Linkage of HLA to myasthenia gravis and genetic heterogeneity depending on anti-titin antibodies.

BACKGROUND: MG is an autoimmune disease of the neuromuscular junction. MG with thymus hyperplasia has been associated with, but not genetically linked to, the HLA-DR3 haplotype. OBJECTIVE: To re-evaluate the association of HLA with MG in 656 patients with generalized disease and to test linkage of HLA to MG with thymus hyperplasia. METHOD: Patients were genotyped for HLA-DRB1. Data analysis included case-control comparisons after subgrouping patients by thymus histopathology. The transmission of parental alleles to MG offspring with thymus hyperplasia was studied in simplex families using the transmission/disequilibrium test (TDT) as a test of linkage. RESULTS: MG with thymus hyperplasia was positively associated with DR3 (OR = 4.5, p = 1 x 10(-6)) and negatively associated with DR7 (OR = 0.28, p = 1 x 10(-6)), based on both case-control comparisons and TDT. No association was detected with thymomas. Conversely, patients who lacked thymus anomalies but expressed anti-titin antibodies (ATA) had an increase of DR7 (OR = 2.08, p = 4 x 10(-3)) and a decrease of DR3 (OR = 0.33, p = 9 x 10(-3)). CONCLUSIONS: The authors established linkage of HLA to MG and thymus hyperplasia, defining the MYAS1 locus. Moreover, DR3 and DR7, or closely linked genes, have opposing effects on MG phenotypes. Nonthymomatous patients with ATA may be a pathogenetically distinct subset of MG patients.

[Auto-antibodies against acetylcholine receptors in myasthenia gravis]. / Auto-anticorps anti-récepteur de l'acétylcholine au cours de la myasthénie.

USEFULLNESS OF DETECTION OF ANTIBODIES AGAINST THE ACETYLCHOLINE RECEPTOR IN THE DIAGNOSIS OF MYASTHENIA GRAVIS (MG): MG is an autoimmune disorder including a fatigability of skeletal muscles and the presence of antibodies against the acetylcholine receptor (AChRAbs). These Abs are pathogenic by blocking the acetylcholine receptor within the neuromuscular junction. A transient neonatal MG occurs in 12% of babies born to myasthenic mothers. AChRAbs have been found in 77 to 89% of systemic MG and in 47 to 60% in ocular form of MG. These Abs are generally directed against the "Main Immunogenic Region" (MIR) within the a subunit of the receptor; others are directed against beta, gamma and epsilon subunits. Eleven to 23% of the MG are negative for the Abs directed against the a subunit reinforcing the interest to detect the other AChRAbs, specially the anti-epsilon subunit Abs and the Abs directed against the muscle-specific receptor tyrosine kinase. MULTICENTER EVALUATION: In order to compare the results of AChRAbs for a same patient from various laboratories, a multicenter study was performed on 26 sera with different titers of AChRAbs using radioimmunoassays. Six french laboratories have participated to this study. The antigen was obtained from TE671 cells for 5 laboratories (3 used a commercial test) or from human muscle (1 laboratory). At the end of the study, the qualitative analysis of the results showed a concordance of 92.3%. The interpretation of AChRAbs assays can thus be done by clinicians whatever the test is used. A same quality control is nowadays used by the six laboratories in order to estimate quantitative results according to the assay performed. USEFULNESS OF THE OTHER ABS: The Abs directed against the striated muscle are good markers of thymoma in MG but are not specific for MG. The Abs directed against titin which is the major target of the anti-striated muscle Abs, are also good biological markers of thymoma specially in MG adults younger than 60 years. The detection of anti-myoid cells and thymic reticuloepithelial cells Abs is no more useful due to a lower sensitivity compared to the Abs directed against the striated muscle.

Anti-titin antibodies in myasthenia gravis: tight association with thymoma and heterogeneity of nonthymoma patients.

BACKGROUND: Titin is the major autoantigen recognized by anti-striated muscle antibodies, which are characteristic of generalized myasthenia gravis (MG). OBJECTIVE: To seek a correlation between anti-titin antibodies and other features of MG patients, including histopathology, age at diagnosis, anti-acetylcholine receptor (anti-AChR), autoantibody titers, and clinical severity. METHODS: A novel, highly specific radioligand assay was performed on a large group of 398 patients with generalized MG. RESULTS: Among thymectomized patients, anti-titin antibodies were present in most patients with thymoma (56/70 [80%]), contrasting with only a minority of patients with thymus atrophy or hyperplasia (17/165 [10%]). They were also present in 64 (41%) of 155 nonthymectomized patients who had a radiologically normal thymus. In these patients and in those who had a histologically normal thymus, anti-titin antibodies were associated with a later age at onset of disease and with intermediate titers of anti-AChR antibodies. After controlling for these 2 variables, disease severity was not significantly influenced by anti-titin antibodies. CONCLUSIONS: Anti-titin antibodies are a sensitive marker of thymoma associated with MG in patients 60 years and younger, justifying the insistent search for a thymoma in MG patients of this age group who have these antibodies. In nonthymoma patients, anti-titin antibodies represent an interesting marker complementary to the anti-AChR antibody titer, identifying a restricted subset of patients. These clinical correlations should prompt further studies to examine the mechanisms leading to the production of anti-titin antibodies.

The activity of immunoregulatory T cells mediating active tolerance is potentiated in nonobese diabetic mice by an IL-4-based retroviral gene therapy.

Splenocytes from nonobese diabetic mice overexpressing murine IL (mIL)-4 upon recombinant retrovirus infection lose their capacity to transfer diabetes to nonobese diabetic-scid recipients. Diabetes appeared in 0-20% of mice injected with mIL-4-transduced cells vs 80-100% of controls injected with beta-galactosidase-transduced cells. Protected mice showed a majority of islets (60%) presenting with noninvasive peri-insulitis at variance with beta-galactosidase controls that exhibited invasive/destructive insulitis. Importantly, in all recipients, the transduced proteins were detected within islet infiltrates. Infiltrating lymphocytes from recipients of mIL-4-transduced cells produced high levels of mIL-4, as assessed by ELISA. In recipients of beta-galactosidase-transduced cells, approximately 60% of TCRalphabeta(+) islet-infiltrating cells expressed beta-galactosidase, as assessed by flow cytometry. The protection from disease transfer is due to a direct effect of mIL-4 gene therapy on immunoregulatory T cells rather than on diabetogenic cells. mIL-4-transduced purified CD62L(-) effector cells or transgenic BDC2.5 diabetogenic T cells still transferred disease efficiently. Conversely, mIL-4 transduction up-regulated the capacity of purified immunoregulatory CD62L(+) cells to inhibit disease transfer. These data open new perspectives for gene therapy in insulin-dependent diabetes using T cells devoid of any intrinsic diabetogenic potential.

[Autoimmune hepatitis. Physiopathologic, clinical, histological, and therapeutic features]. / Hépatites auto-immunes. Aspects physiopathologiques, cliniques, histologiques et thérapeutiques.

Autoimmune hepatitis is characterized by an inflammation of the portal tract with lymphocytes and plasma cells, an hypergammaglobulinemia and a variety of circulating autoantibodies. The presence of smooth muscle antibodies and/or antinuclear antibodies define type 1. Type 2 is characterized by the presence of liver-kidney--microsomal antibodies. Environmental, genetic and infectious factors may explain the autoreactivity of T cells. Different non specific clinical features may be present. Sometimes the presentation may be an acute hepatitis; in the remainder, the disease may not be recognized until liver damage is advanced. Hypergammaglobulinemia and presence of circulating autoantibodies are the key for diagnosis. The association of prednisolone in combination with azathioprine remains the established treatment. If relapse or non response occur, other immunosuppressive therapy such as cyclosporin may be useful. Liver transplantation is reserved for (sub)fulminant forms and end stage liver disease.

Autoantibodies associated with acute rejection after liver transplantation for type-2 autoimmune hepatitis.

Autoantibodies, markers of autoimmune diseases, can also be detected in chronic allograft rejection. However, the appearance of these autoantibodies in acute rejection after orthotopic liver transplantation has not yet been reported. Liver-kidney-microsome type-1 (LKM-1) antibodies directed against the autoantigen cytochrome CYP2D6 define a group of patients with autoimmune hepatitis type-2 (AIH-2), distinct from autoimmune hepatitis type-1 (AIH-1) in which anti-nuclear antibodies and anti-smooth muscle antibodies (SMA) with actin specificity are present in patient sera. Autoantibodies were studied by the quantitative CYP2D6 radioligand assay (RLA) that uses a radiolabeled CYP2D6 as antigen, immunoblotting using recombinant CYP2D6 protein and human liver microsomal and cytosolic fractions, and indirect immunofluorescence (IIF) using rat kidney-stomach-liver cryostat sections. In addition, the specificity of anti-SMA was detected by IIF on HEp2 cell line harvested with colchicin. This report describes the time course of CYP2D6 antibodies and the appearance of anti-SMA (without anti-actin, cytokeratin and vimentin reactivity) associated with acute rejection during a 2-year follow-up, in a patient who underwent transplantation at end-stage type 2 autoimmune hepatitis. In addition, we report a new reactivity against an unknown 40-kDa protein using a rat cytosolic fraction. The detection of autoantibodies in sequential samples may be important to better predict rejection or relapse, and to establish adequate therapy.

Quantitative radioligand assays using de novo-synthesized recombinant autoantigens in connective tissue diseases: new tools to approach the pathogenic significance of anti-RNP antibodies in rheumatic diseases.

OBJECTIVE: To describe new assays for the detection and quantification of antibodies to RNPs in rheumatic diseases, using soluble nuclear antigens synthesized de novo in reticulocyte lysates. METHODS: Sera from 381 patients with various rheumatic diseases, including 212 patients with systemic lupus erythematosus (SLE), were analyzed in order to evaluate the sensitivity and specificity of serum autoantibody reactivities to several recombinant soluble autoantigens: U1-A RNP, Sm-B, SSA/Ro 52 and SSA/Ro 60, SSB/La, and Ku. Radioligand assays (RLAs) were performed following the in vitro transcription and translation of each autoantigen from the corresponding complementary DNA, labeled with 35S-methionine. The radiolabeled protein was then bound by the specific serum autoantibody, forming immune complexes that were captured by protein A-Sepharose beads and quantified by counting the radioactivity. RESULTS: Among the SLE patients, 44% were positive for anti-U1-A RNP activity, 34% for anti-Sm-B, 44% for anti-SSA (32% for Ro 52 and 46% for Ro 60), 32% for anti-SSB/La, and 11% for anti-Ku reactivities. SSA antibodies had a high frequency in patients with mixed connective tissue disease (MCTD) (80%); 65% of these patient sera reacted with Ro 52, 45% with Ro 60, and 45% with U1-A RNP. Twenty percent of the MCTD patients also exhibited antibodies to Sm-B and Ku. In patients with Sjögren's syndrome, anti-SSA was the main anti-RNP antibody (63%), together with SSB/La antibodies (44%). Among patients with inflammatory myopathy, only antibodies against Ro 52 (36%) and Ro 60 (36%) were present. These new RLA allowed observation of a strong correlation (P < 0.0001) between Sm-B antibody levels and the severity of SLE (as measured by the SLE Disease Activity Index), and establishment of a correlation between anti-U1-A RNP antibodies and the occurrence of SLE nephritis (P < 0.02). All RLAs were highly specific for the antigen tested and displayed, in the disease groups studied, a higher sensitivity than conventional immunodiffusion assays. CONCLUSION: These highly sensitive, specific, and quantitative RLAs represent new tools for the detection of autoantibodies to RNP antigens in rheumatic diseases, and may be useful for (differential) diagnosis in clinical practice.

Extrahepatic manifestations associated with hepatitis C virus infection. A prospective multicenter study of 321 patients. The GERMIVIC. Groupe d'Etude et de Recherche en Medecine Interne et Maladies Infectieuses sur le Virus de l'Hepatite C.

From January 1996 to January 1997, 321 patients with an average age of 46 +/- 16 years and chronically infected with hepatitis C virus (HCV) were prospectively enrolled in a study designed to determine the prevalence of extrahepatic manifestations associated with HCV infection in a large cohort of HCV patients, to identify associations between clinical and biologic manifestations, and to compare the results obtained in human immunodeficiency virus (HIV)-positive versus HIV-negative subsets. In a cross-sectional study, clinical extrahepatic manifestations, viral coinfections with HIV and/or hepatitis B virus, connective tissue diseases, and a wide panel of autoantibodies were assessed. Thirty-eight percent (122/321) of patients presented at least 1 clinical extrahepatic manifestation including arthralgia (60/321, 19%), skin manifestations (55/321, 17%), xerostomia (40/321, 12%), xerophthalmia (32/321, 10%), and sensory neuropathy (28/321, 9%). Main biologic abnormalities were mixed cryoglobulins (110/196, 56%), thrombocytopenia (50/291, 17%), and the presence of the following autoantibodies: antinuclear (123/302, 41%), rheumatoid factor (107/280, 38%), anticardiolipin (79/298, 27%), antithyroglobulin (36/287, 13%) and antismooth muscle cell (27/288, 9%). At least 1 autoantibody was present in 210/302 (70%) of sera. By multivariate logistic regression analysis, 4 parameters were significantly associated with cryoglobulin positivity: systemic vasculitis (p = 0.01, odds ratio OR[ = 17.3), HIV positivity (p = 0.0006, OR = 10.2), rheumatoid factor positivity (p = 0.01, OR = 2.8), and sicca syndrome (p = 0.03, OR = 0.27). A definite connective tissue disease was noted in 44 patients (14%), mainly symptomatic mixed cryoglobulinemia and systemic vasculitis, HIV coinfection (23%) was associated with 3 parameters: anticardiolipin (p = 0.003, OR = 4.18), thrombocytopenia (p = 0.01, OR = 3.56), and arthralgia or myalgia (p = 0.017, OR = 0.23). HIV-positive patients presented more severe histologic lesions (p = 0.0004). Extrahepatic clinical manifestations in HCV patients involve primarily the skin and joints. The most frequent immunologic abnormalities include mixed cryoglobulins, rheumatoid factor, antinuclear, anticardiolipin, and antithyroglobulin antibodies. Cryoglobulin positivity is associated with systemic vasculitis and rheumatoid factor and HIV positivity. HIV coinfection is associated with arthralgia or myalgia, anticardiolipin antibodies, and thrombocytopenia.

Sera of children with hepatitis C infection and anti-liver-kidney microsome-1 antibodies recognize different CYP2D6 epitopes than adults with LKM+/HCV+ sera.

BACKGROUND: Liver-kidney microsome type 1 (LKM1) antibodies are specific markers of autoimmune hepatitis (AIH) type 2. Antibodies to LKM1 have been found in 2% to 3% of adults infected with hepatitis C virus (HCV) without AIH. Thirty percent of these antibodies are directed against linear sequences of CYP2D6 protein. LKM1 antibodies in HCV+/LKM1+ sera and in sera of AIH patients do not recognize the same CYP2D6 epitopes. The current study was conducted to determine whether LKM1 antibodies in HCV+/LKM1+ children's sera are the result of the same immune response as the antibodies described in AIH type 2 and in HCV+/LKM1+ adult patients. METHODS: Sera from 10 HCV+/LKM1+ children were tested against human liver microsomal and cytosolic proteins by Western blot analysis and against synthetic peptides of the CYP2D6 sequence between amino acids 200 and 429 by dot blot. The same sera were tested against radiolabeled CYP2D6 by immunoprecipitation. RESULTS: Four of 10 sera tested by Western blot analysis showed immunoglobulin (Ig) G-type antibodies against CYP2D6, and 2 had antibodies against proteins of 58, 66, and 84 kDa. One of the sera also contained IgM-type anti-66-kDa and 84-kDa proteins. The radioligand test detected anti-CYP2D6 antibodies in 9 of 10 patients. Five of the anti-CYP2D6-positive sera recognized a peptide between amino acids 200 and 429 including amino acids 254-271. CONCLUSIONS: Most HCV+/LKM1+ sera from children recognize conformational epitopes of the CYP2D6 antigen, and half recognize linear epitopes. Some HCV+/LKM1+ sera demonstrated antibodies against the AIH type 2 main antigenic site of the CYP2D6. Screening of HCV RNA should be performed before starting treatment of presumed autoimmune hepatitis associated with LKM1.

N-terminus cleavage of bcl-2 by a novel cellular non-ICE cysteine proteinase.

The bcl-2 protein plays an essential role in preventing cell death. Its activity is regulated through association with bcl-2 homologous and nonhomologous proteins and also by serine phosphorylation. We now report that bcl-2 can be proteolytically cleaved towards its N-terminus by a cysteine proteinase present in RL-7 lymphoma cell lysates, yielding a major product of apparent MW 20 kDa, different from the products of bcl-2 cleavage by HIV protease. Moreover, bcl-2 proteins mutated for Asp residues at positions 31 and 34 were efficiently cleaved by RL-7 cell lysates, indicating that this proteolytic activity is distinct from the caspase-3 that cleaves bcl-2 at Asp 34. This bcl-2 cleaving activity is inhibited by E-64 and is therefore distinct from the proteinases of the ICE/Ced-3 family (caspases), whereas reciprocally, ICE (caspase-1) is unable to cleave bcl-2. It is optimally active at pH 5, a feature distinguishing it from calpain, another non-ICE cysteine proteinase which has been associated with apoptosis. This novel bcl-2 cleaving protease, although constitutively present in RL-7 cells and resting peripheral blood lymphocytes (PBL) was upregulated following induction of apoptosis in RL-7 cells or mitogen activation in PBL. The N-terminus of bcl-2 which contains the BH4 domain that binds the kinase Raf-1 and the phosphatase calcineurin is essential for anti-apoptotic activity. Its cleavage might provide a novel post-translational mechanism for regulating bcl-2 function and could amplify ongoing programmed cell death.

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 levels and 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.

Young age and HLA markers enhance the risk of progression to type 1 diabetes in antibody-positive siblings of diabetic children.

The contribution of autoantibodies, HLA markers and age to long-term estimates of risk of type 1 diabetes were examined after a median of 11 years (range 7.5-14) during the follow-up in a cohort of 234 siblings (aged 2-29 years) of French children with recent-onset type 1 diabetes, of whom 12 (5.1%) developed diabetes. We evaluated islet cell antibodies (ICA) by indirect immunofluorescence and autoantibodies to insulin (IAA), to the 65 kDa isoform of glutamic acid decarboxylase (GADA) and to the IA-2 protein (IA-2A) by radioligand assay in sequential serum samples. Among the 234 siblings of type 1 diabetic patients screened, 27 were positive for at least one antibody, 11 of whom progressed to develop type 1 diabetes during the follow-up (sensitivity, 92%, predictive value, 41%). Among the four antibodies tested individually, ICA had the highest sensitivity (83%) but a poor predictive value (59%) and IA-2A the highest predictive value (70%). IAA and GADA both exhibited poor sensitivity and predictive value. Combinations of antibodies achieved better predictive values than antibodies tested individually. Satisfactory predictive values were obtained for the combination of GADA with IA-2A (83%), for any combination of at least two antibodies other than ICA (70%) and for the combination of ICA with at least one other antibody (69%). The risk estimates were highest in the presence of three or four antibodies, whether comprising ICA or not, but with a concomitant loss of sensitivity. For most antibody combinations, cumulative risks showed progression from approximately 50% after 5 years to 100% after 13 years. HLA-DR3/4 was significantly more frequent in siblings developing type 1 diabetes than in non-diabetic siblings (9/12 vs. 39/217, relative risk (RR)=14, P

A new approach to cytochrome CYP2D6 antibody detection in autoimmune hepatitis type-2 (AIH-2) and chronic hepatitis C virus (HCV) infection: a sensitive and quantitative radioligand assay.

Antibodies specific for cytochrome CYP2D6, formally known as liver-kidney-microsome type-1 antibodies (LKM-1), are characteristically found in a subgroup of patients presenting autoimmune hepatitis. They are also found in some patients with chronic HCV infection. These autoantibodies are usually detected by indirect immunofluorescence, immunoblotting and ELISA tests. In an attempt to set up a more sensitive detection assay we developed a quantitative immunoprecipitation radioligand assay using a 35S-methionine-labelled CYP2D6 antigen obtained by in vitro transcription and translation synthesis. All 16 sera from AIH-2 patients strongly bound to this CYP2D6 antigen. Two of the nine sera (22%) from AIH-2 patients that presented only liver cytosol-1 antibodies also bound to CYP2D6. All 24 sera from HCV patients that were positive for LKM-1 antibodies by indirect immunofluorescence were also positive using this CYP2D6 radioligand assay. Lastly, all 15 sera from HCV patients negative for LKM-1 antibodies were negative by this test. The present results support the view that this quantitative radioligand assay is more sensitive than immunoblotting and ELISA CYP2D6 assays, and that it could be used in combination with indirect immunofluorescence assay.

Thymocyte Fas expression is dysregulated in myasthenia gravis patients with anti-acetylcholine receptor antibody.

Myasthenia gravis (MG) is a human autoimmune disease mediated by anti-acetylcholine receptor (AChR) antibodies. The thymus is probably the site where the autoimmune response is triggered and maintained. Recent reports have linked various autoimmune disease with defective Fas expression. We thus analyzed Fas expression in thymocytes and peripheral blood lymphocytes (PBL) from MG patients. The proportion of a thymocyte subpopulation with strong Fas expression (Fas(hi)) was markedly enhanced in MG patients with anti-AChR antibodies (P < .0003, compared with controls). In this group of patients, the proportion of CD4+ Fas(hi) and CD4+ CD8+ Fas(hi) thymocytes were significantly increased (P < .002 for both subsets). Fas(hi) thymocytes were enriched in activated cells and showed intermediate CD3 expression. They were preferentially Vbeta5.1-expressing cells, previously shown to be enriched in potentially autoreactive cells. The proliferative response of thymocytes from MG patients to peptides from the AChR was abolished after depletion of Fas(hi) cells. Fas(hi) thymocytes were sensitive to an agonistic anti-Fas antibody. In peripheral blood, Fas(hi) lymphocytes proportion was not significantly modified in MG patients whatever their anti-AChR antibody titer, compared with controls. Altogether, these results indicate that Fas(hi) thymocytes, which accumulate in MG patients with anti-AChR antibodies, could be involved in the autoimmune response that targets the AChR.

HLA-associated heterogeneity of the humoral response to islet antigens in insulin-dependent diabetes.

Insulin-dependent diabetes mellitus (IDDM) is associated with susceptibility HLA class II alleles. Islet cell antibodies (ICA), detected by indirect immunofluorescence on pancreas sections, represent the best marker of the disease. Autoantibodies to glutamic acid decarboxylase (GADA), one major islet antigen, do not totally account for ICA reactivity, suggesting heterogeneity of the anti-islet humoral response. In 97 patients with IDDM we have correlated ICA heterogeneity with clinical markers and DR and DQ alleles. ICA were found in 81% of the patients, and in 33% the serum blocked the binding to islet cells of reference sera with a granular fluorescence pattern. GADA were found in 62% of cases. Patients with high GADA titers and blocking sera were older at onset and less often had a family history of IDDM, suggesting that these antibodies might be a marker of slow progression to IDDM. ICAs were not associated with particular HLA DR or DQ alleles. Conversely, GADA were less frequent than ICA in DR4 subjects but not in the other groups. Moreover, among DR4 non-DR3 patients, GADA were found almost exclusively in DRB1*0401 patients but not in other DR4 subtypes. There was an association of GADA with DQ alleles but it was secondary to linkage disequilibrium between DR and DQ loci. In conclusion, the heterogeneity of the humoral response in IDDM is controlled by HLA class II genes and correlates with clinical heterogeneity.

Conformational epitopes on CYP2D6 are recognized by liver/kidney microsomal antibodies.

BACKGROUND/AIMS: Four linear antigenic sites have been shown on the CYP2D6 molecule that are recognized by serum positive for liver/kidney microsomal antibody (LKM) type 1. The aim of this study was to search for antibodies against CYP2D6 conformational antigenic sites in LKM-1-positive sera. METHODS: The capacity of four LKM-1-positive sera, before and after absorption with synthetic peptides representing CYP2D6 linear antigenic sites, and rabbit sera against linear antigenic sites between CYP2D6 amino acids 254-271 and 373-389 to inhibit the O-demethylation of dextromethorphan by CYP2D6 was tested in vitro. RESULTS: Inhibition of O-demethylation of dextromethorphan was not modified by absorption of antibodies against linear CYP2D6 antigenic sites. In addition, rabbit sera against two of these sites did not inhibit the reaction. These results strongly suggest that antibodies against CYP2D6 conformational antigenic sites were present in LKM-1-positive sera. CONCLUSIONS: The autoimmune response against CYP2D6 is directed against linear and conformational antigenic sites. These results strengthen the argument that the LKM-1 response is polyclonal and antigen driven.

Cytochrome P450IID6 recognized by LKM1 antibody is not exposed on the surface of hepatocytes.

LKM1 autoantibody, directed against P450IID6, is accepted as a marker of a particular type of autoimmune hepatitis, but its role in the pathogenesis of the disease is controversial. Localization of P450IID6 on the cell surface of rat hepatocytes was previously reported, suggesting that membrane-bound P450IID6 could be the target of LKM1 antibodies, thus allowing immune lysis of hepatocytes. The objective of the present study was to determine, using various methods, the cell localization of P450IID6 in human and rat hepatocytes. Incubation of rat and human hepatocytes with LKM1-positive serum showed slight, if any, cell membrane staining using immunofluorescence, immunoperoxidase and immunoelectron microscopic studies. No staining of the plasma membrane of human hepatocytes was observed when incubations were carried out with immunoaffinity-purified antibody directed against peptide 254-271, the main epitope of P450IID6 recognized by all LKM1 sera tested. Chinese hamster ovary cells, transfected with the complete P450IID6 cDNA and incubated with the supernatant from a B cell lymphoblastoid cell line prepared with the lymphocytes of a LKM1-positive patient, did not show any staining of the cell surface by immunofluorescence. Incubation of rat microsomal fraction vesicles with LKM1-positive serum, followed by protein A-gold immunoelectron microscopy, displayed a staining of almost all vesicles, confirming that P450IID6 is present on the cytoplasmic side of the microsomal membrane, which makes it unable to be expressed on the cell surface even if it were transported from the endoplasmic reticulum (ER). Sulpho NHS Biotin labelling of rat hepatocyte cell membranes did not show the presence of a 50-kD molecule that could have reacted with LKM1 antibody. DNA sequencing of exon 1 of the CYP2D6 gene of a patient positive for LKM1 antibody did not show any difference from that of the normal published sequence of the gene. This does not favour an alteration of the NH2 terminal sequence of the P450IID6 molecule that could explain a translocation of the molecule to the luminal side of the ER, allowing its expression on the cell surface. These results indicate that, in all likelihood, P450IID6 molecule is not present on the cell surface of normal rat and human hepatocytes. Other mechanisms than antibody-mediated cell lysis directed against membrane P450IID6 antigenic determinants must be found to account for the destruction of hepatocytes observed in this disease.

Characterization of anti-liver-kidney microsome antibody (anti-LKM1) from hepatitis C virus-positive and -negative sera.

BACKGROUND: Hepatitis C virus-related antibodies were found in sera positive for antibodies to liver/kidney microsome antibody, usually considered a marker of autoimmune hepatitis. The aim of this study was to analyze the specificity of this autoantibody in sera from patients with and without hepatitis C virus infection. METHODS: Fifteen anti-hepatitis C virus- and anti-liver kidney microsome-positive sera were compared with 11 sera from patients with autoimmune hepatitis, for reactivity against rat and human liver microsomal proteins, P450IID6 recombinant proteins, and various synthetic peptides spanning the 241-429 amino acids sequence of the P450IID6. RESULTS: Ten of 11 sera from patients with autoimmune hepatitis bound to recombinant proteins spanning the P450IID6 region between amino acids 72 and 458. These sera bound to the 254-271 peptide, and some also recognized the 321-351, 373-389 and 410-429 peptides. Four of 15 antihepatitis C virus recognized the fusion protein coded by the full-length P450IID6 complementary DNA; 3 of them also reacted with the P450IID6 region between amino acids 72-456. Only 1 sera recognized the 321-351 peptide. CONCLUSIONS: P450IID6 antigenic sites recognized by anti-hepatitis C virus-positive sera were different from those recognized by sera from patients with autoimmune hepatitis.

Identification and analysis of cytochrome P450IID6 antigenic sites recognized by anti-liver-kidney microsome type-1 antibodies (LKM1).

Anti-liver-kidney microsome type-1 antibodies (LKM1), present in sera from a group of patients with autoimmune hepatitis, are directed against P450IID6. Previous work, using cDNA constructions spanning most of the P450IID6 protein defined the main immunogenic site between the amino acids (aa), 254-271 and predicted the presence of other putative immunogenic sites in the molecule. Fusion proteins from new cDNA constructions, spanning so-far-untested regions between aa 1-125 and 431-522, were not recognized by LKM1-positive sera. Synthetic peptides, representing sequences from putative immunogenic regions or previously untested regions, allowed a precise definition of four antigenic sites located between peptides 257-269, 321-351, 373-389 and 410-429, which were recognized, respectively, by 14, 8, 1 and 2 out of 15 LKM1-positive sera tested. The minimal sequence of the main antigenic site (peptide 257-269) recognized by the autoantibody was established to be WDPAQPPRD (peptide 262-270). In addition, deletion and replacement experiments showed that aa 263 (Asp) was essential for the binding of the autoantibody to peptide 262-270. Analysis of the second most frequently recognized peptide between aa 321-351, was performed using peptides 321-339 and 340-351 in competitive inhibition studies. Complete elimination of antibody binding to peptide 321-351 obtained by absorption of both shorter peptides indicated that peptide 321-351 is a discontinuous antigenic site. LKM1-positive sera reacting against peptide 321-351 recognized either both the shorter peptides or just one of them preferentially. Results of the present study suggest that the production of LKM1 antibodies is an antigen-driven, poly- or oligoclonal B cell response. The identification of antigenic sites will allow: (i) the development of specific diagnostic tests and (ii) further studies on the pathogenic value of LKM1 antibodies in autoimmune hepatitis.