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Anti-mitochondrial antibodies (AMA), directed against the E2 subunits of the 2-oxo acid dehydrogenase complexes, are markers of Primary Biliary Cholangitis (PBC), a chronic autoimmune liver disease. However, it has not been stablished the clinical significance of subunits-specific AMA type PDC-E2 subunit of the pyruvate dehydrogenase complex-, BCOADC-E2 subunit of the branched-chain 2-oxo acid dehydrogenase complex-, OGDC-E2 subunit of the 2-oxo-glutarate dehydrogenase complex- and nPDC -native pyruvate dehydrogenase complex (M2-AMA), and not all AMA specificities are associated with PBC. The aim of the present study was to show the usefulness of the number and combination of subunits-specific AMA positive for the diagnosis of PBC. We detected AMA by indirect immunofluorescence (IIF-AMA) and M2-AMA by dot-blot. We studied the relationship of AMA with some clinical and laboratory variables in 307 patients (37% PBC) with positive dot-blot for M2-AMA. In PBC patients, we detected different E2 subunits of the 2-oxo acid dehydrogenase complexes antibodies (M2-AMA): 82.9% were specific for nPDC, 64.5% for PDC-E2, 44.4% for BCOADC-E2, and 9.6% for OGDC-E2. IIF and dot-blot tests achieved an Area Under the Receiver Operating Characteristic Curve (ROC AUC) of 0.674 (1:320 cut-off titer, Sensibility (Se) 64.7%, Specificity (Sp) 63.4%) and 0.663 (three specificities M2-AMA, Se 43%, Sp 81.2%), respectively. The detection of different E2 subunits of the 2-oxo acid dehydrogenase complexes antibodies (M2-AMA) by dot-blot showed different ROC AUC: anti-PDC-E2 showed an AUC of 0.610, a Se of 43.7%, and a Sp of 76.4%. Finally, the combined detection of nPDC/BCOADC-E2/PDC-E2 reached an AUC of 0.6095, a Se of 59.6%, and a Sp of 70.2%.The identification of two M2-AMA specificities through dot-blot increased PBC odds ratio (OR) by 2.05 (p:0.031), as compared to the identification of one specificity. Moreover, the identification of three and four specificities increased OR by 4.63 (p:0.000) and by 21.53 (p:0.006), respectively. nPDC/OGDC-E2/PDC-E2 and nPDC/OGDC-E2/BCOADC-E2/PDC-E2 combinations increased PBC OR by 10.04 (p:0.034), as compared to any other combination. 1:320 and 1:640 IIF-AMA increased PBC OR by 4.93 (p:0.009) and 7.67 (p:0.001), respectively, as compared to IIF-AMA titers equal to or less than 1:160. M2-AMA dot-blot was less sensitive but more specific than IIF-AMA, with similar predictive capacity for PBC. Increased numbers of M2-AMA specificities clearly increased the risk of PBC. Some combinations were strongly related to PBC (nPDC/BCOADC-E2/PDC-E2), but others were not (one single M2-AMA, and nPDC plus PDC-E2). M2-AMA dot-blot was less sensitive but more specific than IIF-AMA, with similar predictive capacity for PBC. Increased numbers of M2-AMA specificities clearly increased the risk of PBC, being some combinations, such as nPDC/BCOADC-E2/PDC-E2, more related to PBC than others. Finally, the determination of the number of M2-AMA specificities was more useful than the particular subunit target for PBC diagnosis. In conclusion, the study of the number of M2-AMA specificities by dot-blot should definitely be considered for PBC diagnosis., (Copyright © 2021. Published by Elsevier Inc.)

Developing an effective Staphylococcus aureus ( S. aureus ) vaccine has been a challenging endeavor, as demonstrated by numerous failed clinical trials over the years. In this study, we formulated a vaccine containing a highly conserved moonlighting protein, the pyruvate dehydrogenase complex E2 subunit (PDHC), and showed that it induced strong protective immunity against epidemiologically relevant staphylococcal strains in various murine disease models. While antibody responses contributed to bacterial control, they were not essential for protective immunity in the bloodstream infection model. Conversely, vaccine-induced systemic immunity relied on γδ T cells. It has been suggested that prior S. aureus exposure may contribute to the reduction of vaccine efficacy. However, PDHC-induced protective immunity still facilitated bacterial clearance in mice previously exposed to S. aureus . Collectively, our findings indicate that PDHC is a promising serotype-independent vaccine candidate effective against both methicillin-sensitive and methicillin-resistant S. aureus isolates., Competing Interests: Competing interests statement:The authors declare no competing interest.

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.)

The genus Listeria comprises ubiquitous bacteria, commonly present in foods and food production facilities. In this study, three different phage display technologies were employed to discover targets, and to generate and characterize novel antibodies against Listeria: antibody display for biomarker discovery and antibody generation; ORFeome display for target identification; and single-gene display for epitope characterization. With this approach, pyruvate dehydrogenase complex-enzyme 2 (PDC-E2) was defined as a new detection target for Listeria, as confirmed by immunomagnetic separation-mass spectrometry (IMS-MS). Immunoblot and fluorescence microscopy showed that this protein is accessible on the bacterial cell surface of living cells. Recombinant PDC-E2 was produced in E. coli and used to generate 16 additional antibodies. The resulting set of 20 monoclonal scFv-Fc was tested in indirect ELISA against 17 Listeria and 16 non-Listeria species. Two of them provided 100% sensitivity (CI 82.35-100.0%) and specificity (CI 78.20-100.0%), confirming PDC-E2 as a suitable target for the detection of Listeria. The binding region of 18 of these antibodies was analyzed, revealing that ≈ 90% (16/18) bind to the lipoyl domains (LD) of the target. The novel target PDC-E2 and highly specific antibodies against it offer new opportunities to improve the detection of Listeria.

Metabolic reprogramming during macrophage polarization supports the effector functions of these cells in health and disease. Here, we demonstrate that pyruvate dehydrogenase kinase (PDK), which inhibits the pyruvate dehydrogenase-mediated conversion of cytosolic pyruvate to mitochondrial acetyl-CoA, functions as a metabolic checkpoint in M1 macrophages. Polarization was not prevented by PDK2 or PDK4 deletion but was fully prevented by the combined deletion of PDK2 and PDK4; this lack of polarization was correlated with improved mitochondrial respiration and rewiring of metabolic breaks that are characterized by increased glycolytic intermediates and reduced metabolites in the TCA cycle. Genetic deletion or pharmacological inhibition of PDK2/4 prevents polarization of macrophages to the M1 phenotype in response to inflammatory stimuli (lipopolysaccharide plus IFN-γ). Transplantation of PDK2/4-deficient bone marrow into irradiated wild-type mice to produce mice with PDK2/4-deficient myeloid cells prevented M1 polarization, reduced obesity-associated insulin resistance, and ameliorated adipose tissue inflammation. A novel, pharmacological PDK inhibitor, KPLH1130, improved high-fat diet-induced insulin resistance; this was correlated with a reduction in the levels of pro-inflammatory markers and improved mitochondrial function. These studies identify PDK2/4 as a metabolic checkpoint for M1 phenotype polarization of macrophages, which could potentially be exploited as a novel therapeutic target for obesity-associated metabolic disorders and other inflammatory conditions.

The virulence factors of pathogenic microbes often have single functions that permit immune suppression. However, a proportion possess multiple activities and are considered moonlighting proteins. By examining secreted virulence factors of Staphylococcus aureus, we determine that the bacterial lipoic acid synthetase LipA suppresses macrophage activation. LipA is known to modify the E2 subunit of the metabolic enzyme complex pyruvate dehydrogenase (E2-PDH) with a fatty acid derivative, lipoic acid, yielding the metabolic protein lipoyl-E2-PDH. We demonstrate that lipoyl-E2-PDH is also released by S. aureus and moonlights as a macrophage immunosuppressant by reducing Toll-like receptor 1/2 (TLR1/2) activation by bacterial lipopeptides. A LipA-deficient strain induces heightened pro-inflammatory cytokine production, which is diminished in the absence of TLR2. During murine systemic infection, LipA suppresses pro-inflammatory macrophage activation, rendering these cells inefficient at controlling infection. These observations suggest that bacterial metabolism and immune evasion are linked by virtue of this moonlighting protein., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The immune system is a powerful resource for the eradication of cancer, but to overcome the low immunogenicity of tumor cells, a sufficiently strong CD8(+) T cell-mediated adaptive immune response is required. Nanoparticulate biomaterials represent a potentially effective delivery system for cancer vaccines, as they can be designed to mimic viruses, which are potent inducers of cellular immunity. We have been exploring the non-viral pyruvate dehydrogenase E2 protein nanoparticle as a biomimetic platform for cancer vaccine delivery. Simultaneous conjugation of a melanoma-associated gp100 epitope and CpG to the E2 nanoparticle (CpG-gp-E2) yielded an antigen-specific increase in the CD8(+) T cell proliferation index and IFN-γ secretion by 1.5-fold and 5-fold, respectively, compared to an unbound peptide and CpG formulation. Remarkably, a single nanoparticle immunization resulted in a 120-fold increase in the frequency of melanoma epitope-specific CD8(+) T cells in draining lymph nodes and a 30-fold increase in the spleen, relative to free peptide with free CpG. Furthermore, in the very aggressive B16 melanoma murine tumor model, prophylactic immunization with CpG-gp-E2 delayed the onset of tumor growth by approximately 5.5 days and increased animal survival time by approximately 40%, compared to PBS-treated animals. These results show that by combining optimal particle size and simultaneous co-delivery of molecular vaccine components, antigen-specific anti-tumor immune responses can be significantly increased., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Primary biliary cirrhosis is an immune-mediated chronic liver disease whose diagnosis relies on the detection of serum antimitochondrial antibodies directed against a complex set of proteins, among which pyruvate dehydrogenase complex is considered the main autoantigen. We studied the immunological role of the lipoyl domain of this protein using synthetic lipoylated peptides, showing that the lipoyl chain chirality does not affect autoantibody recognition and, most importantly, confirming that both lipoylated and unlipoylated peptides are able to recognize specific autoantibodies in patients sera. In fact, 74% of patients sera recognize at least one of the tested peptides but very few positive sera recognized exclusively the lipoylated peptide, suggesting that the lipoamide moiety plays a marginal role within the autoreactive epitope. These results are supported by a conformational analysis showing that the lipoyl moiety of pyruvate dehydrogenase complex appears to be involved in hydrophobic interactions, which may limit its exposition and thus its contribution to the complex antigenic epitope. A preliminary analysis of the specificity of the two most active peptides indicates that they could be part of a panel of synthetic antigens collectively able to mimic in a simple immunoenzymatic assay the complex positivity pattern detected in immunofluorescence.

Background: Pyruvate dehydrogenase (PDH) activity is altered in many human disorders. Current methods require tissue samples and yield inconsistent results. We describe a modified method for measuring PDH activity from isolated human peripheral blood mononuclear cells (PBMCs). RESULTS/METHODOLOGY: We found that PDH activity and quantity can be successfully measured in human PBMCs. Freeze-thaw cycles cannot efficiently disrupt the mitochondrial membrane. Processing time of up to 20 h does not affect PDH activity with proteinase inhibitor addition and a detergent concentration of 3.3% showed maximum yield. Sample protein concentration is correlated to PDH activity and quantity in human PBMCs from healthy subjects., Conclusion: Measuring PDH activity from PBMCs is a novel, easy and less invasive way to further understand the role of PDH in human disease.

Our understanding of primary biliary cirrhosis has been rapidly growing over the past decade and the disease is now regarded as a model for other female-predominant, organ-specific autoimmune conditions. Primary biliary cirrhosis ensues from a multi-lineage loss of tolerance to the E2 component of the pyruvate dehydrogenase complex. One of the major unanswered questions in the pathogenesis of primary biliary cirrhosis is the specificity of small intrahepatic bile ducts attack while PDC-E2 is present in mitochondria of all nucleated cells. Recent findings suggest that the uniqueness of the primary target tissue, biliary epithelium, may be of considerable importance for understanding primary biliary cirrhosis and that the biliary epithelial cell is more than an innocent victim. Rather, it attracts an immune attack by virtue of the unique apoptotic mechanisms and by the way it handles PDC-E2. Moreover, recent evidence suggests that apoptotic bodies of biliary epithelial cell are able to activate the innate immune system in the presence of anti-mitochondrial antibodies. This review article is intended to provide a critical overview of the role of apoptosis in biliary epithelial cells, the activation of the innate immune system, and its biological and clinical significance in primary biliary cirrhosis., (Copyright © 2013 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Environmental stimulation is a major factor in the initiation and perpetuation of autoimmune diseases. We have addressed this issue and focused on primary biliary cirrhosis (PBC), an autoimmune disease of the liver. Immunologically, PBC is distinguished by immune mediated destruction of the intra hepatic bile ducts and the presence of high titer antimitochondrial autoantibodies (AMA) directed against a highly specific epitope within the lipoic acid binding domain of the pyruvate dehydrogenase E2 subunit (PDC-E2). We submit that the uniqueness of AMA epitope specificity and the conformational changes of the PDC-E2 lipoyl domain during physiological acyl transfer could be the lynchpin to the etiology of PBC and postulate that chemical xenobiotics modification of the lipoyl domain of PDC-E2 is sufficient to break self-tolerance, with subsequent production of AMA in patients with PBC. Indeed, using quantitative structure activity relationship (QSAR) analysis on a peptide-xenobiotic conjugate microarray platform, we have demonstrated that when the lipoyl domain of PDC-E2 was modified with specific synthetic small molecule lipoyl mimics, the ensuing structures displayed highly specific reactivity to PBC sera, at levels often higher than the native PDC-E2 molecule. Hereby, we discuss our recent QSAR analysis data on specific AMA reactivity against a focused panel of lipoic acid mimic in which the lipoyl di-sulfide bond are modified. Furthermore, data on the immunological characterization of antigen and Ig isotype specificities against one such lipoic acid mimic; 6,8-bis(acetylthio)octanoic acid (SAc), when compared with rPDC-E2, strongly support a xenobiotic etiology in PBC. This observation is of particular significance in that approximately one third of patients who have taken excessive acetaminophen (APAP) developed AMA with same specificity as patients with PBC, suggesting that the lipoic domain are a target of APAP electrophilic metabolites such as NAPQI. We submit that in genetically susceptible hosts, electrophilic modification of lipoic acid in PDC-E2 by acetaminophen or similar drugs can facilitate loss of tolerance and lead to the development of PBC., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Primary biliary cirrhosis (PBC) has been often coined a model autoimmune disease based on the homogeneity amongst patients, the frequency and similarity of antimitochondrial antibodies, including the highly directed immune response to pyruvate dehydrogenase (PDC-E2). A significant number of patients with PBC suffer from sicca and amongst these, there are patients who also have classic Sjögren's syndrome. Indeed, both PBC and Sjögren's syndrome are characterized by inflammation of target epithelial elements. Both diseases can be considered on the basis of a number of other related clinical aspects, including proposed unique apoptotic features of the target tissue, the role of secretory IgA, and the frequency with which both diseases overlap with each other. Indeed, PBC may be considered a Sjögren's syndrome of the liver, whereas Sjögren's syndrome can be equally discussed as PBC of the salivary glands. Dissection of the genetic predispositions for both diseases and especially the molecular basis of effector mechanisms, will become critical elements in developing new therapies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.)

Background: In primary biliary cirrhosis (PBC), autoreactivity mainly targets members of the pyruvate dehydrogenase complex (PDC). Because PDC subunits are expressed on the surface of mycoplasma and molecular mimicry may be one aetiological factor, we analysed the presence of mammalian and mycoplasma PDC-specific antibodies in PBC patients., Methods: Antibodies to porcine PDC and Mycoplasma pneumoniae (mp) antigens mpPDH-C (to be designated mpPDC-E2 chain), mpPDH-B (to be designated mpPDC-E1beta chain), mpCARDS TX and mpP1 were investigated in sera from 43 PBC patients, 19 patients with autoimmune hepatitis and 11 healthy controls by an enzyme-linked immunosorbent assay and Western blotting. To study the rate of acute mycoplasma infection, an adhesin P1-specific polymerase chain reaction (PCR) was performed., Results: Immune reactivity to the mpPDC-E2 antigen was significantly enhanced in PBC patients (83.7%) as compared with controls (overall frequency of 36.7%), while antibodies to the porcine PDC-E2 chain were found only in PBC patients (88%) excluding a simple cross-reactivity of PDC-related antibodies. This observation was confirmed by inhibition studies demonstrating that porcine PDC did not inhibit mycoplasma PDC-specific antibodies and vice versa. The occurrence of antibodies to mpPDC seems to precede the occurrence of antibodies to porcine PDC. Infection with mycoplasma was equally distributed in the groups as evidenced by an antibody frequency comparable to CARDS TX and P1 and PCR reactivity., Conclusion: Because PBC patients show a significantly enhanced frequency of mpPDC-E2-related antibodies, besides other factors, molecular mimicry between surface molecules of mycoplasma and epitopes of the autoantigen may play a central role in the aetiopathology of PBC.

Infectious agents have been postulated to play a pathogenic role in the loss of immunological tolerance and the induction of primary biliary cirrhosis, an immune-mediated cholestatic liver disease characterized by progressive destruction of the small intrahepatic bile ducts and subsequent cirrhosis and liver failure. This review discusses emerging issues implicating infectious agents such as Escherichia coli, mycobacteria, chlamydia, helicobacter species, lactobacilli, Novosphingobium aromaticivorans, and betaretroviruses in the pathogenesis of primary biliary cirrhosis. We also review the immunopathological mechanisms responsible for the induction of the disease with special emphasis on the role of molecular mimicry and microbial/self immunological cross-reactivity.

Diagnosis and management of patients with SLE (Systemic Lupus Eritematosus), autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), involves specific diagnostic tests, such as IFA-AMA, IFA anti-dsDNA and immunoblotting for the detection of autoantibodies for specific autoantigens (mitochondria, dsDNA, M2, LKM-1, LC-1, SLA/LP). We established specific correlation between the detected autoantibodies and corresponding clinical findings. The total of 813 serum specimens were probed with IFA-anti-dsDNA, 98 of which tested positive. We also performed dilution analysis to the end point for all the positive specimens. Numerous specimens were tested by IFA, AMA and immunoblotting.

There is growing evidence that the onset of autoimmune disorders can be linked to the inefficient removal of apoptotic cells. Since defects in the elimination of apoptotic cells lead to secondary necrosis and subsequent release of intracellular components, this might explain the generation of autoantibodies against intracellular antigens. Accordingly, we wanted to investigate, whether antibodies from patients with the autoimmune liver disease primary biliary cirrhosis (PBC) recognize self-proteins generated and released during apoptosis. Using Western blot analyses we could detect intracellular antigens with serum IgG from PBC patients but not with serum IgG from healthy donors in lysates of Jurkat T-leukemia, HepG2 hepatoma, and HT-29 colon-carcinoma cells. Interestingly, PBC serum IgG also recognized caspase substrates in cells undergoing apoptosis induced by staurosporine or TRAIL (TNF-related apoptosis inducing ligand). In addition to intracellular antigens, serum IgG from PBC patients detected caspase-dependent antigens in the supernatants of apoptotic (secondary necrotic) cells and antigens on the surface of apoptotic Jurkat cells. Among the caspase substrates recognized by PBC serum IgG we could identify the components PDC-E2 and -E1beta of the known autoantigen PDC (pyruvate dehydrogenase complex). Thus, caspase-mediated processing of intracellular proteins might generate de novo autoantigens that upon release contribute to the generation of autoantibodies and autoimmune diseases as PBC.

Human autoimmune cholangiopathy comprises several intractable liver diseases that ultimately lead to hepatic failure. Primary biliary cirrhosis (PBC), allograft rejection, graft versus host diseases, and, possibly, primary sclerosing cholangitis are representative of immune-mediated cholangiopathies. Among them, PBC is the best-investigated human autoimmune cholangiopathy. The immunological approach to PBC has provided much critical information regarding its pathogenesis. The breakdown of self-tolerance in both B cells and T cells toward E2 components of the pyruvate dehydrogenase complex is evident. However, a number of questions regarding its etiology are unclear, in particular, the mechanisms involved in the selectivity of cholangiocyte destruction. In this brief review, we discuss what we know and we do not know regarding the pathogenesis of PBC.

Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease characterized by inflammation and destruction of intrahepatic biliary epithelial cells, ultimately leading to liver failure. The serological hallmark of PBC is the presence of high-titer antimitochondrial antibodies (AMA) against the inner lipoyl domain of E2 subunits of 2-oxo-acid dehydrogenase complexes, in particular the E2 component of the pyruvate dehydrogenase complex (PDC-E2). The initiating events triggering the autoimmune response are not yet identified but the hypothesis of molecular mimicry is a widely proposed mechanism for the development of autoimmunity in PBC. Several candidates, including bacteria and viruses, have been suggested as causative agents, but also environmental factors, such as chemical xenobiotics, have been implicated in the pathogenesis of primary biliary cirrhosis. In this review, we will discuss our current knowledge of the immunoreactivity of xenobiotically modified PDC peptide antigens. In addition, we will provide a working hypothesis how xenobiotic modification of antigens might occur that ultimately leads to the breaking of self-tolerance and the induction of 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, leading to liver failure in the absence of treatment. Little is known about the etiology of PBC. PBC is characterized by anti-mitochondrial antibodies and destruction of intra-hepatic bile ducts. The serologic hallmark of PBC is the presence of auto-antibodies to mitochondria, especially to the E2 component of the pyruvate dehydrogenase complex (PDC). Current theories on the pathogenesis of PBC favor the hypothesis that the disease develops as a result of an inappropriate immune response following stimulation by an environmental or infectious agent. Some reports suggest that xenobiotics and viral infections may induce PBC. The pathogenetic mechanism is believed to be caused by a defect in immunologic tolerance, resulting in the activation and expansion of self-antigen specific T and B lymphocyte clones and the production of circulating autoantibodies in addition to a myriad of cytokines and other inflammatory mediators. This leads to ductulopenia and persistent cholestasis, by developing end-stage hepatic-cell failure. In this review are given our own and literary data about mechanisms of development of intrahepatic cholestasis and possible ways of its correction.

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.

Mycoplasma gallisepticum is a major pathogen of poultry. Mycoplasma imitans is genetically and antigenically closely related to M. gallisepticum, but so far, only a few proteins of M. imitans have been identified as sharing epitopes with M. gallisepticum. In this study, we identified three proteins of M. gallisepticum that share with M. imitans epitopes defined by monoclonal antibodies (MAbs). MAb 9D4 reacted with the 67-kD hemagglutinin V1hA (previously termed pMGA) of M. gallisepticum and with its continuously expressed 40-kD protein. This MAb also reacted with a 40-kD protein of M. imitans, but not with its putative V1hA. Two-dimensional (2D) immunoblots of M. gallisepticum strains showed that their 40-kD proteins reacting with MAb 9D4 are expressed as major forms with isoelectric points (pI) around 6, and also as less-abundant forms differing in pI. In M. imitans, major forms of 40-kD proteins recognized by MAb 9D4 had pI around 6, whereas minor forms had pI between 5.5 and 5.8. The N-terminal sequence of the M. gallisepticum 40-kD protein recognized by MAb 9D4 strongly indicates that this protein is pyruvate dehydrogenase E1, subunit alpha (PdhA protein, also termed AcoA). The position of elongation factor Tu (EF-Tu), detected by the reference MAb GB8, was very similar in the 2D proteome maps of M. gallisepticum and M. imitans (MW of about 45 kD; pI - 5.6). In both M. gallisepticum and M. imitans, MAb 7G1 reacted with proteins of about 36 kD with similar charges (major forms with pI of about 8). The position of this protein in the proteome map of M. gallisepticum and its N-terminal sequence strongly suggest that MAb 7G1 recognizes lactate (malate) dehydrogenase (Ldh or Mdh). Comparison of 2D proteomes of 10 M. gallisepticum strains indicated that positions of EF-Tu, PdhA, and Ldh proteins are rather consistent and can be used as reference points in further analyses of the M. gallisepticum proteome.

Primary biliary cirrhosis is a chronic cholestatic liver disease of unknown cause that predominantly affects middle-aged women. Distal tubular acidosis is the main renal complication of primary biliary cirrhosis. Tubulointerstitial nephritis and Fanconi syndrome have been reported more rarely. We report on 2 patients with primary biliary cirrhosis who presented with tubulointerstitial nephritis and Fanconi syndrome and review similar cases published previously. Serum from 1 patient exerted an inhibitory effect on pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, 2 mitochondrial enzymes that are the main targets of antimitochondrial antibodies in primary biliary cirrhosis. Antimitochondrial antibodies may have a role in the genesis of tubulointerstitial nephritis and Fanconi syndrome, 2 typical renal features of mitochondrial cytopathies. Tubulointerstitial nephritis and Fanconi syndrome have to be added to the spectrum of renal diseases associated with primary biliary cirrhosis.

The serological hallmark of primary biliary cirrhosis (PBC) is the presence of pyruvate dehydrogenase complex E2 subunit (PDC-E2) antimitochondrial antibodies (AMAs). Anti-PDC-E2 antibodies cross-react specifically with mycobacterial hsp65, and we have demonstrated that the motif SxGDL[ILV]AE shared by PDC-E2(212-226) and hsp's is a cross-reactive target. Having found that this same motif is present only in beta-galactosidase of Lactobacillus delbrueckii (BGAL LACDE), we hypothesized that this homology would also lead to cross-reactivity. The mimics were tested via ELISA for reactivity and competitive cross-reactivity using sera from 100 AMA-positive and 23 AMA-negative PBC patients and 190 controls. An Escherichia coli (ECOLI) PDC-E2 mimic that has been pathogenetically linked to PBC but lacks this motif has been also tested. Anti-BGAL(266-280) LACDE antibodies were restricted to AMA-positive patients (54 of 95, 57%) and belonged to immunoglobulin (Ig) G3. Of the 190 controls, 22 (12%; P < .001) had anti-BGAL(266-280) antibodies, mainly of the IgG4 subclass. ECOLI PDC-E2 reactivity was virtually absent. BGAL(266-280)/PDC-E2(212-226) reactivity of the IgG3 isotype was found in 52 (52%) AMA-positive PBC patients but in only 1 of the controls (P < .001). LACDE BGAL(266-280)/PDC-E2(212-226) reactivity was due to cross-reactivity as confirmed via competition ELISA. Antibody affinity for BGAL(266-280) was greater than for PDC-E2 mimics. Preincubation of a multireactive serum with BGAL(266-280) reduced the inhibition of enzymatic activity by 40%, while marginal effect (12%) or no effect (2%) was observed in human or ECOLI PDC-E2 mimics. In conclusion, IgG3 antibodies to BGAL LACDE cross-react with the major mitochondrial autoepitope and are characteristic of 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.

Novosphingobium aromaticivorans, a unique ubiquitous bacterium that metabolizes xenobiotics and activates environmental estrogens, has been suggested as a pathogenic factor in the development of primary biliary cirrhosis (PBC). To define the molecular basis of PBC sera reactivity, we investigated the characteristic of the bacterial antigens involved. We cloned and sequenced four genes from N. aromaticivorans coding for immunoreactive proteins, arbitrarily named Novo 1 through Novo 4. We subsequently analyzed these proteins for their homology to known mitochondrial proteins and defined their reactivity using monoclonal antibodies (mAbs), rabbit anti-lipoic acid antibody, and PBC/control sera. Moreover, we studied their phylogenetic relation with the known PBC autoantigens. Novo proteins have an extraordinary degree of amino acid homology with all of the major human mitochondrial autoantigens PDC-E2 (Novo 1 and 2), OGDC-E2 (Novo 3), and BCOADC-E2 (Novo 4). Moreover, Novo 1-4 contain a lipoylated domain, are recognized by AMA-positive sera, and react with specific mAbs to mitochondrial antigens. Interestingly, the phylogenetic relation of the proteins emphasizes the conservation of the lipoylated domain. In conclusion, our data provide a high degree of confidence that N. aromaticivorans may potentiate the breakdown of self tolerance in genetically susceptible individuals.

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 availability of monoclonal antibodies (mAbs) against the proteins of the oxidative phosphorylation chain (OXPHOS) and other mitochondrial components facilitates the analysis and ultimately the diagnosis of mitochondrially related diseases. mAbs against each of the five complexes and pyruvate dehydrogenase (PDH) are the basis of a rapid and simple immunocytochemical approach [Hanson, B.J., Capaldi, R.A., Marusich, M.F. and Sherwood, S.W., J. Histochem. Cytochem. 50 (2002) 1281-1288]. This approach can be used to detect if complexes have altered assembly in mitochondrial disease due to mutations in nuclear encoded genes, such as in Leigh's disease, or in mitochondrially encoded genes, e.g., MELAS. Other mAbs have recently been obtained that can immunocapture each of the five OXPHOS complexes, PDH and the adenine nucleotide translocase (ANT) from very small amounts of tissue such as that obtained from cell culture or needle biopsies from patients. When adapted to a 96-well plate format, these mAbs allow measurement of the specific activity of each of the mitochondrial components individually and analysis of their subunit composition and state of posttranslational modification. The immunocapture protocol should be useful not only in the analysis of genetic mitochondrial diseases but also in evaluating and ultimately diagnosing late-onset mitochondrial disorders including Parkinson's disease, Alzheimer's disease, and late-onset diabetes, which are thought to result from accumulated oxidative damage to mitochondrial proteins such as the OXPHOS chain.

A major challenge in vaccine design has been to identify antigen presentation systems that elicit strong T- and B-cell responses. In the authors' laboratory, two new delivery vehicles derived from nonpathogenic prokaryotic organisms were recently designed and investigated. Conserved antigenic determinants were inserted into the N-terminal region of the major pVIII coat protein of bacteriophage fd virions or on the surface of an icosahedral scaffold formed by the acyltransferase component (E2 protein) of the pyruvate dehydrogenase complex of Bacillus stearothermophilus. The data indicate that the antigenic determinant displayed by either fd virions or on the surface of the E2 lattice are accessible to the immune system, and are able to trigger a humoral response as well as a potent helper and cytolytic response in vitro and in vivo. These systems offer the potential for safe and inexpensive vaccines to elicit full-spectrum immune responses.

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.

The identity of reactants for autoantibodies has been successively refined from whole cellular organelles (immunofluorescence), identified molecules (immunoblot; gene expression libraries), epitope regions (truncated cDNAs; peptide scanning) to contact residues, as described here. Most autoantibodies react with conformational epitopes, in which amino acids distant in the linear sequence come into contiguity by protein folding. Identification of contact sites with the antibody paratope requires particular technologies, crystallography, or antibody screening of phage-displayed random peptide libraries. The latter is illustrated by our studies on the autoepitope for anti-PDC-E2 (AMA) in primary biliary cirrhosis (PBC), anti-GAD65 in type 1 diabetes, and anti-C1 of type II collagen in collagen-induced arthritis. More precise definition of the structure of conformational autoepitopes could (a) clarify controversial aspects of autoimmunity including epitope mimicry, epitope spreading, and molecular spatial relationships between B and T cell autoepitopes, and (b) impact on novel diagnostic and therapeutic (vaccine) molecules.

Unlabelled: Primary biliary cirrhosis is a chronic liver disease, characterized by the destruction of the epithelial cells of the sublobular, interlobular and septal bile ducts and with the development of cirrhosis. The presence of anti-mitochondrial antibodies against the subunits of mitochondrial 2-oxoacids dehydrogenases is characteristic for patients with primary biliary cirrhosis. The aim of this work was to study the effect of anti-mitochondrial antibodies upon activity of the isolated mitochondrial pyruvate dehydrogenase complex after the incubation with serum from patients with primary biliary cirrhosis., Group of Patients and Methods: The activity of the purified bovine pyruvate dehydrogenase complex was studied spectrophotometrically in presence of the serum (1: 1000) from five patients with primary biliary cirrhosis and from ten disease free controls., Results: The activity of the pyruvate dehydrogenase was decreased after incubation with the serum from patients with primary biliary cirrhosis. No similar inhibitory effect was found after incubation with serum from controls., Discussion: The inhibitory effect of the anti-mitochondrial antibodies upon activity of pyruvate dehydrogenase may broaden the spectrum of diagnostic methods in patients with primary biliary cirrhosis. Further investigations are necessary to assess the possible application of this method for monitoring of changes during the course of the disease and for assignation of the disease prognosis.

Objective: To identify autoepitopes of E2 subunit of pyruvate dehydrogenase complex (PDC-E2) specific CD8+ CTL in primary biliary cirrhosis (PBC) patients., Methods: An online database SYFPEITHI was applied to predict HLA-A*0201 restricted epitopes which located in PDC-E2 30-50 aa and 150-190 aa where B-cell epitopes clustered with CD4+ T-cell epitopes. T2 cell line reconstitution and stabilization assay, induction of specific CTL lines from peripheral blood mononuclear cells (PBMCs) of patients with PBC and cytotoxicity of peptides-induced CTL were performed to screen the epitopes from those candidates., Results: Five potential epitopes were predicted by database. Of the 5 candidates, two peptides 159-167 aa and 165-174 aa, with highly binding activity to HLA-A*0201 molecules, could stimulate PBMCs from most HLA-A*0201 positive PBC patients to proliferate and peptide-induced CTL lines showed specific cytotoxicity., Conclusion: Peptides of KLSEGDLLA (159-167 aa) and LLAEIETDKA (165-174 aa) in the inner lipoyl domain of PDC-E2 are HLA-A*0201 restricted CD8+ CTL immunodominant epitopes in PBC.

Background: Primary biliary cirrhosis (PBC) is an immune-mediated chronic cholestatic disease characterized by the presence of antibodies directed predominantly against the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2). What provokes tolerance breakdown in PBC remains to be established, though there is evidence to indicate that microbes may induce anti-mitochondrial antibodies (AMA) through a mechanism of molecular mimicry., Methods: Having found that urease beta (UREB)(22-36) antigen of Helicobacter pylori (HELPY) shares extensive (87%) similarity with PDC-E2(212-226), the major mitochondrial autoepitope, it was hypothesized that this would also lead to cross-reactivity. The UREB/PDC-E2 mimics were thus constructed and tested by ELISA in 112 PBC patients and 114 controls., Results: Reactivity to PDC-E2(212-226) was found in 104 patients but to UREB(22-36) in only 2. In these two patients, the double reactivity was not cross-reactive. The lack of surface antibody accessibility to UREB(22-36), as demonstrated through three-dimensional model prediction analysis, may explain this unexpected finding. There was some speculation on whether HELPY UREB(22-36) might act as a cross-reactive CD4 T-cell epitope. All seven PBC patients, tested in a standard proliferation assay against PDC-E2(212-226), gave a positive response. All seven were unresponsive to HELPY UREB(22-36). The pattern of reactivity to HELPY antigens by immunoblot was similar between anti-PDC-E2-positive and negative PBC cases, as well as between PBC patients and controls., Conclusion: Contrary to common belief, extensive sequence homology (molecular mimicry) between self and microbe does not necessarily result in cross-reactivity. It is therefore likely that, when present, cross-reactivity between self and microbes is of biological importance.

The autoimmune liver disease primary biliary cirrhosis (PBC) is characterized by the breakdown of normal immune self tolerance to pyruvate dehydrogenase complex (PDC). How tolerance is broken to such a central and highly conserved self antigen in the initiation of autoimmunity remains unclear. One postulated mechanism is that reactivity arises to an altered form of self antigen with subsequent cross-reactivity to native self. In this murine study, we set out to examine whether sensitization with a covalently modified form of self PDC can give rise to the pattern of breakdown of B-cell and T-cell tolerance to self PDC seen in PBC patients. The notion that altered self can lead to tolerance breakdown was studied by sensitizing SJL/J mice with a covalently modified (biotinylated) preparation of self murine PDC (mP/O-B). Subsequently, antibody and T-cell reactivities to unmodified self mP/O were studied. Sensitization with mP/O-B elicited high-titre, high-affinity antibody responses reactive with both the mP/O-B immunogen and, importantly, native mP/O. In addition, significant MHC class II restricted splenic T-cell responses to native mP/O (i.e., true autoimmune responses) were seen in mP/O-B sensitized animals. The breakdown of T-cell self tolerance to mP/O was not seen in animals sensitized with irrelevant biotinylated antigens. In conclusion, this study provides evidence to support the concept that exposure to covalently modified self PDC can, in the correct proimmune environment, replicate the full breakdown of B-cell and T-cell immune tolerance to PDC seen in PBC. One potential etiological pathway in PBC therefore could be the breakdown of tolerance to self PDC occurring after exposure to self antigen covalently modified in the metabolically active environment of the liver.

Previous studies on Spanish patients with Primary Biliary Cirrhosis (PBC) have shown extensive, disease-specific cross-reactivity between the 65-kDa heat shock protein (hsp65) of Mycobacterium gordonae and pyruvate dehydrogenase complex-E2 (PDC-E2), the major target of anti-mitochondrial antibody (AMA). Studies on a British population were unable to substantiate these findings. Having found that there is an excellent and almost unique match between the PDC-E2 autoepitope and a sequence in mycobacterial hsp65s, we tested the corresponding peptides by ELISA for cross-reactivity using sera from 90 PBC patients, 40 Spanish and 50 British, and 84 pathological controls. Reactivity to the MYCGO hsp65(90-104)/human PDC-E2(212-226)pair was present in 19 (47.5%) Spanish PBC patients and in 2 (4%) of the 50 British. Reactivity was not seen in any of the controls. Simultaneous reactivity to mimics was due to cross-reactivity as confirmed by inhibition studies. Three dimensional modelling predicts mycobacterial hsp65(90-104)to be exposed on the surface of the protein. The affinity of anti-hsp65(90-104)antibody was higher than that of anti-PDC-E2(212-226). Hsp65(90-104)is a target of disease-specific cross-reactivity to PDC-E2(212-226). The geographical confinement of this phenomenon is probably the result of complex genetic, environmental and immunological interaction.

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.

Background/aims: Previous studies on patients with primary biliary cirrhosis (PBC) have shown extensive cross-reactivity between the dominant B- and T-cell epitopes of human pyruvate dehydrogenase complex-E2 (PDC-E2), and microbial mimics. Such observations have suggested microbial infection as having a role in the induction of anti-mitochondrial antibodies, through a mechanism of molecular mimicry. However the biological significance of these cross-reactivities is questionable, because PDC-E2 is so highly conserved among various species., Methods: Interrogating protein databases, ten non-PDC-E2 microbial sequences with high degree of similarity to PDC-E2(212-226) were found in Escherichia coli (6), Helicobacter pylori, Pseudomonas aeruginosa, Cytomegalovirus, and Haemophilus influenzae. We report on a study testing reactivity and competitive cross-reactivity against these respective peptides, and in some cases the parent protein, using sera from 55 patients with PBC, compared to reactivity of 190 pathological and 28 healthy controls., Results: Cross-reactivity to E. coli mimics was commonly seen in PBC, and in a subset of pathological controls except where there was no evidence of urinary tract infection and correlated with anti-mitochondrial reactivity., Conclusions: E. coli/PDC-E2 cross-reactive immunity characterizes primary biliary cirrhosis; the large number of E. coli immunogenic mimics may account for the dominance of the major PDC-E2 autoepitope.

Background & Aims: Although considerable effort has been directed toward the mapping of peptide epitopes by autoantibodies, the role of nonprotein molecules has been less well studied. The immunodominant autoantigen in primary biliary cirrhosis (PBC), E2 components of pyruvate dehydrogenase complexes (PDC-E2), has a lipoate molecule bonded to the domain to which autoantibodies are directed., Methods: We examined sera from patients with PBC (n = 105), primary sclerosing cholangitis (n = 70), and rheumatoid arthritis (n = 28) as well as healthy volunteers (n = 43) for reactivity against lipoic acid. The lipoic acid hapten specificity of the reactive antibodies in PBC sera was determined following incubation of aliquots of the sera with human serum albumin (HSA), lipoylated HSA (HSA-LA), PDC-E2, lipoylated PDC-E2, polyethylene glycol (PEG), lipoylated PEG, free lipoic acid, and synthetic molecular mimics of lipoic acid., Results: Anti-lipoic acid specific antibodies were detected in 81% (79 of 97) of antimitochondrial antibody (AMA)-positive patients with PBC but not in controls. Two previously unreported specificities in AMA-positive sera that recognize free lipoic acid and a carrier-conjugated form of lipoic acid were also identified., Conclusions: We hypothesize that conjugated form(s) of native or xenobiotic lipoic acid mimics contribute to the initiation and perpetuation of autoimmunity by at first breaking self-tolerance and participating in subsequent determinant spreading. The variability in the immunoreactive carrier/lipoate conjugates provides an experimental framework on which potential mechanisms for the breakdown of self-tolerance following exposure to xenobiotics can be investigated. The data have implications for patients taking lipoic acid as a dietary supplement.

Two non-pathogenic scaffolds (represented by the filamentous bacteriophage fd and the dihydrolipoyl acetyltransferase E2 protein of the Bacillus stearothermophilus pyruvate dehydrogenase (PDH) complex) able to deliver human immunodeficiency virus (HIV)-1 antigenic determinants, were designed in our laboratories and investigated in controlled assay conditions. Based on a modification of the phage display technology, we developed an innovative concept for a safe and inexpensive vaccine in which conserved antigenic determinants of HIV-1 reverse transcriptase (RTase) were inserted into the N-terminal region of the major pVIII coat protein of bacteriophagefd virions. Analogously, we developed another antigen delivery system based on the E2 component from the PDH complex and capable of displaying large intact proteins on the surface of an icosahedral lattice. Our data show that both of these systems can deliver B and T epitopes to their respective presentation compartments in target cells and trigger a humoral response as well as a potent helper and cytolytic response in vitro and in vivo.

There is a considerable database on the effector mechanisms for CD8 recognition of PDC-E2 in primary biliary cirrhosis (PBC). In particular, the specific roles of MHC class I, the mitochondrial autoepitope, and the liver-specific T cell precursor frequency, are defined for HLA-A2.1 patients. There is evidence for a role of MHC class I-mediated presentation of exogenous antigens, or cross-presentation, in the development of the antimitochondrial response and a contributory role of Fcgamma receptor-mediated uptake of autoantigen-autoantibody complexes for the induction of a PDC-E2 specific autoreactive CTL response. Based on this background, we examined potential intracellular pathways for processing the immunodominant mitochondrial autoantigen, PDC-E2, by dendritic cells (DC). In particular, we studied the effects of the proteasome inhibitor lactacystin and the endosomal acidification inhibitor bafilomycin on the induction of PDC-E2-specific CTL response in PBC. Importantly, our data indicate that pre-treatment with either lactacystin or bafilomycin inhibits the PDC-E2 immune complex-induced CTL response. The processing and presentation of PDC-E2 by CD8(+)T cells is mediated by proteasomes and facilitated by Fcgamma receptor-mediated endocytosis. This data reflects another layer of interaction between components of the immune system in the development of autoimmunity. Further characterization of autoantigen uptake and processing may lead to potential therapeutic intervention.

Patients with primary biliary cirrhosis develop progressive ductopenia associated with the production of antimitochondrial antibodies that react with a protein aberrantly expressed on biliary epithelial cells and peri-hepatic lymph nodes. Although no specific microbe has been identified, it is thought that an infectious agent triggers this autoimmune liver disease in genetically predisposed individuals. Previous serologic studies have provided evidence to suggest a viral association with primary biliary cirrhosis. Here we describe the identification of viral particles in biliary epithelium by electron microscopy and the cloning of exogenous retroviral nucleotide sequences from patients with primary biliary cirrhosis. The putative agent is referred to as the human betaretrovirus because it shares close homology with the murine mammary tumor virus and a human retrovirus cloned from breast cancer tissue. In vivo, we have found that the majority of patients with primary biliary cirrhosis have both RT-PCR and immunohistochemistry evidence of human betaretrovirus infection in lymph nodes. Moreover, the viral proteins colocalize to cells demonstrating aberrant autoantigen expression. In vitro, we have found that lymph node homogenates from patients with primary biliary cirrhosis can induce autoantigen expression in normal biliary epithelial cells in coculture. Normal biliary epithelial cells also develop the phenotypic manifestation of primary biliary cirrhosis when cocultivated in serial passage with supernatants containing the human betaretrovirus or the murine mammary tumor virus, providing a model to test Koch's postulates in vitro.

Thomas Addison described three of the classical autoimmune diseases, so can justifiably be regarded as one of the fathers of the study of autoimmunity. The least well recognised of these conditions, the autoimmune liver disease primary biliary cirrhosis (PBC), is in some ways the most interesting. As a result of the relatively advanced state of knowledge of its immunopathogenesis, it represents a good model for the study of autoimmune disease. The classical pathological lesion of PBC is apoptotic damage to the biliary epithelial cells lining the small intrahepatic bile ducts. The disease is typified by two symptom sets: fatigue and pruritus, which can occur at any stage of the disease process, and the features of advanced liver disease, which occur when secondary liver damage results from bile retention. Although autoantibodies directed at, in particular, pyruvate dehydrogenase complex (PDC) are almost universally present in PBC (and represent an important diagnostic tool), it appears likely that CD8+ cytotoxic T cells reactive with self-PDC derived epitopes are directly responsible for target cell damage. Recent studies in humans and a novel murine disease model have shed light on the mechanism of breakdown of immune tolerance to self-PDC; they provide important insights into the pathogenesis of PBC in particular and of autoimmunity in general.