Characterization of autoantibodies against uridine-diphosphate glucuronosyltransferase in patients with inflammatory liver diseases.

Uridine diphosphate glucuronosyltransferase (UGT) was identified as an antigenic target in a subgroup of liver-kidney microsomal autoantibodies and was termed LKM-3. To evaluate the nature of LKM-3 antibodies, we screened sera from 80 untreated patients with autoimmune hepatitis (AIH) type 1 and 2, primary biliary cirrhosis (PBC), AIH/PBC, hepatitis C virus (HCV) infection, and 12 healthy individuals (controls) against 7 recombinant human UGT isoenzymes (UGT1A1, UGT1A4, UGT1A6, UGT1A7, UGT1A9, UGT1A10, and UGT2B7). Autoantibodies reacting against various UGT isoenzymes were observed in sera from 3 of 18 AIH type 2 and 1 of 27 of the HCV patients. The anti-UGT-positive sera from AIH type 2 patients revealed the strongest immunoreactivity against UGT1A1, the main UGT-isoform involved in the bilirubin glucuronidation. Additionally, these sera were able to block UGT-mediated substrate glucuronidation in vitro. The prevalence for UGT1A1 was shown by 2 independent techniques: (1) UGT1A1 was identified as the main antigen by Western blotting. Preabsorption of sera with UGT1A1 prevented reaction against all tested UGT-isoforms. (2) In vitro immunoinhibition experiments showed that glucuronidation of the anticancer drug flavopiridol by UGT1A1 was more strongly inhibited than its UGT1A9-mediated biotransformation. In contrast, the serum from the HCV-patient reacted predominately with UGT1A6, and moreover, the immunoreactivity pattern was different from that of the AIH group. To summarize, we show the subtype preference of antibodies against UGT1A1 in a subgroup of AIH type 2 patients. These autoantibodies inhibit UGT-mediated glucuronidation in vitro, but it is unlikely that anti-UGT antibodies will have a marked effect on the patients capacity for drug biotransformation, as serum bilirubin levels in patients remained within the normal range.

Autoantibodies against nuclear pore complexes are associated with more active and severe liver disease in primary biliary cirrhosis.

BACKGROUND/AIMS: Antibodies against nuclear pore complexes (NPCs) have been detected in primary biliary cirrhosis (PBC), but their clinical relevance is still unsettled. METHODS: We tested sera from 171 consecutive PBC patients and 230 control subjects (149 with autoimmune or viral liver diseases, 28 with systemic lupus erythematosus, and 53 healthy) by immunoblotting for antibodies against purified human NPCs. RESULTS: Antibodies to NPCs were detected in 27% of the patients with PBC, were highly specific (97%), and were not associated with antimitochondrial antibodies. Their prevalence was higher in symptomatic patients (36 vs. 16%, P < 0.01) and was associated (P < 0.001) with more severe disease, as assessed by the presence of cirrhosis or its complications (13% prevalence in patients without cirrhosis, 31% in uncomplicated, and 54% in complicated cirrhosis), or by the application of the Mayo prognostic model (12% in the lowest, 21% in the median, 47% in the highest score tertile). Positive patients had higher levels of serum bilirubin (2.2 +/- 3.7 vs. 1.0 +/- 1.1 mg/dl, P < 0.01) and more marked inflammatory infiltrates on liver biopsy (P < 0.05). CONCLUSIONS: Autoantibodies to NPCs are more prevalent in PBC patients than in controls and are strongly associated with more active and severe disease.

Members of the glutathione S-transferase gene family are antigens in autoimmune hepatitis.

BACKGROUND & AIMS: Autoimmmune hepatitis (AIH), a chronic liver disorder, can be classified into two subtypes on the basis of the specificities of circulating autoantibodies. Type I AIH is defined by antibodies to nuclear and/or smooth muscle antigens (SMA), and type II is characterized by antibodies to cytochrome P450IID6. There is an additional type of AIH characterized by antibodies to a cytosolic soluble liver antigen (SLA), which can occur alone or in combination with antinuclear antibodies and SMA. The aim of this study was to identify the reactive antigen in SLA, a heterogenous cytosolic fraction consisting of at least 100 extremely soluble proteins. METHODS: Sera from 31 patients with AIH reacting with SLA and from 30 disease controls were tested. The immunoreactive antigens were determined using immunoprecipitation and immunoblotting after one- and two-dimensional polyacrylamide gel electrophoresis. The antigens were identified by microsequencing of the corresponding protein spots. RESULTS: Twenty-five of 31 anti-SLA-positive sera (80, 7%) reacted with a set of proteins ranging from 25 to 27 kilodaltons that were identified as three subunits of glutathione S-transferases: Ya, Yb1, and Yc. CONCLUSIONS: Glutathione S-transferase subunit proteins represent the major autoantigen in anti-SLA-positive AIH. This new finding permits the establishment of standardized immunoassays for routine diagnosis.

Autoantibodies against nucleoporin p62 constitute a novel marker of primary biliary cirrhosis.

BACKGROUND & AIMS: Patients with primary biliary cirrhosis (PBC) frequently produce autoantibodies against constituents of the nuclear envelope. We have recently observed a reactivity of PBC sera with a nuclear envelope antigen at approximately 60 kilodaltons. The aim of this study was to characterize the reactive antigen. METHODS: Sera from 103 patients with liver disease were tested by immunoblotting after the separation of proteins by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) using proteins of whole nuclei and nuclear pore complexes (NPCs). RESULTS: A nuclear immunofluorescence staining with peripheral accentuation was observed in 25 of 43 sera from patients with PBC. In immunoblotting, the sera displaying a peripheral pattern reacted preferentially with two proteins. Twelve sera showed specificity to an antigen of 210 kilodaltons corresponding to gp210, and 14 sera recognized a protein band at 60 kilodaltons. The intensity of the reactive band at 60 kilodaltons was clearly stronger on blots with immobilized proteins of the NPC than on blots with nuclear proteins, thereby indicating that the sera targeted an antigen as being a component of the NPC. Immunoblotting performed after protein separation by two-dimensional PAGE showed that the sera are directed against nucleoporin p62, a functional protein of the NPC. CONCLUSIONS: Anti-p62 antibodies appear to be a novel marker that is specific for PBC.

Anti-gp210 antibodies in sera of patients with primary biliary cirrhosis. Identification of a 64 kD fragment of gp210 as a major epitope.

Patients with primary biliary cirrhosis (PBC) frequently produce autoantibodies against gp210, an integral glycoprotein of the nuclear pores. this protein consists of three main domains: a large glycosylated lumenal domain, a single hydrophobic transmembrane segment and a short cytoplasmic tail. It has been previously shown that autoantibodies from PBC patients exclusively react with the cytoplasmic tail when recombinant rat gp210 expressed in Escherichia coli was used as antigen. Using human gp210 isolated from HeLa cells we found the lumenal domain as the major target. The aim of this study was to further characterize the dominant autoepitopes of gp210. Sera from 88 patients with autoimmune liver disease and 20 controls were used. Gp210 protein was digested with papain or endoglycosidase H and then subjected to immunoblotting. Autoantibodies against gp210 were detected in 12 of 43 (28%) PBC patients, but in none of the autoimmune hepatitis and control sera. Four of 12 (33%) anti-gp210 positive sera reacted with a fragment consisting of the cytoplasmic tail and 8 (66%) sera targeted an epitope located within the large lumenal domain. Furthermore, our data show that antigenic determinant is restricted to the 64 kD glycosylated amino-terminal fragment and that carbohydrate residues are an essential part of this novel epitope. We suggest that antigens possessing both epitopes namely; the glycosylated lumenal domain and the cytoplasmic tail should be used for screening tests in order to detect all sera with anti-gp210 specificity.

Autoantibodies from patients with primary biliary cirrhosis preferentially react with the amino-terminal domain of nuclear pore complex glycoprotein gp210.

Patients with primary biliary cirrhosis frequently develop autoantibodies directed to gp210, a major glycoprotein of the nuclear pore complex. This protein contains a large glycosylated cisternal domain, a single transmembrane segment, and a short cytoplasmic tail. It has been previously shown that autoantibodies from primary biliary cirrhosis patients exclusively react with the cytoplasmic tail. We demonstrate that autoantibodies against gp210 recognize at least two different epitopes. 4 out of 12 anti-gp210 positive sera reacted with the fragment consisting of the cytoplasmic tail, and 8 sera targeted a novel epitope located within the large glycosylated lumenal domain. Moreover, our data prove that carbohydrate moieties are an essential part of this novel epitope. We propose, therefore, that future screening assays should be performed with antigens possessing both epitopes to detect all sera with anti-gp210 specificity.

Autoantibodies against constituents of nuclear pore complexes in patients with primary biliary cirrhosis and autoimmune hepatitis.

Sera obtained from patients with autoimmune liver disease were screened in indirect immunofluorescence microscopy for the presence of autoantibodies. Patients' sera, which strongly stained nuclei (ANA) with peripheral accentuation, were used for further experiments to define the corresponding antigen(s). Nuclei and nuclear subfractions were isolated from HeLaS3 cells and used as antigen source. Immunoblotting experiments were performed after separation of nuclear proteins by one- and two-dimensional polyacrylamide gel electrophoresis. Some ANA positive sera recognized the nuclear protein with molecular mass of approximately 200 kDa. Further analysis revealed that the patients' sera reacted with gp210, an integral protein of the nuclear pores. The incidence and clinical significance of these antibodies is discussed.

Nucleolar proteins B23 and C23 as target antigens in chronic graft-versus-host disease.

Previously we observed that sera from recipients of allogeneic bone marrow transplants who developed extensive chronic graft-versus-host disease (GVHD) intensively stained the nucleolar region of target cells in indirect immunofluorescence microscopy. To identify the target antigens, immunoblotting experiments were performed using isolated nuclei, isolated nucleoli, and purified nuclear and nucleolar proteins as the antigen source. The nucleolar phosphoproteins B23 and C23 were identified as the main target antigens. Eleven of 19 extensive chronic GVHD sera reacted with these nucleolar phosphoproteins. In addition, four sera recognized histone H1, and two sera recognized the nuclear lamins A and C. Our patients reacting with the nucleolar proteins had symptoms resembling that of scleroderma or Sjögren's syndrome.

Antibodies to nuclear lamin C in chronic hepatitis delta virus infection.

Sera of patients with chronic hepatitis delta virus infection stained the nuclear periphery in indirect immunofluorescence. Using proteins of isolated nuclei, isolated nuclear matrices, the nuclear pore complex-lamina fraction and purified lamins A and C as antigen source in immunoblotting experiments, nuclear lamin C was identified as the reactive antigen. Most sera tested (8 of 10) recognized nuclear lamin C exclusively, but not the nuclear lamins A and B. Antibodies reacting with both nuclear lamins A and C, which share extensive sequence homologies, have been reported to occur in autoimmune hepatitis and primary biliary cirrhosis. The present findings suggest that the novel autoantibody associated with chronic hepatitis delta virus infection recognizes an epitope localized in the short carboxyterminal region of nuclear lamin C.

Autoantibodies against different histone H1 subtypes in systemic lupus erythematosus sera.

The H1 histones represent the most heterogenous class of histone proteins. In this study, we analyzed the specificity of human antibodies against 6 H1 subtypes. H1 histones from rat organs were separated by reverse-phase high performance liquid chromatography and used as antigens in immunoblotting experiments. Sera containing anti-histone H1 antibodies were obtained from patients with systemic lupus erythematosus. Of the 9 sera tested, 2 reacted with only 1 H1 subtype. The other sera recognized different combinations of H1 subtypes. Only 1 serum reacted with all 6 H1 subtypes. Histones H1.5 and H1.1 were the subtypes most frequently recognized by the human autoantibodies. Our data indicate that human anti-H1 antibodies represent a heterogenous population, directed mainly against epitopes localized in the variable region of the H1 molecule.

Antibodies to nuclear lamin proteins in liver disease.

Sera of patients with autoimmune liver disease contained antibodies reactive with nuclear lamins. These antigens were identified in immunoblotting experiments, using isolated nuclei, nuclear matrices, nuclear lamina-pore complexes and purified lamins as antigen source. The lamins were, furthermore, characterized by 2-dimensional gel electrophoresis. Antibodies to nuclear lamins were found in 75 per cent of the active lupoid hepatitis cases, but not in patients with inactive disease. Anti-lamin antibodies were detected in 8 per cent of primary biliary cirrhosis sera. The autoimmune liver disease sera recognized predominantly the nuclear lamins A/C, and less frequently the lamins A/B/C or lamin B.

Antibodies to nuclear lamins in autoimmune liver disease.

Antibodies to nuclear lamins were detected in sera of patients with autoimmune liver disease. In indirect immunofluorescence tests, these sera revealed staining of the nuclear periphery. Using isolated nuclei, nuclear matrices, nuclear lamina-pore complexes, and chromatographically purified lamins as antigen source, the nuclear lamins A, B, and C were identified as reactive antigens in immunoblotting experiments. The lamins were also identified by 2-D gel electrophoresis. Antibodies to nuclear lamins occurred in 12 of 16 cases of active lupoid hepatitis, but not in 35 patients with the disease in remission. However, only 3 of 37 sera of patients with primary biliary cirrhosis contained anti-lamin antibodies. Autoimmune liver disease sera reacted preferentially with lamins A/C and less frequently with lamin B or lamins A/B/C.

Long-term follow-up study of asymptomatic HBsAg-positive voluntary blood donors in Austria: a clinical and histologic evaluation of 242 cases.

Two hundred forty-two voluntary blood donors, referred after detection of HBsAg positivity, underwent clinical evaluation and liver biopsy and were prospectively followed for an average of 3.5 years. At initial testing, 65% of HBsAg carriers had normal laboratory findings; during follow-up, 26% of these carriers developed abnormal test results, at least transiently. Liver histology was normal in 31.4%, revealed nonaggressive liver disease in 63.6% and chronic active hepatitis or cirrhosis in 5%, only. All except one case of chronic active hepatitis or cirrhosis were associated with abnormal blood biochemical tests. Sequential liver biopsies obtained in 56 HBsAg carriers after a minimal interval of 4 years showed mitigation of inflammatory changes in 5.4% and developing chronic active hepatitis in three cases (5.4%). One carrier died of primary hepatocellular carcinoma. Upon follow-up, HBsAg persisted in 98%. Anti-HBe was found in 90% of all carriers already at the initial testing. HBeAg positivity (7.5%) was associated with chronic active hepatitis as well as nonaggressive liver disease; clearance of HBeAg occurred in 40% after 2 to 8 years. Because of the subclinical progression of liver disease and the increased risk for developing primary hepatocellular carcinoma in asymptomatic HBsAg carriers, routine blood testing, including alpha-fetoprotein screening, as well as abdominal ultrasound surveillance are indicated. Liver biopsy, however, should be restricted to carriers with abnormal biochemical findings.

Nuclear antigens recognized by antibodies present in liver disease sera.

Nuclear and nuclear matrix proteins of HeLa cells were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and subsequently transferred onto nitrocellulose. Antibodies present in sera of patients with primary biliary cirrhosis and autoimmune chronic active hepatitis reacted with some of the blotted proteins. The antibodies were mainly directed against chromatin-associated proteins and protein constituents of discrete RNP particles. In addition, antibodies found in autoimmune liver disease sera detected a hitherto undescribed nuclear protein of 54 kD, and a nuclear matrix protein of approximately 150 kD. Antibodies recognizing a nuclear 25 kD doublet apparently constituted a marker antibody for autoimmune liver disease. Those directed at the 17 kD centromere protein were associated with the primary biliary cirrhosis-related CREST syndrome, while those recognizing La antigen were related to cases of sicca syndrome associated with autoimmune liver diseases.