Detection of polyreactive immunoglobulin G facilitates diagnosis in children with autoimmune hepatitis.

OBJECTIVE: The detection of autoantibodies is essential to diagnose autoimmune hepatitis (AIH). Particularly in children, specificity of autoantibodies decreases due to lower titers being diagnostic and being present not only in AIH but also in other liver diseases. Recently, quantification of polyreactive IgG (pIgG) for detection of adult AIH showed the highest overall accuracy compared to antinuclear antibodies (ANA), anti-smooth muscle antibodies (anti-SMA), anti-liver kidney microsomal antibodies (anti-LKM) and anti-soluble liver antigen/liver pancreas antibodies (anti-SLA/LP). We aimed to evaluate the diagnostic value of pIgG for pediatric AIH. DESIGN: pIgG, quantified using HIP1R/BSA coated ELISA, and immunofluorescence on rodent tissue sections were performed centrally. The diagnostic fidelity to diagnose AIH was compared to conventional autoantibodies of AIH in training and validation cohorts from a retrospective, European multi-center cohort from nine centers from eight European countries composed of existing biorepositories from expert centers (n = 285). RESULTS: IgG from pediatric AIH patients exhibited increased polyreactivity to multiple protein and non-protein substrates compared to non-AIH liver diseases and healthy children. pIgG had an AUC of 0.900 to distinguish AIH from non-AIH liver diseases. pIgG had a 31-73% higher specificity than ANA and anti-SMA and comparable sensitivity that was 6-20 times higher than of anti-SLA/LP, anti-LC1 and anti-LKM. pIgG had a 21-34% higher accuracy than conventional autoantibodies, was positive in 43-75% of children with AIH and normal IgG and independent from treatment response. CONCLUSION: Detecting pIgG improves the diagnostic evaluation of pediatric AIH compared to conventional autoantibodies, primarily owing to higher accuracy and specificity.

Evaluation of immunoserological detection of anti-liver kidney microsomal, anti-soluble liver antigen and anti-mitochondrial antibodies.

Autoantibodies are the diagnostic hallmark of autoimmune liver diseases. Indirect immunofluorescence (IFT) is the reference method for the detection of anti-mitochondrial antibodies (AMA) and anti-liver kidney microsomal type-1 (anti-LKM1) antibodies, and inhibition ELISA (iELISA) for anti-soluble liver antigen (anti-SLA) antibodies. Given the complexity of these techniques, commercial ELISAs have emerged as a practical alternative, but without head-to-head validations. This study evaluated the agreement between three commercial ELISAs and the reference techniques and the impact of polyreactive immunoglobulin G (pIgG), a recently described phenomenon in autoimmune hepatitis, on commercial ELISAs. Inter-rater reliability was assessed using Cohen-Kappa coefficient (κ). Forty-eight, 46, and 66 samples were analyzed for AMA, anti-LKM1, and anti-SLA, respectively. For AMA, one commercial assay showed high agreement (κ = 0.91 (0.78-1.00)) with the reference method, while the other two showed weak or moderate agreement. For anti-LKM1, only one commercial assay showed high agreement (κ = 0.86 (0.71-1.0)). For anti-SLA antibodies only moderate agreement was achieved (κ up to 0.71 (0.52-0.89)). There was a trend towards higher pIgG levels in false-positives in the commercial ELISAs. Patients with high suspicion of autoimmune liver diseases should be referred to reference laboratories with the capacity of performing gold standard methods if the initial ELISA-based screening was performed.

Antineutrophil cytoplasmic antibodies in bile are associated with disease activity in primary sclerosing cholangitis.

OBJECTIVE. Primary sclerosing cholangitis (PSC) is an autoimmune cholestatic liver disease of unknown etiology. The role of antineutrophil cytoplasmic antibodies (ANCAs) in the serum of patients with PSC remains unclear. We hypothesized that ANCA may be detectable in bile, potentially providing diagnostic and prognostic information. METHODS. Serum and bile were prospectively collected during endoscopic retrograde cholangiography (ERC) in 72 patients with PSC and other non-PSC obstructive biliary diseases. ANCA measurements were performed by indirect immunofluorescence (IIF). RESULTS. Immunoglobulin G (IgG) ANCA was detected significantly more often in the bile of PSC patients (15/39; 38%) than without (2/33; 6%) (p = 0.001). IgG ANCA in bile was associated with a ten times higher risk of PSC (p = 0.005). In addition, IgG ANCA positivity in bile was associated with the presence of dominant strictures (p = 0.03), cholangiographic severity (p = 0.004), number of ERC (p = 0.01) and interventions performed (p = 0.03). However, IgG ANCA in bile did not correlate with transplantation, cholangiocarcinoma or death. No association was observed between ANCA positivity in sera and ANA and ASCA positivity in sera or bile with the above-mentioned clinical features. CONCLUSIONS. The presence of ANCA in the bile of patients with PSC is a novel finding and highly suggestive of PSC. Biliary IgG ANCA correlates with the severity of bile duct strictures and the ensuing number of ERCs and interventions. Therefore, a positive ANCA status in bile may serve as a diagnostic and prognostic marker of the disease progression and biliary complications.

Epitope spreading of the anti-CYP2D6 antibody response in patients with autoimmune hepatitis and in the CYP2D6 mouse model.

Autoimmune hepatitis (AIH) is a serious chronic inflammatory disease of the liver with yet unknown etiology and largely uncertain immunopathology. The hallmark of type 2 AIH is the generation of liver kidney microsomal-1 (LKM-1) autoantibodies, which predominantly react to cytochrome P450 2D6 (CYP2D6). The identification of disease initiating factors has been hampered in the past, since antibody epitope mapping was mostly performed using serum samples collected late during disease resulting in the identification of immunodominant epitopes not necessarily representing those involved in disease initiation. In order to identify possible environmental triggers for AIH, we analyzed for the first time the spreading of the anti-CYP2D6 antibody response over a prolonged period of time in AIH patients and in the CYP2D6 mouse model, in which mice infected with Adenovirus-human CYP2D6 (Ad-h2D6) develop antibodies with a similar specificity than AIH patients. Epitope spreading was analyzed in six AIH-2-patients and in the CYP2D6 mouse model using SPOTs membranes containing peptides covering the entire CYP2D6 protein. Despite of a considerable variation, both mice and AIH patients largely focus their humoral immune response on an immunodominant epitope early after infection (mice) or diagnosis (patients). The CYP2D6 mouse model revealed that epitope spreading is initiated at the immunodominant epitope and later expands to neighboring and remote regions. Sequence homologies to human pathogens have been detected for all identified epitopes. Our study demonstrates that epitope spreading does indeed occur during the pathogenesis of AIH and supports the concept of molecular mimicry as a possible initiating mechanism for AIH.

Cytochrome P450 2A6: a new hepatic autoantigen in patients with chronic hepatitis C virus infection.

BACKGROUND/AIMS: Cytochromes P4502A6 (CYP2A6) and P4501A2 (CYP1A2) were described as hepatic autoantigens in the autoimmune polyglandular syndrome type-1 (APS-1). We evaluated the significance of anti-CYP2A6 and anti-CYP1A2 in several hepatic diseases in the absence of APS-1. METHODS: A radioligand assay (RLA) based on immunoprecipitation of [(35)S]-methionine-labeled CYP2A6 and CYP1A2 was used. Four hundred and thirty subjects with chronic viral hepatitis (n=185), autoimmune liver diseases (n=181), autoimmune rheumatic diseases (ARD, n=31) and healthy (n=33) were tested. RESULTS: Seven out of 366 patients with liver diseases were anti-CYP2A6 positive. Neither healthy nor ARD patients showed anti-CYP2A6. One out of 181 patients with autoimmune liver diseases tested anti-CYP2A6 positive. A significantly higher prevalence of anti-CYP2A6 (P<0.05) was detected with six out of seven patients positive in the viral hepatitis group. The latter were infected by flaviviruses (1 HGV/GBVC, 5 HCV). 4/5 HCV/anti-CYP2A6 positive sera were positive for anti-LKM-1 by immunofluorescence and for anti-CYP2D6 by RLA. None of the 430 sera recognized CYP1A2. CONCLUSIONS: For the first time CYP2A6 is reported as a hepatic autoantigen in patients with viral hepatitis caused by flaviviruses and in particular in HCV/anti-LKM-1 positive patients. Multicenter studies are needed in order to investigate the clinical importance of this novel finding. This study further supports that anti-CYP2A6 in the absence of flavivirus is rather limited to APS-1.

Characterization of a lipoyl domain-independent B-cell autoepitope on the human branched-chain acyltransferase in primary biliary cirrhosis and overlap syndrome with autoimmune hepatitis.

BACKGROUND AND AIMS: Antimitochondrial antibodies (AMA) which recognize pyruvate acetyltransferase (PDC-E2) represent a highly diagnostic feature of primary biliary cirrhosis (PBC). The analysis of immunofluorescence (IF)-AMA-positive sera in PBC patients indicates a conformational epitope located within the lipoyl binding domain of bovine branched-chain acyltransferase (BCKADC-E2) alone or in combination with AMA directed against PDC-E2 the significance of which is presently unclear. In the present study, immunoreactivities and disease associations of AMA against BCKADC-E2 were analyzed. B-cell autoepitopes on BCKADC-E2 were mapped by immunoprecipitation assay. METHODS: Sera of 96 IF-AMA-positive patients with serological evidence of anti-BCKADC-E2 alone (n = 26), anti-PDC-E2 alone (n = 15), and both anti-BCKADC-E2 and anti-PDC-E2 (n = 55) were analyzed by Western blot and ELISA in addition to an analysis of B cell autoepitopes on BCKADC-E2 by immunoprecipitation using in vitro translated, unmodified human proteins. Ninety-four patients without IF-AMA [blood donors (n = 30), rheumatoid arthritis (n = 40), autoimmune hepatitis (AIH)(n = 10) and primary sclerosing cholangitis (PSC) (n = 14) served as controls. RESULTS: Eighty of 81 (99%) sera positive for BCKADC-E2 recognized the full length, mature protein, while only 2/10 AIH sera and none of the other controls showed reactivity. Of the 68 PBC sera 58 (85%) recognized the N-terminus consisting of aa 1-144 representing the lipoyl domain. Surprisingly, C-terminal sequences (aa 143-421) were recognized by 46 out of 68 sera (68%). Three PBC sera reacted with the C-terminus only. Only 1/7 serum from patients with an "overlap syndrome of PBC and AIH" was reactive with C-terminal sequences. CONCLUSIONS: Our analysis of BCKADC-E2-positive PBC sera identified a novel B cell epitope on the C-terminal part of the human protein. Our data indicate that a distinct subset of AMA recognize sequence(s) on BCKADC-E2 which located outside of the lipoyl binding domain. The absence of immunoreactivity against C-terminal sequences may serve as a marker differentiating patients with PBC and overlap syndrome of PBC with AIH.

Increased incidence of anti-LKM autoantibodies in a consecutive cohort of hepatitis C patients from central Greece.

OBJECTIVES: In Greece, there are insufficient data regarding the presence of non-organ and liver-related autoantibodies in hepatitis C patients. This study in a consecutive cohort of 39 such patients from central Greece investigates (1) the prevalence of non-organ and liver-related autoantibodies, and (2) the reactivity of anti-liver-kidney microsomal type 1 antibodies (in the case of positivity with at least one of the methods used) against their molecularly defined antigens. DESIGN: All serum samples were tested by standard and molecular assays for the presence of anti-nuclear antibodies, smooth muscle antibodies, anti-liver-kidney microsomal type 1 antibodies, antibodies against parietal cells, anti-CYP2A6, anti-CYP1A2 and anti-CYP2D6 autoantibodies. METHODS: Indirect immunofluorescence, competitive enzyme-linked immunosorbent assays, immunoblotting and novel radioligand assays based on immunoprecipitation of [35S]-methionine labelled recombinant CYP2A6, CYP1A2 and CYP2D6 His-taq fusion proteins produced by in vitro transcription/translation were used. RESULTS: Seven out of 39 patients (17.9%) tested positive for smooth muscle antibodies, 2/39 (5.1%) tested positive for anti-nuclear antibodies, 1/39 (2.5%) tested positive for parietal cell antibodies, and 4/39 (10.3%) were found to be anti-liver-kidney microsomal positive (with at least one of the methods used). All sera were negative for anti-CYP2A6 and anti-CYP1A2 autoantibodies. Three out of four anti-liver-kidney microsomal positive samples had the typical liver-kidney microsomal staining pattern shown by indirect immunofluorescence. However, none tested positive for anti-CYP2D6 autoantibodies using the competitive CYP2D6 enzyme-linked immunosorbent assay, the specific CYP2D6 radioligand assay, and western blot using either human microsomes or recombinant CYP2D6. The fourth patient tested negative for anti-liver-kidney autoantibodies by either indirect immunofluorescence or the competitive enzyme-linked immunosorbent assay, but was repeatedly positive for anti-CYP2D6 autoantibodies by the sensitive and specific radioligand assay. Western blot experiments using human microsomes in this patient serum revealed two bands of 50 kDa and 55 kDa that documented as anti-CYP2D6 and anti-uridine triphosphate glucuronosyltransferase autoantibodies when recombinant CYP2D6 and recombinant uridine triphosphate glucuronosyltransferase autoantigens were used for immunoblot, respectively. CONCLUSIONS: A relatively high incidence of anti-liver-kidney microsomal autoantibodies (10.3%) was found in a consecutive sample of Greek patients with hepatitis C. The expanded panel of assays, however, failed to document CYP2D6 as the target autoantigen of anti-liver-kidney microsomal autoantibodies in most patients. We report for the first time the detection of parietal cell antibodies and both anti-CYP2D6 (anti-liver-kidney microsomal type 1) and anti-uridine triphosphate glucuronosyltransferase (anti-liver-kidney microsomal type 3) autoantibodies in patients who were hepatitis C positive/hepatitis D negative. Further studies are needed to confirm our findings and to determine whether these preliminary results have a clinical importance or not.