The mitochondrial adenine nucleotide translocator is an antigen in primary biliary cirrhosis.

Circulating antibodies reacting specifically with the adenine nucleotide translocator from liver mitochondria were detected in sera from 12 patients with proven primary biliary cirrhosis (PBC) by a solid phase double antibody immunoradiometric assay (IRMA). Furthermore these antibodies were absorbed with the isolated adenine nucleotide translocator from liver mitochondria. None of the sera from 20 normal individuals, four patients with anti-mitochondrial positive pseudolupus syndrome (PLE) sera (M-3) and three patients with syphilis (anti-M-l) had antibodies directed against this protein from inner mitochondrial membrane. The adenine nucleotide translocator as antigen in PBC could clearly be distinguished from the ATPase associated PBC specific M-2 antigen. With the present study, for the first time, a well characterized protein from inner mitochondrial membrane has been clearly defined as an autoantigen in primary biliary cirrhosis.     

Inhibition of the adenine nucleotide translocator by organ specific autoantibodies in primary biliary cirrhosis.

Sera from 13 patients with proven primary biliary cirrhosis (PBC) were studied for the capacity to bind to the adenine nucleotide translocator (ANT) isolated from heart, kidney and liver mitochondria. Antibodies against the ANT from liver were detected in the serum of all PBC patients, while 10 of 13 sera were negative when tested with the ANT from heart. None of the sera showed a significant binding to the ANT from kidney. The specific binding and the organ specificity of the autoantibodies against the ANT from liver were also confirmed by immunoabsorption studies on the isolated proteins. To distinguish between antibody titre and antibody activity, we measured the ability of the antisera to inhibit the adenine nucleotide transport across inner mitochondrial membrane using isolated mitochondria from heart, kidney and liver. Six of 13 patient sera tested inhibited the adenine nucleotide transport from liver mitochondria, however, none of the sera inhibited the transport from heart or kidney mitochondria again indicating the organ specificity of the antigen and the autoantibodies.     

Primary biliary cirrhosis: further biochemical and immunological characterization of mitochondrial antigens.

The nature of mitochondrial PBC-related antigens has been investigated with radioimmunoassay (RIA) and immunoblotting methods. The major antigen(s) was located by RIA in beef heart mitochondria, submitochondrial particles, chloroform-extracted F1-ATPase and Complex III. Cross-competition RIA experiments showed that the same antigen is present in all the above samples but at different concentrations. The antigen is not present in purified F1-ATPase, cytochrome oxidase, the oligomycin sensitivity conferring protein (OSCP), Factor6, or the Transhydrogenase. Immunoblot analysis of the above mitochondrial proteins revealed two PBC-related antigens (apparent molecular weights of 70 KD and 60 KD) whose distribution in the various proteins and protein complexes correlated well with the antigens determined by RIA. Immunoblot analysis of mitochondrial antigens was carried out using sera from normal subjects and from patients with PBC and with different autoimmune diseases (AID). Only PBC sera reacted with the 70 KD and 60 KD antigens. The PBC antigen detected by RIA in submitochondrial particles and the chloroform-F1-ATPase could be blocked by Mersalyl, suggesting its relationship to the mitochondrial 'M2' antigen. Furthermore, the antigenicity of the 70 KD peptide was shown by immunoblotting to be dependent upon mercaptoethanol. Thus, not only is the antigenicity of the 70 KD component dependent on a sulphur group, but the sulphur must be in the reduced form.     

Complete loss-of-function of the heart/muscle-specific adenine nucleotide translocator is associated with mitochondrial myopathy and cardiomyopathy

Multiple mitochondrial DNA deletions are associated with clinically heterogeneous disorders transmitted as mendelian traits. Dominant missense mutations were found in the gene encoding the heart and skeletal muscle-specific isoform of the adenine nucleotide translocator (ANT1) in families with autosomal dominant progressive external opthalmoplegia and in a sporadic patient. We herein report on a sporadic patient who presented with hypertrophic cardiomyopathy, mild myopathy with exercise intolerance and lactic acidosis but no ophthalmoplegia. A muscle biopsy showed the presence of numerous ragged-red fibers, and Southern blot analysis disclosed multiple deletions of muscle mitochondrial DNA. Molecular analysis revealed a C to A homozygous mutation at nucleotide 368 of the ANT1 gene. The mutation converted a highly conserved alanine into an aspartic acid at codon 123 and was absent in 500 control individuals. This is the first report of a recessive mutation in the ANT1 gene. The clinical and biochemical features are different from those found in dominant ANT1 mutations, resembling those described in ANT1 knockout mice. No ATP uptake was measured in proteoliposomes reconstituted with protein extracts from the patient's muscle. The equivalent mutation in AAC2, the yeast ortholog of human ANT1, resulted in a complete loss of transport activity and in the inability to rescue the severe Oxidative Phosphorylation phenotype displayed by WB-12, an AAC1/AAC2 defective strain. Interestingly, exposure to reactive oxygen species (ROS) scavengers dramatically increased the viability of the WB-12 transformant, suggesting that increased redox stress is involved in the pathogenesis of the disease and that anti-ROS therapy may be beneficial to patients.     

Primary biliary cirrhosis of the liver

Primary biliary cirrhosis (PBC) of the liver is considered to be an autoimmune liver disease in which an immune aggression is directed against small biliary ducts--mitochondrial antigens of a biliary epithelium. The targets of an immune aggression in PBC may also be the histocompatibility antigens of the biliary ducts epithelium. This makes PBC resembling so-called disease "graft against host". The destruction of bile ducts in PBC is brought about through the mechanisms of an immune cytolysis and is manifested morphologically by the symptoms of the delayed hypersensitivity response. Cholangiole proliferation develops along the ductal destruction, sclerotic changes and cholestasis increase in their intensity. Small-nodular cirrhosis develops at the final stage of disease. Systemic manifestations of PBC are associated with a circulation of immune complexes containing hepatobiliary antigens. Cross-immune reactions are also of importance in the mechanisms of extrahepatic lesions.     

Primary biliary cirrhosis: antigenic specificity of IgM-type mitochondrial antibodies analyzed by immunoblotting and ELISA

The antigenic reactivities of circulating IgM- and IgG-type antimitochondrial antibodies (AMA) from 18 patients with primary biliary cirrhosis (PBC) were compared by the use of immunoblotting and enzyme-linked immunosorbent assay (ELISA). In immunoblotting, the binding patterns of IgM and IgG were very similar when F1-ATPase and mitochondria were used as antigens. The major PBC-specific IgM-reactive antigen was identical with the dominating IgG-reactive antigen, sharing the same molecular weight of 70 kD and the same requirement for reduced thiol groups for expression of antigenicity. Other PBC-related mitochondrial proteins with variable antigenicity had the molecular weights of 60 and 43 kD. Depending on the IgM and IgG reactions in F1-ELISA, PBC patients can be grouped into three categories: patients with IgG and IgM (12/18), IgG alone (5/18) and IgM alone (1/18). By serum fractionation, the IgM reactivity was shown to be a true PBC-related antibody antigen reaction, and not due to interference of rheumatoid factors.     

Mitochondrial antibodies in primary biliary cirrhosis. VI. Association of the complement fixing antigen with a component of the mitochondrial F1-ATPase complex.

The complement fixing antigen of the inner mitochondrial membrane previously shown to be associated with the mitochondrial ATPase could be further purified by subjecting the ATPase extracted from beef heart and brown fat mitochondria to ion exchange and gel filtration chromatography. Although the ATPase activity could be clearly dissociated from the complement fixing activity, subunits of the F1-ATPase complex were always found in the purified fractions. The alpha, gamma, delta and epsilon subunits of the complex could be excluded with high probability as target antigens in contrast to the beta band which was always found in association with the antigen activity. These findings imply that the active centre of the ATPase enzyme is not involved in the antibody reaction but molecules of the ATPase complex may have antigen binding capacity. Treatment of ATPase associated antigen with trypsin did not markedly affect the complement binding, while SMP's treated in the same way lost their antigen activity indicating that sera from patients with primary biliary cirrhosis (PBC) may have mitochondrial antibodies of different specificities reacting with trypsin sensitive as well as trypsin insensitive components of the inner membrane. The purified antigen reacted exclusively with sera from patients with PBC and may be therefore used as a marker antigen.     

Primary biliary cirrhosis in patients with breast cancer: studying the link

Primary biliary cirrhosis (PBC) is a liver disease of unknown etiology characterized by chronic nonsuppurative destructive cholangitis (CNSDC) of intrahepatic septal and interlobular bile ducts. It is generally defined as an autoimmune disease. Characteristically, patients with PBC have a cholestatic serum hepatic profile and circulating antimitochondrial antibodies (AMA). PBC is progressive and ultimately leads to biliary cirrhosis and liver failure. It occurs at least three times more often in women than in men and it is the most common indication for liver transplantation in women around the world. There is no known cure for PBC. Despite the remarkable progress elucidating the genetics of breast cancer, and the effort placed on breast cancer education and screening methods, the mortality of breast cancer remains unacceptably high. In this essay, we describe the similarities between breast cancer and PBC and how their pathogenesis may be related. The hypothesis stated herein has evolved from reports from the early 1980s that linked an increased risk for breast cancer with PBC, and from the author's clinical experience with patients who suffer from both diseases. The association between these two diseases in the USA merits further investigation. If it is confirmed, risk factors involved in their pathogenesis will be identified.     

Primary biliary cirrhosis: an increased incidence of extrahepatic malignancies?

In a retrospective review of 85 patients with primary biliary cirrhosis (PBC), 10 (11.8%) were noted to have extrahepatic malignant neoplasm. In seven female patients the tumour developed within a mean of 3.5 yr after the clinical onset of PBC. This observed number of tumours, 3.5 times more common than the expected age-adjusted incidence, was statistically significant at the 0.5% level.     

Primary biliary cirrhosis in the mouse: induction by human mycoplasma-like organisms

Human intraocular and orbital chronic inflammatory disease with autoimmune features has been reported to be caused by mycoplasma-like organisms (MLO). MLO are intracellular cell-wall deficient pathogenic bacteria, closely related to rickettsia, with a characteristic ultrastrural pleomorphic tubulo-spherical and filamentous appearance. No culture system has been developed for MLO and diagnosis of MLO disease is made by detecting these bacteria within infected cells using a transmission electron microscope. In human MLO ocular and orbital disease the organisms are found in parasitized leucocytes at the disease site. Inoculation of human MLO into mouse eyelids produces a high incidence of orbital and introcular disease. MLO disseminate to produce randomly distributed lethal systemic disease with infected leucocytes found in all disease sites and with similar histologic features in all disease sites. Microvasculitis is the initial lesion. Disease progression results in lysis of vascular and parenchymal structures, stromal lymphocytic infiltrates, granulomas, and fibrosis. This report describes the hepatic portal chronic progressive inflammatory disease in 11 of 100 of those mice versus 0 in 200 controls. MLO parasitized portal leucocytes are present in all 11 inflamed livers versus 0 in 5 control livers (P less than 0.05). The resemblance of the animal liver disease induced by MLO to human primary biliary cirrhosis and rifampin treatment of MLO disease are discussed.     

Mitochondrial antibodies in primary biliary cirrhosis: IV. Significance of membrane structure for the complement-fixing antigen

The effect on the mitochondrial antigen of different agents known to influence the integrity and structure of membranes has been studied using quantitative complement fixation with autoantibodies from the serum of a patient with primary biliary cirrhosis. The susceptibility to proteolytic enzymes suggests that the antigen is a protein. Activity depends upon an association with phospholipids. Addition of phospholipids prevents loss of antigen during artificial ageing of mitochondria at 37°. Activity is lost after treatment with phospholipases or solvents which extract phospholipids. Antigen is also destroyed by surface active agents which dissociate the link with phospholipid but those which weaken bonds between phospholipids and hydrophobic molecules yield fragments of antigen-containing membrane structures which, nonetheless, still react with the mitochondrial autoantibody.     

Mitochondrial antibodies in primary biliary cirrhosis. III. Characterization of the inner-membrane complement fixing antigen

The mitochondrial inner-membrane autoantigen reacting with the serum of patients with primary biliary cirrhosis, was purified up to fifty-fold by centrifugal fractionation of sub-mitochondrial fragments obtained by hypotonic swelling and ultrasonication. Respiration and oxidative phosphorylation as well as swelling and contraction were not affected by the presence of the antibody and it could be shown that a number of readily extractable mitochondrial enzymes, and the components of the electron transport chain were not associated with the antigen. The highly purified antigen fraction, although having a vesicular ultrastructure with phospholipid composition characteristic of inner membranes, was nonetheless devoid of cytochrome oxidase activity. Although the exact location of the antigen on the inner membranes is not yet known, it can be physically dissociated from the components of the respiratory chain.     

Single nucleotide polymorphisms of the mannose-binding lectin are associated with susceptibility to primary biliary cirrhosis.

Although the immunopathogenesis of primary biliary cirrhosis (PBC) remains unknown, familial clustering of patients with PBC suggests an important role for genetic factors. In addition, recent data support the thesis that the mucosal immune response against intraluminal pathogens may be involved with the onset of PBC. Mannose-binding lectin (MBL) is a key factor in innate mucosal defenses and has several key single nucleotide polymorphisms (SNPs). To study whether MBL gene SNPs are associated with susceptibility to PBC, we studied 65 patients with PBC and 218 controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequence specific priming-polymerase chain reaction (SSP-PCR) to examine four polymorphic loci: two (H/L and X/Y) within the promoter region and the other two (P/Q and A/B) within exon-1. We also analyzed serum MBL concentrations. Interestingly, the prevalence of haplotype HYPA, leading to hyper-production of MBL, as well as HYPA/HYPA genotype were significantly increased in PBC compared to controls (0.53 vs. 0.44, P=0.031; 33.9%vs. 17.0%, P=0.003, respectively). Furthermore, individuals homozygous for HYPA had a significantly increased risk for PBC (odds ratio (OR)=2.51, 95% confidence interval (CI)=1.34-4.66). Our results demonstrate that the MBL genotype can be significantly associated with increased risk for PBC, and further, that increased production of MBL plays a critical role in immunopathogenesis.     

Characterization of the mitochondrial inner membrane protein translocator Tim17 from Trypanosoma brucei

Mitochondrial protein translocation machinery in the kinetoplastid parasites, like Trypanosoma brucei, has been characterized poorly. In T. brucei genome database, one homolog for a protein translocator of mitochondrial inner membrane (Tim) has been found, which is closely related to Tim17 from other species. The T. brucei Tim17 (TbTim17) has a molecular mass 16.2kDa and it possesses four characteristic transmembrane domains. The protein is localized in the mitochondrial inner membrane. The level of TbTim17 protein is 6-7-fold higher in the procyclic form that has a fully active mitochondrion, than in the mammalian bloodstream form of T. brucei, where many of the mitochondrial activities are suppressed. Knockdown of TbTim17 expression by RNAi caused a cessation of cell growth in the procyclic form and reduced growth rate in the bloodstream form. Depletion of TbTim17 decreased mitochondrial membrane potential more in the procyclic than bloodstream form. However, TbTim17 knockdown reduced the expression level of several nuclear encoded mitochondrial proteins in both the forms. Furthermore, import of presequence containing nuclear encoded mitochondrial proteins was significantly reduced in TbTim17 depleted mitochondria of the procyclic as well as the bloodstream form, confirming that TbTim17 is critical for mitochondrial protein import in both developmental forms. Together, these show that TbTim17 is the translocator of nuclear encoded mitochondrial proteins and its expression is regulated according to mitochondrial activities in T. brucei.