Editorials Serological Markers of Primary Biliary Cirrhosis: Diagnosis, Prognosis and Subsets
In clinical practice, the most discriminating combination of biochemical and serological tests used to establish the diagnosis of PBC is an elevation of the serum alkaline phosphatase and IgM levels, together with the presence of antimitochondrial antibodies (MAS), which are mandatory for diagnosis. AMAs were first described as serological markers of PBC more than 25 years ago by Walker et al. (1)using immunofluorescence (IF) techniques. They demonstrated antibodies that gave a characteristic pattern of fluorescence staining of composite tissue blocks in the sera of virtually all patients with PBC (2). This non-organ, non-species-specific staining was localized t o the mitochondria by Berg, Doniach and Roitt in 1967 (3). Its usefulness as a marker for PBC was documented by several investigators who reported detectable AMA by indirect IF in 84% to 99% of patients (4-6).AMAs detected by IF were not entirely specific for PBC but could be found in sera from patients with other disorders (e.g., syphilis and myocarditis [7,8]). However, this lack of specificity was seldom a problem, and IF became the established conventional technique for detection of A M A in patients with suspected PBC. In clinical practice, a patient with cholestasis but negative for AMA by IF always arouses suspicion that PBC is not the correct diagnosis; conversely, the finding of AMAs by IF in a patient with normal liver blood test results may indicate early PBC (9). In view of the close association between AMA and PBC, much research has focused on the nature of the mitochondrial autoantigens. Little progress was made until a decade ago, when Berg and colleagues separated inner and outer mitochondrial membranes and showed that autoantibodies in PBC sera reacted with a trypsinsensitive antigen termed M2 on the inner mitochondrial membrane (10). The sera from patients with other disorders who were AMA positive by IF did not react with “purified” M2, whereas 96% of sera from 752 PBC patients did (11). Subsequent characterization by Western immunoblotting showed that M2 consisted of several antigenic determinants that were present not only in a variety of mammalian organs but also in yeast
Address reprint requests to: Dr. M. F. Bassendine, University of Newcastle upon Tyne, Medical Molecular Biology Group, Floor 4, Catherine Cookson Building, The Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. 31/1/35603
and bacteria (12). However, the identity of these non-organ, non-species-specific autoantigens remained elusive until 1988, when two groups independently showed that the major 70-kD M2 antigen that had been cloned by Gershwin and colleagues in 1987 (13)was the E2 component of pyruvate dehydrogenase complex (PDC) (14,15). It rapidly became clear that the other M2 antigenic determinants visualized on immunoblotting were also components of the 2-0x0 acid dehydrogenase multienzyme complexes located in mammalian mitochondria (16). Before the recent identification of the major M2 autoantigens, several groups developed rapid and relatively simple ELISAs for detection of AMA using mitochondrial or submitochondrial fractions as antigens (17-21). Once the identity of the M2 autoantigens was known, similar ELISAs were developed with recombinant fusion proteins or purified enzymes as antigens. Van der Water and colleagues (22) found a sensitivity of 88%in the diagnosis of PBC using recombinant PDC E2 alone as antigen; it increased to 96% when combined with recombinant E2 of branched-chain 2-0x0-acid dehydrogenase complex (BCOADC). A similar sensitivity of 93% in the diagnosis of PBC was found with purified PDC E2 and protein X as antigen (23). In this issue, Leung et al. (24) report a further elegant refinement of the ELISA in an attempt to improve its sensitivity. They have designed a recombinant protein to include the major immunodominant epitopes from the E2s of both rat PDC and bovine BCOADC. They show in a group of 84 PBC patients that the use of this recombinant dual-headed hybrid molecule gives a sensitivity of 93% in the diagnosis of PBC, compared with a sensitivity of 89% using recombinant PDC-E2 fusion protein alone. It should be noted, though, that the antigen used to coat the ELISA plates is not the purified hybrid antigen but an Escherichia coli lysate in which the expressed recombinant protein constitutes 10% to 15% of the whole (24). It has been demonstrated that PBC sera recognized two polypeptides present in crude extracts of wild-type E. coli. Use of deletion strains of E. coli has allowed identification of these polypeptides as the E2 components of PDC and 2-0x0-glutarate dehydrogenase complex (OGDC) (25). Thus in effect a cocktail of recombinant mammalian PDC EBIBCOADC E2 and bacterial PDC and OGDC are used in the ELISA reported by Leung et al in this issue (24). The inclusion of purified recombinant hybrid antigen and lysate of wild-type E. coli in their study would have been
545
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BASSENDINE AND YEAMAN
interesting. Previous work has shown, however, that the titer of AMA against mammalian PDC is higher than that against the bacterial antigen (26). The presence of bacterial antigens in the ELISA also raises the possibility that in a larger study, false-positive reactions will be encountered in non-PBC patients who have antibodies to E. coli. This type of designer ELISA may eventually replace conventional indirect immunofluorescence for the detection of AMA in routine clinical practice, but before it does, further studies are required. A direct blinded comparison of the two techniques should be undertaken to establish whether all patients who are AMA positive by IF can be detected using such a n ELISA. In the study reported by Leung et a1 (241, it is not stated whether the six patients wit,h PBC who were negative on ELISA had detectable AhL4 by IF. Another way of comparing the two techniques would be to assess how much of the fluorescent staining seen on composite tissue blocks could be absorbed out using the cocktail of dual-headed recombinant protein and bacterial antigens. It is possible that a component of the reactivity seen by IF is attributable to other M2 polypeptides (16) or to other reported antigens associated with the outer mitochondrial membrane-M4, M8 and M9 (11, 27). Autoantibodies reacting with these antigens are not found in the sera of all patients with PBC, so detection of these other AMAs would be of little value in diagnosis. However, they are of considerable interest to the clinician because Klein and Berg have presented evidence suggesting that antibodies to M4 and M8 occur preferentially in patients with progressive disease (28, 291, whereas antibodies to M9 may be associated with a benign course (30). These interesting observations have been difficult to confirm in other laboratories because the biochemical identities of the antigens remained elusive. However, Klein and Berg recently demonstrated that M9 antibodies recognize an epitope of glycogen phosphorylase (311, whereas M4 antibodies react with sulphite oxidase (32). Once these observations have been validated, the way is clear for the development of ELISAs to detect AMA, which may be of prognostic relevance in patients in whom PBC has been diagnosed. In addition to the heterogeneity of AMAS, other autoantibodies have been described (33, 34) and are being characterized and identified in patients with PBC. This serves to emphasize that the breakdown in selftolerance in this “model” autoimmune disease is broadly based. Of particular interest are two district antinuclear antibodies (ANAs) that appear to be specific for patients with PBC. One ANA, which reacts with multiple nuclear dots (MNDs) by IF, has been found in 10% to 44% of PBC patients (35-37) (mainly in those with associated sicca syndrome [381). The major antigenic determinant has been characterized as a polypeptide with an apparent molecular weight of 95 to 100 kD, and a full-length complementary DNA encoding this nuclear autoantigen has recently been isolated by
HEPK~I’OI,OGY
Szostecki et a1 (39). This group has used the partially purified recombinant human nuclear antigenifusion protein in an ELISA and detected MND ANA in 50 of 184 (27%) of PBC patients but not in patients suffering from other liver diseases. Moreover, some sera from PBC patients contained MND ANA but did not contain concomitant AMA, raising the possibility that autoantibodies to this MND antigen may define a subgroup of PBC patients. PBC sera may also contain ANAs that produce a nuclear rim staining pattern in immunofluorescence (40). Two families of autoantibodies directed against integral membrane proteins of the nuclear envelope have been described in PBC patients. They appear to be disease specific: one reacts with integral membrane glycoprotein gp210 (41-431, and the other (described to date in only two AMA-negative patients) reacts against the nuclear envelope lamin B receptor (44). Further work on correlating the presence of these PBC-specific ANAs with clinical parameters of the disease is awaited with interest. Leung and colleagues (24) state their belief that “with the increasing use and sophistication of biotechnology for immunodiagnosis, the conventional usage of IF will become a n anachronism.” However, IF has not only demonstrated the heterogeneity of non-organ, non-species-specific autoantibodies found in this puzzling disease but also continues to be useful in the clinic. It is conceivable, though, that in the foreseeable future the clinician, confronted with a patient with suspected PBC, will obtain the results of AMA (and ANA) testing not by IF but by a batch of ELISAs: one to the 2-oxo-acid dehydrogenase complexes or recombinant dual (or multiple!)-headed molecules to establish the diagnosis, one to other mitochondrial antigens (M4, M8 and M9) to indicate the prognosis and another to particular nuclear proteins to define the subgroup. Despite all these exciting advances, however, the bottom line is that we are still no nearer to understanding the etiopathogenesis of PBC. Is the disease caused by a single etiological “agent,” with the different clinical manifestations dependent on the immunogenetic background of the patient, or do the different serological markers reflect a variety of etiologies, as is the case with chronic hepatitis (45)? The good news is that with the application of recombinant DNA technology to the study of the serological markers of PBC, we now have the tools with which to address these outstanding questions. MARGARET F. BASSENDINE, F.R.C.P. Medical Molecular Biology Group Department of Medicine S . J. YEAMAN, Ph.D. Department of Biochemistry and Genetics The Medical School Newcastle upon Tyne N E 2 4HH United Kingdom
Vol. 15, No. 3, 1992
SEROLOGICAL MARKERS OF PBC
REFERENCES 1. Walker JG, Doniach D, Roitt IM, Sherlock S. Serological tests in diagnosis of primary biliary cirrhosis. Lancet 1965;1:827-831. 2. Doniach D, Roitt IM, Walker JG, Sherlock S. Tissue antibodies in primary biliary cirrhosis, active chronic (lupoid) hepatitis, cryptogenic cirrhosis and other liver diseases and their clinical implications. Clin Exp Immunol 1966;1:237-262. 3. Berg PA, Doniach D, Roitt IM. Mitochondrial antibodies in primary biliary cirrhosis. I. Localization of the antigen to mitochondrial membranes. J Exp Med 1967;126:277-290. 4. Goudie RB, MacSween RNM, Goldberg DM. Serological and histological diagnosis of primary biliary cirrhosis. J Clin Pathol 1966;19:527-538. 5. Klatskin G, Kantor FS. Mitochondrial antibody in primary biliary cirrhosis and other diseases. Ann Intern Med 1972;77:533-541. 6. Munoz LE, Thomas HC, Scheuer PJ, Doniach D, Sherlock S. Is mitochondrial antibody diagnostic of primary biliary cirrhosis? Gut 1981;22:136-140. 7. Doniach D, Delharity J, Lindquist HJ, Catterall RD. Mitochondrial and other tissue autoantibodies in patients with biological false positive reactions for syphilis. Clin Exp Immunol 1970;6: 871-884. 8. Klein R, Maisch B, Kochsiek K, Berg PA. Demonstration of organ specific antibodies against heart mitochondria (anti-M7) in sera from patients with some forms of heart diseases. Clin Exp Immunol 1984;58:283-292. 9. Mitchison HC. Bassendine MF. Hendrick AM. Bennet MK. Bird AG, Watson h B , James OFW. Positive antimitochondrial antibody but normal liver function tests: is this primary biliary cirrhosis? HEPATOLOGY 1986;6:1279-1284. 10. Berg PA, Klein R, Lindenborn-Fotinos J , Kloppel W. ATPaseassociated antigen (M2): marker antigen for serological diagnosis of primary biliary cirrhosis. Lancet 1982;2:1423-1426. 11. Berg PA, Klein R, Lindenborn-Fotinos J. Antimitochondrial antibodies in primary biliary cirrhosis. J Hepatol1986;2:123-131. 12. Lindenborn-Fotinos J, Baum H, Berg PA. Mitochondrial antigens in primary biliary cirrhosis: further characterization of the M2 antigen by immunoblotting, revealing species and non-speciesspecific determinants. HEPATOLOGY 1985;5:763-769. 13. Gershwin ME, Mackay IR, Sturgess A, Coppel RL. identification and specificity of a cDNA encoding the 70kDa mitochondrial antigen recognized in primary biliary cirrhosis. J immunol 1987;138:3525-3531. 14. Yeaman SJ, Fussey SPM, Danner DJ, James OFW, Mutimer DJ, Bassendine MF. Primary biliary cirrhosis: identification of two major M2 mitochondrial autoantigens. Lancet 1988;2:10671070. 15. Van der Water J , Fregeau D, Davis P, Ansari A, Danner D, Leung P, Coppel R, et al. Autoantibodies of primary biliary cirrhosis recognize dihydrolipoamide acetyltransferase and inhibit enzyme function. J Immunol 1988;141:2321-2324. 16. Bassendine MF, Yeaman SJ.Primary biliary cirrhosis: nature of autoantigens. Springer Semin Immunopathol 1990;12:73-83. 17. Nagai S, Manns M, Meyer zum Buschenfelde KH, Dienes HP. Detection of mitochondrial antibodies directed against the primary biliary cirrhosis (M2) antigen by an enzyme-linked immunoabsorbent assay (ELISA). J Immunol Methods 1983;60: 77-88. 18. Kenna JG, Neuberger J, Davies E, Eddleston ALWF, Williams R. A simple enzyme linked immunoabsorbent assay for detection of antimitochondrial antibodies. J Immunol Methods 1984;73: 40 1-413. 19. Kaplan MM, Gandolfo JV, Quaroni EG. An enzyme-linked immunoabsorbent assay (ELISA) for detecting antimitochondrial antibody. HEPATOLOGY 1984;4:727-730. 20. Dighiero G, Magnac CH, Saint Martin J de, Abuaf N. Detection of anti-mitochondria1 antibodies by ELISA and western-blot techniques and identification by one and two dimensional gel electrophoresis of M2 target antigens. Clin Exp Immunol 1987;70: 640-648. 21. Fusconi M, Ghadiminejad I, Bianchi FB, Baum H, Bottazzo GF,
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Pisi E. Heterogeneity of antimitochondrial antibodies with M2-M4 pattern by immunofluorescence as assessed by western blotting and enzyme linked immunoabsorbent assay. Gut 1988;29: 440-447. 22. Van de Water J , Cooper A, Surh CD, Coppel R, Danner D, Ansari A, Dickson D, et al. Detection of autoantibodies to recombinant mitochondrial proteins in patients with primary biliary cirrhosis. N Engl J Med 1989;320:1377-1380. 23. Heseltine L, Turner IB, Fussey SPM, Kelly PJ, James OFW, Yeaman SJ, Bassendine MF. Primary biliary cirrhosis: quantitation of autoantibodies to purified mitochondrial enzymes and correlation with disease progression. Gastroenterology 1990;99: 1786-1792. 24. Leung PSC, Iwayama T, Prindiville T, Chuang DT, Ansari AA, Max Wynn R, Dickson R, et al. Use of designer recombinant mitochondrial antigens in the diagnosis of primary biliary cirrhosis. HEPATOLOGY 1992;15:367-372. 25. Fussey SPM, Ali ST, Guest JR, James OFW, Bassendine MF, Yeaman SJ. Reactivity of primary biliary cirrhosis sera with Escherichia coli dihydrolipoamide acetyltransferase (E2p): characterization of the main immunogenic region. Proc Natl Acad Sci USA 1990;87:3987-3991. 26. Fussey SPM, Lindsay JG, Fuller C, Perham RN, Dale S, James OFW, Bassendine MF, et al. Autoantibodies in primary biliary cirrhosis: analysis of reactivity against eukaryotic and prokaryotic 2-0x0 acid dehydrogenase complexes. HEPATOLOGY 1991;13: 467-474. 27. Berg PA, Klein R. Heterogeneity of antimitochondrial antibodies. Semin Liver Dis 1989;9:103-116. 28. Klein R, Kloppel G, Garbe W, Fintelmann V, Berg PA. Antimitochondrial antibody profiles can predict the final outcome of primary biliary cirrhosis already at early stages: a retrospective analysis of 76 patients over 6-18 years. J Hepatol 1990;12:2127. 29. Weber P, Brenner J , Stechemesser E, Klein R, Weckenmann U, Kloppel G, Kirchhof M, et al. Characterization and clinical relevance of a new complement fixing antibody, anti-M8, in patients with primary biliary cirrhosis, HEPATOLOGY 1986;6: 553-559. 30. Klein R, Kloppel G, Fischer R, Fintelmann V, Muting D, Berg PA. The antimitochondrial antibody anti-M9: a marker for the diagnosis of early primary biliary cirrhosis, J Hepatol 1988;6: 299-306. 31. Klein R, Berg PA. Anti-M9 antibodies in sera from patients with primary biliary cirrhosis recognise an epitope of glycogen phosphorylase. Clin Exp Immunol 1990;81:65-71. 32. Klein R, Berg PA. Anti-M4 antibodies in primary biliary cirrhosis react with sulphite oxidase, a n enzyme of the mitochondrial inter-membrane space. Clin Exp Immunol 1991;84:445448. 33. Bernstein RM, Neuberger JM, Bunn CC, Calendar ME, Hughes GRV, Williams R. Diversity of autoantibodies in primary biliary cirrhosis and chronic active hepatitis. Clin Exp Immunol 1984; 551553460. 34. Powell F, Schroeter AL, Dickson ER. Antinuclear antibodies in primary biliary cirrhosis. Lancet 1984;1:288-289. 35. Fritzler MJ, Valencia DW, McCarty GA. Speckled pattern antinuclear antibodies resembling anticentromere antibodies. Arthritis Rheum 1985;27:92-96. 36. Szostecki C, Krippner H, Penner E, Bautz FAS. Autoimmune sera recognise a 100 kD nuclear protein antigen (sp-100). Clin Exp Immunol 1987;68:108-116. 37. Evans J, Reuben A, Craft J. PBC 95k, a 95-kilodalton nuclear autoantigen in primary biliary cirrhosis. Arthritis Rheum 1991; 34:731-736. 38. Fusconi M, Cassani F, Govoni M, Casselli A, Farabegoli F, Lenzi M, Ballardini G, et al. Antinuclear antibodies of primary biliary cirrhosis recognise 78-92 kD and 96-100 kD proteins of nuclear bodies. Clin Exp Immunol 1991;83:291-297. 39. Szostecki C, Guldner HH, Netter HJ, Wills H. isolation and characterization of cDNA encoding a human nuclear antigen
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predominantly recognized by autoantibodies from patients with primary biliary cirrhosis. J Immunol 1990;145:4338-4347. 40. Lozano F, Pares A, Borche L, Plana M, Gallart T, Rodes J, Vives J. Autoantibodies against nuclear envelope-associated proteins in 1988;8:930-938. primary biliary cirrhosis. HEPATOLOGY 41. Lassoued K, Guilly MN, Andre M, Paintrand M, Dhumeaux D, Danon F, Rrouet JC, et al. Autoantibodies to 200kD polypeptidek) of the nuclear envelope: a new serological marker of primary biliary cirrhosis. Clin Exp Immunol 1988;74:283-288. 42. Courvalin J C , Lassoued K, Bartnik E, Rlobel G, Wozniak RW. The 2 10-kD nuclear envelope polypeptide recognised by human au-
HEPATOLOGY
toantibodies in primary biliary cirrhosis is the major glycoprotein of the nuclear pore. J Clin Invest 1990;86:279-285. 43. Wozniak RW, Bartnik E, Blobel G . Primary structure analysis of an integral membrane glycoprotein of the nuclear pore. J Cell Biol 1989;108:2083-2092. 44. Courvalin JC, Lassoued K, Worman HJ, Blobel G. Identification and characterization of autoantibodies against the nuclear envelope lamin B receptor from patients with primary biliary cirrhosis. J Exp Med 1990;172:961-967. 45. Sherlock S. Classifying chronic hepatitis. Lancet 1989;2:11681170.
In clinical practice, the most discriminating combination of biochemical and serological tests used to establish the diagnosis of PBC is an elevation of the serum alkaline phosphatase and IgM levels, together with the presence of antimitochondrial antibodies (MAS), which are mandatory for diagnosis. AMAs were first described as serological markers of PBC more than 25 years ago by Walker et al. (1)using immunofluorescence (IF) techniques. They demonstrated antibodies that gave a characteristic pattern of fluorescence staining of composite tissue blocks in the sera of virtually all patients with PBC (2). This non-organ, non-species-specific staining was localized t o the mitochondria by Berg, Doniach and Roitt in 1967 (3). Its usefulness as a marker for PBC was documented by several investigators who reported detectable AMA by indirect IF in 84% to 99% of patients (4-6).AMAs detected by IF were not entirely specific for PBC but could be found in sera from patients with other disorders (e.g., syphilis and myocarditis [7,8]). However, this lack of specificity was seldom a problem, and IF became the established conventional technique for detection of A M A in patients with suspected PBC. In clinical practice, a patient with cholestasis but negative for AMA by IF always arouses suspicion that PBC is not the correct diagnosis; conversely, the finding of AMAs by IF in a patient with normal liver blood test results may indicate early PBC (9). In view of the close association between AMA and PBC, much research has focused on the nature of the mitochondrial autoantigens. Little progress was made until a decade ago, when Berg and colleagues separated inner and outer mitochondrial membranes and showed that autoantibodies in PBC sera reacted with a trypsinsensitive antigen termed M2 on the inner mitochondrial membrane (10). The sera from patients with other disorders who were AMA positive by IF did not react with “purified” M2, whereas 96% of sera from 752 PBC patients did (11). Subsequent characterization by Western immunoblotting showed that M2 consisted of several antigenic determinants that were present not only in a variety of mammalian organs but also in yeast
Address reprint requests to: Dr. M. F. Bassendine, University of Newcastle upon Tyne, Medical Molecular Biology Group, Floor 4, Catherine Cookson Building, The Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. 31/1/35603
and bacteria (12). However, the identity of these non-organ, non-species-specific autoantigens remained elusive until 1988, when two groups independently showed that the major 70-kD M2 antigen that had been cloned by Gershwin and colleagues in 1987 (13)was the E2 component of pyruvate dehydrogenase complex (PDC) (14,15). It rapidly became clear that the other M2 antigenic determinants visualized on immunoblotting were also components of the 2-0x0 acid dehydrogenase multienzyme complexes located in mammalian mitochondria (16). Before the recent identification of the major M2 autoantigens, several groups developed rapid and relatively simple ELISAs for detection of AMA using mitochondrial or submitochondrial fractions as antigens (17-21). Once the identity of the M2 autoantigens was known, similar ELISAs were developed with recombinant fusion proteins or purified enzymes as antigens. Van der Water and colleagues (22) found a sensitivity of 88%in the diagnosis of PBC using recombinant PDC E2 alone as antigen; it increased to 96% when combined with recombinant E2 of branched-chain 2-0x0-acid dehydrogenase complex (BCOADC). A similar sensitivity of 93% in the diagnosis of PBC was found with purified PDC E2 and protein X as antigen (23). In this issue, Leung et al. (24) report a further elegant refinement of the ELISA in an attempt to improve its sensitivity. They have designed a recombinant protein to include the major immunodominant epitopes from the E2s of both rat PDC and bovine BCOADC. They show in a group of 84 PBC patients that the use of this recombinant dual-headed hybrid molecule gives a sensitivity of 93% in the diagnosis of PBC, compared with a sensitivity of 89% using recombinant PDC-E2 fusion protein alone. It should be noted, though, that the antigen used to coat the ELISA plates is not the purified hybrid antigen but an Escherichia coli lysate in which the expressed recombinant protein constitutes 10% to 15% of the whole (24). It has been demonstrated that PBC sera recognized two polypeptides present in crude extracts of wild-type E. coli. Use of deletion strains of E. coli has allowed identification of these polypeptides as the E2 components of PDC and 2-0x0-glutarate dehydrogenase complex (OGDC) (25). Thus in effect a cocktail of recombinant mammalian PDC EBIBCOADC E2 and bacterial PDC and OGDC are used in the ELISA reported by Leung et al in this issue (24). The inclusion of purified recombinant hybrid antigen and lysate of wild-type E. coli in their study would have been
545
546
BASSENDINE AND YEAMAN
interesting. Previous work has shown, however, that the titer of AMA against mammalian PDC is higher than that against the bacterial antigen (26). The presence of bacterial antigens in the ELISA also raises the possibility that in a larger study, false-positive reactions will be encountered in non-PBC patients who have antibodies to E. coli. This type of designer ELISA may eventually replace conventional indirect immunofluorescence for the detection of AMA in routine clinical practice, but before it does, further studies are required. A direct blinded comparison of the two techniques should be undertaken to establish whether all patients who are AMA positive by IF can be detected using such a n ELISA. In the study reported by Leung et a1 (241, it is not stated whether the six patients wit,h PBC who were negative on ELISA had detectable AhL4 by IF. Another way of comparing the two techniques would be to assess how much of the fluorescent staining seen on composite tissue blocks could be absorbed out using the cocktail of dual-headed recombinant protein and bacterial antigens. It is possible that a component of the reactivity seen by IF is attributable to other M2 polypeptides (16) or to other reported antigens associated with the outer mitochondrial membrane-M4, M8 and M9 (11, 27). Autoantibodies reacting with these antigens are not found in the sera of all patients with PBC, so detection of these other AMAs would be of little value in diagnosis. However, they are of considerable interest to the clinician because Klein and Berg have presented evidence suggesting that antibodies to M4 and M8 occur preferentially in patients with progressive disease (28, 291, whereas antibodies to M9 may be associated with a benign course (30). These interesting observations have been difficult to confirm in other laboratories because the biochemical identities of the antigens remained elusive. However, Klein and Berg recently demonstrated that M9 antibodies recognize an epitope of glycogen phosphorylase (311, whereas M4 antibodies react with sulphite oxidase (32). Once these observations have been validated, the way is clear for the development of ELISAs to detect AMA, which may be of prognostic relevance in patients in whom PBC has been diagnosed. In addition to the heterogeneity of AMAS, other autoantibodies have been described (33, 34) and are being characterized and identified in patients with PBC. This serves to emphasize that the breakdown in selftolerance in this “model” autoimmune disease is broadly based. Of particular interest are two district antinuclear antibodies (ANAs) that appear to be specific for patients with PBC. One ANA, which reacts with multiple nuclear dots (MNDs) by IF, has been found in 10% to 44% of PBC patients (35-37) (mainly in those with associated sicca syndrome [381). The major antigenic determinant has been characterized as a polypeptide with an apparent molecular weight of 95 to 100 kD, and a full-length complementary DNA encoding this nuclear autoantigen has recently been isolated by
HEPK~I’OI,OGY
Szostecki et a1 (39). This group has used the partially purified recombinant human nuclear antigenifusion protein in an ELISA and detected MND ANA in 50 of 184 (27%) of PBC patients but not in patients suffering from other liver diseases. Moreover, some sera from PBC patients contained MND ANA but did not contain concomitant AMA, raising the possibility that autoantibodies to this MND antigen may define a subgroup of PBC patients. PBC sera may also contain ANAs that produce a nuclear rim staining pattern in immunofluorescence (40). Two families of autoantibodies directed against integral membrane proteins of the nuclear envelope have been described in PBC patients. They appear to be disease specific: one reacts with integral membrane glycoprotein gp210 (41-431, and the other (described to date in only two AMA-negative patients) reacts against the nuclear envelope lamin B receptor (44). Further work on correlating the presence of these PBC-specific ANAs with clinical parameters of the disease is awaited with interest. Leung and colleagues (24) state their belief that “with the increasing use and sophistication of biotechnology for immunodiagnosis, the conventional usage of IF will become a n anachronism.” However, IF has not only demonstrated the heterogeneity of non-organ, non-species-specific autoantibodies found in this puzzling disease but also continues to be useful in the clinic. It is conceivable, though, that in the foreseeable future the clinician, confronted with a patient with suspected PBC, will obtain the results of AMA (and ANA) testing not by IF but by a batch of ELISAs: one to the 2-oxo-acid dehydrogenase complexes or recombinant dual (or multiple!)-headed molecules to establish the diagnosis, one to other mitochondrial antigens (M4, M8 and M9) to indicate the prognosis and another to particular nuclear proteins to define the subgroup. Despite all these exciting advances, however, the bottom line is that we are still no nearer to understanding the etiopathogenesis of PBC. Is the disease caused by a single etiological “agent,” with the different clinical manifestations dependent on the immunogenetic background of the patient, or do the different serological markers reflect a variety of etiologies, as is the case with chronic hepatitis (45)? The good news is that with the application of recombinant DNA technology to the study of the serological markers of PBC, we now have the tools with which to address these outstanding questions. MARGARET F. BASSENDINE, F.R.C.P. Medical Molecular Biology Group Department of Medicine S . J. YEAMAN, Ph.D. Department of Biochemistry and Genetics The Medical School Newcastle upon Tyne N E 2 4HH United Kingdom
Vol. 15, No. 3, 1992
SEROLOGICAL MARKERS OF PBC
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