation between HBcAg in the hepatocytes, and VCF and immunoglobulin deposition in the HBsAg-positive livers. In chronic hepatitis, of thirty-nine biopsies which were positive for HBcAg, thirty-five fixed complement in the hepatocytes-twenty-one of these in both the
Hepatitis B core antigen immune complex
19
FIG. 3. In vitro complement fixation in HBcAg-containing nuclei: same area as is Fig. 2, photographed after adjusting the filters specific for TRITC. All the HBcAg containing nuclei fixed complement in vitro. The red fluorescence is stronger and the number of complement-positive nuclei is more than the number of IBcAgpositive nuclei. (Magnification x 386.)
nucleus and cytoplasm. In the more active diseases (CAH and active cirrhosis), HBcAg was found in thirty-four out of forty-two cases, of which thirty-one had been found to stain positively for VCF activity. In less active cirrhosis and chronic persistent hepatitis (CPH) the proportion of both parameters was lower; five out of twelve had HBcAg, of which four had VCF. Of twenty-one cases in which VCF was found throughout the hepatocytes. eleven had a similar distribution of HBcAg. In the single CPH case which was found to be negative for VCF, only a couple of nuclei were positive (faintly) for HBcAg. Three biopsies which were definitely positive for HBcAg (two from CAH and one active cirrhosis) were completely negative for VCF. VCF was only found once in cases of acute hepatitis and this case gave only weak nuclear staining, without coincident HBcAg staining. In three AHTC cases where HBcAg was demonstrated, VCF activity was also found. No HBcAg or VCF were found in the four cases with 'near normal liver'. Relation between VCF, immunoglobulin deposition in the liver and circulating anti-HBc In HBsAg-positive chronic hepatitis, twenty-nine out of thirty-five VCF-positive cases also had immunoglobulin (mostly IgG) deposited in hepatocyte nuclei. In the immunoglobulin depositionpositive group, twenty-three sera were available for study; all had anti-HBc and five had also anti-HBs One patient had both circulating antibodies and yet was negative for immunoglobulin deposition. In the six remaining VCF-positive biopsies, despite the fact that a few hepatocyte nuclei contained HBcAg and there was anti-HBc in the blood, there was no immunoglobulin deposition, nor was there in the HBsAg-positive acute hepatitis group. On the other hand, IgG could be localized in all three VEF-positive cases from AHTC, which also had anti-HBc in the blood. No immunoglobulin deposition was found in the 'HBsAg-carrier' cases, despite circulating anti-HBc. No HBcAg, VCF or immunoglobulin deposition was found in any of the HBsAg-negative cases (Table 1), not even in the two cases of active cirrhosis where intrahepatic HBsAg was found.
20
M. B. Ray et al. '0
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Hepatitis B core antigen immune complex
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Hepatitis B core antigen immune complexes in the liver of hepatitis B patients M. B. RAY,* V. J. DESMET,* A. F. BRADBURNEt J. DESMYTER,t J. FEVERY+ & J. DE GROOTEt *Laboratory of Histochemistry and Cytochemistry, tViral Diseases Laboratory and +Division of Hepatology,
Academisch Ziekenhuis Sint Rafail, University of Leuven, Belgium
(Accepted for publication 20 December 1978) SUMMARY
Single liver biopsies from 102 clinically diagnosed hepatitis patients were examined by immunofluorescence for the presence of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg), complement and immunoglobulin deposition, and for their capacity to fix human complement in vitro. Of the sixty-five HBsAg positive livers, fifty-three were histologically diagnosed as chronic hepatitis, three as acute hepatitis, five as acute hepatitis with signs of transition to chronicity, and four as 'near normal liver'. In the group with chronic hepatitis, HBcAg was observed in thirty-nine livers, all of which also had HBsAg. Thirty-five of these thirty-nine cases also had the ability to fix complement in vitro in the hepatocyte nuclei and/or cytoplasm. Of these thirty-five cases, twenty-nine were positive for immunoglobulin deposition on the nuclei. All of these cases had antibody to HBcAg in the blood, but only five had anti-HBs. The frequency of in vitro complement fixation and immunoglobulin deposition was higher in active forms of the disease, such as chronic aggressive hepatitis and active cirrhosis, than in non-active disease such as chronic persistent hepatitis and mild cirrhosis. By the application of double fluorescent staining techniques, complement fixation was observed in some HBcAg-positive nuclei. In the 'near normal liver' cases there was no intrahepatic accumulation of HBcAg, and despite the presence of anti-HBc in the blood, in vitro complement fixation and immunoglobulin deposition were both absent. The group of three HBsAg positive 'acute hepatitis with signs of transition to chronicity' cases behaved similarly to those with chronic aggressive hepatitis and had circulating anti-HBc, in vitro complement fixation and immunoglobulin deposition in the hepatocytes. None had circulating anti-HBs. In the group with HBs-positive acute hepatitis, anti-HBc in the blood was the only other evidence of hepatitis B virus infection. INTRODUCTION Hepatitis B surface (HBsAg) and hepatitis B core (HBcAg) antigens are two morphologically and immunologically distinct components of the hepatitis B virus (HBV) (Almeida, Rubinstein & Stott, 1971; Barker et al., 1974). The intrahepatic distribution patterns of these antigens are distinct in both acute and chronic hepatitis B (Ray et al., 1976a) but the origin of these patterns of HBV antigen expression and their functional roles in the histological alterations seen in hepatitis B infection remain obscure. Early studies supported the view that HBsAg immune complexes may mediate liver cell damage in hepatitis (Nowoslawski et al., 1972). Several workers have reported the presence of bound IgG with anti-HBc Correspondence: Dr M. B. Ray, Laboratorium voor Histochemie en Cytochemie, Departement Medische Navorsing, Academisch Ziekenhuis Sint Rafael, B-3000 Leuven, Belgium. 0099-9104/79/0070-0015$02.00 (© 1979 Blackwell Scientific Publications
15
16
AM. B. Ray et al.
specificity in HBcAg-positive complexes (Arnold et al., 1975; Gerber, Sarno & Vernace, 1976; Rizzetto et al., 1976; Ten Kate et al., 1974). Such immune complexes were observed in 9000 of HBsAg chronic aggressive hepatitis (CAH) cases and in 17% of cases with acute hepatitis B, (Arnold et al., 1975). Gerber et al. (1976) reported the presence of HBcAg, IgG and in vitro complement fixation (VCF) in CAH but not in acute hepatitis (AH). Ten Kate et al. (1974) demonstrated nuclear IgG in some livers with intranuclear core particles. The present study was designed to investigate further the various intrahepatic localization patterns of HBV components, in vitro complement fixation and immunoglobulins in over 100 patients. These results are correlated with the histological diagnosis and with circulating antibodies to HBs and HBc antigens. MATERIALS AND METHODS 102 patients with clinically diagnosed hepatitis were each subjected once to needle biopsy. Some patients with diagnosed CAH and active cirrhosis received standard therapy with prednisolone alone or in combination with azothioprine. Histological diagnosis was used as the sole classification criterion of the hepatitis patients (see Review by an International Group, 1971, 1977). Acute hepatitis with signs of a possible transition to chronicity (AHTC) is a histological entity combining lobular features of acute hepatitis with periportal piece-meal necrosis as described previously (Review by an International Group, 1971; 1977). Biopsies with minimal inflammatory changes but with numerous 'ground glass' hepatocytes were categorized as 'near-normal liver'. Immunofluorescence of HBsAg and HBcAg. The procedures used for the immunofluorescent demonstration of these antigens have been described previously (Ray et al., 1976a), as have the technical aspects for the demonstration of HBsAg in frozen liver biopsies (Ray, Van Damme & Desmet, 1974). Both the serology and the fluorescent examinations were done under 'double-blind' conditions. In vitro complement fixation (VCF). The technique described by Burkholder (1961) was used, employing fresh human serum of blood group AB and negative for anti-nuclear factor as the source of complement (C3). This serum was incubated with the 4p thick unfixed cryostat sections of biopsy material. Donors of serum used as a C3 source had previously been checked to make sure that all serology for HBV infection was negative. Incubation was for 45 min at 370C, after which the sections were washed and then stained with fluoresceine isothiocyanate (FITC) conjugate rabbit anti-human C3 (Hyland) for 30 min at 370C. As a complement control, the fresh human serum was inactivated by heating at 560C for 1 hr before use. A second control omitted the source of C3 and serial sections were stained directly with the FITC-anti-C3. Immunoglobulin detection. Rabbit antisera directed against human IgG, IgA and IgM were purchased from Organon Teknika, Antwerp. These antisera were used to detect immunoglobulins in biopsy sections in indirect immunofluorescence, using FITC-conjugated goat anti-rabbit globulins (Hyland) as the second layer. Occasionally, immunoglobulins were tested for directly by using FITC-conjugated rabbit anti-human IgG, IgA and IgM (Nordic). The specificity of all these anti-human immunoglobulin antisera was controlled by staining sections of lymph node biopsies from serologically proven monoclonal myelomas and lymphomas. Double staining with contrast colour fluorochromes. Double staining was used to reveal the concomitant presence of HBV antigen and VCF activity. Using both FITC and rhodamine (TRITC) conjugated antisera, serial sections of biopsies were stained to these two schedules: (1) HBsAg and HBcAg: rabbit anti-HBs + goat anti-rabbit globulin/TRITC + human anti-HBc/FITC; (2) HBcAg and VCF: C3 as normal human serum + goat anti-human C3/TRITC + human anti-HBc/ FITC. All TRITC-conjugated antisera were purchased from Nordic, Antwerp. After completing each staining schedule, slides were washed in buffer and mounted as usual. The specimens were examined in a Leitz Ortholux microscope with an epi-illumination arrangement using an Xenon XBO 75 lamp, and equipped with excitation filters for alternative examination at fluorescein and rhodamine excitation wavelengths. Serology. Serum samples from each patient were collected within a week of liver biopsy and tested for HBsAg by radioimmunoassay (Ausria II, Abbott). All of the 102 patients in this study were tested for HBsAg and eighty-eight serum samples were available for further studies on anti-HBs and anti-H1Bc. Anti-HBs was measured by either passive haemagglutination (Electro-nucleonics) or radioimmunoassay (Ausab, Abbott). Anti-HBc was titrated by a solid-phase radioimmunoassay developed in our laboratories (Desmyter & Bradburne, 1975).
RESULTS Demonstration of HBsAg and HBcAg The intrahepatic distribution patterns of HBsAg alone, and in combination with HBcAg in the various histological groups found in biopsies from the 102 patients were similar to those reported previously (Ray et al., 1976a, b; Ray & Desmet, 1976c). HBsAg was observed in both the cytoplasm and membranes of the liver cells. HBcAg was localized mostly in the nuclei of hepatocytes but was, on rare
17 Hepatitis B core antigen immune complex occasions, to be found in the cytoplasm and periphery of the hepatocytes. The distribution patterns and mutual proportions of HBsAg and HBcAg were disease-related. Demonstration of in vitro complement fixation Complement fixation was detected in the nucleus and cytoplasm of the hepatocytes as bright, granular fluorescence. In most hepatocytes the intra- or extranuclear staining patterns were mutually exclusive, but sometimes staining throughout the cell was observed (Fig. 1). On occasions Kupffer cells were also stained positively. The in vitro complement fixation was only detected if fresh human serum was used as the source of complement; heat-inactivation or the omission of C3 abolished any reaction.
FIG. 1. In vitro complement fixation: liver biopsy from a patient with untreated chronic aggressive hepatitis. Positivity is present both in the nuclei and cytoplasm of the hepatocytes. Cytoplasmic complement fixation is coarsely granular and is observed in the periphery of the hepatocytes. (Magnification x 400.)
Localization of immunoglobulin In the majority of cases, immunoglobulin classes were only detected individually. IgG was predominant, being observed exclusively in the hepatocyte nuclei of 900 , of the biopsies positive for immunoglobulins. IgM was detected only twice (accompanying IgG) and IgA only once. The fluorescence varied in intensity and was mostly granular in appearance. Staining of the nuclei was far more common than in the cytoplasm; any in the latter was only very faint. Liver sinusoids were strongly stained, especially for IgG, and this hindered the detection of IgG in the cell membranes. However, membrane-bound IgG was sometimes definitely located. The revelation of HB V antigens by double staining procedures Double staining of positive specimens for both HBsAg and HBcAg produced different patterns of combination. Some liver cells contained nuclear HBcAg alone; others were positive for HBsAg only, either cytoplasmic or membrane-associated. However, particularly if the category of membrane-only HBsAg localization is included a substantial proportion of liver cells expressed both antigens. When using the double staining schedule for both HBcAg and VCF, the majority of cells were found to be positive for both parameters together (Fig. 2 & 3). B
18
M. B. Ray et al.
FIG. 2. HBcAg fluorescence: liver biopsy from a patient with untreated chronic aggressive hepatitis, double stained first with normal human serum followed by goat anti-human C3/TRITC and then with antiHBc/FITC. HBcAg specific green fluorescence of variable intensity is limited to the nuclei of the hepatocytes. (Magnification x 386.)
Incidence of circulating antibodies to HBV antigens Table 1 shows the frequencies of anti-HBs and anti-HBc in the sera of all the patients studied. In HBsAg sero-positive chronic hepatitis, 100% of the sera examined had circulating anti-HBc; six (150%) of them had anti-HBs as well. In twenty-nine HBsAg-negative chronic hepatitis cases, 24% were positive for anti-HBc alone and another 8% also had anti-HBs. Yet another 1200 carried anti-HBs alone. Two patients (with active cirrhosis) had anti-HBc in the serum, but although the liver was positive for HBsAg by fluorescence, the serum was negative by radioimmunoassay. In the twelve cases of acute hepatitis, half were HBsAg-positive and five (83%) had circulating antiHBc with HBsAg. In contrast only one of the five sera studied from HBsAg- negative cases had antiHBc. Out of all the patients with HBsAg, anti-HBs detected only when anti-HBc was present. These cases were confined to the group with chronic hepatitis and the anti-HBs was only detectable by radioimmunoassay. In the AHTC group, the only patient without HBsAg was negative for anti-HBs and anti-HBc as well; all four HBsAg-positive cases examined had anti-HBc, but no anti-HBs. All the HBsAg carriers ('near normal liver') had anti-HBc and no anti-HBs in the blood.
Relationship between membrane-localized HBsAg and nuclear HBcAg In chronic hepatitis and in AHTC there is a direct correlation between the membrane-localized HBsAg and the nuclear HBcAg (Ray et al., 1976a). This finding was confirmed by the present results.
Relationship between HBcAg in the liver cells and in vitro complement fixation Table 2 lists in detail the correlation between HBcAg in the hepatocytes, and VCF and immunoglobulin deposition in the HBsAg-positive livers. In chronic hepatitis, of thirty-nine biopsies which were positive for HBcAg, thirty-five fixed complement in the hepatocytes-twenty-one of these in both the
Hepatitis B core antigen immune complex
19
FIG. 3. In vitro complement fixation in HBcAg-containing nuclei: same area as is Fig. 2, photographed after adjusting the filters specific for TRITC. All the HBcAg containing nuclei fixed complement in vitro. The red fluorescence is stronger and the number of complement-positive nuclei is more than the number of IBcAgpositive nuclei. (Magnification x 386.)
nucleus and cytoplasm. In the more active diseases (CAH and active cirrhosis), HBcAg was found in thirty-four out of forty-two cases, of which thirty-one had been found to stain positively for VCF activity. In less active cirrhosis and chronic persistent hepatitis (CPH) the proportion of both parameters was lower; five out of twelve had HBcAg, of which four had VCF. Of twenty-one cases in which VCF was found throughout the hepatocytes. eleven had a similar distribution of HBcAg. In the single CPH case which was found to be negative for VCF, only a couple of nuclei were positive (faintly) for HBcAg. Three biopsies which were definitely positive for HBcAg (two from CAH and one active cirrhosis) were completely negative for VCF. VCF was only found once in cases of acute hepatitis and this case gave only weak nuclear staining, without coincident HBcAg staining. In three AHTC cases where HBcAg was demonstrated, VCF activity was also found. No HBcAg or VCF were found in the four cases with 'near normal liver'. Relation between VCF, immunoglobulin deposition in the liver and circulating anti-HBc In HBsAg-positive chronic hepatitis, twenty-nine out of thirty-five VCF-positive cases also had immunoglobulin (mostly IgG) deposited in hepatocyte nuclei. In the immunoglobulin depositionpositive group, twenty-three sera were available for study; all had anti-HBc and five had also anti-HBs One patient had both circulating antibodies and yet was negative for immunoglobulin deposition. In the six remaining VCF-positive biopsies, despite the fact that a few hepatocyte nuclei contained HBcAg and there was anti-HBc in the blood, there was no immunoglobulin deposition, nor was there in the HBsAg-positive acute hepatitis group. On the other hand, IgG could be localized in all three VEF-positive cases from AHTC, which also had anti-HBc in the blood. No immunoglobulin deposition was found in the 'HBsAg-carrier' cases, despite circulating anti-HBc. No HBcAg, VCF or immunoglobulin deposition was found in any of the HBsAg-negative cases (Table 1), not even in the two cases of active cirrhosis where intrahepatic HBsAg was found.
20
M. B. Ray et al. '0
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