A major CYP2D6 autoepitope in autoimmune hepatitis type 2 and chronic hepatitis C is a three-dimensional structure homologous to other cytochrome P450 autoantigens.

Liver-kidney microsomal antibodies type 1 (LKM) are a diagnostic marker for autoimmune hepatitis type 2 (AIH-2), however, LKM autoantibodies are also detected in a small percentage of patients with chronic hepatitis C. The major target of LKM antibodies as evidenced by indirect immunofluorescence is cytochrome P4502D6 (CYP2D6). Anti-CYP2D6 titers of 62 LKM positive sera, 196 sera of patients with hepatic and rheumatic diseases and 33 sera of healthy blood donors (BD) were determined by an in vitro transcription/in vitro translation assay (ITT). Twenty five out of 26 AIH-2 sera and 33/36 LKM positive hepatitis C virus (HCV) sera were anti-CYP2D6 positive by ITT and antibody titers were similar in both patient groups. Epitope mapping experiments were performed by a series of truncated CYP2D6 proteins and by single epitopes of 257-269, 321-351, 373-389 and 410-419 amino acid (aa) expressed as DHFR-fusion proteins in Escherichia coli. The major linear epitope consists of 257-269 aa. This epitope is recognized with a significantly higher prevalence (64%) in AIH-2 than in LKM sera from patients with chronic hepatitis C (24%) (p < 0.001). None of the other autoepitopes showed significant differences in the prevalence of recognition by sera from both patient groups. Minor binding sites consisted of 321-351 aa, which was recognized by less than 20% of LKM sera and in the C-terminal region of 350-494 aa, which was recognized by less than 5% of LKM sera Our study revealed an epitope of 321-379 an on CYP2D6, which was shown to be conformation dependent. It was recognized by the vast majority of LKM sera, specifically by 76% of sera from HCV positive LKM patients and also by 76% of sera from patients with AIH-2. This epitope is homologous to three-dimensional epitopes detected by autoantibodies directed against hepatic cytochromes P450s in drug induced hepatitis and to an autoepitope on CYP21B associated with adrenal failure.

Persistence of autoantibodies against recombinant mitochondrial and nuclear pore proteins after orthotopic liver transplantation for primary biliary cirrhosis.

Primary biliary cirrhosis is an autoimmune disease characterized by high titer autoantibodies predominantly against mitochondrial antigens PDC-E2, BCOADC-E2 and OGDC-E2. Currently orthotopic liver transplant (OLT) is the major form of treatment for end-stage primary biliary cirrhosis (PBC), but it is still unclear whether the autoimmune response continues post-transplantation. In this study we took advantage of a well-defined collection of sera collected serially before and after liver transplantation. We assayed these sera for quantitative and isotype-specific titers of antibodies against a set of recombinant mitochondrial autoantigens. We also studied reactivity to gp210. Serum samples were taken before transplantation and at intervals of 6 months, 1, 2, and 3 years after OLT. Before OLT 24/35 patients were AMA-positive, including seven out of the 35 to PDC-E2 alone, eight to both PDC-E2 and OGDC-E2, six to both PDC-E2 and BCOADC-E2, two to BCOADC-E2 alone and one to OGDC-E2. Following OLT, the frequency of sera that responded to PDC-E2 alone increased from seven to 12/35. Similarly, reactivity to BCOADC-E2 slightly increased from two to four out of 35. However, there was an overall decrease in sera that responded to more than one antigen. Neither Ig isotype nor subclass of the autoimmune response changed following OLT. Findings with gp210 were similar, in that reactivity to gp210 was found in nine out of 35 patients pre-OLT; following OLT the frequency decreased to seven out of 35 patients. Overall, the titers of AMAs decline slightly during the first year post-OLT, but are equivalent to pre-OLT values by 6 months. Moreover, the antibody subclass/ isotype remained unchanged. These data suggest that the removal of a diseased PBC liver has little, if any, impact on the serological characteristics of PBC. Moreover, it provides information regarding the natural history of PBC, particularly on the long latency time for disease development.

Identification of cyclin A as a molecular target of antinuclear antibodies (ANA) in hepatic and non-hepatic autoimmune diseases.

BACKGROUND/AIMS: Antinuclear antibodies (ANA) are a diagnostic hallmark of various autoimmune diseases and also of autoimmune hepatitis type 1. The designation ANA describes a heterogeneous group of autoantibodies. In liver diseases, only a few nuclear target antigens have been molecularly identified and characterized. Cyclins play a central role in cell cycle regulation, DNA transcription, and cell proliferation. Cyclin A was also identified as an integration site of the hepatitis B virus in a patient with hepatocellular carcinoma. In this study we identify cyclin A as a novel nuclear target protein of ANA. METHODS: Sera of patients with autoimmune hepatitis (AIH) type 1 (n = 61), type 2 (n = 21), and type 3 (n = 39), primary biliary cirrhosis (PBC) (n = 107), rheumatic diseases (systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), mixed connective tissue disease (MCTD)) (n = 42) and normal controls (n = 100) were evaluated for ANA by indirect immunofluorescence. Baculovirus-generated recombinant human cyclin A protein was used for immunoblotting to study the prevalence of anti-cyclin A autoantibodies in these sera. RESULTS: Sera of patients with AIH type 1 and rheumatic diseases had ANA detected by indirect immunofluorescence. In AIH type 1 12/61 (20%) and in rheumatic diseases 6/42 (14%) were immunoblot positive for autoantibodies against human cyclin A. In PBC, AIH type 3 and normal control sera negative for ANA by immunofluorescence, anti-cyclin A autoantibodies were present in 7-9%; in AIH type 2 and SLE they were undetectable by immunoblot. In some sera a typical cyclin A immunofluorescence was observed. Anti-cyclin A antibodies recognize a 45 and 50 kDa recombinant protein species, providing evidence for the recognition of at least two molecular epitopes. CONCLUSIONS: This study has identified cyclin A as a human autoantigen in hepatic and non-hepatic autoimmune diseases. More studies are required to evaluate the clinical and pathophysiological significance of anti-cyclin A autoantibodies. The identification of human anti-cyclin A autoantibodies may additionally become a valuable tool for studying the function and regulation of cyclin A in mammalian and human cells.

High prevalence of serological markers of autoimmunity in patients with chronic hepatitis C.

The advent of specific antiviral therapy for chronic hepatitis C has increased the importance of establishing the correct etiology of chronic hepatitis in patients, especially because interferon alfa (IFN-alpha) has been reported to exacerbate autoimmune hepatitis (AIH), whereas corticosteroids increase viral replication in chronic hepatitis C. In our medical center, we have treated many patients with apparent chronic hepatitis C and serological or clinical evidence of autoimmunity. Our aim was to estimate the prevalence of this association and to learn whether demographic or clinical features distinguished between patients with or without autoimmune markers. We performed a retrospective review of the records of 244 unselected patients seen at the Clinics and Hospital of the University of Massachusetts between May 1991 and November 1993, who had elevated serum aminotransferases. One hundred seventeen patients had chronic hepatitis C defined by elevations of serum alanine transaminase (ALT) for at least 6 months, positive serum antibodies to hepatitis C virus (HCV; second-generation enzyme immunoassay [EIA2] or recombinant immunoblot assay [RIBA]), and absence of hepatitis B surface antigen in the serum. Records were reviewed for results of autoimmune markers in sera, including anti-nuclear antibodies (ANAs), anti-smooth muscle antibodies (SMAs), rheumatoid factor (RF), antimitochondrial antibodies (AMAs), anti-liver and kidney microsomal (LKM) antibodies, and cryoglobulins. We found a high prevalence of positivity, particularly for anti-SMAs (66%) and RF (76%) in both men and women. Forty of 41 patients tested negative for anti-LKM antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of liver-kidney microsomal autoantibodies in chronic hepatitis C and D virus infection.

BACKGROUND/AIMS: Anti-liver-kidney microsomal (LKM) autoantibodies occur in a proportion of patients with chronic hepatitis C and D infections. Because of different immunofluorescence patterns, antibodies in hepatitis C and D were termed LKM-1 and LKM-3, respectively. The aim of the present study was to evaluate the different specificities of LKM-1 and LKM-3 antibodies. METHODS: Forty-nine samples of LKM-1 sera and 16 samples of LKM-3 sera were studied for reactivity against rat and human liver microsomal proteins by immunofluorescence, enzyme-linked immunosorbent assay, and Western blot. RESULTS: Thirty-four percent of the LKM-1 sera reacted with 50-kilodalton cytochrome P4502D6 in Western blot. In addition, a proportion of the sera recognized either a 59- or 70-kilodalton antigen, and 45% of the sera did not react in Western blot. Recently, the major LKM-3 antigen was identified as an autoepitope expressed on uridine diphosphate-glucuronosyltransferases (UGT). Seven LKM-3-positive sera reacted with recombinant rabbit family one UGT. None of the anti-LKM-1-positive hepatitis C sera reacted with UGT. Antibody reactivity against liver microsomal proteins in enzyme-linked immunosorbent assay ended when antigens were pretreated with sodium dodecyl sulfate, confirming that antibodies recognize conformational epitopes. CONCLUSIONS: LKM-1 antibodies in hepatitis C are more heterogeneous and react with different antigens compared with LKM-3 antibodies in hepatitis D.

Anti-GOR in hepatitis D: specific association with hepatitis C virus superinfection.

A group of 113 patients with chronic hepatitis D was investigated for the presence of anti-GOR and liver kidney microsomal antibodies. Eight patients were anti-GOR positive and also positive for hepatitis C virus-infection. In sera from 16 patients liver-kidney microsomal antibodies were detectable by immunofluorescence. They were classified as LKM-3 due to their fluorescence pattern. Two of the LKM-3-positive sera were also anti-hepatitis C virus and anti-human immunodeficiency virus positive. None of these patients were positive for anti-GOR. Fourteen sera from LKM-3-positive patients reacted in Western blot with a microsomal protein at 55 kDa that differs from the 50-kDa LKM-1 (cytochrome P450IID6) antigen. Our studies demonstrate that hepatitis D virus itself does not induce an autoimmune reaction against the GOR antigen and that autoimmunity to the LKM-3 antigen induced by hepatitis D virus infection does not correlated with anti-GOR. These studies support the specificity of the anti-GOR response for hepatitis C virus infection.

Properdin, a positive regulator of complement activation, is expressed in human T cell lines and peripheral blood T cells.

Properdin plays an important regulatory role in the activation of the complement system. Here we report the biosynthesis of properdin in four different human T cell lines and in T cells purified from peripheral blood. Cell sorting experiments, in conjunction with Northern blotting, showed that both CD4- and CD8-bearing populations of T cells have the potential to synthesize properdin. The functional activity of properdin secreted from these T cell lines was determined in a hemolytic assay. In view of the numerous ways in which complement activation may influence cellular immune response, the present results indicate, for the first time, a possible interaction between the complement and the T cell system.

Human complement factor B: functional properties of a recombinant zymogen of the alternative activation pathway convertase.

The human complement factor B is a centrally important component of the alternative pathway activation of the complement system. Here we report the isolation, characterization and eukaryotic expression of the first full length cDNA transcript for human factor B. In a factor B dependent haemolysis assay, the recombinant human factor B generated by transient COS cell transfection was shown to reconstitute haemolytic activity of factor B depleted human serum. To study the biological activities assigned to factor B, the availability of recombinant polypeptides representing definite portions of the human factor B molecule is desirable.

Involvement of membrane-associated TNF in the killing of Leishmania donovani parasites by macrophages.

A killing process is described where macrophages destroy 3H Thymidine prelabeled Leishmania parasites during the process of phagocytosis. This phagocytosis associated killing may represent a first line of defense and reduces drastically the amount of parasites homing in the macrophages. Treatment of the macrophages with anti-TNF antibody inhibited the phagocytosis associated killing. Soluble TNF had no effect on Leishmania parasites. Macrophages performed the killing process in the complete absence of any secreted TNF. Experiments using fixed macrophages and macrophage membranes revealed the existence of a 26 Kd membrane-TNF molecule. This membrane TNF was active against a TNF sensitive tumor cell line whereas the fixed cells or the isolated membranes had no cytotoxic effect on Leishmania parasites. This data indicates, that the Leishmanicidal effect was mediated through TNF but that apparently TNF itself was not the cytotoxic principle. Experiments using organ macrophages from Leishmania infected susceptible C 57/Bl 6 healer mice demonstrated that during the first 4 weeks of the infection the organ macrophages were totally unable to destroy Leishmania parasites independent of additional activation whereas the mechanism to destroy P815 tumor cells was unaffected. Similar data were obtained in therapy protocols using interferon gamma encapsulated in liposomes in order to target the substance to the macrophages. Also under these in vivo conditions the organ macrophages could not be activated and no beneficial effect was obtained by this activation treatment. In contrast, when therapy was started early (day 0 or 1) or late (5 weeks) after infection a highly significant reduction of the parasite burden was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)