Antimitochondrial Antibodies and Other Antibodies in Primary Biliary Cirrhosis: Diagnostic and Prognostic Value

Clin Liver Dis 12 (2008) 261–276

Antimitochondrial Antibodies and Other Antibodies in Primary Biliary Cirrhosis: Diagnostic and Prognostic Value Luigi Muratori, MD, PhD*, Alessandro Granito, MD, Paolo Muratori, MD, Georgios Pappas, MD, Francesco B. Bianchi, MD Department of Internal Medicine, Cardioangiology, Hepatology, Alma Mater Studiorum - University of Bologna, Policlinico Sant’Orsola-Malpighi, Via Massarenti, 9-40138, Bologna, Italy

Historical overview Antimitochondrial antibodies (AMA) were described for the first time in patients who had primary biliary cirrhosis (PBC) more than 40 years ago [1] and continue to be regarded as the most sensitive and specific immunologic hallmark of the disease [2,3]. The original indirect immunofluorescence (IFL) technique set-up in the laboratory of Ivan M. Roitt and Deborah Doniach is still the most common assay used to reveal the classical immunomorphologic pattern of AMA, corresponding to the cytoplasmic staining of tissues rich in mitochondria, such as rat kidney, stomach, and liver [1]. The antigenic target of AMA initially was identified as a non–organ-specific ATPase-associated antigen (M2) present in the inner mitochondrial membrane [4], consisting of several mitochondrial polypeptides [5]. AMA reactivity tentatively has been classified into nine mitochondrial antigen/ antibody systems (M1 to M9), of which only M2, M4, M8, and M9 are considered PBC specific [6]. The 70-74–kd mitochondrial polypeptide recognized by most AMA-positive sera in immunoblotting [7] was cloned [8] and identified as the E2 component of the pyruvate dehydrogenase multienzyme complex (PDC-E2) [9]. In addition to PDC-E2, other members of the of 2-oxo acid dehydrogenase complex (2-OADC) were recognized as additional targets of AMA, namely the E2 subunit of branched-chain 2-OADC

* Corresponding author. E-mail address: [email protected] (L. Muratori). 1089-3261/08/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cld.2008.02.009 liver.theclinics.com

262

MURATORI

et al

(BCOADC-E2), the E2 subunit of oxoglutarate dehydrogenase complex (OGDC-E2), and the E1a subunit and the E3 binding protein of PDC (PDC-E1a and E3BP, respectively). These enzyme complexes play a central role in the glycolytic pathway, the tricarboxylic acid cycle, and the pathway of branched-chain amino acid metabolism. The E2 enzymes share a common structure in the N-terminal domain containing lipoic acid moieties. In mammals, only five proteins contain lipoic acid, and four of them are PBC autoantigens [10]. The dominant epitope recognized by AMA is located within the lipoyl domain, and the presence of the lipoic acid moiety on the E2 proteins is essential for AMA binding. In addition, anti–lipoic acid antibodies have been detected in the vast majority of PBC sera but not in controls [11]. Methods of antimitochondrial antibody detection IFL on cryostatic unfixed sections of rat kidney, stomach, and liver, originally described in 1965 [1], still is the routine assay to detect AMA. The ubiquitous distribution of the mitochondrial antigens determines the typical granular, coarse cytoplasmic staining of the renal tubular cells, gastric parietal cells, and hepatocytes (Fig. 1). There is another immunofluorescence pattern often misdiagnosed as AMA given by liver/kidney microsomal

Fig. 1. The typical granular ‘‘coarse’’ cytoplasmic reactivity of AMA on different unfixed rat substrates by IFL: renal tubules are all positive, whereas the glomeruli are not (A); gastric parietal cells also are positive (B); liver hepatocytes are homogeneously stained (C).

AMA AND OTHER ANTIBODIES IN PBC

263

antibody type 1 (LKM1). LKM1 stains the cytoplasm of liver and renal tubules but not the parietal cells of the stomach; in addition, the detection of alternating positive and weakly positive or even negative renal tubules is the typical feature of LKM1 renal pattern [12], because its target antigen, the isoform 2D6 of the cytochrome P450, is distributed unevenly along the renal tubule. The misinterpretation of LKM1 as AMA may have important diagnostic and therapeutic consequences, as LKM1 is the serologic marker of type 2 autoimmune hepatitis (AIH) [13], usually an aggressive liver disease, often requiring intensive immunosuppression [14]. The technical guidelines recently issued by the International Autoimmune Hepatitis Group (IAIHG) committee on autoimmune liver serology are a first step toward standardization of autoantibody detection and interpretation using the IFL technique [15]. HEp2 cell lines are an additional substrate for AMA detection by IFL. AMA-positive sera give a typical granular cytoplasmic staining on HEp2 cells (Fig. 2). In addition, IFL on HEp2 cells allows the immunomorphologic definition of antinuclear reactivities, commonly observed in patients who have PBC [16,17]. Western immunoblotting (W-IB) is considered a second-level test to confirm the presence of AMA detected by IFL or to identify AMA-specific

Fig. 2. The ‘‘coarse’’ cytoplasmic staining is the typical pattern of AMA by IFL on HEp2 cells, whereas the nuclei are negative (left panel). W-IB allows further dissection of AMA reactivity, which is directed against members of the 2-OADCs, a family of polypeptides of different molecular size. In this representative picture, AMA react with the 74-kd E2 component of PDC-E2, the 55-kd E3BP, the 52-kd E2 subunit of BCOADC-E2, and the 48-kd E2 subunit of OGDC-E2, in different combinations (right panel). See Table 2.

264

MURATORI

et al

reactivities in patients who have a negative IFL AMA test, given the higher sensitivity of W-IB versus IFL [18]. Mitochondrial preparations from bovine or porcine heart are electrophoretically separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose filters, which then are probed with sera from patients who have PBC and controls [19]. The single serum sample can react with distinct mitochondrial antigens in different associations (see Fig. 2). PDC-E2 and E3BP, with which PDC-E2 is cross-reactive, are recognized in the greater part of cases (93%), followed by OGDC-E2 (40%) and BCOADC-E2 (31%) [19]. A synopsis of the hierarchy of the different AMA reactivities, their frequencies, and associations in the authors’ PBC cases is in Table 1 [19]. The identification and cloning of the different mitochondrial proteins recognized by AMA-positive sera allowed the development of different ELISAs. The standardization of the procedure militates in favor of the ELISAs, using purified [20] or recombinant mitochondrial proteins [21,22]. In the authors’ experience, however, comparing different commercial and in-house assays, the most efficient approach to detecting AMA was classical IFL followed by W-IB with beef heart mitochondrial extract or ELISA with recombinant antigens. The W-IB and the ELISA not only are helpful confirmatory tests but also may identify AMA in the small proportion of sera nonreactive with conventional IFL [18]. The authors’ experience comparing sensitivity, specificity, and positive predictive value of the IFL, W-IB, and in-house and commercial ELISAs for AMA detection is reported in Table 2. Notwithstanding continuous and strenuous efforts to maximize the sensitivity of the assays to detect AMA in otherwise rigorously AMA-negative sera [22–24], it is commonly accepted that a small number (up to 5%–10%) of patients who have PBC may lack AMA, even after investigation with all the currently available techniques. From a clinical standpoint, however, the absence of AMA is almost irrelevant, because patients who have AMAnegative PBC have the same clinical, biochemical, and histologic features of classical (ie, AMA-positive) PBC [25,26], the same clinical outcomes, and the same biochemical response to ursodeoxycholic acid treatment [27]. Table 1 Hierarchy of antimitochondrial antibodies reactivities to different mitochondrial antigens in 147 patients who had primary biliary cirrhosis Mitochondrial antigens

Positive patients

Prevalence

PDC-E2 þ E3BP PDC-E2 þ E3BP þ OGDC-E2 PDC-E2 þ E3BP þ BCOADC-E2 þ OGDC-E2 PDC-E2 þ E3BP þ BCOADC-E2 BCOADC-E2 None

63/147 29/147 17/147 10/147 9/147 19/147

43% 20% 12% 6% 6% 13%

The different patterns of reactivities were detected using W-IB with beef heart mitochondria (see Fig. 2).

265

AMA AND OTHER ANTIBODIES IN PBC

Table 2 Comparison of different techniques for antimitochondrial antibodies detection in patients who had primary biliary cirrhosis

IFL on rat tissue IFL on HEp-2 cells W-IB In-house ELISA Commercial ELISA

Sensitivity (127 cases)

Specificity (166 þ 100 cases)

Positive predictive value

71.6% 72.4% 85% 81.1% 78.8%

97.4% 93.3% 97.8% 97.8% 97.8%

0.938 0.835 0.947 0.944 0.943

The sensitivity was evaluated in 127 patients who had PBC and the specificity in 166 patients who had type 1 autoimmune hepatitis and in 100 patients who had nonalcoholic fatty liver disease.

Clinical significance of antimitochondrial antibodies The detection of AMA is virtually diagnostic of PBC, even in the absence of symptoms and with normal alkaline phosphatase. When AMA is detected in otherwise asymptomatic subjects without cholestasis, PBC already is present histologically in 40% of cases [28], and in the remaining patients it is likely to develop in succeeding years [29–32]. The detection of AMA is important information in the hands of clinicians to confirm the diagnosis of PBC [33]; however, the type and strength of AMA reactivity has no prognostic value. AMA titer may differ by more than 200-fold among patients who have PBC, but in the single patient it remains stable over the years, and its measurements by IFL or by quantitative immunoassays with recombinant mitochondrial proteins are not useful parameters for predicting disease progression [34], an observation in contrast with a previous suggestion that quantitation of IgG AMA to purified mitochondrial enzymes correlates with advancing of liver disease [20]. The preliminary claims that AMA profiles determined at an early stage may discriminate between a benign and a progressive course of PBC [35,36] were not validated on a larger series of patients [37]. The different type and number of mitochondrial antigens recognized by W-IB at presentation is independent of the stage of the liver disease and not associated with peculiar clinical, biochemical, histologic, and immunologic features or with the Mayo risk score [19]. The suggestion that IgA class antibodies to 2-OADC might be predictive markers of histopathologic progression [38] is not confirmed [39]; however, the serum level of antibodies against pyruvate dehydrogenase of the IgA class are lowered after treatment with ursodeoxycholic acid [40]. Patients who have AMA of the IgG3 subclass more frequently are cirrhotic, and a positive correlation is reported between AMA IgG3 titer and the Mayo risk score [41]. IFL AMA titers correlate with the number and intensity of W-IB bands but not with reactivity to PDC-E2 [42]. Type and frequency of AMA reactivity are similar in men and women who have PBC [43], despite the fact that men often are diagnosed later, when the disease has reached a more advanced stage [44].

266

MURATORI

et al

Other autoantibodies in primary biliary cirrhosis The notion that a considerable proportion of patients who have PBC also have antinuclear antibodies (ANA) detectable by IFL dates back to the 1960s [45], but only 2 decades later ANA patterns were characterized further on HEp2 cells [46,47]. ANA are present in nearly half of patients who have PBC irrespective of their AMA status, and overall antinuclear reactivities are predominant (up to 85%) in AMA-negative PBC [48]. Using HEp2 cell lines as IFL substrate, the ANA patterns detected in patients who have PBC are heterogeneous and often different patterns coexist in the same serum (Table 3). In patients who have PBC, the most relevant IFL patterns are anticentromere antibodies (ACA), anti–multiple nuclear dots (anti-MND), and anti–nuclear envelope antibodies. The ACA reactivity is directed against discrete granules in interphase cells and is evident particularly in mitotic cells [49] (Fig. 3). The target antigen of ACA in patients who have PBC is centromere protein B (CENP-B) [50], an 80-kd polyprotein that interacts with centromeric heterochromatin in human chromosomes. The antigenic targets of anti-MND are discrete nuclear dots, different from nucleoli and from the ACA granules by being larger, fewer in number, and not seen in mitotic cells or on metaphase chromosome spreads (see Fig. 3). The molecular target of anti-MND is a 100-kd soluble protein called sp100 [51,52] and, less often, a transformation and cell growth suppressing Table 3 Antinuclear antibodies in 195 patients who had primary biliary cirrhosis

ANA patterns  Speckled  MND  Rim-like/membranous  Centromere  Homogeneous ANA specificities  Anti-sp100  Anti-gp210  Anti-LBR  Anti-CENP-B  SSA-Ro52  Anti-dsDNA

All patients (195)

AMA-positive patients (173)

AMA-negative patients (22)

97 (50%) 56 (30%) 41 (21%) 35 (18%) 37 (18%) 6 (3%) 108 (55%) 65 (33%) 42 (21%) 11 (6%) 41 (21%) 54 (28%) 10/125 (8%)

82 (47%) 47 (24%) 30 (17%) 24 (14%) 34 (20%) 4 (2%) 92 (53%) 53 (31%) 32 (18%) 10 (6%) 38 (22%) 46 (27%) 10/118 (8.4%)

15 (68%) 9 (41%) 11 (50%) 11 (50%) 3 (14%) 2 (10%) 16 (73%) 12 (54%) 10 (45%) 1 (5%) 3 (14%) 8 (36%) 0/7

ANA patterns were evaluated on HEp2 cell lines by IFL. All ANA specificities (except antidsDNA) were evaluated with ELISA or immunoblot assay with recombinant or purified proteins. Anti-dsDNA antibodies were evaluated by IFL on Crithidia luciliae in 125 patients. The sum of the different ANA patterns and specificities appears greater then the cumulative number of positive patients because multiple reactivities were commonly observed in the same serum. Abbreviation: LBR, lamin B receptor.

AMA AND OTHER ANTIBODIES IN PBC

267

Fig. 3. ANA patterns by IFL on HEp2 cells. ACA react with discrete granules in interphase cells and typically in the mitotic cells (A). Anti-MND react with 5–20 nuclear dots distinct from nucleoli and from the ACA targets. Punctate staining of chromosomes in mitosis clearly distinguishes ACA from anti-MND (B). Anti-MND are isolated (B), whereas there is the concomitant AMA cytoplasmic staining (C). A triple reactivity is showed (D): in addition to cytoplasmic AMA and the nuclear anti-MND pattern, the rim-like/membranous reactivity is evident as a thin ring around the nuclear envelope.

protein named PML, aberrantly expressed in promyelocytic leukemia cells [53,54]. The IFL pattern of the anti–nuclear envelope antibodies is called rimlike/membranous, giving the appearance of a thin ring confined to the nuclear membrane (see Fig. 3) [55]. The molecular targets of this rim-like/ membranous IFL pattern are identified as structural components of the nuclear pore complex, such as gp210 [56] and nucleoporin p62 [57], and of the nuclear membrane, such as lamin B receptor [58,59]. With conventional IFL, the concomitant presence of AMA technically may hamper the detection of the rim-like/membranous pattern. To facilitate the detection of ANA patterns in AMA-positive patients, the use of specific antisera to each of the four IgG isotypes as secondary antibody recently has been suggested [60]. Another expedient is the pretreatment of cultured HEp2 or HeLa cells with 1% formaldehyde: the fixative reduces antigenicity and, therefore, intensity of the cytoplasmic AMA signal, without altering the antigenic epitopes on the nuclear envelope, so that the concomitant rim-like/ membranous ANA pattern is revealed more efficiently [61].

268

MURATORI

et al

Diagnostic and prognostic value of antinuclear antibodies in primary biliary cirrhosis At variance with AMA, the fine dissection and classification of the ANA specificities may offer not only diagnostic support but also prognostic information on the disease. ANA reactivities, such as the anti-MND and the rim-like/membranous patterns, which are relatively rare if not absent in normal and pathologic controls, are strongly associated to PBC [62–64], and now there is enough evidence to consider these ANA IFL patterns as surrogate positive markers of the disease in AMA-negative patients [48,65,66]. Furthermore, several ANA specificities, in particular anti-sp100, anti-gp210, and antilamin B receptor, seem predominant in patients who have PBC [16,48,54,64]. Sicca syndrome [67,68], systemic sclerosis [69], and CREST (calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia) syndrome [70] are connective tissue disorders significantly associated with PBC; therefore, the corresponding ‘‘rheumatologic’’ ANA reactivities (anti-SSA/Ro, anti-Scl70, and ACA, respectively) are not unexpected in these patients. In the setting of autoimmune liver disease, however, irrespective of concomitant rheumatologic manifestations, anti-SS-A/ Ro-52kD and ACA/anti-CENP-B may attain diagnostic relevance as additional AMA-independent specific serologic markers of PBC [71]. Some ANA specificities, such as anti-gp210 and possibly anti-p62, also offer prognostic indication, being significantly associated to aggressive disease with poor prognosis [48,63,72,73]. In addition, in Japanese patients who had PBC, not only anti-gp210 is a significant risk factor for disease progression toward hepatic failure but also ACA positivity offers prognostic information, being significantly associated to the development of portal hypertension [74]. An overview of the IFL ANA patterns and ANA specificities detected in the authors’ patients who had PBC is summarized in Table 3.

Other autoantibodies in primary biliary cirrhosis Small ubiquitin-related modifiers (SUMOs) are linked covalently to sp100 and PML, and autoantibodies against SUMOs were exclusively detected in a proportion of anti-MND–positive patients who had PBC [75]. Autoantibodies directed to other components of the nuclear envelope, such as nuclear lamins and translocated promoter region (Tpr), were observed in some patients who had PBC and also in patients who had other systemic autoimmune disorders [76]. Antibodies against SOX13, a transcriptional regulatory protein with DNA-binding capacity, were detected in 18% of patients who had PBC [77] and also are present in AIH, autoimmune cholangitis, and type 1 diabetes mellitus. Anti–lamina-associated polypeptide 2 (anti-LAP2) were detected in 6% of patients who had PBC [73] but are not disease-specific [78].

AMA AND OTHER ANTIBODIES IN PBC

269

Antimitochondrial antibodies and liver transplantation In the first months after liver transplantation for end-stage PBC, AMA titer declines slightly and then rapidly returns to pretransplantation values; the antibody subclass/isotype remains unchanged and AMA fluctuations offer no insight on the possible recurrence of the disease [79]. A similar behavior after liver transplantation is observed for PBC-specific ANA: anti-gp210 and anti-sp100 antibodies levels remain stable irrespective of the clinical outcome and do not reflect recurrent disease activity in the graft [79–81]. Antimitochondrial antibodies, autoimmune hepatitis, and overlap syndrome The erroneous interpretation of LKM1 as AMA in patients suffering from AIH may delay the diagnosis and the correct immunosuppressive treatment. Patients who have genuine AMA and definite diagnosis of AIH exist, however, and AMA presence does not preclude a satisfactory response to corticosteroids [82]. In recent years, an increasing number of studies have reported patients presenting, or developing over time, mixed clinical and laboratory features consistent with the hepatitic (ie, AIH) and the cholestatic (ie, PBC or primary sclerosing cholangitis) forms of autoimmune liver disease [83–87]. In the authors’ experience, a retrospective analysis of 125 patients who had PBC identified six cases with IAIHG score [88] consistent with AIH, a ‘‘hepatitic’’ biochemical picture, and liver histology with aspects of interface hepatitis, all features suggesting a diagnosis of ‘‘overlap AIH-PBC syndrome.’’ The distinctive serologic feature of these six patients who had overlap AIH-PBC, in comparison with the remaining 119 patients who had ‘‘pure’’ PBC, was the positivity of high titers (R1:80) anti–double-stranded DNA (anti-dsDNA) antibodies by IFL on Crithidia luciliae (Fig. 4) (G. Pappas, personal communication). Anti-dsDNA were already observed in nearly one third of patients who had advanced stage AIH [89]. If confirmed in larger series, high-titer anti-dsDNA antibodies could acquire the role of serologic marker of the overlap AIH-PBC syndrome. Antimitochondrial antibodies and chronic graft-versus-host disease Chronic graft-versus-host disease (GVHD) and PBC share many clinical and laboratory features, the most intriguing of all being portal lymphocytic infiltration and destruction of small bile ducts. Several studies reported the occurrence of AMA in a significant proportion of patients who had chronic GVHD, suggesting a potential common pathogenic background [90]. Further investigations, however, using not only IFL but also immunoblotting with beef heart mitochondria and ELISA with recombinant polypeptides of human PDC-E2, bovine BCOADC-E2, and rat OGDC-E2 failed to detect anti–PDC-E2 in bone marrow transplant recipients who had chronic

270

MURATORI

et al

Fig. 4. Anti-dsDNA antibodies are directed against the kinetoplast (arrows) of Crithidia luciliae.

GVHD [91], whereas a variety of other non–organ-specific autoantibodies were observed, in particular, ANA, the presence of which is significantly associated with the development of chronic GVHD [92]. One possible exception is a subgroup of patients who had multiple myeloma who developed high-titer AMA specifically targeting PDC-E2 after allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusions. The epitope specificity of these anti–PDC-E2 antibodies was not located, however, within the lipoyl domain of PDC-E2, which is the typical antigenic target of PBC sera [93]. Antimitochondrial antibodies and fulminant hepatic failure AMA reactivity can be detected transiently in a proportion of patients suffering from acute liver failure, possibly as a consequence of the oxidative stress-induced liver damage: 28 (40.6%) of 69 male and female patients who had fulminant hepatitis resulting from different causes (paracetamol poisoning, drug-induced liver injury, and hepatitis A, hepatitis B) developed AMA; the AMA specificity using immunoblot with recombinant antigens was the same as that observed in patients who had PBC, albeit at lower titer; AMA reactivity remained rather constant during the first week but disappeared rapidly within 1 year [94]. A concomitant study, however, identified only one subject (1.3%) as AMA positive by IFL among 72 patients presenting with acute liver failure [95]. The expected lower sensitivity of the IFL technique might explain such a discrepancy. Concluding remarks AMA remain the serologic cornerstone in the diagnosis of PBC, even if they are not detectable in a proportion of patients, notwithstanding the

AMA AND OTHER ANTIBODIES IN PBC

271

most sensitive and sophisticated technologies used. To fill in the serologic gap in AMA-negative PBC, there now is sound evidence to consider ANA patterns, such as anti-MND and anti-rim-like/membranous, as PBC-specific surrogate hallmarks of the disease, and their detection can be considered virtually diagnostic. Furthermore, particular ANA specificities, such as anti-gp210, anti-p62, ACA, and anti-dsDNA, may provide additional diagnostic and prognostic information. Summary AMA are the most sensitive and specific immunologic markers of PBC; the E2 component of the pyruvate dehydrogenase complex and other members of the 2-OADC are their molecular targets. Even if AMA titers or patterns have no prognostic significance, their presence is diagnostic of PBC and predictive of disease occurrence even in asymptomatic patients who do not have cholestasis. A small proportion of patients believed to have PBC (up to 5%–10%) test AMA negative, even with the most sophisticated techniques available; however, AMA-positive and AMA-negative PBC share the same clinical and prognostic features. ANA are found in nearly 50% of patients who have PBC, irrespective of their AMA status, the most informative immunofluorescence patterns being anti-MND, anti–rim-like/membranous, and anticentromere. The most important ANA specificities are nuclear pore complex proteins (gp210 and nucleoporin p62), a nuclear membrane protein (lamin B receptor), nuclear bodies (sp100 and PML), CENP-B, and double-stranded DNA. Immunofluorescence ANA patterns, such as anti-MND and anti–rimlike/membranous, are extremely PBC-specific and can be considered positive hallmarks of the disease, with the same diagnostic value of AMA. Several ANA specificities have additional diagnostic or prognostic value: anti-gp210 is associated with rapidly evolving liver disease, anticentromere enhances the risk for developing portal hypertension, and anti-dsDNA often is found in patients who have AIH-PBC overlap syndrome. References [1] Walker JG, Doniach D, Roitt IM, et al. Serological tests in diagnosis of primary biliary cirrhosis. Lancet 1965;285:827–31, i. [2] Bogdanos DP, Baum H, Vergani D. Antimitochondrial and other autoantibodies. Clin Liver Dis 2003;7:759–77, vi. [3] Kaplan MM, Gershwin ME. Primary biliary cirrhosis. N Engl J Med 2005;353:1261–73. [4] Berg PA, Klein R, Lindenborn-Fotinos J, et al. ATPase-associated antigen (M2): marker antigen for serological diagnosis of primary biliary cirrhosis. Lancet 1982;320:1423–6, ii. [5] Lindenborn-Fotinos J, Baum H, Berg PA. Mitochondrial antibodies in primary biliary cirrhosis: species and nonspecies specific determinants of M2 antigen. Hepatology 1985;5:763–9. [6] Berg PA, Klein R. Antimitochondrial antibodies in primary biliary cirrhosis and other disorders: definition and clinical relevance. Dig Dis 1992;10:85–101.

272

MURATORI

et al

[7] Frazer IH, Mackay IR, Jordan TW, et al. Reactivity of anti-mitochondrial autoantibodies in primary biliary cirrhosis: definition of two novel mitochondrial polypeptide autoantigens. J Immunol 1985;135:1739–45. [8] Gershwin ME, Mackay IR, Sturgess A, et al. Identification and specificity of a cDNA encoding the 70 kd mitochondrial antigen recognized in primary biliary cirrhosis. J Immunol 1987;138:3525–31. [9] Fussey SP, Guest JR, James OF, et al. Identification and analysis of the major M2 autoantigens in primary biliary cirrhosis. Proc Natl Acad Sci U S A 1988;85:8654–8. [10] Ishibashi H, Shimoda S, Gershwin M. The immune response to mitochondrial autoantigens. Semin Liver Dis 2005;25:337–46. [11] Bruggraber SF, Leung PS, Amano K, et al. Autoreactivity to lipoate and a conjugated form of lipoate in primary biliary cirrhosis. Gastroenterology 2003;125:1705–13. [12] Rizzetto M, Swana G, Doniach D. Microsomal antibodies in active chronic hepatitis and other disorders. Clin Exp Immunol 1973;15:331–44. [13] Homberg JC, Abuaf N, Bernard O, et al. Chronic active hepatitis associated with antiliver/ kidney microsome antibody type 1: a second type of ‘‘autoimmune’’ hepatitis. Hepatology 1987;7:1333–9. [14] Gregorio GV, Portmann B, Reid F, et al. Autoimmune hepatitis in childhood: a 20-year experience. Hepatology 1997;25:541–7. [15] 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. [16] Worman HJ, Courvalin JC. Antinuclear antibodies specific for primary biliary cirrhosis. Autoimmun Rev 2003;2:211–7. [17] Invernizzi P, Selmi C, Ranftler C, et al. Antinuclear antibodies in primary biliary cirrhosis. Semin Liver Dis 2005;25:298–310. [18] Muratori P, Muratori L, Gershwin ME, et al. ‘True’ antimitochondrial antibody-negative primary biliary cirrhosis, low sensitivity of the routine assays, or both? Clin Exp Immunol 2004;135:154–8. [19] Muratori L, Muratori P, Granito A, et al. The Western immunoblotting pattern of antimitochondrial antibodies is independent of the clinical expression of primary biliary cirrhosis. Dig Liver Dis 2005;37:108–12. [20] Heseltine L, Turner IB, Fussey SP, et al. Primary biliary cirrhosis. Quantitation of autoantibodies to purified mitochondrial enzymes and correlation with disease progression. Gastroenterology 1990;99:1786–92. [21] Moteki S, Leung PS, Coppel RL, et al. Use of a designer triple expression hybrid clone for three different lipoyl domain for the detection of antimitochondrial autoantibodies. Hepatology 1996;24:97–103. [22] Miyakawa H, Tanaka A, Kikuchi K, et al. Detection of antimitochondrial autoantibodies in immunofluorescent AMA-negative patients with primary biliary cirrhosis using recombinant autoantigens. Hepatology 2001;34:243–8. [23] Oertelt S, Rieger R, Selmi C, et al. A sensitive bead assay for antimitochondrial antibodies: chipping away at AMA-negative primary biliary cirrhosis. Hepatology 2007;45: 659–65. [24] Gabeta S, Norman GL, Liaskos C, et al. Diagnostic relevance and clinical significance of the new enhanced performance M2 (MIT3) ELISA for the detection of IgA and IgG antimitochondrial antibodies in primary biliary cirrhosis. J Clin Immunol 2007;27:378–87. [25] Invernizzi P, Crosignani A, Battezzati PM, et al. Comparison of the clinical features and clinical course of antimitochondrial antibody-positive and -negative primary biliary cirrhosis. Hepatology 1997;25:1090–5. [26] Zhang FK, Jia JD, Wang BE. Clinical evaluation of serum antimitochondrial antibodynegative primary biliary cirrhosis. Hepatobiliary Pancreat Dis Int 2004;3:288–91.

AMA AND OTHER ANTIBODIES IN PBC

273

[27] Kim WR, Poterucha JJ, Jorgensen RA, et al. Does antimitochondrial antibody status affect response to treatment in patients with primary biliary cirrhosis? Outcomes of ursodeoxycholic acid therapy and liver transplantation. Hepatology 1997;26:22–6. [28] 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. [29] Metcalf JV, Mitchison HC, Palmer JM, et al. Natural history of early primary biliary cirrhosis. Lancet 1996;348:1399–402. [30] Springer J, Cauch-Dudek K, O’Rourke K, et al. Asymptomatic primary biliary cirrhosis: a study of its natural history and prognosis. Am J Gastroenterol 1999;94:47–53. [31] Prince M, Chetwynd A, Newman W, et al. Survival and symptom progression in a geographically based cohort of patients with primary biliary cirrhosis: follow-up for up to 28 years. Gastroenterology 2002;123:1044–51. [32] Prince MI, Chetwynd A, Craig WL, et al. Asymptomatic primary biliary cirrhosis: clinical features, prognosis, and symptom progression in a large population based cohort. Gut 2004;53:865–70. [33] Heathcote EJ. Management of primary biliary cirrhosis. The American Association for the Study of Liver Diseases practice guidelines. Hepatology 2000;31:1005–13. [34] Van Norstrand MD, Malinchoc M, Lindor KD, et al. Quantitative measurement of autoantibodies to recombinant mitochondrial antigens in patients with primary biliary cirrhosis: relationship of levels of autoantibodies to disease progression. Hepatology 1997;25:6–11. [35] Klein R, Kloppel G, Garbe W, et al. Antimitochondrial antibody profiles determined at early stages of primary biliary cirrhosis differentiate between a benign and a progressive course of the disease. A retrospective analysis of 76 patients over 6-18 years. J Hepatol 1991;12:21–7. [36] Klein R, Pointner H, Zilly W, et al. Antimitochondrial antibody profiles in primary biliary cirrhosis distinguish at early stages between a benign and a progressive course: a prospective study on 200 patients followed for 10 years. Liver 1997;17:119–28. [37] Joshi S, Cauch-Dudek K, Heathcote EJ, et al. Antimitochondrial antibody profiles: are they valid prognostic indicators in primary biliary cirrhosis? Am J Gastroenterol 2002;97: 999–1002. [38] Masuda J, Omagari K, Ohba K, et al. Correlation between histopathological findings of the liver and IgA class antibodies to 2-oxo-acid dehydrogenase complex in primary biliary cirrhosis. Dig Dis Sci 2003;48:932–8. [39] Omagari K, Kadokawa Y, Nakamura M, et al. IgA class antibodies to 2-oxo-acid dehydrogenase complex are not predictive markers of histopathological progression in primary biliary cirrhosis. Autoimmunity 2006;39:107–12. [40] Kisand KE, Kisand KV, Karvonen AL, et al. Antibodies to pyruvate dehydrogenase in primary biliary cirrhosis: correlation with histology. APMIS 1998;106:884–92. [41] Rigopoulou E, Davies E, Bogdanos D, et al. Antimitochondrial antibodies of immunoglobulin G3 subclass are associated with a more severe disease course in primary biliary cirrhosis. Liver Int 2007;27:1226–31. [42] Rigopoulou EI, Bogdanos DP, Liaskos C, et al. Anti-mitochondrial antibody immunofluorescent titres correlate with the number and intensity of immunoblot-detected mitochondrial bands in patients with primary biliary cirrhosis. Clin Chim Acta 2007;380:118–21. [43] Nalbandian G, Van de Water J, Gish R, et al. Is there a serological difference between men and women with primary biliary cirrhosis? Am J Gastroenterol 1999;94:2482–6. [44] Muratori P, Granito A, Pappas G, et al. Clinical and serological profile of primary biliary cirrhosis in men. QJM 2007;100:534–5. [45] Doniach D, Roitt IM, Walker JG, et al. Tissue antibodies in primary biliary cirrhosis, active chronic (lupoid) hepatitis, cryptogenic cirrhosis and other liver diseases and their clinical implications. Clin Exp Immunol 1966;1:237–62. [46] Bernstein RM, Neuberger JM, Bunn CC, et al. Diversity of autoantibodies in primary biliary cirrhosis and chronic active hepatitis. Clin Exp Immunol 1984;55:553–60.

274

MURATORI

et al

[47] Kurki P, Gripenberg M, Teppo AM, et al. Profiles of antinuclear antibodies in chronic active hepatitis, primary biliary cirrhosis and alcoholic liver disease. Liver 1984;4:134–8. [48] Muratori P, Muratori L, Ferrari R, et al. Characterization and clinical impact of antinuclear antibodies in primary biliary cirrhosis. Am J Gastroenterol 2003;98:431–7. [49] Bernstein RM, Callender ME, Neuberger JM, et al. Anticentromere antibody in primary biliary cirrhosis. Ann Rheum Dis 1982;41:612–4. [50] Parveen S, Morshed SA, Nishioka M. High prevalence of antibodies to recombinant CENP-B in primary biliary cirrhosis: nuclear immunofluorescence patterns and ELISA reactivities. J Gastroenterol Hepatol 1995;10:438–45. [51] Szostecki C, Guldner HH, Netter HJ, et al. Isolation and characterization of cDNA encoding a human nuclear antigen predominantly recognized by autoantibodies from patients with primary biliary cirrhosis. J Immunol 1990;145:4338–47. [52] Szostecki C, Will H, Netter HJ, et al. Autoantibodies to the nuclear Sp100 protein in primary biliary cirrhosis and associated diseases: epitope specificity and immunoglobulin class distribution. Scand J Immunol 1992;36:555–64. [53] Sternsdorf T, Guldner HH, Szostecki C, et al. Two nuclear dot-associated proteins, PML and Sp100, are often co-autoimmunogenic in patients with primary biliary cirrhosis. Scand J Immunol 1995;42:257–68. [54] Szostecki C, Guldner HH, Will H. Autoantibodies against ‘‘nuclear dots’’ in primary biliary cirrhosis. Semin Liver Dis 1997;17:71–8. [55] Lozano F, Pares A, Borche L, et al. Autoantibodies against nuclear envelope-associated proteins in primary biliary cirrhosis. Hepatology 1988;8:930–8. [56] Wesierska-Gadek J, Hohenauer H, Hitchman E, et al. Autoantibodies from patients with primary biliary cirrhosis preferentially react with the amino-terminal domain of nuclear pore complex glycoprotein gp210. J Exp Med 1995;182:1159–62. [57] Wesierska-Gadek J, Hohenuer H, Hitchman E, et al. Autoantibodies against nucleoporin p62 constitute a novel marker of primary biliary cirrhosis. Gastroenterology 1996;110: 840–7. [58] Ye Q, Worman H. Primary structure analysis and lamin B and DNA binding of human LBR, an integral protein of the nuclear envelope inner membrane. J Biol Chem 1994;269: 11306–11. [59] Courvalin JC, Lassoued K, Worman HJ, et al. Identification and characterization of autoantibodies against the nuclear envelope lamin B receptor from patients with primary biliary cirrhosis. J Exp Med 1990;172:961–7. [60] Rigopoulou EI, Davies ET, Pares A, et al. Prevalence and clinical significance of isotype specific antinuclear antibodies in primary biliary cirrhosis. Gut 2005;54:528–32. [61] Tsiakalou V, Tsangaridou E, Polioudaki H, et al. Optimized detection of circulating antinuclear envelope autoantibodies by immunofluorescence. BMC Immunol 2006;7:20. [62] Nickowitz R, Wozniak R, Schaffner F, et al. Autoantibodies against integral membrane proteins of the nuclear envelope in patients with primary biliary cirrhosis. Gastroenterology 1994;106:193–9. [63] Invernizzi P, Podda M, Battezzati PM, et al. Autoantibodies against nuclear pore complexes are associated with more active and severe liver disease in primary biliary cirrhosis. J Hepatol 2001;34:366–72. [64] Muratori P, Muratori L, Cassani F, et al. Anti-multiple nuclear dots (anti-MND) and antiSP100 antibodies in hepatic and rheumatological disorders. Clin Exp Immunol 2002;127: 172–5. [65] Vergani D, Bogdanos D. Positive markers in AMA-negative PBC. Am J Gastroenterol 2003; 98:241–3. [66] Granito A, Muratori P, Muratori L, et al. Antinuclear antibodies giving the ‘multiple nuclear dots’ or the ‘rim-like/membranous’ patterns: diagnostic accuracy for primary biliary cirrhosis. Aliment Pharmacol Ther 2006;24:1575–83.

AMA AND OTHER ANTIBODIES IN PBC

275

[67] Dorner T, Held C, Trebeljahr G, et al. Serologic characteristics in primary biliary cirrhosis associated with sicca syndrome. Scand J Gastroenterol 1994;29:655–60. [68] Akiyama Y, Tanaka M, Takeishi M, et al. Clinical, serological and genetic study in patients with CREST syndrome. Intern Med 2000;39:451–6. [69] Rigamonti C, Shand LM, Feudjo M, et al. Clinical features and prognosis of primary biliary cirrhosis associated with systemic sclerosis. Gut 2006;55:388–94. [70] Shoji I, Takagi T, Kasukawa R. Anti-centromere antibody and CREST syndrome in patients with primary biliary cirrhosis. Intern Med 1992;31:1348–55. [71] Granito A, Muratori P, Muratori L, et al. Antibodies to SS-A/Ro-52kD and centromere in autoimmune liver disease: a clue to diagnosis and prognosis of primary biliary cirrhosis. Aliment Pharmacol Ther 2007;26:831–8. [72] Itoh S, Ichida T, Yoshida T, et al. Autoantibodies against a 210 kDa glycoprotein of the nuclear pore complex as a prognostic marker in patients with primary biliary cirrhosis. J Gastroenterol Hepatol 1998;13:257–65. [73] Miyachi K, Hankins RW, Matsushima H, et al. Profile and clinical significance of antinuclear envelope antibodies found in patients with primary biliary cirrhosis: a multicenter study. J Autoimmun 2003;20:247–54. [74] 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. [75] Janka C, Selmi C, Gershwin ME, et al. Small ubiquitin-related modifiers: a novel and independent class of autoantigens in primary biliary cirrhosis. Hepatology 2005;41:609–16. [76] Ou Y, Enarson P, Rattner JB, et al. The nuclear pore complex protein Tpr is a common autoantigen in sera that demonstrate nuclear envelope staining by indirect immunofluorescence. Clin Exp Immunol 2004;136:379–87. [77] Fida S, Myers MA, Whittingham S, et al. Autoantibodies to the transcriptional factor SOX13 in primary biliary cirrhosis compared with other diseases. J Autoimmun 2002;19: 251–7. [78] Konstantinov K, Foisner R, Byrd D, et al. Integral membrane proteins associated with the nuclear lamina are novel autoimmune antigens of the nuclear envelope. Clin Immunol Immunopathol 1995;74:89–99. [79] Mattalia A, Luttig B, Rosina F, et al. Persistence of autoantibodies against recombinant mitochondrial and nuclear pore proteins after orthotopic liver transplantation for primary biliary cirrhosis. J Autoimmun 1997;10:491–7. [80] Luettig B, Boeker KH, Schoessler W, et al. The antinuclear autoantibodies Sp100 and gp210 persist after orthotopic liver transplantation in patients with primary biliary cirrhosis. J Hepatol 1998;28:824–8. [81] Dubel L, Farges O, Courvalin JC, et al. Persistence of gp210 and multiple nuclear dots antibodies does not correlate with recurrence of primary biliary cirrhosis 6 years after liver transplantation. J Hepatol 1998;28:169–70. [82] Kenny RP, Czaja AJ, Ludwig J, et al. Frequency and significance of antimitochondrial antibodies in severe chronic active hepatitis. Dig Dis Sci 1986;31:705–11. [83] Czaja AJ. Frequency and nature of the variant syndromes of autoimmune liver disease. Hepatology 1998;28:360–5. [84] Chazouilleres O, Wendum D, Serfaty L, et al. Primary biliary cirrhosis-autoimmune hepatitis overlap syndrome: clinical features and response to therapy. Hepatology 1998; 28:296–301. [85] Lohse AW, zum Buschenfelde KH, Franz B, et al. Characterization of the overlap syndrome of primary biliary cirrhosis (PBC) and autoimmune hepatitis: evidence for it being a hepatitic form of PBC in genetically susceptible individuals. Hepatology 1999;29:1078–84. [86] Heathcote J. Primary biliary cirrhosis with features of autoimmune hepatitis. Curr Gastroenterol Rep 2001;3:60–4.

276

MURATORI

et al

[87] Muratori L, Cassani F, Pappas G, et al. The hepatitic/cholestatic ‘‘overlap’’ syndrome: an Italian experience. Autoimmunity 2002;35:565–8. [88] Alvarez F, Berg PA, Bianchi FB, et al. International Autoimmune Hepatitis Group Report: review of criteria for diagnosis of autoimmune hepatitis. J Hepatol 1999;31:929–38. [89] Czaja AJ, Morshed SA, Parveen S, et al. Antibodies to single-stranded and double-stranded DNA in antinuclear antibody-positive type 1-autoimmune hepatitis. Hepatology 1997;26: 567–72. [90] Siegert W, Stemerowicz R, Hopf U. Antimitochondrial antibodies in patients with chronic graft-versus-host disease. Bone Marrow Transplant 1992;10:221–7. [91] Quaranta S, Shulman H, Ahmed A, et al. Autoantibodies in human chronic graft-versushost disease after hematopoietic cell transplantation. Clin Immunol 1999;91:106–16. [92] Patriarca F, Skert C, Sperotto A, et al. The development of autoantibodies after allogeneic stem cell transplantation is related with chronic graft-vs-host disease and immune recovery. Exp Hematol 2006;34:389–96. [93] Bellucci R, Oertelt S, Gallagher M, et al. Differential epitope mapping of antibodies to PDC-E2 in patients with hematologic malignancies after allogeneic hematopoietic stem cell transplantation and primary biliary cirrhosis. Blood 2007;109:2001–7. [94] Leung PS, Rossaro L, Davis PA, et al. Antimitochondrial antibodies in acute liver failure: implications for primary biliary cirrhosis. Hepatology 2007;46:1436–42. [95] Bernal W, Ma Y, Smith HM, et al. The significance of autoantibodies and immunoglobulins in acute liver failure: a cohort study. J Hepatol 2007;47:664–70.