Anti-Ro/SSA and La/SSB antibodies

The Ro/La system is considered as an heterogeneous antigenic complex, constituted by three different proteins (52?kDa Ro, 60?kDa Ro and La) and four small RNAs particles. Anti-Ro/SSA are the most prevalent specificity among many autoimmune diseases, such as systemic lupus erythematosus (SLE), SS/SLE overlap syndrome, subacute cutaneous LE (SCLE), neonatal lupus and primary biliary cirrhosis. In contrast, anti-La/SSB is more associated with Sjögren's syndrome (SS). The differences between 52?kDa, 60?kDa Ro and La could explain why different assays did not show equivalent performance in anti-Ro and anti-La autoantibodies detection. The RNA precipitation assay had the highest sensitivity and specificity, usually considered as the reference methods. CIE is considered the most reliable to detect anti-Ro/SSA antibodies in routine practice, performing better than immunoblotting (IB) and some ELISAs. It shows a high sensitivity (89%) and specificity (100%). ELISA is generally considered a safe, rapid, sensitive and specific tecnique. Therefore, its high sensitivity often corresponds to a very low clinical specificity and the assay can give false positive results. Therefore, it is very important to search anti-Ro and anti-La only in selected patients, using the assay with high specificity and good predictive value, in order to have clinically significant and true positive results.     

Anti-Ro/SSA and La/SSB antibodies

The Ro/La system is considered as an heterogeneous antigenic complex, constituted by three different proteins (52 kDa Ro, 60 kDa Ro and La) and four small RNAs particles. Anti-Ro/SSA are the most prevalent specificity among many autoimmune diseases, such as systemic lupus erythematosus (SLE), SS/SLE overlap syndrome, subacute cutaneous LE (SCLE), neonatal lupus and primary biliary cirrhosis. In contrast, anti-La/SSB is more associated with Sj?gren's syndrome (SS). The differences between 52 kDa, 60 kDa Ro and La could explain why different assays did not show equivalent performance in anti-Ro and anti-La autoantibodies detection. The RNA precipitation assay had the highest sensitivity and specificity, usually considered as the reference methods. CIE is considered the most reliable to detect anti-Ro/SSA antibodies in routine practice, performing better than immunoblotting (IB) and some ELISAs. It shows a high sensitivity (89%) and specificity (100%). ELISA is generally considered a safe, rapid, sensitive and specific tecnique. Therefore, its high sensitivity often corresponds to a very low clinical specificity and the assay can give false positive results. Therefore, it is very important to search anti-Ro and anti-La only in selected patients, using the assay with high specificity and good predictive value, in order to have clinically significant and true positive results.     

New coupled-particle light-scattering assay for detection of Ro/SSA (52 and 60 kilodaltons) and La/SSB autoantibodies in connective tissue diseases

The diagnostic and analytical performance of the coupled-particle light-scattering assay in detecting anti-Ro/SSA autoantibodies (the 60-kDa [Ro60] and the 52-kDa [Ro52] antibodies) and anti-La/SSB autoantibodies was evaluated. The antigens were obtained by recombinant DNA procedures to include the most immunogenic epitopes for each protein by using a prokaryotic expression system. Serum samples from 151 patients with connective tissue diseases and 52 control subjects (including patients with viral infections, patients with Lyme disease, and healthy subjects) were studied. Sensitivities for detection of anti-Ro/SSA and anti-La/SSB were 88.2 and 95.2%, respectively; specificities were 97.6 and 98.1%, respectively. The intra-assay coefficient of variation (CV) ranged from 4.3 to 10.9% for anti-Ro/SSA and from 2.8 to 12.5% for anti-La/SSB; interassay CVs ranged from 6.5 to 13.2% and from 8.2 to 14.5%, respectively. Among the anti-Ro/SSA-positive samples, Ro60 was recognized by 66% of the test sera and Ro52 was recognized by 95% of the test sera. Thirty-four percent of the Ro/SSA-positive sera were reactive only with the Ro52 antigen, indicating that anti-Ro52 is the most common antibody specificity recognized by anti-Ro/SSA autoantibodies. No differences were found between the prevalences of anti-Ro60 and anti-Ro52 in relation to systemic lupus erythematosus or Sj?gren's syndrome. The results of the present study indicate that this new immunoassay is an efficient diagnostic tool for the detection of anti-Ro/SSA and anti-La/SSB antibodies in patients with autoimmune disorders.     

Detection and occurrence of the 60- and 52-kD Ro (SS-A) antigens and of autoantibodies against these proteins.

The simultaneous detection of anti-La, anti-60-kD Ro and anti-52-kD Ro antibodies by immunoblotting is greatly improved by changing the crosslinking level in the gel to an acrylamide/bisacrylamide ratio of 19:1. Using this method for the analysis of a number of systemic lupus erythematosus (SLE) and Sjögren''s syndrome patient sera it was observed that antibody to the 52-kD Ro protein without anti-60-kD Ro antibody was restricted to Sjögren''s syndrome patients (9/26), whereas antibody to the 60-kD Ro protein without contaminating anti-52-kD Ro antibody was only found in SLE patients (8/38). Moreover, in Sjögren''s syndrome patient sera anti-Ro antibody was found only in combination with anti-La antibody (20/26), whereas in SLE patient sera anti-Ro antibody could be found without detectable anti-La specificity (4/38). Double immunofluorescence microscopy revealed that the 52-kD Ro and the 60-kD Ro proteins co-localize in the cytoplasm as well as in the nucleus, whereas immunoprecipitation of [32P]-labelled HeLa cell extract with monospecific anti-52-kD Ro and anti-60-kD Ro sera showed that both proteins are associated with the Ro RNAs. These data suggest the presence of both the 52-kD and the 60-kD Ro proteins in the same ribonucleoprotein complexes. To study the evolutionary conservation of the 52-kD Ro, the 60-kD Ro and the La proteins, extracts of cell lines derived from various mammalian species were analysed on Western blots using monospecific human antibodies. In contrast to the 60-kD Ro and the La antigens which are well conserved in evolution, the 52-kD Ro antigen could be detected in primate cells only by this immunological approach.     

New Coupled-Particle Light-Scattering Assay for Detection of Ro/SSA (52 and 60 Kilodaltons) and La/SSB Autoantibodies in Connective Tissue Diseases

The diagnostic and analytical performance of the coupled-particle light-scattering assay in detecting anti-Ro/SSA autoantibodies (the 60-kDa [Ro60] and the 52-kDa [Ro52] antibodies) and anti-La/SSB autoantibodies was evaluated. The antigens were obtained by recombinant DNA procedures to include the most immunogenic epitopes for each protein by using a prokaryotic expression system. Serum samples from 151 patients with connective tissue diseases and 52 control subjects (including patients with viral infections, patients with Lyme disease, and healthy subjects) were studied. Sensitivities for detection of anti-Ro/SSA and anti-La/SSB were 88.2 and 95.2%, respectively; specificities were 97.6 and 98.1%, respectively. The intra-assay coefficient of variation (CV) ranged from 4.3 to 10.9% for anti-Ro/SSA and from 2.8 to 12.5% for anti-La/SSB; interassay CVs ranged from 6.5 to 13.2% and from 8.2 to 14.5%, respectively. Among the anti-Ro/SSA-positive samples, Ro60 was recognized by 66% of the test sera and Ro52 was recognized by 95% of the test sera. Thirty-four percent of the Ro/SSA-positive sera were reactive only with the Ro52 antigen, indicating that anti-Ro52 is the most common antibody specificity recognized by anti-Ro/SSA autoantibodies. No differences were found between the prevalences of anti-Ro60 and anti-Ro52 in relation to systemic lupus erythematosus or Sjögren's syndrome. The results of the present study indicate that this new immunoassay is an efficient diagnostic tool for the detection of anti-Ro/SSA and anti-La/SSB antibodies in patients with autoimmune disorders.     

Autoantibodies to the Ro/SSA antigen are conformation dependent. I: Anti-60 kD antibodies are mainly directed to the native protein; anti-52 kD antibodies are mainly directed to the denatured protein.

Recent studies have shown that Ro/SSA autoantigen is heterogeneous and the autoanti-Ro/SSA response is correspondingly heterogeneous. There are two isoform families; the 60 kD forms and the 52 kD forms. We studied the antigenic difference between the native and denatured Ro/SSA isoforms and found that the autoanti-Ro/SSA response to the native 60 kD antigen is quite homogeneous. All anti-Ro/SSA sera recognize the native kD antigen regardless of the reactivities to the 60 kD band on the Western blot. Surprisingly, no anti-Ro/SSA sera without anti-La/SSB reacts with the native 52 kD Ro/SSA, although sera with both precipitating anti-Ro/SSA and anti-La/SSB can immunoprecipitate the native 52 kD antigen. Anti-Ro/SSA sera exist which react exclusively with the native 60 kD Ro/SSA protein (10/43, 23%) while no anti-Ro/SSA sera have been found which react exclusively with the denatured 52 kD Ro/SSA antigen. In sera with anti-Ro/SSA precipitins alone, only antibody to the denatured 52 kD Ro/SSA molecule is found! In sera with anti-Ro/SSA and anti-U1 RNP precipitins, no antibody to either native or denatured 52 kD Ro/SSA is found, while in sera with both anti-Ro/SSA and anti-La/SSB precipitins, antibodies to both the native and denatured forms of 52 kD Ro/SSA are present. These data suggest that the anti-Ro/SSA response to the 60 kD molecule is driven by the native 60 kD Ro/SSA molecule while the molecular identification of the antigen drive in the anti-52 kD Ro/SSA response is unknown.     

IgM and IgG Subclass Distribution of Human Anti-Ro/SSA 60 kDa Autoantibodies

The Ro/SSA and La/SSB antigens are common targets for autoantibodies found in the sera of patients with Sjögren's syndrome and SLE. The anti-Ro/SSA and anti-La/SSB antibodies often appear together but are not cross-reactive. This paper describes the humoral autoimmune response to the Ro/SSA 60 kDa protein moiety with respect to the presence of IgM and IgG1-4 antibodies. IgM antibodies to the Ro 60 kDa protein coexisted with IgG anti-Ro 60 kDa antibodies in nearly half of the sera. A similar fraction also contained IgM anti-La/SSB antibodies. The frequency of sera with IgM antibodies of both specificities was that expected from random overlap.
A predominating igGl anti-Ro 60 kDa response was found in all patients, but anti-Ro 60 kDa antibodies of the other IgG subclasses were present also in a high number of sera. This is in contrast to the reported IgG subclass distribution of anti-La/SSB antibodies.
Mapping of IgM and IgG 1–4 antibody recognition of different parts of the Ro 60 kDa protein was also performed. IgM and IgGl-4 antibodies of all sera reacted with the central part of the Ro 60 kDa protein, encompassing amino acid residues 181–320.     

Epitope mapping with synthetic peptides of 52-kD SSA/Ro protein reveals heterogeneous antibody profiles in human autoimmune sera.

The reactivity of autoantibodies present in the sera of 489 patients with Sjögren's syndrome (SS), systemic lupus erythematosus (SLE) and other autoimmune diseases was investigated by ELISA using recombinant 52-kD SSA/Ro protein (rRo52) and 39 overlapping synthetic peptides representing the entire sequence of Ro52. We report that IgG antibodies reacting with rRo52 were present in the sera of a large number of patients with SS (67% of patients with primary SS and 46% of patients with SS associated with SLE), whereas they were less frequent (10-25%) in SLE, rheumatoid arthritis (RA), juvenile chronic arthritis (JCA) and mixed connective tissue disease (MCTD), and absent in scleroderma. Among the 39 peptides tested, five were recognized by sera from 30-65% of patients with SS, namely peptides representing residues 2-11, 107-122, 107-126, 277-292 and 365-382. Patients with JCA had raised levels of IgG antibodies reacting with peptides 2-11 and 365-382, and 51% of patients with MCTD had raised levels of IgG antibodies reacting with peptide 365-382. None of the five peptides was recognized by more than 20% of sera from patients with SLE and RA. Interestingly, and of importance in the field of diagnostic tests based on peptides, the reactivity of antibodies to the Ro52 synthetic peptides varied greatly according to the origin of sera. Inhibition experiments using either patients' sera or antibodies induced in rabbits against Ro52 peptides showed that the four domains 2-11, 107-122, 277-292 and 365-382 are accessible on the surface of the Ro52 protein. These regions may thus be involved in the induction of specific antibodies in autoimmune patients.     

Antibodies to EYRKK vesicular stomatitis virus-related peptide account only for a minority of anti-Ro60kD antibodies.

Previous studies demonstrated a possible antigenic relation between the carboxyl terminal portion of anti-Ro60kD autoantigen and a nucleocapsid protein (N) of vesicular stomatitis virus (VSV). In order to investigate whether anti-Ro60kD autoantibodies react with the VSV homologous region of the Ro60kD protein we synthesized, according to Merrifield's method, the EYRKKMDI octapeptide (8p) sharing a common sequence with the N protein of VSV. Sera from 61 patients with autoimmune rheumatic diseases (34 systemic lupus erythematosus (SLE), 21 Sjörgren's syndrome (SS) and six rheumatoid arthritis (RA)) as well as 59 from normal blood donors were tested for the presence of anti-Ro60kD autoantibodies by ELISA and immunoblot (IB) and anti-8p antibodies by ELISA. Antibodies to 8p were found in 9/31 of anti-Ro60kD IB-positive sera, 5/30 of anti-Ro60kD-negative sera and 2/59 of normal control sera. The concordance between the anti-8p ELISA and the anti-Ro60kD IB was very poor (chi 2 = 0.71, P = 0.4) in contrast to the anti-Ro60kD ELISA and the anti-Ro IB (chi 2 = 27.6, P = 10(-7)). Subsequent affinity purification of the anti-8p antibodies from a strong positive anti-8p and anti-Ro60kD SLE serum yielded 95% depletion of the anti-8p activity and 37% reduction of the anti-Ro60kD activity. Inhibition assays with the affinity-purified anti-8p antibodies demonstrated that the octapeptide gave 94.5% inhibition of the anti-Ro60kD activity, while Ro60kD protein led to 42.3% inhibition of the anti-8p. Preincubation of the serum with the octapeptide produced 4% inhibition of anti-Ro60kD ELISA. These results indicate that the anti-8p antibodies account only for a minority of the anti-Ro60kD autoantibodies.     

Comparison of the anti-Ro/SSA autoantibody profile between patients with primary and secondary Sjögren's syndrome

OBJECTIVE: To measure serum levels of anti-Ro52-kD/SSA, anti-Ro60-kD/SSA and anti-La/SSB autoantibodies in patients with primary and secondary Sj?gren's syndrome. To examine if there is any connection between the disease and the subtype-spectrum of these antibodies. METHODS: We measured serum levels of anti-Ro52-kD/SSA, anti-Ro60-kD/SSA and anti-La/SSB autoantibodies by ELISA, in the sera of patients with primary Sj?gren's syndrome with or without extraglandular manifestations and with or without anti-La/SSB positivity and of patients with systemic lupus erythematosus/Sj?gren's syndrome overlapping disease with or without anti-La/SSB positivity. RESULTS: Differences of the distribution of the anti-Ro52-kD/SSA and the anti-Ro60-kD/SSA were found between the primary and secondary Sj?gren's syndrome patients' groups; when Sj?gren's syndrome is accompanied by systemic lupus erythematosus, the occurrence of anti-Ro60-kD/SSA autoantibodies is significantly higher than in primary Sj?gren'syndrome. CONCLUSION: Our results suggest that there is a possible connection between the distribution of the subtypes of the anti-Ro/SSA autoantibodies and the disease type in primary/secondary Sj?gren's syndrome.     

Heterogeneity of the Ro/SSA antigen and autoanti-Ro/SSA response: evidence of the four antigenically distinct forms.

Precipitating antibodies to the Ro/SSA antigen occur in the sera of 40% of patients with sytemic lupus erythematosus (SLE) and in 40–70% of the sera of patients with primary Sjögren's syndrome. Previous work has shown that lymphocyte extracts contain two Ro/SSA antigens with protein moieties of 60 kD and 52 kD and that erythrocyte haemolysate contain two analogous but antigenically distinct Ro/SSA molecules of 60 kD and 54 kD. Frequency analysis of the various specificities in 43 sera with precipitating anti-Ro/SSA and studies with affinity-eluted antibodies suggest that the lymphocyte 60 kD and erythrocyte 60 kD Ro/SSA molecules are related as are the lymphocyte 52 kD and erythrocyte 54 kD Ro/SSA proteins. Anti-Ro/SSA sera when accompanied by other precipitins (anti-La/SSB and anti-U1RNP) react preferentially with certain Ro/SSA isoforms. Evidence is also presented for a 45 kD form of Ro/SSA. These data suggest that the antigenic heterogeneity of the Ro/SSA antigen is immunologically relevant and that there are two families of Ro/SSA antigens: one comprising of the two 60-kD proteins in the erythrocyte and lymphocyte and the other the erythrocyte 54 kD and lymphocyte 52 kD Ro/SSA proteins, respectively.     

Anti-Ro52 antibodies frequently co-occur with anti-Jo-1 antibodies in sera from patients with idiopathic inflammatory myopathy

We analysed 112 idiopathic inflammatory myopathy (IIM) sera for the presence of anti-Ro, anti-La and anti-histidyl-tRNA synthetase (Jo-1) autoantibodies, and subsequently mapped B cell epitopes on the Ro52 protein recognized by anti-Ro52⁺ IIM sera. Sera were characterized by immunoblotting, ELISA and RNA precipitation. Both anti-Ro60 and anti-La activity was found in 4% of IIM sera. Anti-Ro52 antibodies were present in 20% of IIM sera. However, in anti-Jo-1⁺ IIM sera (21%), the frequency of the anti-Ro52 antibodies was found to be much higher (58%). No cross-reactivity between anti-Ro52 and anti-Jo-1 antibodies could be detected in these sera. To learn more about the nature of anti-Ro52 antibodies occurring in IIM sera, we analysed the major epitopes of the Ro52 protein targeted by anti-Ro52⁺ IIM sera by immunoprecipitation of in vitro translated Ro52 deletion mutants. The major epitope was mapped in the region bordered by amino acids 126 and 252. This part of the protein includes a long α-helical region which contains two potential coiled-coil domains as well as a leucine zipper motif. Although no difference in Ro52 epitope recognition between anti-Jo-1⁺ and anti-Jo-1⁺IIM sera could be observed, our results suggest that the autoimmune response against Ro52 and Jo-1 in IIM patients is coupled.     

The detection of anti-Ro/SS-A and anti-La/SS-B antibodies. A comparison of counterimmunoelectrophoresis with immunoblot, ELISA, and RNA-precipitation assays.

The presence in serum of anti-Ro/SS-A and/or anti-La/SS-B autoantibodies is a characteristic of autoimmune diseases such as Sj?gren's syndrome and systemic lupus erythematosus. To evaluate different assays currently available for the detection of these antibodies 50 sera were tested using the four different assay methods: counterimmunoelectrophoresis (CIE), RNA precipitation assay, immunoblotting technique and ELISA. The RNA-precipitation assay showed the highest sensitivity and specificity. The CIE for the detection of anti-Ro/SS-A antibodies gave comparable results whereas the Ro/SS-A ELISA and Ro/SS-A or HeLa immunoblot showed lower sensitivities (96% and 80% respectively). Sensitivity was even lower (66%) when only reactivity towards the 60 kDa Ro/SS-A protein was considered. The ELISA for the detection of anti-La/SS-B antibodies showed a sensitivity of 98%, the immunoblotting technique of 86% and the CIE only 67%. The high sensitivity of the La/SS-B ELISA went together with a low specificity of 14%. We conclude from these data that for the detection of anti-Ro/SS-A and anti-La/SS-B antibodies the RNA precipitation assay shows the highest sensitivity and highest specificity. For routine screening purposes the CIE is the most convenient and reliable assay to detect anti-Ro/SS-A antibodies. For the detection of anti-La/SS-B antibodies the immunoblot corresponds most closely to the RNA precipitation assay.     

Human Anti-Ro Autoantibodies Bind Peptides Accessible to the Surface of the Native Ro Autoantigen

The relationship between fine specificity of linear epitopes and conformational determinants has been explored in a naturally arising human autoimmune response. In particular, the hypothesis tested is that the linear epitopes of the human Ro autoantigen are components of its conformational epitopes. Twenty groups among the 531 overlapping octapeptides 60kDa Ro are variably bound by anti-Ro precipitin positive lupus sera whose reactivity was easily distinguished from sera of normal controls and of anti-Ro precipitin negative lupus patients. The specific activities of anti-peptide antibodies and of anti-native Ro autoantibodies are similarly increased after affinity enrichment using native human Ro as ligand. Moreover, affinity-enriched anti-native Ro autoantibodies bind virtually the same 20 groups of epitopes recognized by whole anti-Ro positive sera. Two peptides (residues 274–290 and 480–494) from the defined 60 kDa Ro octapeptide epitopes have been prepared and used as ligands for affinity purification of peptide specific autoantibodies. The binding of both whole IgG and affinity-enriched peptide specific autoantibodies is inhibited by native Ro autoantigen. Thus, none of the available data can be construed to support the existence of cryptic linear epitopes in this system. Indeed, the data are only consistent with the conclusion that all of the anti-Ro octapeptide autoantibodies are part of the population of anti-native Ro autoantibodies in this naturally arising autoimmune response.     

The development of a quantitative assay for the detection of anti-Ro/SS-A and anti-LA/SS-B autoantibodies using purified recombinant proteins.

A characteristic of patients with autoimmune diseases such as Sj?gren's syndrome and systemic lupus erythematosus is the presence of anti-Ro/SS-A and anti-La/SS-B autoantibodies in their circulation. In order to investigate specific autoantibody levels in the sera of these patients quantitative assays for the detection of both anti-Ro/SS-A and anti-La/SS-B reactivity were developed. Ro/SS-A (60 kDa) and La-SS-B (50 kDa) cDNAs were cloned and expressed in E. coli as non-fusion proteins. These were purified to homogeneity using two different purification protocols. With these recombinant antigens, specific enzyme-linked immunosorbent assays (ELISAs) were developed. 40 sera positive for anti-Ro/SS-A autoantibodies in counterimmunoelectrophoresis (CIE) were tested in both the Ro/SS-A and La/SS-B ELISA. Activity values reproducibly ranged from 1536 to 120,000 U in the Ro/SS-A ELISA and from 763 to 2,500,000 U in the La/SS-B ELISA. The suitability of these ELISAs as screening assays was further investigated by testing 200 sera sent to our laboratory for routine detection of autoantibodies to extractable nuclear antigen (ENA: anti-Sm, anti-RNP, anti-Ro/SS-A and anti-La/SS-B). Both ELISAs showed a high sensitivity and specificity (Ro/SS-A ELISA 85% and 94%, La/SS-B ELISA 100% and 98% respectively), when compared to the standard assays, the RNA-precipitation assay and the HeLa immunoblotting test. From these data we conclude that a quantitative analysis of both anti-Ro/SS-A and anti-La/SS-B autoantibodies is now possible using purified recombinant non-fusion proteins. For screening purposes the La/SS-B ELISA showed a great improvement in sensitivity for the detection of anti-La/SS-B activity in comparison to the La/SS-B CIE, while the Ro/SS-A ELISA almost equalled the performance of the Ro/SS-A CIE.     

A common autoepitope near the carboxyl terminus of the 60-kD Ro ribonucleoprotein: Sequence similarity with a viral protein

The Ro ribonucleoprotein is composed of hY RNA and a 60.7-kD peptide that is antigenic for autoantibodies produced by many patients with systemic lupus erythematosus or Sjögren's syndrome and mothers of newborns with complete congenital heart block. A major immunoreactive fragment (13 kD) of the 60-kD Ro is bound by 28 of 45 (62%) of the anti-Ro sera tested. Amino acid sequence analysis localizes this fragment to the carboxyl end of the 60-kD Ro peptide. All possible overlapping octapeptides of this 13-kD peptide of 60-kD Ro have been assessed for antigenicity. Sera that bind the 13-kD peptide fragment in immunoblot generally also bind the octapeptides of Ro spanning the sequence AIALREYRKKMDIPA (P<0.01). Inhibition studies with synthetic peptides and purified Ro have established specificity for reference serum antibody binding to an antigenic octapeptide, EYRKKMDI, from this region. The closely related sequence EYRKKLMD is found in the nucleocapsid protein of vesicular stomatitis virus and may portend an immunologic link to this or a related viral antigen. These results also demonstrate that despite fine specificity variation between human sera, there are recurring patterns of anti-Ro binding shared by some patients who have precipitating anti-Ro autoantibodies.     

Clinical constellation of annular erythema associated with anti-Ro/La autoantibodies

Annular erythema (AE) associated with anti-Ro (SSA) and/or La (SSB) autoantibody in patients with Sjögren syndrome (SS) or with SS/systemic lupus erythematosus overlap syndrome (SS/SLE), has recently been described in Orientals, and it may be a counterpart of annular skin lesion of the subacute cutaneous LE seen mostly in Caucasians. The author examined five Korean AE patients in respect to dinical diversity. In this small-sample study, subtle differences appeared between individual cases regarding the serologic features and the diagnoses of the disease. Among the five cases, four had circulating anti-Ro and anti-La antibodies, and one had only anti-La. Regarding the diagnosis, one was SS/SLE, two were primary SS, and the remaining two were only "AE associated with anti-Ro/La antibody". There seem to be a wide clinical spectrum in the disease expression of AE associated with anti-Ro/La autoantibody than previously thought.     

Sjögren's syndrome: autoantibodies to cellular antigens. Clinical and molecular aspects

Autoantibodies to cellular autoantigens are usually found in sera of patients with systemic autoimmune rheumatic diseases. Patients with Sj?gren's syndrome (SS) frequently present autoantibodies to both organ and non-organ-specific autoantigens. The most commonly detected autoantibodies are those directed against the ribonucleoproteins Ro/SSA and La/SSB. The presence of the antibodies in SS is associated with early disease onset, longer disease duration, parotid gland enlargement, higher frequency of extraglandular manifestations and more intense lymphocytic infiltration of the minor salivary glands. Over the past several years, the structure and function of these autoantigens have been extensively studied. Several centers, using different techniques, have investigated the B cell epitopes on the protein components Ro 60 kD, Ro 52kD, and La 48 kD. Finally, increased evidence of direct involvement of anti-Ro/SSA and anti-La/SSB autoantibodies in the pathogenesis of tissue injury has been contributed by several studies.     

Anti-argonaute2 (Ago2/Su) and -Ro antibodies identified by immunoprecipitation in primary anti-phospholipid syndrome (PAPS)

Objectives. Primary anti-phospholipid syndrome (PAPS) is an autoimmune condition defined by anti-phospholipid antibodies (aPL) and thrombotic or obstetric events. Some PAPS can evolve into systemic lupus erythematosus (SLE) during follow-up. Few studies systematically examined lupus autoantibodies and their clinical significance in PAPS. The aim of our study is to analyze the clinical and laboratory correlations with lupus-related autoantibodies, detected by immunoprecipitation (IP), a technique not yet systematically applied to investigate autoantibodies in this condition.

Methods. Sera from 52 PAPS patients were screened by indirect immunofluorescence (IIF) antinuclear antibodies (ANA), IP of ³⁵S-labeled K562 cell extract, and ELISA [anti-Argonaute2 (Ago2, Su), 60kRo, 52kRo, La, dsDNA)]. Anti-Ago2/Su positive sera were also tested for anti-GW bodies (GWBs) by IIF double staining, using rabbit anti-Rck/p54 serum.

Results. First, 56% of PAPS patients (29/52) were ANA positive, mainly with speckled pattern. Anti-Ago2/Su antibodies were found in 13% (7/52), anti-Ro/SSA in 10% (5/52), anti-La in one case. The clinical profile of patients did not seem to be related to the presence of these antibody specificities. However, levels of IgG anti-β2 glycoprotein I antibodies were lower in anti-Ago2/Su positive patients (p = 0.02). None of anti-Ago2/Su or —Ro patients developed SLE during a 2-year follow-up. Ago2 is a key component of GWBs, however, only 1/7 anti-Ago2/Su serum showed a typical cytoplasmic GWBs staining.

Conclusions. Anti-Ago2/Su and -Ro antibodies are the two autoantibodies detected by IP in our PAPS cohort. Clarifying why Ago2/Su and Ro are specific targets of autoimmunity may help to understand the mechanisms of autoantibody production.     

The homogeneous multiplexed system--a new method for autoantibody profile in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a multi-systemic autoimmune disease leading to immunological aberrations and excessive multiple autoantibody production. The aim of this study was to investigate the prevalence of multiple autoantibodies in SLE patients utilizing the multiplex system method. We analyzed the presence of elevated titers of anti-Ro, anti-La, anti-RNP, anti-Sm, anti-Jo1, anti-centromere, anti-Scl-70, anti-histone, and anti-dsDNA antibodies in 199 serum samples (113 SLE patients, 86 healthy donors). We compared the type, level and number of autoantibodies and the correlation between the autoantibody profile and disease severity utilizing the SLEDAI score. Elevated titers of at least one autoantibody were detected in 48% of 42 SLE patients. Elevated titers of anti-Ro antibodies were most commonly detected. The distribution of specific autoantibodies was: anti-Ro- 23.8%, anti-dsDNA- 19%, antihistone- 19%, anti-RNP- 14.2%, anti-La antibodies- 11.9%, anti-Sm- 7.1%, anti-Scl 70-4.7%, and anti-centromere- 2.4%. Utilizing ROC analysis, the sensitivity and specificity of anti-DNA antibodies at a cutoff value of 34 IU/ml were 87.1% and 79.4% respectively. Elevated titers of anti-Jo1 antibody were not detected. There was a correlation with the titer of anti-Ro antibodies and disease activity by the SLEDAI score. Seven patients harbored one autoantibody only, 15 patients harbored 2-3 autoantibodies, 3 patients harbored 4-5 autoantibodies, and one patient harbored 6 autoantibodies. A correlation between the number of autoantibodies per patient and disease severity was found. One patient with a multitude of autoantibodies had severe lupus and a myriad of clinical manifestations. In conclusion, the multiplex system is specific and sensitive, provides an autoantibody profile in a single test, and may be useful as a diagnostic test for SLE. Elevated anti-Ro antibodies are associated with severe disease. An autoantibody load may be indicative of more severe disease.