Identification of New Autoantigens by Protein Array Indicates a Role for IL4 Neutralization in Autoimmune Hepatitis

Autoimmune hepatitis (AIH) is an unresolving inflammation of the liver of unknown cause. Diagnosis requires the exclusion of other conditions and the presence of characteristic features such as specific autoantibodies. Presently, these autoantibodies have relatively low sensitivity and specificity and are identified via immunostaining of cells or tissues; therefore, there is a diagnostic need for better and easy-to-assess markers. To identify new AIH-specific autoantigens, we developed a protein microarray comprising 1626 human recombinant proteins, selected in silico for being secreted or membrane associated. We screened sera from AIH patients on this microarray and compared the reactivity with that of sera from healthy donors and patients with chronic viral hepatitis C. We identified six human proteins that are specifically recognized by AIH sera. Serum reactivity to a combination of four of these autoantigens allows identification of AIH patients with high sensitivity (82%) and specificity (92%). Of the six autoantigens, the interleukin-4 (IL4) receptor fibronectin type III domain of the IL4 receptor (CD124), which is expressed on the surface of both lymphocytes and hepatocytes, showed the highest individual sensitivity and specificity for AIH. Remarkably, patients'' sera inhibited STAT6 phosphorylation induced by IL4 binding to CD124, demonstrating that these autoantibodies are functional and suggesting that IL4 neutralization has a pathogenetic role in AIH.Autoantibodies specific for proteins or nonprotein antigens (dsDNA, snRNP, carbohydrates) are often the serological hallmark of autoimmune diseases. Autoantibodies can be simply an epiphenomenon secondary to a chronic inflammatory milieu (1), but they can also play a direct pathogenetic role, as antithyroglobulin antibodies do in Hashimoto''s thyroiditis (2).Autoimmune hepatitis (AIH)¹ is a chronic necro-inflammatory disease of unknown etiology that affects predominantly women with an incidence of 1 to 2 per 100,000 per year and a prevalence of 10 to 20 out of 100,000 (3, 4). AIH is subdivided into two major types on the basis of autoantibody reactivity (5). Antibodies to nuclei and/or to smooth muscle characterize type 1 AIH, whereas antibodies to a liver-kidney microsomal constituent define patients with type 2 AIH. Because the detection of these autoantibodies is done by means of immunofluorescence on rodent multi-organ sections (liver, kidney, stomach), there are problems with the standardization and interpretation of the immunostaining patterns (6). To overcome these methodological problems, the International Autoimmune Hepatitis Group established an international committee to define guidelines and develop procedures and reference standards for more reliable testing (7, 8). Although ELISA and bead assays with purified or recombinant autoantigens are under development (9), they actually represent a complementary, rather than alternative, approach to traditional immunofluorescence. Moreover, serological overlap is frequently observed between AIH and other non-autoimmune liver diseases such as chronic viral hepatitis (10). Therefore, new, highly specific markers represent an unmet medical need for the more accurate diagnosis and classification of AIH.Besides the potential diagnostic application, the discovery of novel AIH autoantigens could provide insights into the disease pathogenicity mechanism. Although some AIH target-autoantigens have been identified and characterized, little is known about their pathogenetic role, and other autoantigens are probably still unknown. Autoantibodies, to be considered pathogenetic, must have at least two features: (i) the target-autoantigen should be either expressed on the plasma membrane of target cells or secreted by cells (i.e. should be exposed to autoantibodies), and (ii) binding of the autoantibodies to the target antigen should disturb a cellular function directly or indirectly. A possible pathogenetic role in AIH has been put forward for autoantibodies specific for cytochrome P450 2D6 (CYP2D6) or Asialoglycoprotein receptor 1 (AGPR-1), which are both present on the hepatocyte cell membrane (10).Protein microarrays are a powerful technology, as they allow the simultaneous screening of thousands of analytes (11). In the present study, to identify new autoantigens with potential diagnostic and/or pathogenetic roles in AIH, we printed a microarray with 1626 human proteins whose main feature was being either secreted or membrane associated (i.e. potentially exposed to autoantibody recognition). We used this microarray to screen panels of sera from patients with AIH and identified six new protein antigens that are recognized with high sensitivity and specificity. One of these six autoantigens is the interleukin-4 (IL4) receptor fibronectin type III (FNIII) domain of the IL4 receptor (CD124), and, interestingly, patients'' autoantibodies specific for CD124 neutralize IL4 signaling, suggesting a possible pathogenetic role for IL4 neutralization in AIH.     

Autoimmune liver disease 2007

Autoimmune liver disease (ALD) includes a spectrum of diseases which comprises both cholestatic and hepatitic forms: autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC) and the so called "overlap" syndromes where hepatitic and cholestatic damage coexists. All these diseases are characterized by an extremely high heterogeneity of presentation, varying from asymptomatic, acute (as in a subset of AIH) or chronic (with aspecific symptoms such as fatigue and myalgia in AIH or fatigue and pruritus in PBC and PSC). The detection and characterization of non organ specific autoantibodies plays a major role in the diagnostic approach of autoimmune liver disease; anti nuclear reactivities (ANA) and anti smooth muscle antibodies (SMA) mark type 1 AIH, liver kidney microsomal antibody type 1 (LKM1) and liver cytosol type 1 (LC1) are the serological markers of type 2 AIH; antimitochondrial antibodies (AMA) are associated with PBC, while no specific marker is found in PSC, since anticytoplasmic neutrophil antibodies with perinuclear pattern (atypical p-ANCA or p-ANNA) are also detected in a substantial proportion of type 1 AIH cases. Treatment options rely on immunosoppressive therapy (steroids and azathioprine) in AIH and on ursodeoxycholic acid in cholestatic conditions; in all these diseases liver transplantation remains the only therapeutical approach for the end stage of liver disease.     

Pediatric autoimmune liver diseases: the molecular basis of humoral and cellular immunity

Pediatric autoimmune liver disease is mainly represented by two similar liver disorders: autoimmune hepatitis (AIH) and autoimmune sclerosing cholangitis (ASC), both characterized by hypergammalobulinemia, interface hepatitis and the presence of a wide range of circulating autoantibodies. Although similar features are seen in AIH and inflammatory bowel disease, histological biliary changes are more common in ASC. In addition to their role as diagnostic markers, autoantibodies, such as anti-extractable nuclear antigen (ENA) antibodies and liver kidney microsomal antibody type 1 (LKM1) may be involved directly in inducing aggressive liver diseases. Although the cellular immune response in pediatric autoimmune liver disease has been less intensively investigated than humoral immunity, the importance of antigen specific T cells has been explored. Both alphabeta and gammadelta T cells derived from either peripheral blood and liver biopsies have highly heterogeneous TCR gene usage and cytolytic activity has been demonstrated. There have been attempts to seek triggers of liver autoimmunity and several sequences shared in common between autoantigens and hepatotropic viruses, namely hepatitis B, C and cytomegalovirus have been identified. The presence of cross-reactivity between homologous sequences, especially between HCV and cytochromes, supports the possibility that molecular mimicry plays a role in the induction of autoantibodies and autoreactive cytotoxic T cells.     

Two of Them Do It Better: Novel Serum Biomarkers Improve Autoimmune Hepatitis Diagnosis

Background

Autoimmune hepatitis (AIH) is a chronic liver disease of unknown aetiology and characterized by continuing hepatocellular inflammation and necrosis. Autoantibodies represent accessible markers to measure the adaptive immune responses in the clinical investigation. Protein microarrays have become an important tool to discriminate the disease state from control groups, even though there is no agreed-upon standard to analyze the results.

Results

In the present study 15 sera of patients with AIH and 78 healthy donors (HD) have been tested against 1626 proteins by an in house-developed array. Using a Partial Least Squares Discriminant Analysis (PLS-DA) the resulting data interpretation led to the identification of both new and previously identified proteins. Two new proteins AHPA9419 and Chondroadherin precursor (UNQ9419 and CHAD, respectively), and previously identified candidates as well, have been confirmed in a validation phase by DELFIA assay using a new cohort of AIH patients. A receiver operating characteristic analysis was used for the evaluation of biomarker candidates. The sensitivity of each autoantigen in AIH ranged from 65 to 88%; moreover, when the combination of the two new autoantigens was analyzed, the sensitivity increased to 95%.

Conclusions

Our findings demonstrate that the detection of autoantibodies against the two autoantigens could improve the performance in discriminating AIH patients from control classes and in combination with previously identified autoantigens and they could be used in diagnostic/prognostic markers.     

Cytochrome P450 enzymes and UDP-Glucuronosyltransferases as hepatocellular autoantigens

Cytochromes P450 and UDP-Glucuronosyltransferases (UGT) are targets of microsomal autoantibodies in liver and kidney (LKM). LKM autoantibodies are observed in autoimmune hepatitis, in some patients with viral hepatitis, drug-induced hepatitis and autoimmune hepatitis as disease component of the autoimmune polyglandulars syndrome type 1 (APS-1). In autoimmune hepatitis LKM antibodies are markers of autoimmune hepatitis type 2. The major target of LKM-1 antibodies is cytochrome P450 2D6; a second less frequent target was the described UGTs of family 1. In autoimmune hepatitis LKM-1 autoantibodies are usually directed against small linear epitopes. LKM autoantibodies are also associated with infection with hepatitis viruses C and D. In hepatitis C about 1–2% of patients develop LKM-1 autoantibodies. About 60% of these autoantibodies are conformation dependent. The presence of LKM autoantibodies in hepatitis C may be associated with an increased risk in interferon treatment. LKM-3 autoantibodies are found in about 8% of patients with hepatitis D and are directed against conformational epitopes. Patients treated with certain drugs may develop drug induced hepatitis. In hepatitis induced by tienilic acid, tienilic acid is activated by and covalently bound to cytochrome P450 2C9. Activation of the immune system results in the formation of autoantibodies against cytochrome P450 2C9 (LKM-2) and infiltration of the liver with immune cells. A similar mechanism has been described for dihydralazine induced hepatitis, where autoantibodies are directed against P450 1A2 (LM). Autoantibodies directed against cytochrome P450 1A2 also are found in patients suffering from hepatitis as a disease component of APS-1.Abbreviations AIH autoimmune hepatitis - APS1 autoimmune polyendocrine syndrome type 1 - APS-1 autoimmune polyglandular syndrome type 1 - LKM microsomal autoantibodies in liver and kidney - HSV-1 herpes simplex virus type 1 - UGT UDP-glucuronosyltransferases     

Hepatitis B Virus with X Gene Mutation Is Associated with the Majority of Serologically “Silent” Non-B,Non-C Chronic Hepatitis

Hepatitis B virus (HBV) with X gene mutations has been a putative pathogen of chronic hepatitis without serological markers of known hepatitis viruses. The aim of this study was to reconfirm whether the HBV with the X gene mutation is associated with these serologically “silent” non-B, non-C (NBNC) chronic hepatitis, alcoholic liver disease (ALD) and autoimmune hepatitis (AIH). HBV DNA was amplified from serum and sequenced in 30 patients with NBNC chronic hepatitis in comparison with 20 patients with ALD and 5 patients with AIH. HBV DNA was identified in 21 patients (70%) in NBNC chronic hepatitis by nested polymerase chain reaction while only one patient (5%) in ALD and none in AIH showed HBV DNA. Eighteen (85.7%) of the 21 identified HBV DNAs had an identical 8-nucleotide deletion mutation at the distal part of the X region. This mutation affected the core promoter and the enhancer II sequence of HBV DNA and created a translational stop codon which truncated the X protein by 20 amino acids from the C-terminal end. All the HBV DNAs had a precore mutation at the 83rd nucleotide resulting in disruption of HBe antigen synthesis. These results indicate that HBV mutants are closely associated with the majority of serologically “silent” NBNC chronic hepatitis cases and the population of such mutant HBV DNAs is not uniform.     

New autoantibodies in early rheumatoid arthritis

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease causing articular cartilage and bone destruction. Since irreversible joint destruction can be prevented by intervention at the early stages of disease, early diagnosis of RA is important. In this study, we identified new autoantibodies in the sera of patients with early (less than one year) RA.

Methods

We screened the sera of 20 RA patients with disease duration less than one year, 19 RA patients with disease duration more than five years and 23 controls on 8,268 human protein arrays. We confirmed the validity of protein array detection by ELISA assays. We then performed epitope mapping with overlapping 15-mers to analyze RA sera reactivity.

Results

WIBG (within BGCN homolog (Drosophila)), GABARAPL2 (GABA(A) receptor associated protein like 2) and ZNF706 (zinc finger protein 706) proteins are preferentially recognized by autoantibodies from early RA patients. Of interest, autoantibodies to WIBG are very specific for early RA. Indeed, 33% of early RA patients'' sera recognize WIBG versus 5% of RA patients with disease duration more than 5 years and 2% of controls. We identified three linear peptides on WIBG GABARAPL2 and ZNF706 that are preferentially recognized by sera of early RA patients.

Conclusions

We identified new autoantibodies associated with RA with disease duration less than one year. These autoantibodies could be used as diagnosis markers in RA patients.     

Absence of autoantibodies against correctly folded recombinant fibrillin-1 protein in systemic sclerosis patients

Autoantibodies against short recombinant fragments of fibrillin-1 produced in bacterial expression systems have been found in tight-skin mouse, systemic sclerosis, mixed connective tissue disease, and primary pulmonary hypertension syndrome. In patients with scleroderma, the frequency of anti-fibrillin-1 antibodies was 42% in Caucasians. Until now it has been unclear whether this immune response has a primary function in disease pathogenesis or is a secondary phenomenon. In the present study we analyzed the frequency of autoantibodies against two overlapping recombinant polypeptides spanning the N-terminal and C-terminal halves of human fibrillin-1, which were produced in human embryonic kidney (HEK-293) cells. Correct three-dimensional structures of the recombinant fibrillin-1 polypeptides were shown by electron microscopy and immunoreactivity with antibodies. Screening of fibrillin-1 antibodies was performed in 41 sera from systemic sclerosis patients and in 44 healthy controls with a Caucasian background. Microtiter plates were coated with the recombinant polypeptides of fibrillin-1 and incubated with 1:100 diluted sera. Positive binding was defined as being more than 2 SD above the mean of the control group. ELISAs showed that none of the sera of patients with systemic sclerosis contained autoantibodies against the N-terminal or C-terminal recombinant fibrillin-1 polypeptide. The data show the absence of autoantibodies against recombinant fibrillin-1 protein in Caucasian systemic sclerosis patients. Because the correct three-dimensional folding of the recombinant proteins has been substantiated by several independent methods, we conclude that autoantibodies against correctly folded fibrillin are not a primary phenomenon in the pathogenesis of systemic sclerosis.     

Oxidant stress in type I autoimmune hepatitis: the link between necroinflammation and fibrogenesis?

Autoimmune hepatitis (AIH) is a chronic liver disease of unknown aetiology characterized by circulating autoantibodies, hyperglobulinaemia and interface hepatitis. The mechanisms of progression from initial autoimmune attack to fibrosis and cirrhosis are unclear but oxidant stress may be involved. Markers of lipid peroxidation, antioxidant status, hepatic fibrogenesis and liver function were measured in blood and urine in 35 controls and in 33 patients with type-1 AIH; histology was assessed in 18 patients. In AIH, markers of lipid peroxidation were significantly elevated (8-isoprostane in both plasma and urine P < 0.001; plasma malondialdehyde P = 0.017). Total antioxidant capacity in protein-free serum and total glutathione in both whole blood and plasma were significantly reduced (P = 0.007, P = 0.037, P < 0.001, respectively). The antioxidants selenium, vitamin A and vitamin E were significantly decreased (P = 0.007, P < 0.001, P = 0.025, respectively); vitamin C was unchanged. Urinary 8-isoprostane correlated positively with interface hepatitis and necroinflammatory score and with hepatic fibrogenesis (type III procollagen peptide). Interface hepatitis correlated negatively with vitamin A and whole blood total glutathione. Oxidant stress, as reflected in blood and urine by a wide range of pro- and antioxidant markers, is a significant feature of AIH and provides a probable mechanism linking hepatic necroinflammation to fibrogenesis and disease progression.     

Serum soluble IL-2 receptor as a marker of lymphocyte activation in some autoimmune diseases. Effect of immunosuppressive therapy

Our experience regarding serum soluble interleukin-2 receptor (sIL-2R) measurement as a marker of lymphocyte activation consists of patients with autoimmune disease: 37 with systemic lupus erythematosus (SLE), 23 with autoimmune hepatitis (AIH), 74 with inflammatory bowel disease and six with Wegener's granulomatosis (WG). The influence of immunosuppressive therapy has also been assessed. Serum sIL-2R in SLE is significantly higher than in healthy controls and good correlation is found between sIL-2R and disease activity. Severity of kidney inflammation in lupus nephritis can be reflected by the increased excretion of sIL-2R. It was found that sIL-2R level significantly falls when the disease becomes clinically inactive after immunosuppressive therapy, but in many cases (up to 50%) it does not reach normal levels. The last finding suggests that lymphocyte activation may still be present even though the disease is considered inactive under clinical criteria and support the need of prolonged immunosuppression after the first signs of remission. In AIH the serum levels of sIL-2R are elevated in all patients with active disease; all cases with "highly active" disease have significantly higher concentrations than patients with "mild activity". A good correlation has been demonstrated between elevated serum sIL-2R values and anti-asialoglycoprotein receptor (ASGPR) titer (the specific marker of AIH). The follow-up study showed a significant decrease of both sIL-2R levels and anti-ASGPR titer after 3-9 month immunosuppressive therapy. The findings support that sIL-2R and anti-ASGPR titer could serve as reliable humoral markers for disease-specific activity. Compared with inactive ulcerative colitis (UC) and Crohn's disease (CD), significantly higher levels of sIL-2R were present in the serum of patients with active disease, and in inactive disease than in healthy age-matched controls. Methotrexate (MTX) therapy of patients with refractory UC resulted in sIL-2R decrease at the end of therapeutic period (20 i.m. injections of once a week 25 mg), good responders showing > 50% decrease even at 5-7 weeks of treatment. Serum sIL-2R is elevated in all six patients with WG. Contrary to anti-neutrophil cytoplasmic antibodies (ANCA), sIL-2R remains elevated above cut-off for normal range, despite clinical improvement following immunosuppressive treatment. The last observation suggests that serum sIL-2R is not a good measure of the disease activity and argue for the need of longer immunosuppressive therapy just after the first days of clinical remission.     

The specificity of disease-associated anti-fibrillarin autoantibodies compared with that of HgCl2-induced autoantibodies

Autoantibodies against nucleolar components are a common serological feature of patients suffering from scleroderma, a collagen vascular autoimmune disease. An important target of these autoantibodies is a protein with an apparent molecular weight of 36 kDa and a pI value of 8.5, located in the dense fibrillar component of the nucleolus and therefore termed fibrillarin. Animal models in which abundant anti-nucleolar antibodies appear spontaneously have not yet been described; however, high levels of anti-fibrillarin antibodies can be induced by treating susceptible strains of mice with sub-toxic amounts of mercuric chloride. In this study, we have analysed the specificity of anti-fibrillarin autoantibodies of human and murine origin. Our results suggest that both species have similar, if not identical conformational epitopes that are the target of anti-fibrillarin autoantibodies; these epitopes require the presence of a 30-kDa fragment of the fibrillarin molecule. Post-translational modifications such as the dimethylation of arginines in the N terminus of the protein are not essential for antibody recognition.Abbreviations ANA Antinuclear autoantibodies - ANolA Antinucleolar autoantibodies - snoRNA small nucleolar RNA - snoRNP small nucleolar ribonucleoprotein     

What autoantibody tests should become widely available to help scleroderma diagnosis and management?

Anti-Th/To autoantibodies have been recognized as serological markers of systemic sclerosis (SSc) for more than 20 years. However, validated immunoassay kits to test this specificity have not been commercially available. SSc autoantibodies are basically mutually exclusive and are associated with a certain subset of the disease and/or with organ involvement. Anti-Th/To are generally considered to be markers of the limited cutaneous type of SSc with the involvement of certain internal organs. The excellent correlation between anti-Rpp25 as detected by their novel chemiluminescent method and anti-Th/To as detected by immunoprecipitation suggest that the new assays may become widely available tests for clinicians in future and could help to clarify the clinical significance of anti-Th/To in SSc as well as other conditions over different races or countries.In a previous issue of Arthritis Research & Therapy, Mahler and colleagues reported a novel immunoassay for detecting anti-Th/To autoantibodies [1], which has the potential to make this testing widely available to clinicians using the newly developed ELISA and chemiluminescent immunoassay (CLIA). Serum from patients with systemic autoimmune rheumatic diseases often contains clinically important autoantibodies that react with various intracellular antigens. Detection of anti-nuclear antibodies by indirect immunofluorescence is widely accepted as a screening test for the diagnosis of many systemic autoimmune rheumatic diseases.Systemic sclerosis (SSc) is a disease characterized by collagen deposition and subsequent fibrosis in skin and internal organs. The extent of fibrosis shows considerable variation in severity, which classifies SSc into two distinct subtypes: the limited cutaneous form and the diffuse cutaneous form. Among SSc-associated autoantibodies, anti-centromere antibodies are widely accepted to be associated with limited cutaneous SSc while anti-topoisomerase I and anti-RNA polymerase III (anti-RNAP) antibodies are associated with diffuse cutaneous SSc [2]. In contrast to these three specific antibodies that have been widely used in the diagnosis and management of SSc patients, SSc autoantibodies showing nucleolar immunofluorescence staining pattern have not been utilized. There are two kinds of anti-nucleolar antibodies (ANoA): anti-U3-RNP (fibrillarin) and anti-Th/To [3]. Although they are mainly found in patients with SSc, information on their clinical significances is limited and inconclusive, partly due to a lack of commercially available antibody tests.Mahler and colleagues detected anti-Th/To antibodies using recombinant Rpp25, which is a component of the more than 10 proteins in the Th/To RNA-protein complex [4]. They utilized full-length, purified, recombinant human Rpp25 antigen for the quantitative measurement of anti-Th/To antibodies, which were defined by immunoprecipitation as the reference method. The newly established CLIA showed high sensitivity (100.0% = 8/8) and specificity (99.5% = 365/367) comparable with or better than ELISA. In another cohort, an ELISA indicated three cases showing false-negative anti-Th/To detection, which may be resolved by adding other antigenic components to the coated antigens. In CLIA, two false-positive cases were found, which were ANoA-negative. Evaluation with simultaneous performing anti-nuclear antibodies by indirect immunofluorescence is still important.The coexistence of SSc-marker antibodies in the same patient is rare. ANoA-positive SSc or SSc-suspected patients, who do not have anti-centromere, anti-topoisomerase I, or anti-RNAP antibodies, will be a subset with high probability of having anti-Th/To or antiU3-RNP antibodies. Anti-Th/To antibodies are frequently found in limited cutaneous SSc patients, often those with interstitial lung disease and pulmonary hypertension [2,5], whereas anti-U3-RNP antibodies are associated with pulmonary hypertension, renal disease and myositis in diffuse cutaneous SSc patients [2]. The prevalence of these antibodies in patients with SSc varies for different ethnic backgrounds. For example, in the US SSc cohort, all African-American patients with ANoA-dominant antibodies had anti-U3-RNP, which was found in 30% of all patients, whereas anti-Th/To was found in only 3% [6]. In Caucasian patients, however, anti-Th/To was more frequently found than anti-U3-RNP (9% vs. 4%). In Italian patients, anti-Th/To was found in 4% whereas anti-U3RNP was not found [6]. These antibodies were detected by complicated RNA analysis using immunoprecipitation and silver staining of RNAs or a radioisotope-based immunoprecipitation assay.Some autoantibodies have great diversity in their prevalence among different races and countries, besides the above instances. Recent studies showed that the prevalence of anti-RNAP antibodies was lower in French patients than in the US patients [7]. Anti-Mi-2 antibodies, which are first described as a serological marker of dermatomyositis, were the most common in patients with adult-onset dermatomyositis in Mexico City (59%) [8]. This finding is surprising to us because only 5% of Japanese dermatomyositis patients had anti-Mi-2 antibodies in our study [9]. Commercially available immunoassay kits would make it easier to perform international studies. Since immunoassays using CLIA have very high sensitivity (for example, chemiluminescent ELISA [10]) and are time-saving (for example, beads assay in a liquid phase [1]), the development of such assays would be a reasonable direction for autoantibody immunoassays.SSc specificity and the clinical association of anti-RNAP antibodies have been known for over 20 years; however, until recently, the detection of these antibodies had been utilized only at a few institutions that can perform radioimmunoprecipitation. Since the anti-RNAP ELISA kit became widely available to clinicians, it has now become a part of routine serological tests in SSc. More and more data from different countries have become available, which have illustrated the difference in prevalence and clinical association of anti-RNAP antibodies. Anti-Th/To immunoassay may become utilized in a similar way in future. The newly established method for measuring autoantibodies should be carefully evaluated using large international cohorts of SSc patients and other autoimmune conditions, to establish its utility and clinical significance. In SSc clinics, it will be ideal to have reliable, commercially available immunoassays for major ANoA, anti-Th/To antibodies and anti-U3-RNP antibodies, in addition to anti-PM-Scl antibodies, which are a marker of polymyositis/scleroderma overlap syndrome [1].     

Relationship between haemolytic and sphingomyelinase activities in a partially purified beta-like toxin from Staphylococcus schleiferi

beta-Toxins of staphylococcal species possess dual activity in that they can both lyse erythrocytes (by 'hot-cold' lysis) and catalyse hydrolysis of membrane-associated sphingomyelin. However, the precise relationship between these two activities has not been extensively studied. We have partially purified a beta-like toxin from culture supernatants of Staphylococcus schleiferi N860375 which exhibits both 'hot-cold' lysis of erythrocytes and neutral sphingomyelinase activities. This toxin has a strong preference for sheep erythrocytes, the membranes of which are rich in sphingomyelin. Kinetic analysis suggests that haemolysis and sphingomyelinase activities are very closely associated obeying identical Michaelis-Menten kinetics. However, pre-treatment with antibodies to Staphylococcus aureus beta-toxin, Ca(2+), dithiothreitol and phenylmethylsulfonyl fluoride appear to inhibit sphingomyelinase activity significantly more strongly than haemolysis while Mg(2+) activates sphingomyelinase activity more strongly than haemolysis. We attribute these effects to differences in binding properties in the two assays. Micropurification by both sphingosylphosphocholine-agarose affinity chromatography and preparative electrophoresis revealed that the 34-kDa toxin associates non-covalently with individual proteins.     

Features of autoantigens

The major cellular antigens recognized by autoantibodies in SLE and other systemic autoimmune diseases have been identified and characterized over the past 25 years. The pioneering studies of Eng Tan demonstrate the importance of autoantibodies as diagnostic markers. However, why certain autoantibodies, such as anti-Sm, are pathognomonic of SLE, while others are markers of othe autoimmune disease subsets, remains unanswered. This central question continues to drive much current research into the pathogenesis of SLE. Features of the autoantigens recognized by autoantibodies may provide important clues to the causes of lupus. Most autoantigens in systemic autoimmunity are multicomponent nucleoprotein complexes. These particles are encountered by the immune system as units, resulting in the tandem production of autoantibodies recognizing several components of the same complex. However, the intermolecular-intrastructural spreading of autoimmunity is regulated by mechanisms that at present are defined poorly. Also unexplained is the observation that the antigenic determinants recognized by autoantibodies are restricted and frequently correspond to active sites or functional domains. Analysis of experimental models of autoimmunity suggests that altering the structure of autoantigens, due to abnormal protein-protein interactions, hapten binding, altered degradation, or other mechanisms, could help to explain both the restricted patterns of autoantibody spreading and the selective targeting of antigenic sites. This may be a worthwhile area for further investigation of the pathogenesis of systemic autoimmune diseases.Abbreviations MCTD mixed connective tissue disease - PM/DM polymyositis / dermatomyositis - SLE Systemic lupus erythematosus - SSc systemic sclerosis - SVT simian virus 40 large T antigen     

Effects of rottlerin on silica-exacerbated systemic autoimmune disease in New Zealand mixed mice

Environmental crystalline silica exposure has been associated with formation of autoantibodies and development of systemic autoimmune disease, but the mechanisms leading to these events are unknown. Silica exposure in autoimmune-prone New Zealand mixed (NZM) mice results in a significant exacerbation of systemic autoimmunity as measured by increases in autoantibodies and glomerulonephritis. Previous studies have suggested that silica-induced apoptosis of alveolar macrophages (AM) contributes to the generation of the autoantibodies and disease. Rottlerin has been reported to inhibit apoptosis in many cell types, possibly through direct or indirect effects on PKCdelta. In this study, rottlerin reduced silica-induced apoptosis in bone marrow-derived macrophages as measured by DNA fragmentation. In NZM mice, RNA and protein levels of PKCdelta were significantly elevated in AM 14 wk after silica exposure. Therefore, rottlerin was used to reduce apoptosis of AM and evaluate the progress of silica-exacerbated systemic autoimmune disease. Fourteen weeks after silica exposure, NZM mice had increased levels of anti-histone autoantibodies, high proteinuria, and glomerulonephritis. However, silica-instilled mice that also received weekly instillations of rottlerin had significantly lower levels of proteinuria, anti-histone autoantibodies, complement C3, and IgG deposition within the kidney. Weekly instillations of rottlerin in silica-instilled NZM mice also inhibited the upregulation of PKCdelta in AM. Together, these data demonstrate that in vivo treatment with rottlerin significantly decreased the exacerbation of autoimmunity by silica exposure.     

Human anti-asparaginyl-tRNA synthetase autoantibodies (anti-KS) increase the affinity of the enzyme for its tRNA substrate

Autoantibodies directed against specific human aminoacyl-tRNA synthetases have been associated with a clinical picture including myositis, arthritis, interstitial lung disease and other features that has been referred to as the "anti-synthetase syndrome". Anti-asparaginyl-tRNA synthetase autoantibodies (anti-KS), the most recently described anti-synthetase autoantibodies, are directed against human cytosolic asparaginyl-tRNA synthetase and neutralize specifically its activity. Here we show that these antibodies recognize two epitopes on the human enzyme, an N-terminal epitope reactive in immunoblot experiments and a heat-labile epitope in the catalytic domain. In contrast to the well studied anti-Jo-1 autoantibodies anti-KS when bound to the synthetase increase the affinity of the synthetase for its tRNA substrate and prevent aminoacylation without interfering with the amino acid activation step.     

SEREX identification of the autoantibodies that are prevalent in the cerebrospinal fluid of patients with moyamoya disease

We performed SEREX (serological analysis of recombinant cDNA expression library) to identify autoantibodies that are prevalent in the cerebrospinal fluid of patients with moyamoya disease. These autoantibodies include PC326 (of unknown function), SRY (sex determining region Y), and peroxisomal D3,D2-enoyl-CoA isomerase.     

Fetal programming of adipose tissue function: an evolutionary perspective

Obesity is an escalating threat of pandemic proportions and has risen to such unrivaled prominence in such a short period of time that it has come to define a whole generation in many countries around the globe. The burden of obesity, however, is not equally shared among the population, with certain ethnicities being more prone to obesity than others, while some appear to be resistant to obesity altogether. The reasons behind this ethnic basis for obesity resistance and susceptibility, however, have remained largely elusive. In recent years, much evidence has shown that the level of brown adipose tissue thermogenesis, which augments energy expenditure and is negatively associated with obesity in both rodents and humans, varies greatly between ethnicities. Interestingly, the incidence of low birth weight, which is associated with an increased propensity for obesity and cardiovascular disease in later life, has also been shown to vary by ethnic background. This review serves to reconcile ethnic variations in BAT development and function with ethnic differences in birth weight outcomes to argue that the variation in obesity susceptibility between ethnic groups may have its origins in the in utero programming of BAT development and function as a result of evolutionary adaptation to cold environments.