Search

Glycosylation of antibodies, particularly in the Fc domain, critically modulate the ability of antibodies to bind to FcRs, maintaining immune quiescence to achieve a finely orchestrated immune response. The removal of sialic acid and galactose residues dramatically alters the physiological function of IgGs, and alterations of Ig glycosylation have been associated with several autoimmune disorders. However, Ig glycosylation has not been extensively studied in autoimmune cholangitis. We applied triple quadruple mass spectroscopy with subsequent multiple reaction monitoring to elucidate the profile, composition and linkage of sugar residues of antibody glycans in patients with primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) and healthy controls (HC). Agalactosylated, HexNAc terminated IgG1 glycoforms were enriched in both PBC and PSC. Levels of IgM glycans at site N439 and fucosylated glycans in J chain, were significantly decreased in PBC compared to PSC and HC. PSC patients had decreased bisecting glycoforms and increased biantennary glycoforms on IgA compared to PBC. Importantly, our data demonstrate the association of distinct branching and composition patterns of Ig glycoforms with disease severity and liver cirrhosis, which highlight the importance of glycan biology as a potential mechanism and/or a disease specific signal of inflammation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background and Aims: The most highly directed and specific autoantibody in human immunopathology is the serologic hallmark of primary biliary cholangitis (PBC), antimitochondrial antibodies (AMAs). However the clinical significance of finding a positive AMA, with normal alkaline phosphatase (ALP) remains enigmatic., Methods: We took advantage of 169 consecutive outpatients who were identified as having a positive AMA, but normal ALP levels between January 2012 and January 2018. A liver biopsy was performed on 67/169 of these AMA positive normal ALP patients., Results: In all 169 patients we reconfirmed the AMA and also performed anti-gp210 and anti-sp100, liver stiffness (LSM) assessed by vibration-controlled transient elastography (VCTE), an abdominal computed tomography (CT) scan, and either a magnetic resonance imaging (MRI) or ultrasound. The liver biopsies were reviewed by two unbiased observers. 87.6% of the 169 patients were females with a mean age of 46; the median AMA titer 1:320; an elevated serum IgM was found in 53.3%. Importantly, in patients with a liver biopsy, 55（82.1%）out of 67 had varying degrees of cholangitis activity, diagnostic of PBC., Conclusion: In patients who were AMA-positive but had normal ALP levels, more than 80% were associated with histological classic PBC. These data emphasize the importance of a positive AMA, even with a normal ALP and also question the role of ALP as a sole surrogate marker of cholangitis., (Copyright © 2019. Published by Elsevier Ltd.)

The immunomodulatory effect of IL-10 as an immunosuppressive and anti-inflammatory cytokine is well known. Taking advantage of our established mouse model of autoimmune cholangitis using 2-octynoic acid conjugated ovalbumin (2-OA-OVA) induction, we compared liver pathology, immune cell populations and antimitochondrial antibodies between IL-10 knockout and wild type mice immunized with 2-OA-OVA. At 10 weeks post immunization, portal inflammation and fibrosis were more severe in 2-OA-OVA immunized IL-10 knockout mice than in wild type mice. This was accompanied by significant higher levels of collagen I and III expression, T, NK and NKT subsets in liver and IgG anti-mitochondrial autoantibodies (AMAs) compared to 2-OA-OVA immunized wild type mice, suggesting that endogenous IL-10 is necessary for the maintenance of immune tolerance in primary biliary cholangitis (PBC). Further, we investigated whether administration of exogenous IL-10 could prevent PBC by administration of IL-10 expressing recombinant adeno-associated virus (AAV-IL-10) either 3 days before or 3 weeks after the establishment of liver pathology. Interestingly, administration of AAV-IL-10 resulted in increased liver inflammation and fibrosis, accompanied by increases in IFN-γ in liver CD4 + T cell, granzyme B, FasL, and CD107a in liver CD8 + T and NKT cells, and granzyme B and FasL in liver NK cells of AAV-IL-10 administered mice compared with control mice. Furthermore, administration of AAV-IL-10 significantly increased levels of proinflammatory cytokines and chemokines (IFN-γ, TNF-α, CXCL9 and CXCL10) and collagen I and III production in naïve mice, together with increase in immune cell infiltration and collagen deposition in the liver, suggesting a role of IL-10 in fibrosis. In conclusion, our data demonstrate that endogenous IL-10 is critical in the maintenance of immune tolerance but exogenous administration of IL-10 exacerbates liver inflammation and fibrosis. Furthermore, the distinctive presence of inflammatory immune cell populations and collagen expression in AAV-IL-10 treated naïve mice cautions against the clinical use of exogenous IL-10 in patients with autoimmune cholangitis., (Copyright © 2018. Published by Elsevier Ltd.)

Autoimmune serology is key to the diagnosis and management of autoimmune liver diseases. Its correct use in clinical practice requires a basic knowledge of the laboratory techniques used for autoantibody detection. Indirect immunofluorescence (IIF) on triple rodent tissue is still the gold standard screening procedure for liver-relevant autoantibodies, while HEp2 cells and human ethanol-fixed neutrophils are used as substrates to characterize nuclear reactivities and to detect anti-neutrophil cytoplasm antibody, respectively. Assays based on purified or recombinant antigens are increasingly used, having the main advantage of being observer-independent and the disadvantage of detecting only autoantibodies whose antigenic target has been identified. The AIH-specific anti-soluble liver antigen antibody cannot be detected by IIF and a molecular-based assay should be used at the screening level. Since autoantibodies may be present in the context of viral hepatitides and other inflammatory liver diseases it is important to exclude these conditions before diagnosing autoimmune liver disease. Anti-nuclear antibody (ANA), most often with a homogeneous IIF pattern on HEp2 cells, characterizes type 1 autoimmune hepatitis (AIH), and is found in association with anti-smooth muscle antibody in about half of the cases. Two IIF ANA patterns are specific for primary biliary cholangitis, namely the rim-like/membranous pattern, and the multiple nuclear dots pattern. Anti-liver kidney microsomal antibody type 1 is the serological hallmark of type 2 AIH, often in association with anti-liver cytosol type 1 antibody. Atypical perinuclear anti-neutrophil antibody, referred to as perinuclear anti-neutrophil nuclear antibody, is frequently detected in primary sclerosing cholangitis, in AIH type 1 and in inflammatory bowel diseases. The anti-asiaglycoprotein receptor antibody is liver-specific but not disease-specific, and reliable commercial assays for its detection are lacking. Anti-mitochondrial antibody is the hallmark of primary biliary cholangitis (PBC), being disease-specific and present in about 95% of the PBC patients. Its incidental detection presages the future development of PBC., (Copyright © 2018 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.)

Our laboratory has hypothesized that xenobiotic modification of the native lipoyl moiety of the major mitochondrial autoantigen, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), may lead to loss of self-tolerance in primary biliary cirrhosis (PBC). This thesis is based on the finding of readily detectable levels of immunoreactivity of PBC sera against extensive panels of protein microarrays containing mimics of the inner lipoyl domain of PDC-E2 and subsequent quantitative structure-activity relationships (QSARs). Importantly, we have demonstrated that murine immunization with one such mimic, 2-octynoic acid coupled to bovine serum albumin (BSA), induces anti-mitochondrial antibodies (AMAs) and cholangitis. Based upon these data, we have focused on covalent modifications of the lipoic acid disulfide ring and subsequent analysis of such xenobiotics coupled to a 15mer of PDC-E2 for immunoreactivity against a broad panel of sera from patients with PBC and controls. Our results demonstrate that AMA-positive PBC sera demonstrate marked reactivity against 6,8-bis(acetylthio)octanoic acid, implying that chemical modification of the lipoyl ring, i.e. disruption of the S-S disulfide, renders lipoic acid to its reduced form that will promote xenobiotic modification. This observation is particularly significant in light of the function of the lipoyl moiety in electron transport of which the catalytic disulfide constantly opens and closes and, thus, raises the intriguing thesis that common electrophilic agents, i.e. acetaminophen or non-steroidal anti-inflammatory drugs (NSAIDs), may lead to xenobiotic modification in genetically susceptible individuals that results in the generation of AMAs and ultimately clinical PBC., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A dual isotype (IgG, IgA) enzyme-linked immunosorbent assay (ELISA) designed to provide enhanced detection of primary biliary cirrhosis (PBC)-specific autoantibodies against both major mitochondrial and nuclear antigens has been developed and recently become commercially available. The assay (PBC Screen) simultaneously detects IgG and IgA autoantibodies to the immunodominant portions of the 3 major mitochondrial (MIT3) and nuclear (gp210, and sp100) antigens. The aim of this study was to compare the performance of the PBC Screen to the combined performance obtained with individual IgG ELISAs to MIT3, gp210, and sp100 on a large group of selected patients from multiple centers. A total of 1175 patients with PBC and 1232 subjects without PBC were evaluated. Non-PBC groups included healthy controls (624) as well as individuals with autoimmune hepatitis (281), primary sclerosing cholangitis (77), viral hepatitis (91 hepatitis B and 98 hepatitis C), other liver diseases (31), and other infectious or autoimmune diseases (30). The PBC Screen at the receiver operator characteristic optimized cutoff of 27.8 units, had an overall sensitivity of 83.8%, specificity of 94.7% and area under curve of 0.9212. This was similar to the specificity of 96.1% obtained by the combined results of individual MIT3, sp100, and gp210 IgG ELISAs (kappa index at 0.898). Of the 253 PBC patients without AMA detectable by immunofluorescence, 113 (44.7%) were interpreted as positive for PBC-specific autoantibodies. In conclusion, the PBC Screen is an appropriate first-line test for the diagnosis of PBC, including for patients negative for markers assessed using conventional methods., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

In primary biliary cirrhosis (PBC) serum markers other than anti-mitochondrial antibodies (AMA) are promising in terms of disease severity and comorbidities, as well represented by anti-nuclear antibodies (ANA). The aim of the present study was thus to evaluate the prevalence and clinical significance of a large profile of serum autoantibodies in PBC sera. We utilized 69 sera from European patients with PBC (including 20 AMA-negative) and 297 sera from geographically and sex-matched healthy controls. All sera were tested for the presence of ANA and autoantibodies associated with thrombophilia, vasculitis, and gastrointestinal disease. Autoantibodies other than AMA were detected in 53/69 (76%) PBC sera vs. 105/297 (35%) among controls. The prevalence of ANA (targeting dsDNA, Sm, chromatin, ribosomal-P, RNP, SmRNP, SSA, SSB, and centromere) and thrombophilia-associated autoantibodies (i.e. anti-beta2GPI, phosphatydilserine, prothrombin) was common among patients with PBC. When clinical features were compared, the presence of anti-prothrombin IgM was associated with a worse prognosis as represented by a higher Mayo score. We demonstrate an increased prevalence of ANA and thrombophilia-associated autoantibodies in PBC sera and an association between the latter autoantibodies and PBC stage. The role of thrombophilia-associated antibodies will warrant further studies, based in particular on the incidence of portal hypertension at early stages of PBC., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Primary biliary cirrhosis (PBC) is a chronic cholestatic autoimmune liver disease characterized by selective destruction of the intrahepatic bile ducts and highly specific serum anti-mitochondrial autoantibodies (AMA). Several studies have attempted to determine the cytokine pattern characterizing PBC, yet no definitive data have been gathered. The present study was designed to evaluate pro-inflammatory cytokines (IL-1beta, IL-6, TNFalpha), soluble IL-2 receptor (sIL-2R, e.g. soluble CD25), and complement components (C1q, C3, factor B, properdin) levels in sera from 84 patients with PBC and 41 controls. PBC was characterized by significantly higher levels of all pro-inflammatory cytokines when compared to controls; these included IL-1beta (433.3 +/- 13.2 vs. 316.6 +/- 14.7 pg/ml, P < 0.001), IL-6 (701 +/- 17.4 vs. 158 +/- 22.5 pg/ml, P < 0.001), TNFalpha (3.38 +/- 0.6 pg/ml vs. undetectable, P = 0.001), and sIL-2R (1527.1 +/- 106 vs. 566.4 +/- 28.7 U/ml, P < 0.001). Similarly, all complement components were also significantly higher in PBC compared to control sera. In conclusion, PBC sera manifest higher levels of sIL-2R and complement components and this may reflect a perpetuated immune activation. As expected, we also report that all major pro-inflammatory cytokine levels are enhanced in PBC. Further longitudinal analyses could demonstrate a correlation between these markers and disease stage or inflammatory activity, to predict histological staging, disease activity, and response to treatment.

Primary biliary cirrhosis is characterized by chronic hepatic inflammation and immune mediated apoptosis of bile duct epithelial cells. Delayed macrophage phagocytosis of opsonized apoptotic cells, noted in other autoimmune diseases, may promote inflammation. Recent studies suggest serum anti-CD16 autoantibodies contribute to impaired macrophage phagocytosis by blocking complement receptor 3 (CR3) signaling via CD16. Therefore, serum anti-CD16 levels and the ability of monocyte derived macrophages from individuals with PBC to phagocytosis apoptotic cells were compared to controls. The mean level of anti-CD16 IgM autoantibodies (0.86+/-0.62 v. 0.35+/-0.22, respectively, p=0.031) was increased in PBC compared to control sera, and mean PBC phagocytosis of opsonized apoptotic cells was significantly decreased compared to controls (23.9+/-12.2% v. 43.9+/-14.4%, respectively, p=0.020). However, PBC phagocytosis of opsonized apoptotic cells was not significantly affected by the presence or absence of autologous serum (20.8+/-13.5% v. 23.9+/-12.2%, respectively, p=0.560). PBC phagocytosis of opsonized apoptotic cells inversely correlated with CD16 (and CR3) expression levels on Day 5 after culture in the presence or absence of autologous serum (r=-0.546, p=0.033 and r=-0.519, p=0.042, respectively). Phagocytosis of non-opsonized apoptotic cells did not correlate with CD16 or CR3 expression (p>0.050). In conclusion, PBC macrophage phagocytosis of opsonized apoptotic cells is impaired, irrespective of serum factors and may increase hepatic inflammation.

Although the pathogenesis of primary biliary cirrhosis (PBC) remains enigmatic, the immune system plays a key role in the initiation and subsequent development of pathology. Previous studies have indicated a critical role of the innate immune system. Importantly, natural killer (NK) cells are abundant in liver where they serve as sentinels of the immune system. In addition, NK cells have significant biologic activity based on their production of immunoregulatory cytokines. To address this issue, we have investigated several qualitative and quantitative activities of NK cells in patients with PBC as well as normal and liver diseased controls. We report herein a marked increase in the frequency and absolute number of blood and liver NK cells in PBC patients. Moreover, the cytotoxic activity and perforin expression by isolated NK cells were significantly increased in PBC patients associated with increased levels of plasma IL-8 and the expression of CD128a (IL-8 receptor) on NK cells. In contrast, the levels of IFN-gamma, IL-6 and IL-8 synthesized by NK cells were significantly decreased in PBC patients as compared to controls. In conclusion, data from this study provide compelling evidence supporting a biologic role of NK cells in the immunopathogenesis of PBC.

Infiltrating memory T cells play an important role in the destruction of the biliary tract in primary biliary cirrhosis (PBC) and inflammatory chemokines control lymphocyte traffic through their interactions with T cell chemokine receptors. In the present study, we measured plasma levels of chemokines interferon-gamma-inducible protein-10 (IP-10) and monokine induced by gamma interferon (MIG), and also studied the expression of CXCR3 chemokine receptors in 105 subjects, including 53 patients with PBC, 26 first degree relatives and 26 healthy controls. Interestingly, plasma IP-10 and MIG levels in PBC were increased significantly compared to controls and appeared to increase with disease progression. By immunohistochemistry, IP-10 and MIG expressions were evident in the portal areas in PBC. Further, the frequency of CXCR3-expressing cells in peripheral blood was also significantly higher in PBC, and CXCR3-positive cells were also found in the portal areas of diseased livers, primarily on CD4+ cells. Finally, the daughters and sisters of PBC patients also demonstrated increased plasma levels of IP-10 and MIG, but, in contrast, displayed normal frequency of CXCR3+ expressing peripheral blood lymphocytes. Our data imply a role for specific chemokine-chemokine receptor interactions in the pathogenesis of PBC and also highlight the familial risk factor.

There are few case reports on the association between autoimmune hepatitis (AIH) and anticardiolipin antibodies (anti-CLAbs) and/or antiphospholipid syndrome (APLS). We studied the anti-CLAbs prevalence in AIH and other hepatic diseases. We also investigated whether anti-CLAbs are co-factor dependent and which is their avidity since co-factor dependency or increased resistance is associated with APLS. Fifty-nine AIH patients, 228 HCV, 50 HBV, 123 with other non-viral and non-autoimmune liver disorders (nV-nALD) and 267 healthy people were investigated for anti-CLAbs and antibodies against beta-2-glycoprotein I (anti-beta2-GPI). Resistance of IgG anti-CLAbs was evaluated using 2 M urea. IgG anti-CLAbs detected in 39% of AIH, 19.7% of HCV (p=0.006), 14% of HBV (p=0.01), 8.1% of nV-nALD (p=0.000) and 1.1% of healthy (p=0.000). IgG anti-CLAbs were associated with the presence of cirrhosis and active AIH while their resistance to urea was high. Anti-beta2-GPI was detected in two AIH patients. We demonstrated a significantly higher prevalence of anti-CLAbs in patients with AIH compared to other diseases and healthy people. Anti-CLAbs were associated with AIH stage but no association was found with APLS clinical manifestations (thrombosis, pregnancy morbidity, thrombocytopenia). However, their avidity was comparable with that of APLS indicating the need for prospective studies in order to address whether anti-CLAbs in AIH may contribute to the progression of liver disease or APLS development.

A bacteriological aetiology is suspected to be the triggering factor in primary biliary cirrhosis. We studied lipid A, the toxic and immunogenic moiety of gram-negative bacteria lipopolysaccharide, which accumulates abnormally in Kupffer cells, hepatocytes, and biliary epithelial cells in primary biliary cirrhosis patients. Anti-lipid A antibody levels from serum samples from 36 primary biliary cirrhosis patients, drawn before and after ursodeoxycholic acid treatment, were compared to those from patients with other liver diseases (n=236), non-hepatic diseases (n=249), and healthy subjects (n=75). In primary biliary cirrhosis patients, the prevalence of IgM anti-lipid A antibodies was higher before than after ursodeoxycholic acid therapy (64% vs 22%, respectively; P<0.001). Patients with anti-lipid A antibodies had significantly higher IgM levels than those without antibodies (8.7+/-1.1 g/l vs 4.4+/-0.8 g/l, P<0.02). Total IgM levels were correlated with anti-lipid A antibody levels (r=0.65, P<0.02). After therapy, the serum IgM levels decreased significantly (P<0.03). These results indicate that bacterial antigens may participate in the observed increase of serum IgM levels, and support an aetiological role of a gut-derived endotoxin antigen in the pathogenesis of primary biliary cirrhosis.

Previous studies documenting the existence of cross-reactivity between the lipoated (but not unlipoated) forms of the inner lipoyl domain (E2L2) of PDC-E2 [the major autoantigen in Primary biliary cirrhosis (PBC)] and trifluoroacetylated (TFA) proteins, led us to hypothesize that PBC may be due to an initial insult with an environmental agent that cross-reacts with TFA. Therefore, we performed a comparative study of the reactivity of rabbit anti-TFA antibody and anti-lipoic acid (LA) antibody against the mitochondrial autoantigens of human PBC and various TFA and LA conjugated proteins. Whereas both anti-TFA and anti-LA reacted with PDC-E2, the wild-type lipoated form of E2L2, OGDC-E2, E3-BP and LA-KLH, neither reacted with BCOADC-E2 or the non-lipoated form of E2L2. Of interest was that while anti-TFA reacted with PDC-E2, TFA-RSA and LA-KLH, it failed to inhibit PDC-E2 enzyme function. In contrast, anti-LA demonstrated cytoplasmic and mitochondrial staining, and inhibited PDC enzyme activity. Hence, although considerable cross reactivity exists between anti-TFA and anti-LA, the molecular nature of the interaction is clearly different. One of 14 PBC sera reacted weakly with TFA-albumin, whereas four of 14 PBC sera reacted with LA-KLH. Immunohistochemically, both anti-TFA and anti-LA antibodies reacted focally with periportal hepatocytes and bile ducts in both PBC and controls. However, anti-LA produced much stronger focalized staining of the bile ducts of diseased liver. This study suggests that while anti-TFA antibody recognizes lipoic acid-linked enzymes and proteins, the epitope recognized differs from that of anti-LA antibody and PBC autoantibodies. It is unlikely that a response to TFA is the triggering event in PBC. Anti-LA antibodies share a higher degree of similarity to PBC sera providing suggestive evidence that anti-LA antibodies or anti-LA like antibodies (mimotopes) may help define the initiator of the autoimmune response., (Copyright 2000 Academic Press.)

Naturally occurring autoantibodies are ubiquitous and may serve physiological functions. We examined the relationship of natural and disease-associated autoantibodies in the context of autoantibodies to dihydrolipoamide acetyltransferase, the 74 kDa E2 sub-unit of the mitochondrial pyruvate dehydrogenase complex (PDC-E2), characteristic of primary biliary cirrhosis (PBC). We tested for natural autoantibodies to PDC-E2 in normal sera, and compared epitopes recognised by natural and disease-associated autoantibodies. Methods included affinity purification of anti-PDC-E2 from normal and PBC sera, ELISA and immunoblotting, capacity of antibodies to inhibit the enzyme function of the pyruvate dehydrogenase complex (PDC), use of F(ab)2 fragments of anti-PDC-E2 in inhibition assays, and testing affinity purified anti-PDC-E2 on peptide fragments of PDC-E2. We found that natural auto-antibodies to PDC-E2 of IgG class were demonstrable in all healthy human sera (10/10). However, their reactivity differed from that of disease-associated autoantibodies, in that anti-PDC-E2 from normal serum failed to inhibit the catalytic activity of PDC; and F(ab)2 fragments from PBC sera potently blocked the binding of anti-PDC-E2 from PBC sera to PDC-E2, but not the binding of natural anti-PDC-E2 to PDC-E2. Immunoblotting on fragments of PDC-E2 using affinity-purified preparations from PBC sera and normal sera failed to provide evidence for gross differences in epitope reactivity. We conclude that normal human sera contain natural IgG autoantibodies to the immunodominant inner lipoyl domain of PDC-E2, as seen characteristically in PBC. However, there is evidence for differences in fine epitope recognition.

The identification and cloning of the mitochondrial autoantigens in primary biliary cirrhosis (PBC) have provided new clues in disease pathogenesis. The two major autoantigens are the E2 subunits of pyruvate dehydrogenase and branched-chain ketoacid dehydrogenase (BCKD). Interestingly, one of these complexes, BCKD-E2, is already well known to clinical medicine based on its association with genetic mutations in maple syrup urine disease (MSUD). Patients with this disease have an inability to metabolize branched-chain amino acids. In the present study, we have taken advantage of the known sequence of BCKD-E2 from normal humans, and addressed the issue of whether there is an altered autoantigen sequence in hepatocytes of individuals with primary biliary cirrhosis. In particular, we examined both the leader sequence and the B-cell immunodominant epitope, the lipoic acid domain. In addition, because patients with PBC have autoantibodies to the BCKD-E2 complex, we have quantitated plasma levels of alpha-ketoacids potentially affected in maple syrup urine disease. These include pyruvic acid (PY), phenylpyruvic acid (PP), alpha-ketoisocaproic acid (KIC) alpha-ketoisovalerate (KIV) and alpha-keto-beta-methylvaleric acid (KMV). The levels of these alpha-ketoacids were compared in patients with primary sclerosing cholangitis and normal volunteers. The sequence of BCKD-E2 obtained from PBC hepatocytes showed homology with normal BCKD. Further studies of autoantigen structure and sequence are clearly indicated, including those involved in mitochondrial transport and localization. Finally, we noted a statistically significant increase in all plasma alpha-ketoacids except alpha-keto-beta-methylvaleric acid in PBC patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Idiotype-anti-idiotype interactions were investigated in the sera of patients with primary biliary cirrhosis (PBC), which is characterized by the presence of circulating anti-mitochondrial antibodies (AMA). A mouse monoclonal antibody, CPZ674, has been raised to the E2 component of the pyruvate dehydrogenase complex (PDC), which is the target antigen of AMA. CPZ674 recognizes one of the epitopes recognizable by AMA, as demonstrated by competitive Western blotting. Anti-idiotypic antibodies in PBC sera were detected either by their specific binding to CPZ674 in an ELISA or by the formation of idiotype-anti-idiotype complexes with CPZ674, detected using chromatography on a Sephacryl-300 column and by a polyethylene glycol precipitation method. The specificity of the anti-idiotypic antibodies to AMA was shown by their ability to inhibit the binding of AMA to PDC, but not the binding of other autoantibodies to their relevant autoantigens. We have therefore produced evidence for the existence of idiotype-anti-idiotype interactions in PBC, but whether these anti-idiotypic antibodies are involved in the control of AMA is unknown.