Search

Primary biliary cholangitis (PBC) is characterized by the presence of serum anti-mitochondrial autoantibodies (AMAs). To date, four antigens among the 2-oxo-acid dehydrogenase complex family, which commonly have lipoyl domains as an epitope, have been identified as AMA-corresponding antigens (AMA-antigens). It has recently been reported that AMAs react more strongly with certain chemically modified mimics than with the native lipoyl domains in AMA-antigens. Moreover, high concentrations of circulating immune complexes (ICs) in PBC patients have been reported. However, the existence of ICs formed by AMAs and their antigens has not been reported to date. We hypothesized that AMAs and their antigens formed ICs in PBC sera, and analyzed sera of PBC and four autoimmune diseases (Sjögren's syndrome, systemic lupus erythematosus, systemic scleroderma, and rheumatoid arthritis) using immune complexome analysis, in which ICs are separated from serum and are identified by nano-liquid chromatography-tandem mass spectrometry. To correctly assign MS/MS spectra to peptide sequences, we used a protein-search algorithm that including lipoylation and certain xenobiotic modifications. We found three AMA-antigens, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the E2 subunit of the 2-oxo-glutarate dehydrogenase complex (OGDC-E2) and dihydrolipoamide dehydrogenase binding protein (E3BP), by detecting peptides containing lipoylation and xenobiotic modifications from PBC sera. Although the lipoylated sites of these peptides were different from the well-known sites, abnormal lipoylation and xenobiotic modification may lead to production of AMAs and the formation ICs. Further investigation of the lipoylated sites, xenobiotic modifications, and IC formation will lead to deepen our understanding of PBC pathogenesis., (© 2021 British Society for Immunology.)

Autoantibodies to intracellular targets in mitochondria and nuclei are serological hallmarks of primary biliary cirrhosis (PBC). One of the most recently identified cellular targets of PBC autoantibodies is a novel cytoplasmic structure referred to as GW bodies [GWB, G (glycine) W (tryptophan)-containing bodies (GWB)]. GWB are indentified as discrete cytoplasmic domains that are involved in mRNA processing via the RNA interference (RNAi) pathway. Key components of GWB include the proteins GW182, Ago2, RNA-associated protein 55 (RAP55) and Ge-1/Hedls. The primary objective was to study the frequency and clinical association of antibodies directed to GWB components, in 109 PBC patients. Autoantibodies to mitochondrial antigen-pyruvate dehydrogenase complex (M2), branched-chain 2-oxo-acid dehydrogenase complex and 2-oxo glutarate dehydrogenase complex (3E-BPO), gp210, sp100, promyelocytic leukaemia cell antigen (PML) and liver kidney microsomal-1 antigen (LKM-1) were detected by a line immunoassay and antibodies to GWB (GW182, RAP55, Ge-1, GW2, GW3) and glutamate receptor interacting protein (GRIP)-associated protein-1 (GRASP-1), by an addressable laser bead immunoassay (ALBIA). The most common GWB autoantigen targets were: RAP55-28%, GW182-12%, GW2-2% and antibodies to GRASP-1-17%. By comparison, the frequency of reactivity to established PBC autoantigens was: gp210, 27%; sp100, 27% and PML, 17%. None of the autoantibodies were associated with differences in Mayo risk score or liver decompensation. This study is the first study to show that antibodies to RAP55, GW182 and GRASP-1 are the most common GWB targets in PBC., (© 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.)

We sequentially analyzed the serum IgG response against normal mouse brain during experimental autoimmune encephalomyelitis in SJL/J mice injected with CFA, Bordetella pertussis toxin (BPT) and proteolipid protein 139-151 peptide, compared with mice that received CFA and BPT or were uninjected. Dynamic changes were observed from day 0 to day 28 in the 3 groups. Six highly discriminant antigenic bands (kappa=0.974) were identified. Three non-myelin proteins were characterized (mitochondrial aconitase hydratase 2, phosphoglycerate mutase 1, brain specific pyruvate deshydrogenase). The IgG response against two of them was less frequent in EAE whereas it was associated with multiple sclerosis in our previous work.

Background: Primary biliary cirrhosis (PBC) is characterized by the presence of antimitochondrial antibodies (AMA). Autoantibodies specific for the mitochondrial M4 antigen can be detected by a complement fixation test (CFT) but not by immunoblotting. The aim of this study was to elucidate the identity of the M4 antigen., Patients and Methods: M4 proteins were purified by affinity chromatography using IgG fractions of PBC marker sera being CFT positive (n=5) or negative (n=5) and identified by Western blotting, silver staining and sequence analysis. Further, a cohort of 57 PBC patients was tested for the reactivity to M4 and pyruvate dehydrogenase complex (PDC)., Results: Two AMA patterns of the marker sera were visualized: CFT-positive sera were defined as PDC-E2(+)/E1(+) and the CFT-negative sera as PDC-E2(+)/E1(-). The major proteins in the M4 fraction could be related to the PDC-E1 subunits. A clear-cut association between anti-M4 reactivity in the CFT and the reactivity to both PDC subunits could also be documented in the cohort of 57 PBC patients showing anti-PDC-E1alpha and E1beta antibodies at a frequency of 74% and 67%., Conclusions: CFT reactivity against M4 antigens could be preferentially identified as a reaction against PDC-E1. As PDC-E1 subunits as compared with PDC-E2 lack lipoyl-binding sites, they probably have to be considered as an independent and important target.

Antimitochondrial antibodies (AMA) are the serum hallmark of primary biliary cirrhosis (PBC). However, AMA-positivity can be found in non-PBC sera when lower dilutions are used, thus raising issues about the specificity and sensitivity of the test. AMA reacts primarily with the lipoylated domains of pyruvate dehydrogenase-E2 (PDC-E2) which is highly conserved across species, including bacteria. We studied 77 serum samples, including 24 from patients with anti-PDC-E2-positive PBC and 53 controls (16 with autoimmune hepatitis (AIH), 10 with primary sclerosing cholangitis (PSC), and 27 healthy individuals) for their reactivities at serial dilutions (1:10, 1:20 and 1:40) against Escherichia coli DH5 alpha lysate overexpressing human PDC-E2 using immunoblotting (IB). A murine anti-human PDC-E2 monoclonal antibody (mAB) was used as control. We further studied positive sera using adsorption with a synthetic E. coli peptide sharing similarity with human PDC-E2. Finally, we verified whether a unique buffer for E. coli preparation could reduce non-specific serum reactivity. Results demonstrated that 100% of anti-PDC-E2-positive PBC and up to 38% of control sera at 1:10 dilution recognized E. coli PDC-E2 at IB while dilution tests indicated that the overall potency of PBC reactivity was 100-fold higher compared to controls. In fact, a subgroup (20-38%) of non-PBC sera were positive at low titers but lost the reactivity when absorbed with the synthetic E. coli peptide. Finally, our unique buffer reduced the reactivity of non-PBC sera as measured by ELISA. In conclusion, we demonstrated that weak cross-reactivity with E. coli PDC-E2 occurs in non-PBC sera at lower dilutions and that such reactivity is not due to AMA-positivity. The use of a specific buffer might avoid the risk of false positive AMA determinations when E. coli-expressed recombinant antigens are used.

Background: Autoantibody responses reactive with the E2 and E3BP components of pyruvate dehydrogenase complex (PDC), which characterise primary biliary cirrhosis (PBC) crossreact, precluding the identification, from serological studies, of the antigen to which the principal breakdown of tolerance occurs. Although autoreactive T-cell responses to PDC-E2 have been well characterised it is, at present, unclear whether T-cell tolerance breakdown also occurs to PDC-E3BP. The aims of this study were to characterise autoreactive T-cell responses to PDC-E3BP in PBC and potential factors regulating their expression., Methods: Peripheral blood T-cell proliferative responses to purified recombinant human PDC-E2 and PDC-E3BP at a range of concentrations were characterised in PBC patients and control subjects., Results: T-cell proliferative responses to both E2 and E3BP were absent from control subjects (median peak stimulation index (SI) to PDC-E2 1.2 [range 0.3-1.9], 0/10 positive (SI>2.32), median peak SI to PDC-E3BP 1.1 [0.7-2.1]], 0/10 positive). Significant responses to PDC-E2 were seen in the majority of patients (median peak SI 11.4 [0.4-24.4], 17/20 (85%) positive) but to PDC-E3BP in only a minority (median peak SI 1-9 [0.6-9.95], 8/20 (40%) positive). Where responses to PDC-E3BP were seen they were universally secondary to responses to PDC-E2., Conclusions: Despite the presence of antibodies reactive with PDC-E3BP in the majority of PBC patients this self-protein is not a dominant T-cell autoantigen in PBC.

Multiple classes of proteins are modified to tailor them for specific physiological roles. The nature of these posttranslational modifications of proteins, as well as the relationships between them including those of the immune system proteins themselves, and immune system responses are reviewed. Aspects of protein posttranslational modification and their relationship to the pathogenesis of several autoimmune diseases and primary biliary cirrhosis are highlighted.

The key immunological event in the pathogenesis of the autoimmune liver disease primary biliary cirrhosis is breakdown of T-cell self-tolerance to pyruvate dehydrogenase complex (PDC). The mechanism resulting in this breakdown of tolerance remains unclear. Mice exposed to self-PDC mount no immune response; however, animals coexposed to self-PDC and PDC of foreign origin (which in isolation induces a cross-reactive antibody but not an autoreactive T-cell response) show breakdown of T-cell as well as B-cell tolerance. This observation raises the possibility that a cross-reactive antibody response to self-PDC can promote breakdown of T-cell tolerance. The aim of this study was to address the hypothesis that breakdown of T-cell tolerance to PDC can be driven by the presence of B cells and/or antibodies cross-reactive with this self-antigen. Naive female SJL/J mice were exposed to self-PDC alone and in the presence of purified splenic B cells from animals primed with foreign PDC (or controls) or purified immunoglobulin (Ig) G from the same animals. Breakdown of T-cell tolerance was assessed by splenic T-cell proliferative response to antigen at 5 weeks. CD4(+) T-cell proliferative responses indicative of breakdown of T-cell tolerance to self-PDC were seen in the majority (7 of 9, 78%) of animals receiving self-PDC together with purified PDC-reactive B cells. Tolerance breakdown was not seen in animals receiving self-PDC with purified anti-PDC IgG or with B cells from animals sensitized with an irrelevant antigen. In conclusion, breakdown of T-cell tolerance to the highly conserved self-antigen PDC may be mediated by high-level presentation of self-derived epitopes by activated cross-reactive B cells.

Emerging evidence has suggested environmental factors as causative agents in the pathogenesis of primary biliary cirrhosis (PBC). We have hypothesized that in PBC the lipoyl domain of the immunodominant E2 component of pyruvate dehydrogenase (PDC-E2) is replaced by a chemical xenobiotic mimic, which is sufficient to break self-tolerance. To address this hypothesis, based upon our quantitative structure-activity relationship data, a total of 107 potential xenobiotic mimics were coupled to the lysine residue of the immunodominant 15 amino acid peptide of the PDC-E2 inner lipoyl domain and spotted on microarray slides. Sera from patients with PBC (n = 47), primary sclerosing cholangitis (n = 15), and healthy volunteers (n = 20) were assayed for Ig reactivity. PBC sera were subsequently absorbed with native lipoylated PDC-E2 peptide or a xenobiotically modified PDC-E2 peptide, and the remaining reactivity analyzed. Of the 107 xenobiotics, 33 had a significantly higher IgG reactivity against PBC sera compared with control sera. In addition, 9 of those 33 compounds were more reactive than the native lipoylated peptide. Following absorption, 8 of the 9 compounds demonstrated cross-reactivity with lipoic acid. One compound, 2-octynoic acid, was unique in both its quantitative structure-activity relationship analysis and reactivity. PBC patient sera demonstrated high Ig reactivity against 2-octynoic acid-PDC-E2 peptide. Not only does 2-octynoic acid have the potential to modify PDC-E2 in vivo but importantly it was/is widely used in the environment including perfumes, lipstick, and many common food flavorings.

The E2 component of mitochondrial pyruvate dehydrogenase complex (PDC-E2) is the immunodominant autoantigen of primary biliary cirrhosis. Whereas lipoylation of PDC-E2 is essential for enzymatic activity and predominates under normal conditions, other biochemical systems exist that also target the lysine residue, including acylation of fatty acids or xenobiotics and ubiquitinylation. More importantly, the immunogenicity can be affected by derivatization of the lysine residue, as the recognition of lipoylated PDC-E2 by patient autoantibodies is enhanced compared with octanoylated PDC-E2. Furthermore, our laboratory has shown that various xenobiotic modifications of a peptide representing the immunodominant region of PDC-E2 are immunoreactive against patient sera. The only purported regulatory system that prevents the accumulation of potentially autoreactive PDC-E2 is glutathionylation, in which the lysine-lipoic acid moiety is further modified with glutathione during apoptosis. Interestingly, this system is found in several cell lines, including HeLa, Jurkat, and Caco-2 cells, but not in cholangiocytes and salivary gland epithelial cells, both of which are targets for destruction in primary biliary cirrhosis. Hence, the failure of this or other regulatory system(s) may overwhelm the immune system with immunogenic PDC-E2 that can initiate the breakdown of tolerance in a genetically susceptible individual. In this review the authors survey the data available on the biochemical life of PDC-E2, with particular emphasis on the lysine residue and its known interactions with machinery involved in various posttranslational modifications.

Previous work has demonstrated that immunization of rabbits with the xenobiotic 6-bromohexanoate coupled to BSA breaks tolerance and induces autoantibodies to mitochondria in rabbits. Such immunized rabbits develop high-titer Abs to pyruvate dehydrogenase complex (PDC)-E2, the major autoantigen of primary biliary cirrhosis. In efforts to map the fine specificity of these autoantibodies, rabbits were immunized biweekly with 6-bromohexanoate-BSA and screened for reactivity using a unique xenobiotic-peptide-agarose microarray platform with an emphasis on identifying potential structures that mimic the molecular image formed by the association of lipoic acid with the immunodominant PDC-E2 peptide. Essentially, a total of 23 xenobiotics and lipoic acid were coupled to the 12-mer peptide backbones, PDC, a mutant PDC, and albumin. As expected, we succeeded in breaking tolerance using this small organic molecule coupled to BSA. However, unlike multiple experimental methods of breaking tolerance, we report in this study that, following continued immunization, the rabbits recover tolerance. With repeated immunization, the response to the rPDC-E2 protein increased with a gradual reduction in autoantibodies against the lipoic acid-peptide, i.e., the primary tolerance-breaking autoantigen. Detailed analysis of this system may provide strategies on how to restore tolerance in patients with autoimmune disease.

While the pathologic mechanisms responsible for organ-specific tissue damage in primary biliary cirrhosis (PBC) remain an enigma, it has been suggested that the pathology is mediated by autoreactive T cells infiltrating the intrahepatic bile ducts. Previously, we have documented that there is 100-fold enrichment in the frequency of CD4(+) autoreactive T cells in the liver that are specific for peptides encoded by the E2 components of the pyruvate dehydrogenase complexes (PDC-E2). We have also recently characterized the first MHC class I-restricted epitope for PDC-E2, namely amino acid 159-167, a region very similar to the epitope recognized by MHC class II-restricted CD4(+) cells and by autoantibodies. The effector functions of these PDC-E2(159-167)-specific CD8(+) cytotoxic T lymphocytes (CTLs) are not well understood. We have taken advantage of tetramer technology and report herein that there is tenfold increase in the frequency of PDC-E2(159-167)-specific CTLs in the liver as compared with the blood in PBC. In addition, the precursor frequency of the CTLs in blood was significantly higher in early-stage PBC. Of interest was the fact that, upon stimulation with the peptide, the response of PDC-E2(159-167) tetramer-positive cells is heterogeneous with respect to IFN-gamma synthesis. These data, we believe for the first time, document the enrichment of autoantigen-specific CD8(+) T cells in the PBC liver, suggesting that CD8(+) T cells play a significant role in the immunopathogenesis of PBC.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by autoreactive T- and B-cell responses to the highly conserved enzyme pyruvate dehydrogenase complex (PDC). In this study we have examined the breakdown of T-cell tolerance to self-PDC using a mouse model. Female SJL/J mice were sensitized intraperitoneally with foreign-PDC (bovine) and/or self-PDC (murine) in complete Freund's adjuvant, and serum, spleen, and liver tissue was taken 8 weeks later. Animals sensitized with foreign-PDC produced IgG antibodies that were reactive with both foreign and self-PDC, but splenic T cells from these animals only responded to stimulation with foreign PDC. Sensitization with self-PDC elicited neither antibodies nor reactive T cells. Significantly, cosensitization with mixed self-PDC and foreign-PDC resulted in a full breakdown of self-tolerance, with generation of both antibody and T-cell responses to self-PDC of the type seen exclusively in human PBC patients. Mild bile duct lesions deficient in CD8(+) T cells were seen 8 weeks after sensitization with either foreign or self-PDC. However, after sensitization with mixed self-PDC and foreign-PDC, these lesions were significantly larger and heavily infiltrated by CD8(+) T cells. Liver-infiltrating T cells derived from the self-PDC and foreign-PDC cosensitized but not from control animals showed reactivity with self-PDC, suggesting a possible role for autoreactive PDC-specific T-cell responses in the pathogenesis of the observed histologic changes. It is likely that B-cell cross-reactivity between foreign and self-PDC enhances the potential for breakdown of T-cell self-tolerance by allowing efficient presentation of self-antigens in the inoculum. This model may provide a useful system for investigating the etiology and treatment of PBC.

The immunodominant antimitochondrial antibody (AMA) response in primary biliary cirrhosis (PBC) is directed against the E2 component of pyruvate dehydrogenase (PDC-E2). The nature of the clonal selection process is unclear, and to address this issue, we took advantage of a transgenic technology, XenoMouse, that contains 80% of the human immunoglobulin (Ig) variable gene repertoire and can produce high-affinity human antibodies to virtually any immunogen without evidence of clonal bias. We immunized mice with PDC-E2 to obtain 13 HmAbs, including 4 IgG(2) and 9 IgM isotypes. Immunoglobulin gene analysis was unique and demonstrated a clonal bias; the immunoglobulin gene usage was considerably different from other antibody responses analyzed in XenoMouse systems. Four of the 13 mAbs recognized the inner lipoyl domain of PDC-E2, 2 of 13 recognized the entire PDC-E2 molecule, 4 of 13 recognized PDC-E2 and OGDC-E2, 1 of 13 recognized OGDC only, 1 recognized BCOADC-E2 only, and 1 recognized an unidentified 100-kd mitochondrial protein. Immunohistochemical staining using these HmAbs produced mitochondrial staining of septal bile ducts in both PBC and control livers. Ig gene analysis showed that 7 of 13 HmAbs used the V(H)3 and 4 of 13 used VH4 gene repertoire, respectively. Three of 7 V(H)3 antibodies used the same Ig VH3-21 gene family found in human AMA from patients with PBC. The CDRs of these autoantibodies were slightly mutated when compared with the sequences present within the Ig germline genes. In conclusion, the XenoMouse not only recapitulates the unique specificity and restriction of PBC patients, but indicates that the autoantibodies are derived from a restricted clonal selection process. Such data suggest that the original immunogen leads to somatic mutation without subsequent development of determinant spreading.

The close association between autoantibodies against pyruvate dehydrogenase-E2 (PDC-E2), a ubiquitous mitochondrial protein, and primary biliary cirrhosis (PBC) is unexplained. Many autoantigens are selectively modified during apoptosis, which has focused attention on apoptotic cells as a potential source of "neo-antigens" responsible for activating autoreactive lymphocytes. Since increased apoptosis of bile duct epithelial cells (cholangiocytes) is evident in patients with PBC, we evaluated the effect of apoptosis on PDC-E2. Autoantibody recognition of PDC-E2 by immunofluorescence persisted in apoptotic cholangiocytes and appeared unchanged by immunoblot analysis. PDC-E2 was neither cleaved by caspases nor concentrated into surface blebs in apoptotic cells. In other cell types, autoantibody recognition of PDC-E2, as assessed by immunofluorescence, was abrogated after apoptosis, although expression levels of PDC-E2 appeared unchanged when examined by immunoblot analysis. Both overexpression of Bcl-2 and depletion of glutathione before inducing apoptosis prevented this loss of autoantibody recognition, suggesting that glutathiolation, rather than degradation or loss, of PDC-E2 was responsible for the loss of immunofluorescence signal. We postulate that apoptotic cholangiocytes, unlike other apoptotic cell types, are a potential source of immunogenic PDC-E2 in patients with PBC.

The anti-mitochondrial antibody response in primary biliary cirrhosis (PBC) is primarily directed at E2 components of PDC, OGDC, and BCOADC, and E3BP. Previous work has shown that the immunodominant autoreactive T- cell epitope is the PDC-E2 163-176 peptide, restricted by HLA DR53. To address molecular mimicry and cross-recognition among mitochondrial autoantigens, we analyzed reactivity, including agonism and antagonism assays, to a series of single amino acid-substituted peptides using cloned T-cell lines in PBC and controls. Interestingly, fine specificities were unique for every single T-cell clone, but the clones could be categorized into two distinct groups based on recognition motifs of the T-cell receptor (TCR) ligand: group A (170)ExDK(173) and group B (168)EIExD(172). (170)E is the most critical TCR contact residue for both groups of cloned T-cell lines, whereas (173)K and (168)E are the critical TCR contact residues for group A and group B cloned T-cell lines, respectively. More importantly, some group A-cloned T-cell lines cross-reacted to human E3BP 34-47, human OGDC-E2 100-113, and several peptides derived from various microbial proteins carrying an ExDK motif, whereas group B-cloned T-cell lines reacted only to E3BP 34-47 carrying an EIExD motif. Furthermore, an RGxG motif was exclusively found in the complementarity-determining region (CDR3) of the TCR Vbeta in the group B-cloned T-cell lines, while G, S, and/or R were frequently found in the CDR3 of the TCR Vbeta in the group A-cloned T-cell lines. These data provide a framework for understanding molecular mimicry among mitochondrial antigens.

Mitochondrial autoantigens and their B and T cell autoepitopes have been well defined in primary biliary cirrhosis (PBC). However, the relationships of the antimitochondrial antibodies and the mechanisms of bile duct destruction in PBC remain an enigma. The serological hallmark of PBC remains the presence of antibodies to mitochondria, particularly to the E2 component of the pyruvate dehydrogenase complex (PDC-E2). However, several mechanisms may now be proposed which may explain the immune-mediated bile duct damage in PBC. These include the possible role of T cell-mediated cytotoxicity as well as the interaction between the IgA class of antimitochondrial antibodies and the mitochondrial autoantigens. A prominent feature in this discussion is the highly directed and specific immune response to the mitochondrial antigens, including PDC-E2 as well as other members of the 2-oxo-acid dehydrogenase complexes. Ultimately, the mechanisms that lead to this immune reaction should provide data on other questions in PBC, including the reasons for female predominance, the absence of PBC in children and the relative ineffectiveness of immunosuppressive agents., (Copyright 2000 Harcourt Publishers Ltd.)

Autoantibodies to mitochondria (AMA, anti-M2) are a serologic hallmark of primary biliary cirrhosis (PBC). These react with three structurally and functionally related multienzymic complexes, the 2-oxoacid dehydrogenase complexes, but chiefly with the E2 subunit of pyruvate dehydrogenase complex (PDC-E2). Their very dose (95%) and specific association with PBC underpins the autoimmune concept of pathogenesis of that disease, notwithstanding several non-congruent features. Detailed studies, including structural analysis of epitopes, do not disclose how these autoantibodies originate. Their ubiquity in PBC has overshadowed the existence of a second set of relatively PBC-specific autoantibodies to nuclear antigens for which reactants have been cloned and characterized. These include centromeric proteins; proteins of the nuclear pore complex; nuclear dot proteins, which include Sp-100 and the promyelocytic leukemia antigen; and a recently identified autoantigen, SOX13. Certain of these reactants are DNA-binding proteins with transcriptional regulatory activity. Thus serum from individuals with the same clinical syndrome can have autoimmune reactivity to disparate mitochondrial and nuclear constituents in different cellular compartments. Antibody probing of phage displayed random peptide libraries, together with epitope scanning using overlapping sequential octameric peptides from the PDC-E2 sequence, showed that the discontinuous motifs MH, FV(E) and SYP contributed to a predicted conformational antibody epitope in the inner lipoyl domain of PDC-E2.

Primary biliary cirrhosis (PBC) is a cholestatic liver disease characterised by immune-mediated destruction of the biliary epithelial cells (BEC) lining the intrahepatic bile ducts (non-suppurative destructive cholangitis (NSDC)). Autoantibody and autoreactive T-cell responses specific for the self-antigen pyruvate dehydrogenase complex (PDC) are almost ubiquitous in PBC patients, leading to the view that the disease has an autoimmune aetiology. Autoreactive responses in PBC appear to be directed at the E2 and at the E3-binding protein (E3BP) (protein X) components of PDC, with the dominant B-cell and T-cell epitopes in E2 (fewer data are available for E3BP) spanning the inner (of two) lipoic acid-binding domains. The causal link between the breakdown of self-tolerance to PDC (particularly at the T-cell level) and the development of NSDC has been emphasised by the demonstration, in a murine model (experimental autoimmune cholangitis), that sensitisation with PDC of mammalian origin results in a breakdown of both B-cell and T-cell tolerance to murine PDC accompanied by the development of NSDC. An increasing understanding of the role played by PDC-specific autoreactive T cells in the pathogenesis of PBC has led us to examine the role played by the target cells in PBC (BEC) in both the inducer and effector mechanisms responsible for PBC.

Primary biliary cirrhosis (PBC) is an organ-specific autoimmune disease that predominantly affects women and is characterized by chronic progressive destruction of small intrahepatic bile ducts with portal inflammation and ultimately fibrosis. The serologic hallmark of PBC is the presence of antibodies to mitochondria, especially to the E2 component of the pyruvate dehydrogenase complex. The mechanisms by which (and if) such antibodies produce liver tissue injury are unknown. However, the presence of these antibodies has allowed detailed immunological definition of the antigenic epitopes, the nature of reactive autoantibodies and the characterization of T-cell responses. Several mechanisms may now be proposed regarding the immune-mediated bile duct damage in PBC, including the possible role of T-cell-mediated cytotoxicity and intracellular interaction between the IgA class of antimitochondrial antibodies and mitochondrial autoantigens. There are major questions which remain unanswered, including, of course, etiology, but also the reasons for female predominance, the absence of PBC in children, the relative ineffectiveness of immunosuppressive drugs, and the specific role of mitochondrial antigens. The data so far provide suggestive evidence that PBC is a mucosal disease; this thesis provides a basis for discussion of etiology via the enterohepatic circulation of toxins and/or infection.

Primary biliary cirrhosis (PBC) is often considered to be a dry gland disease caused by frequent involvement of salivary and lacrimal glands. Although high titers of antimitochondrial autoantibodies (AMA) have long been recognized in PBC, little is known about the presence of mitochondrial autoantigens in mucosal compartments such as saliva. We investigated saliva and sera in PBC patients and controls for the presence of AMA and mitochondrial antigens. In PBC saliva, AMA were detected in 45 of 49 (92%), with specificity directed against pyruvate dehydrogenase complex (PDC-E2) alone in 22 of 49 (45%), against PDC-E2 and branched-chain 2-oxo-acid dehydrogenase complex E2 (BCOADC-E2) in 4 of 49 (8%), to PDC-E2 and 2-oxoglutarate dehydrogenase complex E2 (OGDC-E2) in 9 of 49 (18%), and to the 3 antigens together in 10 of 49 (20%). Isotyping of the saliva AMA showed that 80% of the patients had immunoglobulin A (IgA) against PDC-E2, 18% had IgM-specific PDC-E2, and 35% had IgG specific PDC-E2. Similar to serum and bile anti-PDC-E2 IgA antibodies, the saliva autoantibodies localized their reactivity to the inner lipoyl domain of PDC-E2. Furthermore, saliva from patients with PBC but not controls inhibited pyruvate dehydrogenase enzyme activity in vitro. In addition, and of particular interest, we detected a molecule with a molecular weight corresponding to PDC-E2 (74 kd) in PBC but not control saliva. These findings make several important points: first, there appears to be localized mucosal immunity in the secretory system of PBC; second, AMA are readily detected in PBC saliva; and third, PDC-E2 may be present in the saliva of PBC.

PBC is characterized by T cell-mediated destruction of the biliary epithelial cells lining the small intrahepatic bile ducts. The E2 and E3 binding protein (E3BP (protein X)) components of pyruvate dehydrogenase complex (PDC) are disease-specific autoantigens in PBC. Attempts to localize the T cell autoepitopes within PDC-E2 have, however, generated contradictory results. One study has suggested the presence of T cell epitopes throughout PDC-E2, whilst another has identified a single dominant 14 amino acid T cell epitope (p163) spanning the lipoic acid binding lysine residue in the inner lipoyl domain (ILD) of PDC-E2. The aim of the current study was to determine the prevalence of T cell responses to p163 and PDC-E2 ILD, and the role played by lipoylation of these antigens in their immunogenicity, in a UK PBC population. We found that the majority of the PBC patients showing a 6-day peripheral blood T cell proliferative response to native human PDC also responded, in a MHC class II-restricted fashion, to biochemically purified PDC-E2 and E3BP (which co-purify) (9/10 positive (SI > 2.76), mean SI 5.74 +/- 5.04 (PDC-E2/E3BP) versus 6.67 +/- 3.84 (PDC), P = NS), implying that the important PBC-specific T cell epitopes are contained within the PDC-E2 or E3BP components of PDC. Only a minority of patients responsive to PDC, however, responded to either lipoylated recombinant PDC-E2 ILD (4/10 positive, mean SI 1.98 +/- 1.24, P < 0.005 versus PDC response) or lipoylated p163 (4/12 positive, mean SI 1.90 +/- 1.58, P < 0.001). The lipoylation state did not affect the T cell response to either ILD or p163. Our findings suggest that in some UK patients with PBC there are immunodominant T cell autoepitopes within PDC-E2/E3BP which are outside the ILD of PDC-E2.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of antimitochondrial Abs (AMA). The autoantigens recognized by AMA are the E2 components of the pyruvate dehydrogenase complex (PDC-E2), the branched chain 2-oxoacid dehydrogenase complex E (BCOADC-E2), and the 2-oxoglutarate dehydrogenase complex E (OGDC-E2). Previous studies using murine monoclonal and human combinatorial Abs to PDC-E2 have demonstrated an intense linear staining pattern in the apical region of biliary epithelial cells (BEC) in PBC but not control liver. We therefore examined whether mAbs to the other mitochondrial autoantigens BCOADC-E2 and OGDC-E2 demonstrated disease-specific patterns of reactivity. Using an expressed recombinant "trihybrid" protein containing the lipoyl domains of PDC-E2, OGDC-E2, and BCOADC-E2, we immunized BALB/c mice to produce 35 mAbs specific for one or more of the above mitochondrial autoantigens. Seven of these mAbs uniquely stained the apical region of BEC in PBC. Of these seven, one was reactive to PDC-E2, two recognized BCOADC-E2, three were reactive to OGDC-E2, and one recognized all three Ags. Our current data demonstrate that, similar to our previous studies regarding PDC-E2, mAbs to BCOADC-E2 and OGDC-E2, or a molecule that cross-reacts with the inner lipoyl domain of all three enzymes, also show a uniquely intense staining pattern in the apical region of BEC in patients with PBC when compared with diseased controls. The abundance of such disease-specific determinants in the target cells of PBC raises interesting possibilities regarding the role of these autoantigens in the pathogenesis of this disease.

The immunodominant antimitochondrial antibody response in patients with primary biliary cirrhosis (PBC) is directed against the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Based on our earlier observations regarding peripheral blood mononuclear cell (PBMC) T cell epitopes, we reasoned that a comparative analysis of the precursor frequencies of PDC-E2 163-176-specific T cells isolated from PBMC, regional hepatic lymph nodes, and from the liver of PBC patients would provide insight regarding the role of T cells in PBC. Results showed a disease-specific 100-150-fold increase in the precursor frequency of PDC-E2 163-176-specific T cells in the hilar lymph nodes and liver when compared with PBMC from PBC patients. Interestingly, autoreactive T cells and autoantibodies from PBC patients both recognize the same dominant epitope. In addition, we demonstrated cross-reactivity of PDC-E2 peptide 163-176-specific T cell clones with PDC-E2 peptide 36-49 and OGDC-E2 peptide 100-113 thereby identifying a common T cell epitope "motif" ExETDK. The peptide 163-176-specific T cell clones also reacted with purified native PDC-E2, suggesting that this epitope is not a cryptic determinant. These data provide evidence for a major role for PDC-E2 peptide 163-176 and/or peptides bearing a similar motif in the pathogenesis of PBC.

Analysis of the primary structure of the lipoyl domain of the dihydrolipoamide acetyltransferase (PDC-E2) component of the porcine pyruvate dehydrogenase complex (PDC) reveals a high degree of homology with M2 antigen and human PDC-E2. The porcine PDC-E2 and the dihydrolipoamide succinyltransferase (OGDC-E2) component of the porcine 2-oxoglutarate dehydrogenase complex (OGDC) were identified as mitochondrial autoantigen with sera from patients with primary biliary cirrhosis (PBC). Immunodominant regions (autoepitopes) on the porcine-PDC-E2 component have been mapped to two regions around Lys-46 (outer lipoyl domain) and Lys-173 (inner lipoyl domain), which contained covalently bound lipoic acid prosthetic group. When these lipoyl domains were cleaved at Asp-45 or Asp-172 with endoproteinase Asp-N, the autoantigenicities of the two domains completely disappeared; this suggested the requirement of Asp-45 or Asp-172 residues for the immunoreaction with PBC sera. In addition, a single 14-amino acid epitope peptide histidine-substituted at Asp-172 did not exhibit competitive inhibition of autoantigen binding. Fragmentation of lipoyl domain of the porcine PDC-E2 by limited proteolysis and BrCN-cleavage after alkylation resulted in rapid loss of autoantigenicity. Enzymatic delipoylation and relipoylation of the complexed and free PDC-E2 and OGDC-E2 components did not influence immunoreactivity with PBC sera.

A serological feature of primary biliary cirrhosis (PBC) is the presence of high-titer antimitochondrial autoantibodies (AMA) in patient sera. Five different target mitochondrial autoantigens recognized by sera from PBC patients have been identified as subunits of the 2-oxoacid dehydrogenase complex (2-OADC), of which pyruvate dehydrogenase complex-E2 (PDH-E2) is the most prominent antigenic component. Extensive molecular and immunological studies in PBC such as cloning of mitochondrial autoantigens, mapping of both T and B cell epitopes and immunohistochemical studies have provided valuable reagents in the understanding of immunopathogenesis of PBC. We mapped the epitope recognized by AMA specific to 2-oxoglutarate dehydrogenase complex-E2 (OGDC-E2) in patients with PBC using full-length rat OGDC-E2 cDNA and a series of expression clones spanning the entire molecule. It appears that the epitope is dependent on conformation and includes the lipoic acid-binding region. Furthermore we have taken advantage of the antigenic mapping studies of PDC-E2, OGDC-E2 and branched chain 2-oxoacid dehydrogenase complex-E2 (BCOADC-E2) subunits and designed a hybrid clone, pML-MIT3, that expressed three different immunodominant epitopes. Our results indicate that an immunoassay using recombinant, cloned autoantigen is a powerful and very specific method for detecting AMA in PBC.

Auto-antibodies specific to various antigens in chronic hepatitis (CH) have been detected but their specificities and implications were uncertain. The aims of the present study were to investigate the frequency and the significance of seropositivity of antibodies to P450IID6 or liver/kidney microsome 1 (LKM1), soluble liver antigen (SLA), pyruvate dehydrogenase (PDH) and branched-chain keto acid dehydrogenase (BCKD) in 188 Japanese patients with different forms of CH by western blot or enzyme immunoassay (EIA). Anti-LKM1 was also measured by indirect immunofluorescent test. Anti-P450IID6 was found in 6/188 (3.2%) CH patients including 5/104 (4.8%) with hepatitis C virus (C) infection and 1/12 (8.3%) CH-C patients with antibodies to nuclear and smooth muscle antigens and hypergammaglobulinaemia (> 2.5 g/dL). This patient was the only one diagnosed with autoimmune hepatitis (AIH). All CH patients with hepatitis B (B), hepatitis non-B non-C (NBNC) and AIH were seronegative for anti-LKM1. Antibodies to soluble liver antigen were found in two of 188 (1%) patients, one with AIH and one with CH-B. Anti-BCKD-E2 but not anti-PDH-E2 was found in four patients (2.5%), one with AIH, two with CH-C, and one with NBNC. There was no obvious difference in age, sex ratio and laboratory findings in patients with or without anti-SLA and anti-BCKD-E2. Antibodies to P450IID6, SLA, PDH-E2 and BCKD-E2 are uncommon in adult CH-C, CH-B, CH-NBNC and AIH patients in Japan. Some of these patients positive for auto-antibodies appear to have autoimmune features and might require a careful follow up. The heterogeneity of these antibodies in CH preclude further justification for subtyping of AIH by the presence of the distinct auto-antibodies.

Background & Aims: Patients with primary biliary cirrhosis (PBC) have autoantibodies that react with components of mitochondrial multienzyme complexes. In addition to binding to mitochondria, patients' autoantibodies to the assumed major autoantigen pyruvate dehydrogenase complex (PDC) dihydrolipoamide acetyltransferase (E2) bind to the plasma membrane of biliary epithelial cells (BECs) specifically in PBC. The aim of this study was to characterize BEC plasma membrane antigens recognized by patients' autoantibodies in PBC., Methods: Antigens prepared from intracellular and plasma membrane-enriched fractions of BECs purified from PBC and control liver were immunoblotted with anti-PDC., Results: In the intracellular fraction, anti-PDC recognized BEC protein bands corresponding to the molecular weight value of E2 and X components of human heart PDC on Western blots. No difference was observed between PDC-E2 in BECs from PBC and controls. However, in PBC but not controls, a 50-kilodalton antigen was detected in the plasma membrane-enriched fraction. This antigen comigrated with component X of purified human heart PDC and was recognized by antibodies specific for PDC-X., Conclusions: The data suggest that PDC-X or a cross-reactive 50-kilodalton antigen is the BEC plasma membrane antigen recognized by patients' autoantibodies in PBC. Furthermore, this antigen, rather than PDC-E2, may be a major B-cell target antigen in PBC.

The role of biliary epithelial cell (BEC) antigens in immune recognition and damage of biliary epithelium in primary biliary cirrhosis (PBC) is unknown. The major autoantigen in PBC (the mitochondrial enzyme pyruvate dehydrogenase dihydrolipoamide acetyltransferase [PDC-E2]) is abnormally distributed in the biliary epithelium of patients with PBC relative to controls. The antigen is not only present in mitochondria but also associated with the BEC plasma membrane. This atypical distribution of PDC-E2 is present both in early (stages I-II) and advanced (stages III-IV) disease, suggesting a role for the antigen in progression and/or etiology of PBC. The identity of the plasma membrane antigen remains unknown, but there is evidence to suggest that it is an antigen that cross reacts with antibodies to PDC-E2. Use of BEC purified from human liver may help in deciphering the possible importance of BEC plasma membrane antigens in immune recognition and toxicity toward BEC in PBC.

The study of T cell responses to autoantigens in human autoimmunity has been hampered by difficulties, firstly in identifying significant autoantigens, and secondly in the purification of authentic human proteins in sufficient quantities to allow characterization of antigen-specific T cell responses. In this study we have purified a human autoantigen, pyruvate dehydrogenase, retaining its enzymatic activity, and characterized autoreactive T cell responses to it in a human autoimmune disease, primary biliary cirrhosis. T cell responses to a mixture of the E2 and protein X subunits of human pyruvate dehydrogenase complex are seen in most affected patients, but in only a small minority of normal and chronic liver disease controls. By contrast, responses to whole pyruvate dehydrogenase complex occur with equal frequency in both groups. This suggests that responses to the E2 component/protein X of pyruvate dehydrogenase complex play a role in the pathogenesis of primary biliary cirrhosis. The availability of significant quantities of the human autoantigen in primary biliary cirrhosis makes this condition an interesting model in which to study true autoreactive human T cell responses.

Affinity mass spectrometry (AMS) was used to evaluate the structural diversity of the E2 component of pyruvate dehydrogenase complex (PDC) in normal and diseased liver cells, including those from patients with the autoimmune disease primary biliary cirrhosis (PBC). Two different antibodies to PDC-E2, the immunodominant mitochondrial autoantigen in patients with PBC, were used. AMS was performed directly on frozen liver sections and purified bile duct epithelial cells. Mass spectrometric signals associated with the molecular recognition of PBC-specific antigenic determinants were enhanced by an in situ enzyme-linked signal amplification process. Samples from patients with PBC gave strong positive signals for the antigen(s) recognized by the monoclonal antibody C355.1. Conversely, tissues from normal and disease controls showed only a minimal signal. AMS was used to identify specific antigenic determinants within the E2 component of PDC for comparison with unknown antigenic determinants observed by affinity capture with C355.1 monoclonal antibody from PBC samples. PDC components bound to C355.1 were mapped and identified by mass before dissociation from the E2 component. A similar approach was used to identify unknown antigenic determinants associated with PBC. We believe AMS may be an important new approach with wide application to the identification of molecules associated with a number of disease states.

Dihydrolipoamide acetyltransferase, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), is the autoantigen most commonly recognized by autoantibodies in primary biliary cirrhosis (PBC). We identified a peptide mimotope(s) of PDC-E2 by screening a phage-epitope library expressing random dodecapeptides in the pIII coat protein of fd phage using C355.1, a murine monoclonal antibody (mAb) that recognizes a conformation-dependent epitope in the inner lipoyl domain of PDC-E2 and uniquely stains the apical region of bile duct epithelium (BDE) only in patients with PBC. Eight different sequences were identified in 36 phage clones. WMSYPDRTLRTS was present in 29 clones; WESYPFRVGTSL, APKTYVSVSGMV, LTYVSLQGRQGH, LDYVPLKHRHRH, AALWGVKVRHVS, KVLNRIMAGVRH and GNVALVSSRVNA were singly represented. Three common amino acid motifs (W-SYP, TYVS, and VRH) were shared among all peptide sequences. Competitive inhibition of the immunohistochemical staining of PBC BDE was performed by incubating the peptides WMSYPDRTLRTS, WESYPDRTLRTS, APKTYVSVSGMV, and AALWGVKVRHVS with either C355.1 or a second PDC-E2-specific mAb, C150.1. Both mAbs were originally generated to PDC-E2 but map to distinct regions of PDC-E2. Two of the peptides, although selected by reaction with C355.1, strongly inhibited the staining of BDE by C150.1, whereas the peptide APKTYVSVSGMV consistently inhibited the staining of C355.1 on biliary duct epithelium more strongly than the typical mitochondrial staining of hepatocytes. Rabbit sera raised against the peptide WMSYPDRTLRTS stained BDE of livers and isolated bile duct epithelial cells of PBC patients more intensively than controls. The rabbit sera stained all size ducts in normals, but only small/medium-sized ductules in PBC livers. These studies provide evidence that the antigen present in BDE is a molecular mimic of PDC-E2, and not PDC-E2 itself.

The detection of antimitochondrial autoantibodies (AMAs) is critical in the diagnosis of primary biliary cirrhosis (PBC). However, conventional laboratory assays to detect AMA are dependent on the time-consuming method of immunofluorescence microscopy, a method often plagued by problems of nonspecificity. AMAs react against mitochondrial autoantigens including the E2 components of the pyruvate dehydrogenase complex (PDC-E2), the branched-chain 2-oxo-acid dehydrogenase complex (BCOADC-E2), and the 2-oxo-glutarate dehydrogenase complex (OGDC-E2). Interestingly, the immunodominant epitopes of PDC-E2, BCOADC-E2, and OGDC-E2 are all conformational lipoate binding sites, but antibodies against them do not cross-react. Although 80% to 90% of sera from patients with PBC react to PDC-E2, approximately 10% patients with PBC react only to BCOADC-E2 and/or OGDC-E2. We have taken advantage of our epitope-mapping studies of the E2 components of PDC, BCOADC, and OGDC, and constructed a "designer" hybrid clone, designated as pML-MIT3, that coexpresses the immunodominant epitopes within the three distinct lipoyl domains. We examined a total of 321 sera, including 186 sera from patients with PBC, to test the immunoreactivity of pMIT3. Of 186 sera from patients with PBC, 152 sera (81.7%) reacted with recombinant fusion protein of PDC-E2, whereas 171 sera (91.9%) showed positive reactivities when probed by immunoblotting against the recombinant fusion protein expressed from the pML-MIT3 clone. Of 34 PBC sera that did not react with recombinant PDC-E2, 18 sera contained BCOADC-E2-specific AMA and 1 serum possessed only OGDC-E2-specific AMA. We also developed an enzyme-linked immunosorbent assay (ELISA), using affinity-purified recombinant fusion protein of pML-MIT3 clone as the antigen source, to quantify specific AMAs in patients with PBC. None of the 135 control sera from patients with primary sclerosing cholangitis (PSC), chronic autoimmune hepatitis (CAH), systemic lupus erythematosus (SLE), or healthy volunteers showed significant reactivity against pML-MIT3 recombinant fusion protein in the ELISA assay. Our results indicate that an ELISA using recombinant, cloned autoantigen of pML-MIT3 is a powerful and very specific method for the detection of AMA.

The immune system is normally tolerant to mitochondrial self-antigens, but responsive against bacteria. Low-titre anti-mitochondrial antibodies (AMA) might be involved in this discrimination. Tolerance is broken in diseases characterised by high titre AMA. Some of these AMA, against cardiolipin, cross-react with DNA. The best studied AMA are those characterising primary biliary cirrhosis (PBC). These are directed against E2 subunits of the oxo-acid dehydrogenase complexes, and also against subunits E1 alpha, E1 beta and X of the pyruvate dehydrogenase complex. AMA of PBC patients also react with bacterial E2s. Reactivities are primarily peptide-specific but with cross-reactivity between mitochondrial and microbial antigens and between E2s of respective complexes. Immunodominant epitopes, for anti E2 AMA, include the conserved sequence flanking the site of lipoyl attachment. It is proposed that the initial stimulus for antibody production is chronic urinary tract infection. AMA themselves are not pathogenic, but CD4+ T-cells would be primed, recognising the lipoyl domain epitope in association with class II HLA. Inappropriate expression of class II antigens on bile duct epithelia, (as found in PBC), might lead to presentation of a particular fragment of HLA-DR alpha, known to be a major MHC presented self-peptide in the mouse. That sequence strongly mimics the lipoyl domain and might be recognised by primed T-cells, initiating the autoimmune cascade. In the mouse, a peptide of ND1 of Complex I is presented in association with class I MHC. Cells exhibiting somatic mutation of such a peptide might thus be subject to attack by CD8+ T-cells. If such peptides were presented by class II HLA, autoimmune diseases might arise, related to mimicry between such peptides and microbial sequences and/or self-antigens. These considerations might apply in Leber's disease and in age-related pathology.

Primary biliary cirrhosis is a chronic cholestatic liver disease associated with autoimmune disorders. Antimitochondrial autoantibodies and granulomatous portal lesions are characteristic in primary biliary cirrhosis. Since granuloma may be induced by Mycobacteria, and there is evidence implicating Mycobacteria as infectious agents capable of initiating autoimmunity, a study was performed to determine the presence of antibodies against 10 atypical Mycobacteria in 19 patients with primary biliary cirrhosis, and in 35 controls (25 patients with other chronic liver diseases and 10 healthy subjects). All primary biliary cirrhosis sera and none of the controls reacted with the extract from Mycobacterium gordonae, showing identical recognition profiles with two polypeptides of 70-65 and 55 kDa. No other reaction was found in primary biliary cirrhosis patients and in controls with the extracts from the other nine atypical Mycobacteria tested. Eluted immunoglobulins which reacted with the 70-65 and 55 kDa polypeptides from M. gordonae, bound to the mitochondrial antigens PDH-E2 and BCKDH-E2. Furthermore, when the extract from M. gordonae was tested with eluted immunoglobulins from recognized PDH-E2 and BCKDH-E2 by primary biliary cirrhosis patients, we observed both 70-65 and 55 kDa polypeptides. These data indicate that antibodies to M. gordonae, found in all primary biliary cirrhosis patients, cross-react with the major mitochondrial targets of the disease. We suggest that M. gordonae may play a potential pathogenic role in primary biliary cirrhosis.

Trifluoroacetylated (CF3CO-) proteins, elicited upon exposure of animals or humans to halothane, were recognized by anti-CF3CO antibody, monospecific for the hapten derivative N6-trifluoroacetyl-L-lysine. Anti-CF3CO antibodies cross-reacted with the dihydrolipoamide acetyltransferase (E2 subunit) of pyruvate dehydrogenase, indicating that epitopes on the E2 subunit of pyruvate dehydrogenase molecularly mimic those on CF3CO-proteins. Lipoic acid, the prosthetic group of the E2 subunit of pyruvate dehydrogenase was essential in this process, in that only the lipoylated form of the recombinantly expressed inner lipoyl domain of the human E2 subunit of pyruvate dehydrogenase, but not the unlipolyated form, was recognized by anti-CF3CO antibody. Furthermore, based on a high degree of structural relatedness, both CF3CO-Lys and (6RS)-lipoic acid, as well as the lipoylated peptide ETDK(lipoyl)ATIG specifically inhibited the recognition by anti-CF3CO antibody of the E2 subunit of pyruvate dehydrogenase, of trifluoroacetylated rabbit serum albumin and of human liver CF3CO-proteins. In sera of patients with halothane hepatitis, autoantibodies with properties identical to those of anti-CF3CO antibody were identified which could not discriminate between CF3CO-proteins and the E2 subunit of pyruvate dehydrogenase. These data suggest that the E2 subunit pyruvate of dehydrogenase is an autoantigen in halothane hepatitis and that molecular mimicry of CF3CO-proteins by the E2 subunit of pyruvate dehydrogenase is due to the similar structures of CF3CO-Lys and lipoic acid.

Approximately 95% of patients with primary biliary cirrhosis have antimitochondrial antibodies against the E2 component of the pyruvate dehydrogenase complex (E2p). Immunodominant sites on E2p have been localized to the inner lipoyl domain, which serves as a covalent attachment site for the essential cofactor, lipoic acid. However, it is not clear whether the presence of lipoic acid is necessary for autoimmune recognition of human E2p. To facilitate further studies on the inner lipoyl domain and to assess the importance of lipoic acid in antibody binding, we used the previously cloned human E2p cDNA in the construction and high-level expression in Escherichia coli of a subgene encoding the domain. Purification and analysis of the gene product revealed that both lipoylated and unlipoylated forms of the intact domain are generated. Immunoblotting, enzyme-linked immunosorbent assay inhibition experiments and antibody affinity measurements using isolated lipoylated and unlipoylated domains demonstrated that the presence of the lipoyl residue is crucial for effective recognition by primary biliary cirrhosis patients' autoantibodies, which have a higher relative affinity for the lipoylated form. Contrary to some previous suggestions, these results indicate that antibodies in primary biliary cirrhosis patients' sera bind most effectively to a unique peptide-cofactor conformation in the lipoyl domain of the human E2p polypeptide. Moreover, the availability of large amounts of human lipoyl domain will permit further studies into the role of the antigen (if any) in disease pathogenesis.

Peripheral B lymphocytes from a patient with primary biliary cirrhosis were infected with Epstein-Barr virus, and Epstein-Barr virus-transformed B lymphocytes producing large amounts of IgG antibodies to pyruvate dehydrogenase complex were selected, expanded and fused with the human-mouse heteromyeloma cell line F3B6. The resulting Epstein-Barr virus-transformed B-cell hybrids were repeatedly cloned by limiting dilution, and three stable hybridoma clones producing human monoclonal antibodies to pyruvate dehydrogenase complex were generated. These monoclonal antibodies, designated M18GP8, M37GP11 and M82GP8, specifically bound to pyruvate dehydrogenase complex, and their dissociation constant with pyruvate dehydrogenase complex was calculated to be 2.4 x 10(-11), 2.3 x 10(-10) and 2.6 x 10(-11) mol/L, respectively. These three monoclonal antibodies stained the mouse stomach/kidney cryostat sections in a typical immunofluorescence pattern of antimitochondrial antibody. Furthermore, the enzymatic activity of pyruvate dehydrogenase complex was almost completely inhibited by the three monoclonal antibodies. Western blotting analysis revealed that M18GP8 and M82GP8 reacted with only pyruvate dehydrogenase complex-E2 in contrast to M37GP11, which reacted with both pyruvate dehydrogenase complex-E2 and protein X. The binding of monoclonal antibody M37GP11 to solid-phase pyruvate dehydrogenase complex was partially inhibited by two different synthetic peptides corresponding to both the inner and outer lipoyl-binding domains of pyruvate dehydrogenase complex-E2. These monoclonal antibodies, which are the first human monoclonal antibodies to pyruvate dehydrogenase complex generated from a patient with primary biliary cirrhosis, will be a valuable tool for studying the B-cell autoepitopes in PDC and the mechanism of autoantibody production in primary biliary cirrhosis.

1. The sera of more than 90% of patients with primary biliary cirrhosis contain antimitochondrial antibodies which react with the E2 component of the pyruvate dehydrogenase complex, identified as the major autoantigen in primary biliary cirrhosis. All previous studies in this area have utilized protein derived from animal tissue or have used recombinant human pyruvate dehydrogenase complex E2 expressed in Escherichia coli. 2. We report the preparation and characterization of native pyruvate dehydrogenase complex and pyruvate dehydrogenase complex E2 from human heart tissue and its application in studies of immune reactivity with the sera of patients with primary biliary cirrhosis. 3. The immune reactivity of sera from patients with primary biliary cirrhosis versus the bovine and human E2/X components of pyruvate dehydrogenase complex was indistinguishable in both immunoblotting and the more sensitive e.l.i.s.a. 4. These findings suggest that the reactivity of sera from patients with primary biliary cirrhosis against the major autoantigen of the disease is a property of that antigen, independent of its human or bovine origin. Furthermore, this justifies the use of bovine pyruvate dehydrogenase complex in past and future work on primary biliary cirrhosis antibody reactivity.

Antimitochondrial autoantibodies are present in sera from close to 95% of patients with primary biliary cirrhosis. The so-called primary biliary cirrhosis-specific antigen, named M2, was found to be associated with an enzyme complex of the inner mitochondrial membrane and, more precisely, with the E2 component, dihydrolipoamide acetyltransferase, of the pyruvate dehydrogenase complex. We recently established that an immunodominant epitope recognized in direct enzyme-linked immunosorbent assay by primary biliary cirrhosis M2+ sera, but not by non-primary biliary cirrhosis M2+ sera, could be mimicked by a synthetic peptide encompassing residues 167-184 of the E2 component and associated with lipoic acid. This fragment is present in the natural inner lipoyl-binding site of the human enzyme, and the presence of lipoic acid located on lysine 173 was found to be essential to allow IgG antibody binding. In this study we have improved the enzyme-linked immunosorbent assay test based on the synthetic peptide-lipoic acid conjugate by using a multiple antigen peptide system containing eight copies of the peptide as antigen. This approach avoids the use of a peptide conjugated to a carrier protein and was found to be particularly efficient because 23 of 27 primary biliary cirrhosis M2+ sera (85%) could be identified. A multiple antigen peptide without lipoic acid was not recognized by primary biliary cirrhosis antibodies. The peptide used in the multiple antigen peptide construction was a short 13-mer peptide encompassing a highly conserved sequence present in both the outer (residues 40-52) and the inner (residues 167-179) lipoyl-binding sites of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Further to define the epitopes of PDC-E2, the major autoantigen in primary biliary cirrhosis (PBC), we have developed and characterized five human monoclonal antibodies. These antibodies were derived by fusing a regional hepatic lymph node from a patient with PBC with the mouse human heterohybrid cell line F3B6. Previous studies of epitope mapping of PDC-E2 have relied on whole sera and have suggested that the immunodominant epitope lies within the inner lipoyl domain of the molecule. However, selective absorption studies using whole sera and a series of overlapping recombinant peptides of PDC-E2 have suggested that the epitope may also include a large conformational component. Moreover, several laboratories have suggested that autoantibodies against the 2-oxo acids dehydrogenase autoantigens are cross-reactive. The five monoclonal antibodies generated included three IgG2a and two IgM antibodies and were studied for antigen specificity using recombinant PDC-E2, recombinant BCKD-E2, histone, dsDNA, IgG (Fc), collagen and a recombinant irrelevant liver specific control, the F alloantigen. The antibodies were also used to probe blots of human, bovine, mouse and rat mitochondria. Finally, fine specificity was studied by selective ELISA and absorption against overlapping expressing fragments of PDC-E2. All five monoclonals, but none of the other mitochondrial autoantigens were specific for PDC-E2. In fact, although affinity purified antibodies to PDC-E2 from patients with PBC cross-reacted with protein X, the human monoclonals did not, suggesting that protein X contains an epitope distinct from that found on PDC-E2. Additionally, all three IgG2 monoclonals recognized distinct epitopes within the inner lipoyl domain of PDC-E2.

Naturally-occurring autoantibodies to a family of mitochondrial enzymes, the 2-oxoacid dehydrogenase complexes (2-OADC), characterize the human liver disease primary biliary cirrhosis. The immunodominant epitope for these autoantibodies is associated with the lipoyl-binding domain of the E2 subunit of the enzymes. The reactivity of these disease-associated autoantibodies was compared with that of antibodies raised in rats and rabbits, by immunization with various preparations derived from the 2-OADC enzymes, using immunization protocols that have successfully induced various organ-specific autoimmune diseases in animals. The immunogens included the intact pyruvate dehydrogenase complex (PDC) from bovine heart, human recombinant PDC-E2, and short synthetic peptides representing the immunodominant lipoic acid binding sequences of the 2-OADC enzymes. The techniques for antibody analysis included immunofluorescence, immunoblotting on mitochondrial extracts, ELISAs using entire PDC, PDC-E2, or synthetic peptides, epitope mapping by peptide scanning on overlapping octameric peptides representing the human PDC-E2 sequence, affinity purification on PDC-E2, and inhibition in vitro by sera of the catalytic function of PDC. Experimental immunization did not elicit any evidence of autoimmune disease. Moreover, the experimentally-induced antibodies in striking contrast to the natural autoantibodies showed preferential reactivity with PDC-E2 rather than with intact PDC, failed to inhibit in vitro the catalytic function of PDC, and, on peptide scanning, reacted with discrete epitopes, but at sites other than the lipoyl-binding region of PDC-E2. Our data indicate that 'multisystem' autoimmune diseases including primary biliary cirrhosis may not be elicitable experimentally because a critical disease-relevant autoepitope is not engaged by the immune system.

Close to 95% of patients with established clinical, biochemical and histologic features of primary biliary cirrhosis (PBC) possess antimitochondrial M2 antibodies reacting with the E2 component, dihydrolipoamide acetyltransferase, of the pyruvate dehydrogenase complex. We examined the ability of synthetic peptides of E2 to be recognized in ELISA by sera from patients with PBC and autoimmune-related disorders. Sera from 14 PBC M2+ patients, 1 PBC M2- patient, 5 non-PBC M2+ patients, and 6 patients with chronic active hepatitis were studied. Among the seven E2 synthetic peptides tested (namely peptides 87-119, 167-184, 169-202, 267-302, 456-477, 498-513 and 530-543), only peptide 167-184 used as OVA conjugate and prepared with lipoic acid (LA) located on lysine 173 (natural inner lipoyl-binding site) was recognized in direct ELISA by PBC M2+ sera. The conjugated peptide 167-184 LA was not recognized in direct ELISA by non-PBC M2+ sera or by sera from patients with chronic active hepatitis. The free peptide 167-184 LA inhibited the ELISA reaction of PBC antibodies to PDH and totally abolished the typical immunofluorescence reaction of PBC sera on rat kidney, stomach and liver, or human HEp-2 cell substrates. No inhibition of ELISA or immunofluorescence reaction was found with the other E2 fragments including peptide 167-184 without LA. Our results show that the lipoyl moiety forms an integral part of a dominant conformational epitope recognized by PBC sera. Inasmuch as the peptide 167-184 LA was not recognized by non-PBC sera in direct ELISA, it could be used as a valuable probe for PBC diagnosis.

Primary biliary cirrhosis (PBC) has been described among various ethnic and racial populations in all parts of the world. However, the incidence and prevalence of PBC varies considerably in different geographic areas. It has the highest frequency in Northern Europe, is considerably lower in Japan and still lower in other parts of Asia. There has not hitherto been a detailed immunological profile of antimitochondrial antibodies according to geographic region. We have used recombinant or purified preparations from the 2-oxo-acid dehydrogenase enzyme complexes, the major mitochondrial autoantigens in PBC (PDC-E2, BCOADC-E2, OGDC, protein X and PDC-E1 alpha) to compare the reactivity of sera from either similarly staged sera from Japanese (n = 23) or American-Caucasian patients (n = 39) with PBC. In all cases, the first available sera following diagnosis was selected. Interestingly, only 65% of Japanese patients reacted by ELISA with PDC-E2 compared with more than 95% of the North American group. Moreover, the level of enzyme-inhibitory antibodies to PDC was lower in the Japanese. Our findings prompt the need for characterization of specific susceptibility genes and environmental factors in various parts of the world to clarify the etiology of PBC.

The mitochondrial autoantibodies present in primary biliary cirrhosis (PBC) react with the 2-oxoacid dehydrogenase enzymes that include the pyruvate dehydrogenase complex (PDC). All epitopes so far demonstrable, including the inner lipoyl domain of PDC-E2, have been revealed by immunoblotting. To identify other epitopes, advantage was taken of the capacity of PBC sera to inhibit in vitro the catalytic function of the PDC enzyme. PBC sera were analyzed by affinity chromatography, using columns containing either recombinant PDC-E2 or intact PDC. Fractions that bound to the column (B) and nonbinding effluent fractions (NB) were tested by immunoblotting and ELISA and for their capacity to inhibit enzyme function. After separation on the PDC-E2 column the B fractions were reactive with PDC-E2 and intact PDC, whereas the NB fractions did not react by immunoblotting or ELISA with PDC-E2 but did react strongly by ELISA with PDC and did strongly inhibit the enzyme function. After separation of sera on the PDC column, the B fractions reacted more strongly with PDC than PDC-E2 by ELISA and strongly inhibited the enzyme function, whereas the NB fractions were nonreactive. Thus we describe a hitherto undetected population of autoantibodies in PBC sera that react only with intact PDC but not with the recombinant PDC-E2 subunit that contains the lipoyl epitope, are demonstrable by ELISA but not by immunoblotting, and notably, inhibit enzyme function. These nonblotting inhibitory autoantibodies in PBC are presumed to react with an exclusively conformational determinant perhaps presented by the tertiary structure of the entire enzyme complex.

Primary biliary cirrhosis is an evolutive and chronic human liver disease characterized by presence of antimitochondrial autoantibodies in the serum. We present the biochemical definition of these autoantigens and show that purified pyruvate dehydrogenase complex contains three of the major mitochondrial antigens of M2 type i.e. the E2 subunit (dihydrolipoamide acetyl transferase), the X subunit and the E1 alpha subunit (pyruvate dehydrogenase), by immunoblotting experiments and inhibition of enzyme activity with several types of serums.

The E2 component (acetyltransferase) of the pyruvate dehydrogenase (PDH) complex is the major mitochondrial autoantigen recognized by autoantibodies in patients with primary biliary cirrhosis (PBC). Previous work, using only a partial length rat liver cDNA clone of PDH-E2, demonstrated that the immunodominant epitope was localized to the lipoic acid binding site. Human PDH-E2, in contrast to rat PDH-E2, has two lipoic acid binding sites. By using a full length human cDNA for PDH-E2, and by preparation of multiple overlapping recombinant fragments, we have determined that three autoreactive determinants are present on human PDH-E2: two cross-reactive lipoyl domains, and an area surrounding the E1/E3 binding region. The dominant epitope was localized to the inner lipoyl domain whereas the outer lipoyl domain only showed a weak cross-reactivity, and only 1/26 PBC sera reacted weakly to the E1/E3 binding region area. By probing recombinant fusion proteins expressed from small restriction fragments of the inner lipoyl domain, we have found that a minimum of 75 amino acids (residues 146-221) were required for detectable autoantibody binding, and that 93 amino acids (residues 128-221) were necessary for characteristically strong antimitochondrial autoantibody recognition. Such a requirement for a large region suggests the possibility that a conformational autoepitope may be recognized. In addition, we have found that absorption of PBC sera with the purified mammalian PDH complex does not remove reactivity against Escherichia coli Ag. The possible implications for such results are discussed.

Antibodies to the mitochondrial autoantigen M2, characteristic of the autoimmune liver disease primary biliary cirrhosis (PBC), react with the E2 subunit of the pyruvate dehydrogenase enzyme (PDH-E2). We examined the effect of disease sera on the enzyme activation catalysed by the PDH complex. Inhibition of enzyme activity was observed in 19 of 24 sera of patients with PBC with a level of greater than 90% inhibition in 14 at a serum dilution of 1/50. The onset of inhibition by serum was rapid, within the time of mixing, and the inhibitory activity was shown to reside in the immunoglobulin fraction of the serum. The immunoglobulin fraction of control sera from patients with other liver diseases (n = 26) and healthy persons (n = 8) failed to produce inhibitory activity. In addition sera from four rabbits, intensively immunized with a recombinant human M2 autoantigen, gave anti-M2 reactions by fluorescence, ELISA and immunoblotting, but did not inhibit the activity of PDH. The failure of experimentally induced M2 antibodies in rabbits to inhibit is interesting in view of the reactivity of the natural M2 autoantibodies of PBC with the highly conserved site on the enzyme which carries the essential lipoic acid cofactor.