Liver Kidney Microsomal Type Research Articles

Formiminotransferase cyclodeaminase is an organ-specific autoantigen recognized by sera of patients with autoimmune hepatitis

Background & Aims: Anti–liver cytosol type 1 autoantibodies have been reported in association with anti–liver-kidney microsome type 1 autoantibodies in 30% of patients with autoimmune hepatitis type II. In 10% of cases, anti–liver cytosol type 1 antibodies are the only liver-related circulating autoantibodies. The liver cytosol antigen is a liver-specific 62-kilodalton protein present in the cell as an oligomer of ~240 kilodaltons. The aim of this study was to identify the antigen recognized by anti–liver cytosol antibody. Methods: To identify the liver cytosol antigen, an anti–liver cytosol type 1–positive serum was used for the screening of a complementary DNA library from HepG2 cells. Double immunodiffusion method was used to show the identity between the cytosolic and the cloned protein. Results: The sequence of two isolated clones showed 85.2% homology with the formiminotransferase cyclodeaminase (FTCD) enzyme from pig liver. Antibodies purified by affinity with the recombinant protein and sera from mice immunized with FTCD recognized a 62-kilodalton human cytosolic protein when tested by immunoblot. The identity of precipitation lines was found between the cytosolic antigen and FTCD. Conclusions: This enzyme is a liver-specific antigen recognized by the sera of patients with autoimmune hepatitis. GASTROENTEROLOGY 1999;116:643-649

Interferon therapy in LKM-1 positive patients with chronic hepatitis C: follow-up by a quantitative radioligand assay for CYP2D6 antibody detection

Background/Aims: Liver/kidney microsomal type 1 (LKM-1) antibodies described by indirect immunofluorescence using frozen sections of kidney, stomach and rat liver define a group of patients with type 2 autoimmune hepatitis. Sera react with a non-glycosylated 50-kD protein of the endoplasmic reticulum, which was recently identified as cytochrome P4502D6 (CYP2D6). LKM-1 antibodies may also be associated with hepatitis C virus infection (HCV+/LKM-1+). For this subset of patients, the choice of steroids or interferon alpha therapy may be difficult because of the association of hepatitis C virus infection and autoimmune manifestations. Recently we developed a quantitative immunoprecipitation radioligand assay using 35S-methionine-labeled CYP2D6 protein produced by in vitro transcription and translation reaction. This method detects antibodies against linear and conformational epitopes in both AIH-2 and HCV+/LKM-1+ patients. The aim of this study was to analyze the time-course of HCV+/LKM-1+ patients, applying our radioligand assay over a long follow-up. Methods: We studied five patients who were positive for CYP2D6 antibodies from among 235 chronic hepatitis C virus hepatitis patients (2.1%) treated with interferon alpha for a minimal follow-up of 2 years. We analyzed LKM-1 antibody titer sequentially by radioligand assay, HCV RNA titer and alanine aminotransferase activity in these patients. Results: We found no aggravation of liver disease in this group of patients. Three of these patients showed a sustained biochemical and virological response after interferon. Two others responded partially to interferon therapy. Alanine aminotransferase level sand HCV-RNA decreased during interferon therapy in responder patients. CYP2D6 antibodies did not change in three responder patients during follow-up. One responder patient decreased CYP2D6 antibody level by radioligand assay, but indirect immunofluorescence titers showed a similar pattern. One partial responder patient decreased CYP2D6 antibody level but was negative by indirect immunofluorescence. Conclusions: Our results show that patients with hepatitis C virus who are positive for CYP2D6 antibodies may be treated with interferon, and respond in the same way as CYP2D6 antibody negative patients. Radioligand assay could be helpful for monitoring HCV+/LKM-1+ patients receiving interferon therapy.

Immunological liver diseases in children.

Autoimmune liver diseases comprise a number of disorders in which inflammatory damage to the liver is believed to derive from an autoimmune attack. These include autoimmune hepatitis (AIH), characterised by positive smooth muscle and/or nuclear (SMA/ANA) or liver kidney microsomal type 1 (LKM1) antibodies, autoimmune sclerosing cholangitis (ASC), usually SMA/ANA positive, and AIH after liver transplantation, which is positive for SMA, ANA, or atypical LKM. These disorders often present with symptoms indistinguishable from prolonged acute hepatitis. Less commonly the onset is insidious, with nonspecific symptoms, or with complications of portal hypertension. For AIH and ASC, experimental evidence suggests that usually in individuals genetically predisposed to autoimmunity, a liver self antigenic peptide is recognized by T lymphocytes which promote a cascade of autoaggressive processes. For AIH after liver transplantation, the pathogenic mechanisms remain to be elucidated. All types of autoimmune liver disorders appear to respond favourably to early treatment with prednisolone with or without azathioprine. For patients presenting with fulminant hepatic failure or with already advanced cirrhosis, immunosuppression is rarely effective and the only mode of treatment is liver transplantation. The role of other immunosuppressant or immunomodulatory drugs, like cyclosporin A, tacrolimus or ursodeoxycholic acid, in the treatment of autoimmune liver disorders remains to be defined.

A novel assay for detecting antibodies to cytochrome P4502D6, the molecular target of liver kidney microsomal antibody type 1

A novel assay for detecting antibodies to cytochrome P4502D6, the molecular target of liver kidney microsomal antibody type 1

A 12-year-old girl with antimitochondrial antibody-positive autoimmune hepatitis

Autoimmune hepatitis is rare and, in childhood, is typically associated with either the nuclear and/or smooth muscle antibody or with the liver kidney microsomal type 1 antibody. Antimitochondrial antibodies, which are considered diagnostic of primary biliary cirrhosis, have not been described in the paediatric age. We report for the first time a 12-year-old girl with antimitochondrial-antibody-positive autoimmune hepatitis. Antimitochondrial antibody positivity was determined by immunofluorescence and immunoblot. The patient's age, clinical, biochemical and histological features and response to immunosuppressive treatment support the diagnosis of autoimmune hepatitis.

Present status of autoimmune hepatitis in Japan - correlating the characteristics with international criteria in an area with a high rate of HCV infection

Background/Aims: A nationwide survey of autoimmune hepatitis (AIH) was carried out in Japan. Methods: Four hundred and ninety-six patients were enrolled by questionnaires sent to 101 hospitals with hepatology specialists. Results: The clinical features of Japanese AIH were as follows: most patients were middle-aged women; serum autoantibodies, especially antinuclear antibody, were frequently positive, serum IgG level was high, and HLA-DR4 was the major HLA allotype. Liver-kidney microsomal type 1 antibody was positive in nine of 79 patients tested. Eight of these antibody positive patients were also positive for antinuclear antibody and five for anti-smooth muscle antibody. Ninety-two percent of the patients showed piecemeal necrosis and 60% bridging necrosis; plasma cell infiltration in the portal areas was observed in 50% of the patients. Only 12.3% were diagnosed as having liver cirrhosis. A favorable effect of corticosteroid, normalization of serum transaminases, was observed in 89% of 317 patients, who were treated with an initial dose of over 30 mg/day. Sixty-two patients were positive for hepatitis C virus (HCV) markers. In these patients, however, only one patient was liver-kidney microsomal type 1 antibody positive. Corticosteroid was effective in 30 (81%) of 37 HCV-marker-positive patients treated with this agent. Thus the efficacy of corticosteroid did not differ from that in AIH patients without HCV infection (90%). Similarly, interferon treatment was used in 20 patients, all of whom were positive for HCV-RNA, and resulted in 50% efficacy as determined by normalization of the serum transaminase level 6 months after treatment. The International Diagnostic Scoring System for the diagnosis of AIH worked well in these patients, except for HCV-infected individuals, that is, approximately 10% of the total of AIH patients

Establishment of a novel radioligand assay using eukaryotically expressed cytchrome P4502D6 for the measurement of liver kidney microsomal type 1 antibdy in patients with autoimmune hepatitis and hepatitis C virus infection

Background/Aims: Liver kidney microsomal type 1 antibody (LKM1) is the diagnostic marker of autoimmune hepatitis (AIH) type 2 and is also found in patients with hepatitis C virus (HCV) infection. Cytochrome P4502D6 (CYP2D6) is the documented target antigen of LKM1 in AIH, but not in HCV infection. To compare the reactivity in the two conditions, we established a radioligand assay using eukaryotically expressed CYP2D6 as target. Methods: A 1.2-kb human CYP2D6 cDNA was isolated from a human liver cDNA library and subcloned into an vitro transcription vector pSP64 Poly(A). Recombinant CYP2D6 was then produced by in vitro transcription/translation, metabolically labelled with 35S methionine and used in the immunoprecipitation assay. Antibodies that bound radiolabelled CYP2D6 were immunoprecipitated and their levels assessed as cpm. Sera from 50 LKM1-positive patients (26 with AIH; 24 with HCV infection ), 128 LKM1-negative patients and 57 normal controls were tested. Results: Reactivity to 35S labelled CYP2D6 was observed in all LKM1-positive sera from patients with AIH and HCV infection, but in none of the controls. The cpm in both conditions were significantly higher than in normal controls ( p<0.0001), and were correlated with the immunofluorescence titres of LKM1 ( r 0.87, p<0.001 and r=0.64, p<0.001 for AIH and HCV infection, respectively.). Reactivity to 35S labelled CYP2D6 was inhibited by addition of an excess of eukaryotically expressed CYP2D6. Conclusions: CYP2D6 is a major target antigen of both AIH and HCV infection. The novel radioligand assay is highly sensitive and specific.

Autoimmune hepatitis type 2 and hepatitis C virus infection: study of HLA antigens

Background/Aims: Markers for hepatitis C virus are often detectable in patients suffering chronic hepatitis with liver-kidney microsomal type 1 antibodies. Several authors have suggested that two subsets of those patients can be defined: a) hepatitis C virus negative and b) hepatitis C virus positive. The aim of this work was to further analyze the possible genetic association, HLA class I and II, in these two groups of patients. Methods: HLA was analyzed in 49 patients. Class I was studied using a standard lymphocytotoxicity test and in class II a reverse hybridization-based test for DRB1 typing and PCR-SSO for DQB1 typing were used. Sixty healthy Spanish subject and 39 chronic hepatitis C subjects without anti-LKM1 antibodies were used as control groups for the “a” and “b” subsets, respectively. Results: No significant association was found with class I specificities in either group. DQB1 typing showed a very significant increase of DQ2 in the “a” group (93.3% vs. 48%; RR=15; p c=0.0025), and DRB1 typing from the “b” group reversed a high association with DR7 (82.3% vs. 43.6%; RR=6; p c=0.0086). Conclusions: Our stuides revealed a strong association with DQ2 for the “a” group and for the first time an extremely high association with DR7 antigen for the “b” subset. Hence it is possible to establish a different genetic profile in these two patient groups.

Anti-neutrophil cytoplasmic antibodies in type 1 and 2 autoimmune hepatitis.

Perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) have been recently defined as the most sensitive autoantibody of type 1 autoimmune hepatitis (AIH-1). Their prevalence in type 2 autoimmune hepatitis (AIH-2) has not yet been evaluated. The aim of the present study was to verify the association of pANCA with AIH-1 in an Italian series and to investigate the prevalence of the antibodies in AIH-2 and in proper control groups represented by cases of chronic hepatitis C (CH-C) with similar autoimmune features. pANCA were found in 30 of 46 (65%) AIH-1 and in 4 of 30 (13%) ANA/smooth muscle antibody (SMA) positive CH-C (P = .0000006). Nineteen AIH-2, 29 liver kidney microsomal antibody type 1/liver cytosol antibody type 1 (LKM1/LC1) positive CH-C cases and 50 healthy controls were all negative. In AIH-1, pANCA were significantly (P = .009) more frequent in males (8 of 9, 89%) than in females (22 of 37, 59%). All pANCA positive sera showed SMA of the antiactin type. The present data confirm that pANCA, although less prevalent in our series than in other reports, do associate with AIH-1 also in the Mediterranean area and show that it can identify a small subgroup (13%) of ANA/SMA positive chronic hepatitis C, in which autoimmune reactions might play a more relevant role than viral infection. They also show the antibodies are absent in AIH-2. In conclusion, pANCA appear to be mutually exclusive of LKM1 positivity, either hepatitis C virus-related or not, thus representing a further valuable tool to differentiate the two types of autoimmune hepatitis.

Prevalence of antibodies to hepatitis C and herpes simplex virus type 1 is not increased in children with liver kidney microsomal type 1 autoimmune hepatitis.

Prevalence of antibodies to hepatitis C and herpes simplex virus type 1 is not increased in children with liver kidney microsomal type 1 autoimmune hepatitis.

Autoantibodies against glucuronosyltransferases differ between viral hepatitis and autoimmune hepatitis

BACKGROUND & AIMS: Approximately 13% of patients with chronic hepatitis D virus (HDV) infection have liver-kidney microsomal antibodies type 3 (LKM-3) directed against family 1 uridine 5'-diphosphate-glucuronosyl- transferases (UGT-1). The aim of this study was to characterize the prevalence and specificity of LKM-3 by recombinant antigen testing systems. METHODS: Enzyme-linked immunosorbent assay (ELISA) and Western blot were performed using baculovirus-generated human UGT-1.1 and -1.6 and rabbit UGT-1.6. Sera from patients with HDV (n = 50), autoimmune hepatitis (AIH) type 2 (n = 50), hepatitis B virus (n = 26), hepatitis C virus (HCV) (n = 25), and LKM-1 autoantibody-positive HCV (n = 14) and sera from normal controls (n = 50) and italian patients with HDV and known LKM-3 autoantibodies were studied. RESULTS: Six percent of patients with HDV from Germany and 8% of patients with type 2 AIH had LKM-3. Sera from italian patients with HDV and patients with AIH type 2 recognized all three recombinant UGT-1. HDV sera from Germany selectively recognized human UGT-1. LKM-3 titers were lower in HDV than in AIH. One patient with AIH had LKM-3 as the only marker of AIH. CONCLUSIONS: This study indicates a molecular target and titer difference of LKM-3 autoantibodies in German subjects with HDV and AIH. It also suggests a geographic target and titer difference of LKM-3 in HDV. LKM-3 are identified as a rare and previously undescribed independent marker of AIH. (Gastroenterology 1996 Dec;111(6):1576-86)

Non-organ specific autoantibodies in children with chronic hepatitis C

Background/Aims: Recent studies in adult patients have established a relationship between hepatitis C virus infection and the presence of liver-kidney microsomal autoantibody type 1 (LKM1). Conversely, little is known regarding the relationship between hepatitis C and autoimmunity in children. In this study, we investigated non-organ specific autoantibodies in 40 otherwise healthy Italian children with chronic hepatitis C. Methods: All but four patients included in the study were asymptomatic. Liver histology, obtained in 35, showed features ranging from minimal to mild chronic hepatitis. Autoantibodies were investigated by indirect immunofluorescence. HCV RNA was assayed by the polymerase chain reaction in 34 cases and viral genotypes were determined. Results: Antinuclear antibodies were detected in three (7.5%) cases, one with a homogeneous pattern; smooth muscle autoantibodies in seven (17.5%) cases, always with V (vessels only) specificity and LKM1 in four (10%), at titers ranging from 1:20 and 1:2560. Clinical and virologic features did not significantly differ between autoantibody positive and negative cases, although infections with HCV genotypes 1a and 2 were more frequent in LKM1-positive patients. During observation, the child with the highest LKM1 titre was unsuccessfully treated with alpha interferon but responded to steroids. Twelve LKM1 negative children were also treated with interferon and one developed low LKM1 titers concomitant with an alanine aminotransferase flare. The sera of the five LKM1-positive children were investigated by immunoblotting with a human microsomal fraction and peptide 257–269 of cytochrome P450IID6. Only the serum of the child with the highest LKM1 titers was reactive. Conclusions: These results show that a consistent proportion of children with chronic hepatitis C circulate non-organ specific autoantibodies. The prevalence of LKM1 is greater than in adults and this could raise problems for the treatment of the disease with interferon. The analysis of LKM1 target antigens might help to identify putative cases of “true” autoimmune hepatitis with concomitant HCV infection that could benefit from steroid treatment.

Autoantibody prevalence in chronic hepatitis B virus infection: Effect in interferon alfa

The most effective treatment of chronic hepatitis B virus (HBV) infection is interferon alfa (IFN-alpha), a potentially severe side effect of which is the induction of autoimmunity. To assess whether IFN- alpha causes clinical or serological autoimmune manifestations, we studied 61 children randomized to receive 5 MU/m2 of IFN-alpha three times per week for 12 weeks, with or without steroid priming or no treatment. Autoantibodies to antinuclei (ANA), smooth muscle (SMA), gastric parietal cell (GPC), liver kidney microsomal type 1, mitochondrial, liver cytosolic antigen, thyroid microsomal, and thyroid globulin were detected by standard techniques. Over a median of 4 years (range, 1-5 years) from randomization, no clinical signs of autoimmunity were observed. Autoantibody positivity for nuclei, smooth muscle, and/or gastric parietal cells was observed on at least one occasion in 42 of 61 children (69%), with no overall difference in the prevalence between patients treated with interferon alone (19 of 24 [79%]), steroids plus interferon (13 of 20 [65%]), or untreated controls (10 of 17 [59%]). There was also no difference in the autoantibody prevalence before, during, and at follow-up after cessation of treatment in both interferon-treated and interferon- untreated patients. Autoantibodies are common in chronic HBV infection, and their prevalence is uninfluenced by IFN-alpha. (Hepatology 1996 Sep;24(3):520-3)

97 PREVALENCE OF ANTIBODIES TO HEPATITIS C AND HERPES SIMPLEX VIRUS TYPE 1 IS NOT INCREASED IN LKM- 1 POSITIVE AUTOIMMUNE HEPATITIS

Hepatitis C (HCV) and herpes simplex type 1 (HSV1) have been implicated in the pathogenesis of liver kidney microsomal type 1 positive autoimmune hepatitis (LKM1 +ve AIH) because the putative target of LKM1, cytochrome P4502D6, shares sequences in common with these viruses. Cross-reactive immunity have been proposed as the cause of liver damage. Were this to be the case, serological evidence of infection with either virus should characterise LKM1 +ve AIH. To determine if signs of HCV or HSV1 infection are more prevalent in LKM1 +ve AIH as compared to patients with autoimmune liver disease who are LKM1 negative, antinuclear and/or smooth muscle antibody positive (ANA/SMA +ve), we have tested the presence of anti-HCV and anti-HSV1 IgG in 47 [median age: 12 (2-16) yrs; 11 boys; 25 LKM-1 +ve; 22 ANA/SMA +ve] children with AIH and 21 [12 (2.5-16) years; 15 boys] with ANA/SMA +ve sclerosing cholangitis (ASC), using enzyme linked immunosorbent assays. 95 children with non-autoimmune liver disease [20 α-1 antitrypsin deficiency, 20 Wilson's disease, 12 Alagille's syndrome, 20 chronic hepatitis B virus (HBV) infection and 21 extrahepatic biliary atresia (EHBA)] and 52 age-matched healthy controls were also tested. Anti-HCV was detected in only one patient with EHBA. There was no difference in the prevalence of anti-HSV1 IgG between LKM1 +ve and ANA/SMA +ve AIH (28% vs 45%) nor in LKM1 +ve AIH vs ASC (45%), non-autoimmune liver disease (29%) and controls (13%). As compared with healthy controls, an increase in the prevalence of anti-HSV1 IgG was noted in ANA/SMA +ve AIH, EHBA, HBV and Alagille's syndrome (13% vs 45%, vs 38%, vs 50%, vs 42% respectively, p<0.05 for all). Our results do not support the reported association between HCV or HSV1 infection, positivity for LKM1 and development of LKM1 +ve AIH. Whether theres is an as yet unidentified virus that triggers LKM1 +ve AIH remains to be established.

Impact of international autoimmune hepatitis group scoring system in definition of autoimmune hepatitis. An Italian experience.

We have reclassified 110 patients with autoantibody-positive cryptogenic chronic hepatitis according tot he aggregate scoring system proposed by the International Autoimmune Hepatitis Group for signs of hepatitis C virus (HCV) infection and the newly proposed terminology of "unclassified" chronic hepatitis. Anti-HCV and HCV viremia were assessed by second-generation assays and reverse transcription-polymerase chain reaction. Immunomorphological and immunochemical characterizations of antinuclear, smooth, muscle, liver-kidney microsomal type 1, and liver cytosol type 1 autoantibodies were also performed. All 45 anti-HCV negative patients fulfilled the score criteria for the diagnosis of "definite" or "probable" autoimmune hepatitis (AIH). Eight anti-HCV-positive cases reached the score of "probable" AIH, whereas the remaining 57 cases were diagnosed as unclassified chronic hepatitis. The scoring system allows the correct identification of all autoimmune cases without HCV infection. Autoimmune hepatitis runs a more severe disease course than unclassified chronic hepatitis, whose clinical and histological features are similar to those of autoantibody-negative chronic hepatitis C.

Recognition of uridine diphosphate glucuronosyl transferases by LKM-3 antibodies in chronic hepatitis D: Philipp T, Durazzo M, Trautwein C, Britta Alex B, Straub P, Lamb JG, Johnson EF, et al. Lancet 1994; 344: 578–581

Patients with chronic hepatitis D often have liver-kidney microsomal antibodies type 3 (LKM-3). These antibodies react with several microsomal antigens that have a molecular weight of 55 HDa and an isoelectric point of about 8. We studied the molecular nature of the antigen and, by immunoscreening a human liver cDNA expression library with KM-3 sera, found that uridine diphosphate glucuronosyl transferase (UGT) appeared as candidate antigens. We confirmed the identity of UGT as an antigen by reacting the sera with recombinant rabbit liver UGT proteins. Some sera reacted with rabbit UGT-2 proteins, but UGT-1 proteins were more sensitive and specific in detecting LKM-3 autoantibodies in patients sera. Anti-UGT-1 antibodies were detected in all LKM-3 positive sera from patients with hepatitis D and 1 out of 11 patients with autimmune hepatitis type 2. Sera from patients who had hepatitis B only did not react with UGT proteins.

Uridine diphosphate glucoronosyl transferases, candidate antigens of liver-kidney microsomal antibodies in hepatitis delta virus-infected patients

Patients with chronic hepatitis D often have liver-kidney microsomal antibodies type 3 (LKM-3). These antibodies react with several microsomal antigens that have a molecular weight of 55 HDa and an isoelectric point of about 8. We studied the molecular nature of the antigen and, by immunoscreening a human liver cDNA expression library with KM-3 sera, found that uridine diphosphate glucuronosyl transferase (UGT) appeared as candidate antigens. We confirmed the identity of UGT as an antigen by reacting the sera with recombinant rabbit liver UGT proteins. Some sera reacted with rabbit UGT-2 proteins, but UGT-1 proteins were more sensitive and specific in detecting LKM-3 autoantibodies in patient sera. Anti-UGT-1 antibodies were detected in all LKM-3 positive sera from patients with hepatitis D and 1 out of 11 patients with autoimmune hepatitis type 2. Sera from patients who had hepatitis B only did not react with UGT proteins.

Recognition of uridine diphosphate glucuronosyl transferases by LKM-3 antibodies in chronic hepatitis D

SummaryPatients with chronic hepatitis D often have liver-kidney microsomal antibodies type 3 (LKM-3). These antibodies react with several microsomal antigens that have a molecularweight of 55 KDa and an isoelectric point of about 8.We studied the molecular nature of the antigen and, by immunoscreening a human liver cDNA expression library with KM-3 sera, found that uridine diphosphate glucuronosyl transferases (UGT) appeared as candidate antigens. We confirmed the identity of UGT as an antigen by reacting the sera with recombinant rabbit liver UGT proteins. Some sera reacted with rabbit UGT-2 proteins, but UGT-1 proteins were more sensitive and specific in detecting LKM-3 autoantibodies in patient sera. Anti-UGT-1 antibodies were detected in all LKM-3 positive sera from patients with hepatitis D and 1 out of 11 patients with autoimmune hepatitis type 2. Sera from patients who had hepatitis B only did not react with UGT proteins.The UGT proteins are part of the phase II enzymes of drug metabolism and are the first such enzymes to be identified as human autoantigens.