Liver International ISSN 1478-3223
Editorials DOI:10.1111/liv.12732 Liver Int. 2015; 35: 299–301
Towards the serological diagnosis of primary biliary cirrhosis In the current issue of the Journal, Norman et al. describe two new autoantibodies–anti-kelch-like 12 (KLHL12) and hexokinase-1 (HK1)–which are specific for primary biliary cirrhosis, and are especially important for patients who are seronegative for antimitochondrial antibody (AMA). But let’s put the newly described specificities in the wider context of the serological diagnosis of PBC. The autoimmune nature of PBC, a disease first recognized by Addison and Gull in 1851, became clear when its unequivocal association (1), with antimitochondrial antibody was described (Fig. 1). It was in 1965 that Walker, Doniach, Roitt and Sherlock (2) observed the presence of AMA in all their patients with PBC and in none of their control group which included patients with extra-hepatic bile duct obstruction, drug induced cholestasis and viral hepatitis. Soon it was appreciated that positivity for AMA was able to guide towards the diagnosis of PBC in the work up of cholestatic conditions, at times avoiding invasive procedures such as explorative laparotomy. AMA in the original paper was detected by indirect immunofluorescence in the laboratory of Professor Deborah Doniach(2); almost fifty years on, this technique is still widely used for routine autoantibody detection, though molecularly based assays are gaining increasing relevance. The presence of AMA at a titre equal or exceeding 1:40 is one of the three objective criteria for the diagnosis of PBC, the second criterion being an unexplained elevation of alkaline phosphatase (ALP) ≥ 1.5 times the upper normal value for over 24 weeks and the third a compatible histological picture, specifically non-suppurative cholangitis and interlobular bile duct injury (3, 4). The diagnosis of PBC rests on the positivity of two of these three criteria though PBC patients often also have elevations of transaminases and of immunoglobulins, particularly IgM. Over the last five decades, there has been a thorough dissection of the AMA specificity. Cloning and identification of the autoantigens targeted by AMA represented a major step forward. In early studies, it was noted that AMA positive sera reacted with several mitochondrial antigens, but a 74 kDa protein was the main target. Collectively these antigens constitute the ‘M2’ family of mitochondrial antigens and have been identified as components of the 2-oxo-acid dehydroge-
Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
nase multienzyme complexes: pyruvate, 2-oxoglutarate and branched-chain 2-oxo acid dehydogenase complexes (PDC, OGDC and BCOADC respectively) (5). Each of these three multifunctional complexes catalyses a set of chain reactions and occupies a key position in energy metabolism in the cell (5). This catalytic function is blocked in vitro by AMA. Somewhat surprisingly, the titre of AMA does not correlate with disease severity (6). In 1987, the cDNA for the 74 kDa mitochondrial autoantigen was cloned and sequenced leading to the identification of the E2 subunit of PDCE2, located within a lipoyl domain, as the major autoantigen in PBC (7–9). It was soon established that serum autoantibodies from some 90–95% of PBC patients reacted with PDC-E2. The reactivity against E2 subunits of the other enzymes–OGDC and BCOADC–was less frequent, ranging from 50 to 70% (10). As mentioned above, AMAs can be detected not only by indirect immunofluorescence (IFL), but also by immunoblotting and enzyme-linked immunosorbent assay (ELISA). IFL using rodent tissue sections has been the mainstay of AMA detection and is still widely utilized because when used with a substrate composed of a combination of rodent tissues (Fig. 1), enables the detection of several autoantibodies including antismooth muscle, antinuclear and liver kidney microsomal antibodies, all important in the diagnosis of autoimmune hepatitis (11). The identification of AMA target antigens as subunits of the 2-OADC has favoured the development of immunoblotting, but this technique is time consuming, labour intensive, and requires, as immunofluorescence, a degree of interpretation. Additional approaches are now well-established e.g. ELISA kits produced commercially for which the intervention of a skilled observer in the interpretation is reduced to a minimum, making them ideal for use in non-specialized centres. Moreover, ELISAs permit the evaluation of numerous sera in a single run. In a single centre study, based on the use of biochemically purified OADC proteins as the ELISA antigenic preparation, the assay achieved a sensitivity of 93% and 96% specificity for the diagnosis of PBC. Antimitochondrial antibody is a good predictor of disease. Twenty-nine patients with normal liver function tests and no symptoms suggestive of liver disease
299
Editorial
Fig. 1. Autoantibodies diagnostic of Primary Biliary Cirrhosis (PBC) detected by indirect immunofluorescence. An antimitocondrial antibody (AMA) stains the mitochondria-rich renal tubules and gastric parietal cells of rodent kidney and stomach. PBC-specific antinuclear antibodies give the multinuclear dot (MND) and the rimlike (RML) patterns when tested on HEp-2 cells.
were ‘accidentally’ found to be positive for AMA–as result of an autoantibody profile testing: the histology was compatible with PBC in 24 (83%) of them (12). 10 years later, three quarters of these subjects became clinically symptomatic with persistently cholestatic liver function tests (13). The median follow-up was 17.8 years, with one subject positive for AMA 23 years before the diagnosis of PBC was made. But not all patients with PBC are positive for AMA: there are cases with typical biochemical and histological features of PBC, but no detectable AMA. This AMA-negative PBC, also referred to as ‘primary autoimmune cholangitis’ or ‘autoimmune cholangiopathy’ (14), represents approximately 6% of the PBC patient population as shown by a study at the Mayo Clinic conducted on some 600 cases (15). In addition to AMA, other autoantibodies are detectable in PBC, and some of them are especially frequent in AMA-negative patients. Antinuclear antibodies (ANAs), are a consistent finding, belonging to two distinct families. The first consists of non-disease specific ANAs targeting antigens such as centromere, Ro/SSA, La/SSB, Scl-70 and histones; the second and diagnostically more important family comprises ANAs highly specific for PBC (Fig. 1). These latter give two distinct immunofluorescence patterns, the perinuclear (rimlike) (16) and the multiple nuclear dot (MND) pattern (17). Their molecular targets have been identified, with the antibodies responsible for the rimlike pattern recognizing constituents of the nuclear envelope including gp210, a 210 kDa transmembrane glycoprotein of the nuclear pore complex (NPC), lamin B receptor and nucleoporin p62, while those responsible
300
Vergani
for the MND pattern recognizing the sp100 and promyelocytic leukaemia proteins. The PBC-specific ANAs are not confined to AMA negative PBC, even though they are over-represented in this form of PBC. This over-representation is partially due to technical reasons since ANA positivity can be obscured by the confounding simultaneous presence of AMA. These methodological limitations are disappearing, however, since knowledge of the molecular targets responsible for a given immunofluorescent pattern is leading to the establishment of ELISAs or similar molecularly based assays. Such assays allow an accurate estimate of ANA specificities even in the presence of AMA (18). Measuring simultaneously the reactivity to three PBC-specific ANA targets, namely anti-sp100, anti-gp210 and anti-LBR, 85% of the AMA-negative PBC patients were found positive for one or more of the specificities, suggesting that the clinicians have at their disposal in the panel of PBC specific ANAs a ‘positive’ tool for the diagnosis of AMA-negative PBC. Earlier immunofluoscence studies where the majority or the totality (14) of AMA negative PBC patients was found to be ANA positive, give support to the notion that PBC-specific ANAs are an essential marker for AMA-negative PBC. Of interest, patients negative for MND or rimlike reactivity by immunofluorescence may be positive for anti-sp100 and anti-gp210 by ELISA–their molecular equivalents (19), confirming the general rule that ELISA has a higher sensitivity than immunofluorescence. Repeated observations have shown that PBC-specific ANAs, especially those directed to NPC proteins and in particular to gp210, are associated with a more active and more advanced liver disease as indicated by the presence of cirrhosis and its complications, or a higher Mayo risk score. Thus, although AMA-negative PBC patients appear to have a similar course as AMApositive cases, cross-sectional and longitudinal data indicate an association between PBC-specific ANA positivity and more severe disease (20, 21). In the present issue of the journal, Norman et al., using high-density human recombinant protein microarrays (22), identified two potential new PBC biomarkers, namely kelch-like 12 (KLHL12, a nuclear protein) and hexokinase-1 (HK1), an enzyme linked to the outer membrane of mitochondria. They went on to test the clinical value of these specificities and found that anti-KLHL12 and anti-HK1 antibodies are detected significantly more frequently in PBC than in non-PBC disease controls, with a high specificity for the disease. By combining anti-HK1 and anti-KLHL12 with available PBC diagnostic markers the diagnostic sensitivity for PBC has increased. Moreover, the addition of these two biomarkers to conventional PBC assays considerably improved the serological diagnostic sensitivity in AMA-negative PBC patients. But what merits attention is how the new autoantigens have Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Vergani
been discovered. The first step was to obtain a PBC profile of potential autoantigens. This was achieved by screening a commercial human proteome microarray containing some 8000 recombinant, eukaryotically expressed, human proteins–printed on a ‘chip’ the size of a microscope slide–with PBC and control sera. Proteins giving higher scores with PBC sera were thus recognized, but still too numerous to handle. The use of profiling software and of statistical prioritization using a z-score analysis cut down the vast array to a manageable number. The potential autoantigens were expressed in a cell free system, with the addition of an HSV tag. The eukaryotically expressed, tagged proteins were then captured by an anti-HSV antibody coated onto the wells of a mictotitre plate. The potential autoantigens could now be probed with PBC sera: the binding of such sera to the autoantigens was revealed by the use of a labelled antihuman immunoglobulin G, the immunoglobulin class to which the autoantibodies belong. At the end of this arduous, yet innovative tour de force, the two new autoantigens were discovered. This study by Norman et al. is the result of close cooperation between academia and industry. This leads us to hope that new assays containing all the relevant PBC diagnostic antigens will soon be available to provide the clinician with a powerful, non-invasive diagnostic tool for the diagnosis of the disease. This novel technical approach offers real opportunities for the discovery of the target antigens of those autoantibodies for which the immunofluorescent pattern, but not the molecular identity, is known. Acknowledgements
Conflict of interest: The authors do not have any disclosures to report.
Diego Vergani Institute of Liver Studies, King’s College Hospital, London, UK
References 1. Walker JG, Doniach D, Roitt IM, Sherlock S. Serological tests in diagnosis of primary biliary cirrhosis. Lancet 1965; 1: 827–31. 2. Doniach D, Roitt I, Walker J, Sherlock S. Tissue antibodies in primary biliary cirrhosis, active chronic (lupoid) hepatitis, cryptogenic cirrhosis and other diseases and their clinical implications. Clin Exp Immunol 1966; 1: 237–62. 3. Lindor KD, Gershwin ME, Poupon R, et al. Primary biliary cirrhosis. Hepatology 2009; 50: 291–308. 4. European Association for the Study of the L. EASL Clinical Practice Guidelines: management of cholestatic liver diseases. J Hepatol 2009; 51: 237–67.
Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Editorial
5. Yeaman SJ. The 2-oxo acid dehydrogenase complexes: recent advances. Biochem J 1989; 257: 625–32. 6. Bowlus CL, Gershwin ME. The diagnosis of primary biliary cirrhosis. Autoimmun Rev. 2014; 13: 441–4. 7. Gershwin ME, Mackay IR, Sturgess A, Coppel RL. Identification and specificity of a cDNA encoding the 70 kd mitochondrial antigen recognized in primary biliary cirrhosis. J Immunol 1987; 138: 3525–31. 8. Van de Water J, Gershwin ME, Leung P, Ansari A, Coppel RL. The autoepitope of the 74-kD mitochondrial autoantigen of primary biliary cirrhosis corresponds to the functional site of dihydrolipoamide acetyltransferase. J Exp Med 1988; 167: 1791–9. 9. Yeaman SJ, Fussey SP, Danner DJ, et al. Primary biliary cirrhosis: identification of two major M2 mitochondrial autoantigens. Lancet 1988; 1: 1067–70. 10. Leung PS, Coppel RL, Ansari A, Munoz S, Gershwin ME. Antimitochondrial antibodies in primary biliary cirrhosis. Semin Liver Dis 1997; 17: 61–9. 11. Vergani D, Alvarez F, Bianchi FB, et al. Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group. J Hepatol 2004; 41: 677– 83. 12. Mitchison HC, Bassendine MF, Hendrick A, et al. Positive antimitochondrial antibody but normal alkaline phosphatase: is this primary biliary cirrhosis? Hepatology. 1986;6:1279–84. 13. Metcalf JV, Mitchison HC, Palmer JM, et al. Natural history of early primary biliary cirrhosis. Lancet 1996; 348: 1399–402. 14. Bogdanos DP, Baum H, Vergani D. Antimitochondrial and other autoantibodies. Clin Liver Dis 2003; 7: 759–77. 15. Lacerda MA, Ludwig J, Dickson ER, Jorgensen RA, Lindor KD. Antimitochondrial antibody-negative primary biliary cirrhosis. Am J Gastroenterol 1995; 90: 247–9. 16. Courvalin JC, Worman HJ. Nuclear envelope protein autoantibodies in primary biliary cirrhosis. Semin Liver Dis 1997; 17: 79–90. 17. Szostecki C, Guldner HH, Will H. Autoantibodies against “nuclear dots” in primary biliary cirrhosis. Semin Liver Dis 1997; 17: 71–8. 18. Vergani D, Bogdanos DP. Positive markers in AMA-negative PBC. Am J Gastroenterol 2003; 98: 241–3. 19. Muratori P, Muratori L, Ferrari R, et al. Characterization and clinical impact of anti-nuclear antibodies in primary biliary cirrhosis. Am J Gastroenterol. 2003; 98: 431–7. 20. Wesierska-Gadek J, Penner E, Battezzati PM, et al. Correlation of initial autoantibody profile and clinical outcome in primary biliary cirrhosis. Hepatology 2006; 43: 1135–44. 21. Nakamura M, Kondo H, Mori T, et al. Anti-gp210 and anti-centromere antibodies are different risk factors for the progression of primary biliary cirrhosis. Hepatology 2007; 45: 118–27. 22. Zhu H, Bilgin M, Bangham R, et al. Global analysis of protein activities using proteome chips. Science 2001; 293: 2101–5.
301
Editorials DOI:10.1111/liv.12732 Liver Int. 2015; 35: 299–301
Towards the serological diagnosis of primary biliary cirrhosis In the current issue of the Journal, Norman et al. describe two new autoantibodies–anti-kelch-like 12 (KLHL12) and hexokinase-1 (HK1)–which are specific for primary biliary cirrhosis, and are especially important for patients who are seronegative for antimitochondrial antibody (AMA). But let’s put the newly described specificities in the wider context of the serological diagnosis of PBC. The autoimmune nature of PBC, a disease first recognized by Addison and Gull in 1851, became clear when its unequivocal association (1), with antimitochondrial antibody was described (Fig. 1). It was in 1965 that Walker, Doniach, Roitt and Sherlock (2) observed the presence of AMA in all their patients with PBC and in none of their control group which included patients with extra-hepatic bile duct obstruction, drug induced cholestasis and viral hepatitis. Soon it was appreciated that positivity for AMA was able to guide towards the diagnosis of PBC in the work up of cholestatic conditions, at times avoiding invasive procedures such as explorative laparotomy. AMA in the original paper was detected by indirect immunofluorescence in the laboratory of Professor Deborah Doniach(2); almost fifty years on, this technique is still widely used for routine autoantibody detection, though molecularly based assays are gaining increasing relevance. The presence of AMA at a titre equal or exceeding 1:40 is one of the three objective criteria for the diagnosis of PBC, the second criterion being an unexplained elevation of alkaline phosphatase (ALP) ≥ 1.5 times the upper normal value for over 24 weeks and the third a compatible histological picture, specifically non-suppurative cholangitis and interlobular bile duct injury (3, 4). The diagnosis of PBC rests on the positivity of two of these three criteria though PBC patients often also have elevations of transaminases and of immunoglobulins, particularly IgM. Over the last five decades, there has been a thorough dissection of the AMA specificity. Cloning and identification of the autoantigens targeted by AMA represented a major step forward. In early studies, it was noted that AMA positive sera reacted with several mitochondrial antigens, but a 74 kDa protein was the main target. Collectively these antigens constitute the ‘M2’ family of mitochondrial antigens and have been identified as components of the 2-oxo-acid dehydroge-
Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
nase multienzyme complexes: pyruvate, 2-oxoglutarate and branched-chain 2-oxo acid dehydogenase complexes (PDC, OGDC and BCOADC respectively) (5). Each of these three multifunctional complexes catalyses a set of chain reactions and occupies a key position in energy metabolism in the cell (5). This catalytic function is blocked in vitro by AMA. Somewhat surprisingly, the titre of AMA does not correlate with disease severity (6). In 1987, the cDNA for the 74 kDa mitochondrial autoantigen was cloned and sequenced leading to the identification of the E2 subunit of PDCE2, located within a lipoyl domain, as the major autoantigen in PBC (7–9). It was soon established that serum autoantibodies from some 90–95% of PBC patients reacted with PDC-E2. The reactivity against E2 subunits of the other enzymes–OGDC and BCOADC–was less frequent, ranging from 50 to 70% (10). As mentioned above, AMAs can be detected not only by indirect immunofluorescence (IFL), but also by immunoblotting and enzyme-linked immunosorbent assay (ELISA). IFL using rodent tissue sections has been the mainstay of AMA detection and is still widely utilized because when used with a substrate composed of a combination of rodent tissues (Fig. 1), enables the detection of several autoantibodies including antismooth muscle, antinuclear and liver kidney microsomal antibodies, all important in the diagnosis of autoimmune hepatitis (11). The identification of AMA target antigens as subunits of the 2-OADC has favoured the development of immunoblotting, but this technique is time consuming, labour intensive, and requires, as immunofluorescence, a degree of interpretation. Additional approaches are now well-established e.g. ELISA kits produced commercially for which the intervention of a skilled observer in the interpretation is reduced to a minimum, making them ideal for use in non-specialized centres. Moreover, ELISAs permit the evaluation of numerous sera in a single run. In a single centre study, based on the use of biochemically purified OADC proteins as the ELISA antigenic preparation, the assay achieved a sensitivity of 93% and 96% specificity for the diagnosis of PBC. Antimitochondrial antibody is a good predictor of disease. Twenty-nine patients with normal liver function tests and no symptoms suggestive of liver disease
299
Editorial
Fig. 1. Autoantibodies diagnostic of Primary Biliary Cirrhosis (PBC) detected by indirect immunofluorescence. An antimitocondrial antibody (AMA) stains the mitochondria-rich renal tubules and gastric parietal cells of rodent kidney and stomach. PBC-specific antinuclear antibodies give the multinuclear dot (MND) and the rimlike (RML) patterns when tested on HEp-2 cells.
were ‘accidentally’ found to be positive for AMA–as result of an autoantibody profile testing: the histology was compatible with PBC in 24 (83%) of them (12). 10 years later, three quarters of these subjects became clinically symptomatic with persistently cholestatic liver function tests (13). The median follow-up was 17.8 years, with one subject positive for AMA 23 years before the diagnosis of PBC was made. But not all patients with PBC are positive for AMA: there are cases with typical biochemical and histological features of PBC, but no detectable AMA. This AMA-negative PBC, also referred to as ‘primary autoimmune cholangitis’ or ‘autoimmune cholangiopathy’ (14), represents approximately 6% of the PBC patient population as shown by a study at the Mayo Clinic conducted on some 600 cases (15). In addition to AMA, other autoantibodies are detectable in PBC, and some of them are especially frequent in AMA-negative patients. Antinuclear antibodies (ANAs), are a consistent finding, belonging to two distinct families. The first consists of non-disease specific ANAs targeting antigens such as centromere, Ro/SSA, La/SSB, Scl-70 and histones; the second and diagnostically more important family comprises ANAs highly specific for PBC (Fig. 1). These latter give two distinct immunofluorescence patterns, the perinuclear (rimlike) (16) and the multiple nuclear dot (MND) pattern (17). Their molecular targets have been identified, with the antibodies responsible for the rimlike pattern recognizing constituents of the nuclear envelope including gp210, a 210 kDa transmembrane glycoprotein of the nuclear pore complex (NPC), lamin B receptor and nucleoporin p62, while those responsible
300
Vergani
for the MND pattern recognizing the sp100 and promyelocytic leukaemia proteins. The PBC-specific ANAs are not confined to AMA negative PBC, even though they are over-represented in this form of PBC. This over-representation is partially due to technical reasons since ANA positivity can be obscured by the confounding simultaneous presence of AMA. These methodological limitations are disappearing, however, since knowledge of the molecular targets responsible for a given immunofluorescent pattern is leading to the establishment of ELISAs or similar molecularly based assays. Such assays allow an accurate estimate of ANA specificities even in the presence of AMA (18). Measuring simultaneously the reactivity to three PBC-specific ANA targets, namely anti-sp100, anti-gp210 and anti-LBR, 85% of the AMA-negative PBC patients were found positive for one or more of the specificities, suggesting that the clinicians have at their disposal in the panel of PBC specific ANAs a ‘positive’ tool for the diagnosis of AMA-negative PBC. Earlier immunofluoscence studies where the majority or the totality (14) of AMA negative PBC patients was found to be ANA positive, give support to the notion that PBC-specific ANAs are an essential marker for AMA-negative PBC. Of interest, patients negative for MND or rimlike reactivity by immunofluorescence may be positive for anti-sp100 and anti-gp210 by ELISA–their molecular equivalents (19), confirming the general rule that ELISA has a higher sensitivity than immunofluorescence. Repeated observations have shown that PBC-specific ANAs, especially those directed to NPC proteins and in particular to gp210, are associated with a more active and more advanced liver disease as indicated by the presence of cirrhosis and its complications, or a higher Mayo risk score. Thus, although AMA-negative PBC patients appear to have a similar course as AMApositive cases, cross-sectional and longitudinal data indicate an association between PBC-specific ANA positivity and more severe disease (20, 21). In the present issue of the journal, Norman et al., using high-density human recombinant protein microarrays (22), identified two potential new PBC biomarkers, namely kelch-like 12 (KLHL12, a nuclear protein) and hexokinase-1 (HK1), an enzyme linked to the outer membrane of mitochondria. They went on to test the clinical value of these specificities and found that anti-KLHL12 and anti-HK1 antibodies are detected significantly more frequently in PBC than in non-PBC disease controls, with a high specificity for the disease. By combining anti-HK1 and anti-KLHL12 with available PBC diagnostic markers the diagnostic sensitivity for PBC has increased. Moreover, the addition of these two biomarkers to conventional PBC assays considerably improved the serological diagnostic sensitivity in AMA-negative PBC patients. But what merits attention is how the new autoantigens have Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Vergani
been discovered. The first step was to obtain a PBC profile of potential autoantigens. This was achieved by screening a commercial human proteome microarray containing some 8000 recombinant, eukaryotically expressed, human proteins–printed on a ‘chip’ the size of a microscope slide–with PBC and control sera. Proteins giving higher scores with PBC sera were thus recognized, but still too numerous to handle. The use of profiling software and of statistical prioritization using a z-score analysis cut down the vast array to a manageable number. The potential autoantigens were expressed in a cell free system, with the addition of an HSV tag. The eukaryotically expressed, tagged proteins were then captured by an anti-HSV antibody coated onto the wells of a mictotitre plate. The potential autoantigens could now be probed with PBC sera: the binding of such sera to the autoantigens was revealed by the use of a labelled antihuman immunoglobulin G, the immunoglobulin class to which the autoantibodies belong. At the end of this arduous, yet innovative tour de force, the two new autoantigens were discovered. This study by Norman et al. is the result of close cooperation between academia and industry. This leads us to hope that new assays containing all the relevant PBC diagnostic antigens will soon be available to provide the clinician with a powerful, non-invasive diagnostic tool for the diagnosis of the disease. This novel technical approach offers real opportunities for the discovery of the target antigens of those autoantibodies for which the immunofluorescent pattern, but not the molecular identity, is known. Acknowledgements
Conflict of interest: The authors do not have any disclosures to report.
Diego Vergani Institute of Liver Studies, King’s College Hospital, London, UK
References 1. Walker JG, Doniach D, Roitt IM, Sherlock S. Serological tests in diagnosis of primary biliary cirrhosis. Lancet 1965; 1: 827–31. 2. Doniach D, Roitt I, Walker J, Sherlock S. Tissue antibodies in primary biliary cirrhosis, active chronic (lupoid) hepatitis, cryptogenic cirrhosis and other diseases and their clinical implications. Clin Exp Immunol 1966; 1: 237–62. 3. Lindor KD, Gershwin ME, Poupon R, et al. Primary biliary cirrhosis. Hepatology 2009; 50: 291–308. 4. European Association for the Study of the L. EASL Clinical Practice Guidelines: management of cholestatic liver diseases. J Hepatol 2009; 51: 237–67.
Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Editorial
5. Yeaman SJ. The 2-oxo acid dehydrogenase complexes: recent advances. Biochem J 1989; 257: 625–32. 6. Bowlus CL, Gershwin ME. The diagnosis of primary biliary cirrhosis. Autoimmun Rev. 2014; 13: 441–4. 7. Gershwin ME, Mackay IR, Sturgess A, Coppel RL. Identification and specificity of a cDNA encoding the 70 kd mitochondrial antigen recognized in primary biliary cirrhosis. J Immunol 1987; 138: 3525–31. 8. Van de Water J, Gershwin ME, Leung P, Ansari A, Coppel RL. The autoepitope of the 74-kD mitochondrial autoantigen of primary biliary cirrhosis corresponds to the functional site of dihydrolipoamide acetyltransferase. J Exp Med 1988; 167: 1791–9. 9. Yeaman SJ, Fussey SP, Danner DJ, et al. Primary biliary cirrhosis: identification of two major M2 mitochondrial autoantigens. Lancet 1988; 1: 1067–70. 10. Leung PS, Coppel RL, Ansari A, Munoz S, Gershwin ME. Antimitochondrial antibodies in primary biliary cirrhosis. Semin Liver Dis 1997; 17: 61–9. 11. Vergani D, Alvarez F, Bianchi FB, et al. Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group. J Hepatol 2004; 41: 677– 83. 12. Mitchison HC, Bassendine MF, Hendrick A, et al. Positive antimitochondrial antibody but normal alkaline phosphatase: is this primary biliary cirrhosis? Hepatology. 1986;6:1279–84. 13. Metcalf JV, Mitchison HC, Palmer JM, et al. Natural history of early primary biliary cirrhosis. Lancet 1996; 348: 1399–402. 14. Bogdanos DP, Baum H, Vergani D. Antimitochondrial and other autoantibodies. Clin Liver Dis 2003; 7: 759–77. 15. Lacerda MA, Ludwig J, Dickson ER, Jorgensen RA, Lindor KD. Antimitochondrial antibody-negative primary biliary cirrhosis. Am J Gastroenterol 1995; 90: 247–9. 16. Courvalin JC, Worman HJ. Nuclear envelope protein autoantibodies in primary biliary cirrhosis. Semin Liver Dis 1997; 17: 79–90. 17. Szostecki C, Guldner HH, Will H. Autoantibodies against “nuclear dots” in primary biliary cirrhosis. Semin Liver Dis 1997; 17: 71–8. 18. Vergani D, Bogdanos DP. Positive markers in AMA-negative PBC. Am J Gastroenterol 2003; 98: 241–3. 19. Muratori P, Muratori L, Ferrari R, et al. Characterization and clinical impact of anti-nuclear antibodies in primary biliary cirrhosis. Am J Gastroenterol. 2003; 98: 431–7. 20. Wesierska-Gadek J, Penner E, Battezzati PM, et al. Correlation of initial autoantibody profile and clinical outcome in primary biliary cirrhosis. Hepatology 2006; 43: 1135–44. 21. Nakamura M, Kondo H, Mori T, et al. Anti-gp210 and anti-centromere antibodies are different risk factors for the progression of primary biliary cirrhosis. Hepatology 2007; 45: 118–27. 22. Zhu H, Bilgin M, Bangham R, et al. Global analysis of protein activities using proteome chips. Science 2001; 293: 2101–5.
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