Circulating Anti-Nuclear Antibodies in Systemic Sclerosis: Utility in Diagnosis and Disease Subsetting.

The presence of circulating anti-nuclear antibodies (ANAs) is a hallmark of immune dysregulation in patients with systemic sclerosis (SSc). Currently, a variety of SSc-specific ANAs, including anticentromere, anti-topoisomerase I, and anti-RNA polymerase III antibodies, have been well characterized, and their commercial kits are available worldwide. Since these autoantibodies are specifically detected in SSc patients and are associated with unique sets of disease manifestations, they are widely used in routine clinical practice for diagnosis, clinical subgrouping, and prediction of future organ involvements and prognosis. In addition, SSc-specific ANAs are also useful in predicting future development of SSc in patients with Raynaud's phenomenon without any scleroderma skin changes, because their production often precedes onset of SSc symptoms. Application of circulating SSc-specific ANA measurement to clinical practice has greatly improved patient care, but utility of the autoantibody testing could be maximized by combining other clinical information, such as degree and extent of skin thickness and disease duration.

[Non-identified antinuclear antibodies in systemic sclerosis]. / Facteurs anti-nucléaires " non-identifiés " dans la sclérodermie systémique.

INTRODUCTION: Systemic sclerosis is a rare auto immune disease characterized by a local or diffuse skin condition and a variable visceral impairment. Anti nuclear antibodies (ANA) can be found in 95 % of patients. The most frequent are the anti topoisomerase 1 or anti Scl 70 and the anti-centromeres. Other antibodies have been reported but they are not conventionally sought in clinical practice. They are referred to as " non identified " ANA. OBJECTIVE: To seek the " non identified " antibodies in patients with scleroderma at Erasme Hospital, to assess their prevalence in this cohort and to correlate their presence with the clinical characteristics. METHODS: 89 patients out of the cohort of Erasme hospital patients with scleroderma have been looked at. Their clinical and biological data have been identified and a detection of antibodies have been performed by first an immonudot technique and second an EliA technique. RESULTS: 17 out of the 89 patients of our cohort had " non identified " ANA. Among them, antibodies in 11 patients have been identified by the immunodot, among which 7 anti-PmScl 75 and/or 100,3 RNA polymerase III and 1 antifibrillarin. The EliA technique identif ied antibodies in 10 patients among which 5 anti- PmScl, 2 anti RNA polymerase, 2 anti-fibrillarin and 1 anti-centromere. CONCLUSION: Auto antibodies other than the antitopoisomerase and anti-centromere have been found in patients with scleroderma in our cohort. Certain links exist between the presence of a given antibody and clinical features. We still have to define whether there exist other auto antibodies of which we still are unaware since in some patient no antibodies were detected.

INTRODUCTION: La sclérodermie systémique est une maladie auto-immune rare caractérisée par une atteinte cutanée locale ou diffuse ainsi qu'une atteinte viscérale variable. On retrouve des facteurs anti-nucléaires (FAN) chez environ 95 % des malades, dont les plus fréquents sont les anti-topoisomérase I (ou anti-Scl 70) et les anticentromères. D'autres auto-anticorps ont été décrits mais ceux-ci ne sont pas classiquement recherchés en pratique clinique, on les dénomme alors " FAN non identifiés ". OBJECTIF: Rechercher les anticorps " non identifiés " chez les patients sclérodermiques de l'hôpital Erasme, établir leur prévalence dans cette cohorte et corréler leur présence avec les caractéristiques cliniques. METHODES: Quatre-vingt-neuf patients issus de la cohorte des patients sclérodermiques de l'hôpital Erasme ont été étudiés. Leurs données cliniques et biologiques ont été relevées, puis une détection d'anticorps a été effectuée d'une part par une technique d'immunodot (topoisomérase I, centromères, ARN polymérase III, fibrillarine, Pm/Scl 75 et 100, PDGFR, NOR 90, Ku et Ro 52) et d'autre part par une technique EliA (topoisomérase I, centromères, ARN polymérase III, fibrillarine et PmScl). RESULTATS: Dix-sept des 89 patients de notre cohorte avaient des FAN " non identifiés ". Parmi ceux-ci, l'immunodot a identifié des anticorps chez 11 patients dont 7 anti-PmScl 75 et/ou 100, 3 anti-ARN pol III et 1 anti-fibrillarine. La technique EliA a permis d'identifier des anticorps chez 10 patients dont 5 anti-PmScl, 2 anti-ARN pol III, 2 anti-fibrillarine et 1 anti-centromère. CONCLUSION: Des auto-anticorps autres que les anti-topoisomérase I et anti-centromères ont été retrouvés chez les patients sclérodermiques de notre cohorte. Il existe certains liens entre la présence d'un anticorps donné et la clinique associée. Il reste à définir s'il existe d'autres auto-anticorps qui ne sont pas encore connus puisque chez certains patients, aucun anticorps n'a été mis en évidence.

[Immunosuppressive effect of bone marrow MSC transplantation in patients with ulcerative colitis with six kinds of one family autoantibodies with cross-relationship between each other].

The 6 types of cross-linked autobodies of one family where identified during relapsing course of Ulcerativ Colities (UC) acompanied with deterioration of clinical and endoscopic activity and increasing rate of acut inflammatory phase (CRP, number of leukocytes and erythrocyte sedimentation rate) of the disease. On the background of transplantation of mesenchymal bone marrow stromal cells (BM MSC), and despit the identification of six types of autoantibodies to antigens of neutrophils, was observed moderate activity of UC and low concentration of autoantibodies than in immunosuppressive therapy without BM MSC transplantation. Discovered anti-inflammatory effect of BM MSCs transplantation in UC may be explained by the systemic influence of immunosuppressive effect: it is known that the BM MSCs inhibit dendritic cells, T-and B-lymphocytes participating in the immune response, activate regulatory T-cells, which produce antinflammatory cytokines, IL-10 and TGF-1beta, which suppress the inflammatory process.

Antinuclear antibodies in rheumatic disease: a proposal for a function-based classification.

Antinuclear antibodies (ANAs) are a diverse group of autoantibodies that bind macromolecular components of the cell nucleus. While some ANAs occur in normal individuals, others are expressed almost exclusively in patients with rheumatic disease and serve as markers for diagnosis and prognosis. Despite the clinical associations of ANAs, the relationship of these antibodies to specific disease manifestations is often unknown because the target antigens are intracellular molecules that are ubiquitously expressed. In systemic lupus erythematosus, the role of ANAs in disease manifestations is better understood, especially for antibodies to DNA and related nucleosomal antigens. These antibodies can promote nephritis by the formation of immune complexes that are deposited in the kidney. In addition, anti-DNA, along with antibodies to RNA-binding proteins such as anti-Sm, can induce non-specific immune abnormalities based on the induction of type interferon 1 by plasmacytoid dendritic cells. Despite ANA expression in rheumatic disease, studies in animal models of inflammation and tissue injury indicate that antibodies to certain nuclear molecules such as HMGB1 have protective effects. Together, these considerations suggest a function-based classification of ANAs based on their expression in normal and autoimmune individuals as well as their capacity to induce or attenuate immunological disturbances. This classification provides a framework to elucidate the serological features of rheumatic disease and the often uncertain relationship between ANA expression and disease manifestations.

Antinuclear antibodies: a contemporary nomenclature using HEp-2 cells.

The choice of terms used to describe indirect immunofluorescence (IIF) staining patterns of autoantibodies binding to HEp-2 cells is at present quite varied and disordered because no accurate consensus on names and descriptions exist. The aim of our study was to propose a logical and ordered IIF classification taxonomy based on 29 different selected IIF patterns. In a preliminary project carried out at Statens Serum Institut it was first shown by use of a software programme named DOORS developed by Percepton Ltd, that reading of digitized images of HEp-2 patterns on an LCD monitor could be used instead of traditional microscopy. Digitized images of HEp-2 patterns were then used in the EU supported project named CANTOR (June 1998-July 2000) aiming to reach consensus among three clinical immunology expert centres and collaborating to attain a classification version that could be used to qualitatively and quantitatively test and train image recognitions skills of laboratory technicians against expert consensus. The usability of this classification version was then tested in a course consisting of training and certification. The conclusion was that participants in the training programme clearly increased their perceptive skills using images, terms, descriptions and the graphic and statistic tools in the self-administered DOORS programme and that software-assisted training could achieve a common and accurate level of visual pattern interpretation. All results from this project were reported to the European Commission but have not previously been published in scientific literature. This communication presents the final results of agreed image classifications.

Aggregation of classifiers for staining pattern recognition in antinuclear autoantibodies analysis.

Indirect immunofluorescence is currently the recommended method for the detection of antinuclear autoantibodies (ANA). The diagnosis consists of both estimating the fluorescence intensity and reporting the staining pattern for positive wells only. Since resources and adequately trained personnel are not always available for these tasks, an evident medical demand is the development of computer-aided diagnosis (CAD) tools that can support the physician decisions. In this paper, we present a system that classifies the staining pattern of positive wells on the strength of the recognition of their cells. The core of the CAD is a multiple expert system (MES) based on the one-per-class approach devised to label the pattern of single cells. It employs a hybrid approach since each composing binary module is constituted by an ensemble of classifiers combined by a fusion rule. Each expert uses a set of stable and effective features selected from a wide pool of statistical and spectral measurements. In this framework, we present a novel parameter that measures the reliability of the final classification provided by the MES. This feature is used to introduce a reject option that allows to reduce the error rate in the recognition of the staining pattern of the whole well. The approach has been evaluated on 37 wells, for a total of 573 cells. The measured performance shows a low overall error rate ( 2.7%-5.8%), which is below the observed intralaboratory variability.

[Patterns of anti-nuclear antibodies in patients with primary biliary cirrhosis and primary Sjögren syndrome].

OBJECTIVE: To investigate the anti-nuclear antibody (ANA) patterns in the diagnosis of primary biliary cirrhosis (PBC) and primary Sjögren syndrome (SS). METHODS: Serum samples from 61 cases of PBC, 28 cases of primary SS, and 11 cases of overlap syndrome of PBC and SS were collected to detect the ANA with indirect immunofluorescence staining, and the difference in ANA patterns was analyzed between the PBC and SS patients. RESULTS: Antinuclear antibodies were detected in 85.2% of the PBC patients, with the following hierarchy of specificities: 37.7% being rim-like, 21.3% being discrete speckled, 18.0% being speckled, 8.2% being multiple nuclear dots, and 4.9% being anti-lamin; 89.3% of the primary SS patients was ANA positive with speckled antibody, only one of them presented discrete speckles (3.6%); and all of the PBC patients overlapped with SS (100%) were positive in ANA, and their contexture of ANAs was similar to those of the PBC patients: 45.5% being rim-like, 18.2% being discrete speckled, 18.2% being speckled, 18.2% being anti-lamin, and 9.1% being multiple nuclear dots. Rim-like pattern and multiple nuclear dots were statistically significant for the diagnosis of PBC, compared with primary SS (chi(2) = 14.236, 3.781, P < 0.01, < 0.05). CONCLUSION: The ANA patterns of PBC are different from those of primary SS. Among them, rim-like pattern and multiple nuclear dots are highly specific nuclear patterns of PBC and may be useful in diagnosing individuals without anti-mitochondrial antibodies.

Anti-dsDNA antibody subtypes and anti-C1q antibodies: toward a more reliable diagnosis and monitoring of systemic lupus erythematosus and lupus nephritis.

The putative distinct diagnostic and pathogenic potential of aDNA-Ab subtypes, differing in their affinity or epitope specificity, was subject of several studies with controversial results. Comparing five assays, characterized by different reaction conditions and nature/source of dsDNA, we investigated the abovementioned problem in a retrospective study on 100 systemic lupus erythematosus (SLE) patients and 100 controls (other CTD, autoimmune hepatopathies). As demonstrated, only assay 3 (Farrzyme, TBS, UK) and 5 (Farr-RIA, Trinity Biotech, Ireland) are really suitable to detect primarily high avidity aDNA-Ab. Both were significantly linked to lupus nephritis (specificity 84%) and highly specific for SLE (95 and 96%). Thereby, assay 3 was found to be the first solid phase ELISA probably suitable to replace the Farr-RIA. Classical ELISAs (assay 1, Orgentec, Germany, and 2, Bindazyme, TBS, UK), detecting aDNA-Ab more or less independent from their avidity, or tests with only intermediate specificity for high avidity Ab (assay 4, ELIAdn, Sweden Diagnostics, Germany), were less specific for SLE (83, 79, 91%, respectively) and not associated with renal involvement (specificity 54-57%). At least in the patients studied here, obvious antigen-related differences could not be observed. With slight differences, all assays were suitable to monitor disease activity and therapy in SLE, agreeing with the ECLAM score in about 70-80% of cases. For lupus nephritis, aC1q-Ab are as specific as high avidity aDNA-Ab and capable to close a diagnostic gap in some cases. Thus, to enhance the specificity (up to 98%) and to consider the distinct diagnostic/pathogenic potential of aDNA-Ab subtypes in SLE, under routine clinical laboratory conditions it should be recommended to combine a sensitive screening test with a more specific second assay.

Anti-dsDNA and anti-Sm antibodies do not predict damage in systemic lupus erythematosus.

We aimed to determine whether anti-dsDNA and anti-Sm antibodies predict damage in systemic lupus erythematosus (SLE). Five-hundred inception patients from the University of Toronto Lupus Clinic were studied. Predictors assessed for the entire study period were: (1) raised anti-dsDNA on two consecutive occasions; (2) anti-dsDNA levels (normal, mildly or highly elevated); (3) presence of antiSm on any occasion. To account for disease duration, the following were assessed at three years post-inception: raised anti-dsDNA on two consecutive occasions; anti-dsDNA levels. These predictors were correlated with the following outcomes: (1) overall SLICC/ACR Damage Index (SDI) at the end of the study period; (2) frequency of damage in the cardiovascular, neuropsychiatric, musculoskeletal and renal components of SDI; ((3) SDI at five years for the predictors assessed at three years post-inception. In the multivariate analysis, presence of anti-DNA antibodies or of anti-SM were non-significant but sex, age at SLE diagnosis, disease duration, corticosteroid use and cumulative dose were strong predictors of damage. Raised anti-dsDNA on two occasions or anti-dsDNA levels in the three years post-inception patients did not predict damage at five years. The presence and levels of anti-dsDNA and anti-Sm antibodies do not predict damage in SLE.

Identification of a SmD3 epitope with a single symmetrical dimethylation of an arginine residue as a specific target of a subpopulation of anti-Sm antibodies.

Anti-Sm antibodies, identified in 1966 by Tan and Kunkel, are highly specific serological markers for systemic lupus erythrematosus (SLE). Anti-Sm reactivity is found in 5-30% of SLE patients, depending on the autoantibody detection system and the racial background of the SLE population. The Sm autoantigen complex comprises at least nine different polypeptides. All of these core proteins can serve as targets of the anti-Sm B-cell response, but most frequently the B and D polypeptides are involved. Because the BB'Sm proteins share cross-reactive epitopes (PPPGMRPP) with U1 specific ribonucleoproteins, which are more frequently targeted by antibodies that are present in patients with mixed connective tissue disease, the SmD polypeptides are regarded as the Sm autoantigens that are most specific to SLE. It was recently shown that the polypeptides D1, D3 and BB' contain symmetrical dimethylarginine, which is a component of a major autoepitope within the carboxyl-terminus of SmD1. In one of those studies, a synthetic dimethylated peptide of SmD1 (amino acids 95-119) exhibited significantly increased immunoreactivity as compared with unmodified SmD1 peptide. Using immobilized peptides, we confirmed that the dimethylated arginine residues play an essential role in the formation of major SmD1 and SmD3 autoepitopes. Moreover, we demonstrated that one particular peptide of SmD3 represents a more sensitive and more reliable substrate for the detection of a subclass of anti-Sm antibodies. Twenty-eight out of 176 (15.9%) SLE patients but only one out of 449 (0.2%) control individuals tested positive for the anti-SmD3 peptide (SMP) antibodies in a new ELISA system. These data indicate that anti-SMP antibodies are exclusively present in sera from SLE patients. Thus, anti-SMP detection using ELISA represents a new serological marker with which to diagnose and discriminate between systemic autoimmune disorders.

Paraneoplastic antibodies coexist and predict cancer, not neurological syndrome.

We investigated coexisting autoantibodies in sera of 553 patients with a neurological presentation and one or more paraneoplastic neuronal nuclear or cytoplasmic autoantibodies: antineuronal nuclear autoantibody type 1 (ANNA-1), ANNA-2, ANNA-3; Purkinje cell cytoplasmic autoantibody type 1 (PCA-1), PCA-2; and CRMP-5-immunoglobulin G or amphiphysin-immunoglobulin G. Except for PCA-1, which occurred alone, 31% of sera had more than one of these autoantibodies. In addition, 25% of sera had neuronal calcium channel (P/Q-type or N-type), potassium channel, ganglionic acetylcholine receptor, muscle acetylcholine receptor, or striational antibodies. The autoantibody profiles observed in patients with paraneoplastic disorders imply the targeting of multiple onconeural antigens and predict the patient's neoplasm, but not a specific neurological syndrome.

Structure of an anti-DNA fab complexed with a non-DNA ligand provides insights into cross-reactivity and molecular mimicry.

Antibodies that recognize DNA (anti-DNA) are part of the autoimmune response underlying systemic lupus erythematosus. To better understand molecular recognition by anti-DNA antibodies, crystallographic studies have been performed using an anti-ssDNA antigen-binding fragment (Fab) known as DNA-1. The previously determined structure of a DNA-1/dT5 complex revealed that thymine bases insert into a narrow groove, and that ligand recognition primarily involves the bases of DNA. We now report the 1.75-A resolution structure of DNA-1 complexed with the biological buffer HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid). All three light chain complementarity-determining regions (CDRs) and HCDR3 contribute to binding. The HEPES sulfonate hydrogen bonds to His L91, Asn L50, and to the backbone of Tyr H100 and Tyr H100A. The Tyr side-chains of L32, L92, H100, and H100A form nonpolar contacts with the HEPES ethylene and piperazine groups. Comparison to the DNA-1/dT5 structure reveals that the dual recognition of dT5 and HEPES requires a 13-A movement of HCDR3. This dramatic structural change converts the combining site from a narrow groove, appropriate for the edge-on insertion of thymine bases, to one sufficiently wide to accommodate the HEPES sulfonate and piperazine. Isothermal titration calorimetry verified the association of HEPES with DNA-1 under conditions similar those used for crystallization (2 M ammonium sulfate). Interestingly, the presence of 2 M ammonium sulfate increases the affinities of DNA-1 for both HEPES and dT5, suggesting that non-polar Fab-ligand interactions are important for molecular recognition in highly ionic solvent conditions. The structural and thermodynamic data suggest a molecular mimicry mechanism based on structural plasticity and hydrophobic interactions.

Simultaneous identification of various antinuclear antibodies using an automated multiparameter line immunoassay system.

The objective was to determine the sensitivity and specificity of an automated multiparameter line immunoassay system compared with other techniques for the identification of autoantibodies in rheumatic diseases. We studied sera from 90 patients. Anti-U1RNP, anti-Sm, anti-Ro/SS-A, anti-La/SS-B, anti-Jo 1 and anti-Scl 70 antibodies were identified by counterimmunoelectrophoresis (CIE) techniques, enzyme-linked immunosorbent assay (ELISA), immunoblotting (IB) using extracts of rabbit thymus and human placenta, and an automated multiparameter line immunoassay system (INNO-LIA ANA UPDATE K-1090) that detects nine different antibodies simultaneously (anti-U1RNP, anti-Sm, anti-Ro/SS-A, anti-La/SS-B, anti-Scl 70, anti-Jo 1, anticentromere, antihistone, and antiribosomal P protein). The line immunoassay system equaled or surpassed the other techniques in the identification of anti-Sm, anti-La/SS-B, anti-Jo 1 and anti-Scl 70 antibodies (sensitivity 100%, specificity 94-100%) and was similarly effective in the case of anti-U1RNP (sensitivity 87.5%, specificity 93.9%) and anti-Ro/SS-A (sensitivity 91.4%, specificity 87.2%) antibodies. In addition, this technique detected more 52 and 60 kD anti-Ro/SS-A sera than IB. Nine antibodies can be detected with this method at a cost of 25.38 Euros per serum sample. In five hours, 19 sera can be studied. The approximate cost of detecting these nine antibodies with an automated ELISA system would be 28.93 Euros, which allows 10 sera to be studied in four hours. In conclusion, the automated multiparameter line immunoassay system is a valid method for the detection of autoantibodies in rheumatic diseases. Its most notable advantages are automated simultaneous detection of several autoantibodies in the same serum and its lower cost compared with ELISA techniques.

[Anti-DNA antibodies: structure and pathogenic role]. / Les anticorps anti ADN: structure et rôle pathogène.

Anti DNA antibodies are generally classified into two major groups: Anti ds DNA (anti double .. stranded DNA) antibody and anti ss-DNA (anti single-stranded DNA) antibody. Anti-ds DNA antibodies are highly specific to systemic lupus erythematosus (SLE) and are probably involved in the pathogenesis of lupus nephritis. There are numerous serological tests for detecting anti-ds DNA. The detection of anti-ds DNA antibodies in the circulation of patients is one of the major criteria for the diagnosis of SLE; moreover, exacerbation of the disease are proceeded by increasing anti-DNA levels and the development of lupus nephritis, one of the most serious complications of the disease, strongly correlates with the presence of high avidity anti-DNA. It was reported that even normal individuals express anti-ds DNA. However, anti ds DNA found in healthy individuals is usually of the immunoglobulin M (IgM) isotype and shows a low affinity to ds-DNA. These natural antibodies are characterized by a wide cross-reactivity and are usually encoded by gene segments in the germ line configuration. In contrast, the ds-DNA antibodies involved influenced by various findings supporting the proposition that anti-ds DNA is involved in the pathogenesis of SLE, an enormous amount of scientific investigation has failed to reveal the exact mechanism through which this occurs.