Michael Mahler

Latest update on the Ro/SS-A autoantibody system.

Anti-SS-A (Ro52/Ro60) autoantibodies have been described as serological marker for Sjögrens syndrome but are also found in patients with other systemic autoimmune diseases. Historically, these autoantibodies were considered as a uniform autoantibody-system. However, recent studies provided evidence that Ro60 and Ro52 are not part of a stable macromolecular complex and that anti-Ro52 and anti-Ro60 (SS-A) antibodies have different clinical associations. The prevalence of anti-Ro52 in systemic sclerosis and myositis is significantly higher than anti-Ro60 (SS-A) and isolated anti-Ro52 can be found in up to 37% of myositis patients, often correlated with anti-Jo-1 reactivity (p=0.0002). Furthermore, recent developments have made significant improvements in the quality of recombinant Ro60 showing excellent performance in Ro60 (SS-A) ELISA (Dr. Fooke Laboratorien). Of note, single reactivity to either Ro52 or Ro60 (SS-A) can be missed when measured with a classical SS-A ELISA based on a mixture of both antigens. Approximately 20% of anti-Ro52 or Ro60 (SS-A) positive samples may remain undetected using a mixture of both antigens. Moreover, the international reference sera from the Centers for Disease Control and Prevention (CDC 2, 3, 7, 10) were further characterized. It was concluded that Ro60 (SS-A) and Ro52 represent two distinct autoantibody systems and that separate detection is desirable in a clinical diagnostic setting.

Epitope specificity and significance in systemic autoimmune diseases

Autoimmune diseases are characterized by self‐reactive immune processes mediated by B and T cells. These disorders exhibit a spectrum of clinical features that range from local or organ specific to systemic diseases. Although a variety of putative mechanisms that trigger the loss of tolerance and thus the genesis of autoimmunity have been identified, for the most part the precise mechanisms remain illusive. Nevertheless, it is widely appreciated that autoantibodies are useful both in the diagnosis of autoimmune disorders and as molecular biological tools to study cellular processes in which the target antigens are involved. Several methods and technologies, including protein fragments, synthetic peptides, phage display, or structural analyses have been developed for the characterization of the specificity of the autoimmune reactions. The present review provides an overview of the autoantibody epitopes in systemic autoimmune diseases as they relate to the clinical relevance and applications of certain autoepitopes and the technologies that are used to classify and identify them.

Closing the serological gap: promising novel biomarkers for the early diagnosis of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation and damage of the joints affecting about 0.5% of the general population. Early treatment in RA is important as it can prevent disease progression and irreversible damage of the joints. Despite the high diagnostic value of anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF), there is a strong demand for novel serological biomarkers to further improve the diagnosis of this abundant disease. During the last decades, several autoantigens have been described in RA including Ra33 (hnRNP A2), fibrinogen, fibronectin, alpha-enolase, type II collagen, immunoglobulin binding protein (BiP), annexins and viral citrullinated peptide (VCP) derived from Epstein Barr Virus-encoded protein (EBNA-2). More recent discoveries include antibodies to carbamylated antigens (anti-CarP), to peptidyl arginine deiminase type 4 (PAD4), to BRAF (v raf murine sarcoma viral oncogene homologue B1) and to 14 autoantigens identified by phage display technology. This review provides a current overview of novel biomarkers for RA and discusses their future potential to improve the diagnosis of the disease.

Autoantibodies to domain 1 of beta 2 glycoprotein 1: a promising candidate biomarker for risk management in antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by frequent clotting in arteries and veins and/or miscarriages. Autoantibodies to phospholipids and to beta 2 glycoprotein 1 (β(2)GP1) play an important role in the pathogenesis of APS. Antibodies to the domain 1 of β(2)GP1 (β(2)GP1-D1) have been suggested as a risk marker for thrombosis and to a lesser extent for pregnancy complications in patients suffering from APS. Despite significant interest in anti-β(2)GP1-D1 antibodies and a considerable research history, the number of studies is still limited and acceptance of the clinical significance of this biomarker is still evolving. The present review summarizes the current knowledge of anti-β(2)GP1-D1 antibodies and provides insights on recent discoveries. Moreover, we present a suggested guideline for future studies to better understand and verify the clinical utility of anti-β(2)GP1-D1 antibodies.

International multicenter evaluation of autoantibodies to ribosomal P proteins.

ABSTRACT Autoantibodies to the ribosomal phosphoproteins (Rib-P) are a serological feature of patients with systemic lupus erythematosus (SLE). The reported prevalence of anti-Rib-P antibodies in SLE ranges from 10 to 40%, being higher in Asian patients. The variation in the observed frequency may be related to a number of factors but is dependent in large part on the test system used to detect the autoantibodies. An association of anti-Rib-P with central nervous system involvement and neuropsychiatric manifestations of SLE has been controversial. In the present international multicenter study, we evaluated the clinical accuracy of a new sensitive Rib-P-specific enzyme-linked immunosorbent assay based on recombinant Rib-P polypeptides. The results showed that 21.3% of 947 SLE patients, but only 0.7% of 1,113 control patients, had a positive test result (P < 0.0001). The sensitivity, specificity, positive and negative predictive values, and diagnostic efficiency were determined to be 21.3%, 99.3%, 95.6%, 62.2%, and 65.3%, respectively. When evaluated in the context of participating centers, the prevalence of anti-Rib-P antibodies was found in descending frequency, as follows: China (35%) > Poland (34%) > Japan (28%) > United States (26%) > Germany (Freiburg; 23.3%) > Denmark (20.5%) > Germany (Berlin; 19%) > Mexico (15.7%) > Israel (11.7%) > Brazil (10%) > Canada (8%). The substantial data from this study indicate that the prevalence of anti-Rib-P antibodies may not be restricted to the genetic background of the patients or to the detection system but may depend on regional practice differences and patient selection. We confirm previously reported associations of antiribosomal antibodies with clinical symptoms and serological findings. Remarkably, we found a lower occurrence of serositis in Rib-P-positive lupus patients.

Advances in B-cell epitope analysis of autoantigens in connective tissue diseases.

The characterization of autoantibody specificities in rheumatic diseases is important in both diagnostic and basic research areas. Identification of the epitopes recognized by autoantibodies and their clinical and biological significance is not a trivial task. Epitopes may range in complexity from simple linear sequences of amino acids to complex quaternary structures. In addition to this structural complexity the frequency with which an autoantigen and its epitopes are recognized in a patient population may be useful in diagnosis, defining disease subgroups, and may offer information on disease prognosis. In this review recent advances in the epitope mapping of autoantigens in connective tissue diseases are discussed, with particular emphasis placed on the methodologies used to identify epitopes and the classification of the structural features of epitopes. To illustrate the identification of epitope structure, clinically relevant autoantigens, including CENP-A, PM/Scl-100, fibrillarin, filaggrin, Ro-52, and dsDNA, are discussed as examples of each type of epitope.

Anti-DFS70/LEDGF Antibodies Are More Prevalent in Healthy Individuals Compared to Patients with Systemic Autoimmune Rheumatic Diseases

Objective. Antinuclear antibodies (ANA) are a serological hallmark of systemic autoimmune rheumatic diseases (SARD) such as systemic lupus erythematosus (SLE). While a number of ANA patterns detected by indirect immunofluorescence (IIF) have diagnostic significance, autoantibodies producing the dense fine speckled (DFS) pattern have been reported to be more prevalent in healthy individuals than in SARD. Methods. Sequential samples submitted for ANA testing were screened for anti-DFS antibodies by IIF (n = 3263). Samples with the DFS pattern were tested for anti-DFS70/lens epithelium–derived growth factor (LEDGF) antibodies by ELISA and by a novel chemiluminescence assay (CIA, Quanta Flash DFS70). Sera from patients with various diseases and healthy individuals were tested for anti-DFS70/LEDGF antibodies by CIA. A cohort of 251 patients with SLE was used to analyze serological and clinical associations of anti-DFS70 antibodies. Results. The frequency of anti-DFS antibodies by IIF was 1.62%. The prevalence of anti-DFS70/LEDGF antibodies as detected by CIA in the different cohorts was 8.9% in healthy individuals, 2.8% in SLE, 2.6% in rheumatoid arthritis, 4.0% in asthma, 5.0% in interstitial cystitis, 1.7% in Graves’ disease, and 6.0% in Hashimoto’s thyroiditis. Of note, the prevalence of anti-DFS70/LEDGF antibodies was significantly higher in healthy individuals compared to patients with SARD (p = 0.00085). In SLE results, anti-DFS70/LEDGF antibodies were not significantly associated with clinical features or other autoantibodies typically found in SLE. Only 1/7 SLE sera showed anti-DFS70/LEDGF, but no other autoantibody reactivity. Conclusion. “Monospecific” anti-DFS70/LEDGF antibodies may represent a biomarker for differentiating SARD from non-SARD individuals, but there is a need for a reliable assay to ensure reactivity to DFS70.

Characterization of the human autoimmune response to the major C-terminal epitope of the ribosomal P proteins

Autoantibodies to the ribosomal phospho (-P) proteins P0, P1, and P2, collectively referred to as Rib-P, are specifically found in 10–40% of patients with systemic lupus erythematosus (SLE). These antibodies are believed to be correlated with lupus nephritis, hepatitis, and central nervous system involvement. The major immunoreactive epitope of these ribosomal antigens has been localized to the carboxy terminus, which is a highly conserved domain of all three proteins and contains two phosphorylated serine residues. The phosphorylated amino acids of the P proteins are known not to be critical epitope determinants. Furthermore, epitope-mapping studies have shown that the major epitope is located within the last 11 C-terminal amino acids. Using peptide arrays we identified more precisely this shared epitope as the six C-terminal amino acids GFGLFD and elucidated the molecular recognition events of anti-Rib-P antibodies at the amino acid level. We identified Phe111 and Phe114 of Rib-P2 as the key residues for the interaction, with further contributions of Gly-112 and Asp-115. This amino acid stretch is also present in proteins of several pathogenic micro-organisms such as Trypanosoma cruzi, Brugia malayi, Pseudomonas aeruginosa, Candida albicans, several Leishmania species, and Bartonella henselae. Using newly developed ELISA systems with a C-terminal peptide (C22) and the recombinant proteins (P0, P1, and P2) as antigens we found a high specificity of anti-Rib-P antibodies for SLE and demonstrated positive correlations with anti-U1-C, anti-Sm-B/B′ and anti-D and anti-dsDNA antibodies. The sensitivity and specificity in the peptide (C22) based assay varied between 12.8%/100% and 23.4%/96.7% for SLE, depending on the assigned cutoff. In contrast to other studies, we found no significant correlation of anti-Rib-P reactivity with central nervous system manifestations or renal involvement in SLE patients. We conclude that the epitope motif GFGLFD in the C-termini of the ribosomal P proteins is the key determinant of anti-Rib-P antibodies, and that the C22 peptide and the recombinant proteins can be used equally well for the detection of anti-Rib-P antibodies. The role of the major Rib-P epitope in the development of anti-ribosomal P antibodies and in the pathogenesis of SLE remains a subject of further investigation.

Importance of the dense fine speckled pattern on HEp-2 cells and anti-DFS70 antibodies for the diagnosis of systemic autoimmune diseases

The presence of anti-nuclear antibodies (ANA) is a hallmark of systemic autoimmune rheumatic diseases (SARD). The indirect immunofluorescence (IIF) assay on HEp-2 cells is a commonly used test for the detection of ANA and was recently recommended as the screening test of choice by a task force of the American College of Rheumatology. However, up to 20% of serum samples from healthy individuals (HI) have been reported to have a positive ANA test, the majority of which are directed to the dense fine speckles 70 (DFS70) antigen. Even more important, the DFS IIF pattern has been reported in 33% of ANA positive HI, but not in ANA positive SARD sera. Since the intended use of the ANA HEp-2 test is to aid in the diagnosis of SARD, the reporting of anti-DFS70 antibodies and their associated pattern (DFS) as a positive test, significantly reduces the specificity and the positive likelihood of the ANA test. This has significant implications for diagnostic algorithms involving the detection of ANA. We summarize the current knowledge of anti-DFS70 antibodies and their impact on ANA testing. We also suggest a test algorithm which considers the DFS pattern and the presence of anti-DFS70 antibodies. In addition, we describe a novel method based on immunoadsorption of anti-DFS70 antibodies, which increases the specificity of the ANA HEp-2 test for SARD and which has the potential to overcome a significant limitation of the ANA HEp-2 assay.

The clinical significance of the dense fine speckled immunofluorescence pattern on HEp-2 cells for the diagnosis of systemic autoimmune diseases.

Antinuclear antibodies (ANAs) are a serological hallmark in the diagnosis of systemic autoimmune rheumatic diseases (SARD). The indirect immunofluorescence (IIF) assay on HEp-2 cells is a commonly used test for the detection of ANA and has been recently recommended as the screening test of choice by a task force of the American College of Rheumatology. However, up to 20% of apparently healthy individuals (HI) have been reported to have a positive IIF ANA test, primarily related to autoantibodies that target the dense fine speckles 70 (DFS70) antigen. Even more important, the DFS IIF pattern has been reported in up to 33% of ANA positive HI, but not in ANA positive SARD sera. Since the intended use of the ANA HEp-2 test is to aid in the diagnosis and classification of SARD, the detection and reporting of anti-DFS70 antibodies and their associated pattern (DFS) as a positive test significantly reduce the specificity and the positive likelihood of the ANA test. This has significant implications for medical management and diagnostic algorithms involving the detection of ANA. Recently, a novel immunoadsorption method has been developed that specifically blocks anti-DFS70 antibodies and, therefore, significantly increases the specificity of the ANA test for SARD. This immunoadsorption method has the potential to overcome a significant limitation of the ANA HEp-2 assay. The present paper summarizes the current knowledge about anti-DFS70 antibodies and their clinical impact on ANA testing.