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.     

B cell apotopes of the 60-kDa Ro/SSA and La/SSB autoantigens

Apoptosis has been proposed to influence the initiation and diversification of autoimmunity to the Ro (SSA)/La (SSB) ribonucleoprotein (RNP) particle and serve as a target for autoantibody-mediated tissue injury. We have developed a new approach to B cell epitope mapping which identifies "apotopes," defined as epitopes expressed on the surface of apoptotic cells. Preliminary studies support a role for apotopes as diagnostic markers in systemic lupus erythematosus (SLE) and primary Sj?gren's syndrome. For example, apotopes within the NH(2)-terminal and central regions of La react with the majority of sera from mothers of infants with congenital heart block. Furthermore, a Ro60 apotope is specific for a subset of SLE with isolated anti-Ro60 responses. The mapping of B cell apotopes may prove superior to standard epitope mapping by suggesting novel pathways of autoantibody production and identifying pathogenic species of autoantibodies.     

Cross-reaction between antibodies to the major epitope of Ro60 kD autoantigen and a homologous peptide of Coxsackie virus 2B protein

Coxsackie virus RNA has recently been detected in biopsy specimens of minor salivary glands from patients with primary Sjögren's Syndrome (pSS). A peptide derived from Coxsackie virus 2B protein (pepCoxs) presents 87% sequence homology with the 222–229 region of the major linear B‐cell epitope of Ro60 kD autoantigen (pep216–232). Synthetic peptides corresponding to pep216–232: ²¹⁶KALSVETEKLLKYLEAV²³² and pepCoxs: ³¹MVTSTITEKL LKNLVKI⁴⁷, were prepared. Sera from 42 patients with pSS and 43 patients with systemic lupus erythematosus (SLE) as well as sera from 27 healthy individuals (normal controls) and sera from 30 patients with rheumatoid arthritis (disease controls) were tested against the two homologous peptides. Twenty‐five percent of SLE sera and 33·3% of pSS sera reacted against pep216–232, whereas 28% of SLE sera and 37% of pSS sera recognized the pepCoxs. The sera reacting with pep216–232 were apparently the same as those reacting with pepCoxs. Normal sera and disease control sera presented only a limited reactivity against both peptides (ranging from 3·7% to 10%). Both peptides reacted more prominently with anti‐Ro/La (+) sera from pSS patients. Thus, pep216–232 was recognized by 17% of the anti‐Ro (+) sera and by 42% of the anti‐Ro/La (+) sera, whereas pepCoxs was recognized by 28·5% and 38% of the a‐Ro(+) and a‐Ro/La(+) sera, respectively. Purified anti‐pep216–232 antibodies readily reacted with both peptides while inhibition experiments revealed the specificity of this reaction. These results suggest a possible cross‐reaction between antibodies to the major linear B‐cell epitope of Ro60 kD autoantigen and the homologous pepCoxs in pSS patients. This cross‐reaction might potentially play a role in autoantibody formation and the perpetuation of the autoimmune response against Ro/SSA and La/SSB.     

IgG antibodies from patients with primary Sjögren''s syndrome and systemic lupus erythematosus recognize different epitopes in 60-kD SSA/Ro protein.

Five synthetic peptides corresponding to the N-, the C- and a central domain in 60-kD SSA/Ro protein were prepared and tested with sera from 112 patients with systemic lupus erythematosus (SLE), 55 with primary Sjögren's syndrome (pSS) and 29 with rheumatoid arthritis. Among these five fragments, one representing residues 21-41, was recognized by antibodies in 57% of pSS patients. Interestingly, this peptide was recognized by only a few (≤7%) of SLE sera, while 63% of pSS sera and 46% of SLE sera tested in parallel possessed antibodies reacting in ELISA with purified 60-kD SSA protein. The ELISA results were compared with the pattern of reactivity obtained in immunodiffusion and immunoblotting. The results indicate that the sensitivity of ELISA using peptide 21-41 and pSS sera was in the same range as immunoblotting and higher than immunodiffusion. Thus the peptide 21-41 proved useful for the detection of anti-SSA antibodies in the sera of patients with pSS. Furthermore, a positive ELISA using peptide 21–41 could be of potential use to discriminate pSS with systemic features from SLE. The fact that peptide 21–41 is recognized by antibodies in pSS but only by very few SLE sera implies that different mechanisms are involved in the anti-SSA immune response in these two autoimmune diseases.     

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.     

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.     

Epitope mapping of the 52-kD Ro/SSA autoantigen.

Autoantibodies to Ro/SSA are commonly found in sera of patients with systemic lupus erythematosus (SLE) and primary Sjögren's syndrome. The presence of these antibodies is related to lymphopenia, photosensitive dermatitis, and pulmonary and renal disease, suggesting that they have an immunopathologic role [1-6]. We previously isolated a cDNA clone which encodes the 52-kD human Ro/SSA protein. In this study we have determined the number and location of epitopes recognized by SLE sera using recombinant proteins encoded by the full-length or overlapping subclones of this cDNA. An immunodominant epitope was detected using Western blots and ELISA on the NH2-terminal side of this protein's putative leucine zipper. The data suggest that 11 amino acids are critical for the recognition of this molecule by these autoantibodies. Although the titres of anti-52-kD Ro/SSA antibodies vary between different patient sera, no heterogeneity in the location of antigenic epitopes to which their autoantibodies bound was detected. This homogeneous pattern of reactivity to a single rather than multiple regions of this protein is unusual for lupus autoantigens which have been identified, and suggests that these antibodies may have arisen as by a cross-reaction to an epitope on another molecule.     

Sera from patients with rheumatic diseases recognize different epitope regions on the 52-kD Ro/SS-A protein.

Patients suffering from systemic lupus erythematosus (SLE) or Sjögren's syndrome (SS) often contain autoantibodies directed to the Ro(SS-A) complex. In this study the antigenic determinants on two of the components of the Ro complex, i.e. the Ro60 and the Ro52 polypeptides, were investigated. Anti-Ro+ sera were selected by counter-immunoelectrophoresis. Depending on the detection method, 59-68% of the SLE patients produced anti-Ro but not anti-La antibody, while 72-81% of the SS patients produced both anti-Ro and anti-La antibody. Immunoprecipitation of recombinant Ro-proteins showed that 61 sera (87%) were reactive with both Ro proteins, seven sera with Ro60 only, one serum with Ro52 only, and one serum did not precipitate the proteins at all. The anti-Ro60 reactivity of human sera is strongly associated with the native form of Ro60, suggesting that conformational autoepitopes are an important feature of Ro60. In the case of Ro52, frequently the residues located between amino acids 216 and 292 were essential for reactivity with the antibodies. With 70% of the lupus sera tested this appeared to be the only region important for reactivity. The antibodies of SS patients generally recognized multiple B cell epitopes located between amino acids 55 and 292. The results of this study indicate that the antigenic determinants on Ro52 are different for autoantibodies produced by lupus patients compared with those of SS patients.     

Detection of anti-Ro(SSA) antibodies by gel double diffusion and a 'sandwich' ELISA in systemic and subacute cutaneous lupus erythematosus and Sj?gren's syndrome

A newly described Ro 'sandwich' ELISA was compared to the gel double diffusion technique to detect anti-Ro(SSA) antibodies in Sj?gren's syndrome, systemic and subacute cutaneous lupus erythematosus patients. This study demonstrates that the ELISA assay increased the frequency of detection of anti-Ro(SSA) antibodies in these well defined connective tissue disease patients by approximately 5-10% compared to the gel double diffusion anti-Ro(SSA) antibody assay. The study also confirms that some patients make anti-Ro(SSA) antibodies directed solely at unique human Ro(SSA) antigen epitopes. We also detected the existence of a significant Sj?gren's syndrome patient population failing to make significant anti-Ro(SSA) antibodies. We conclude from our study that the gel double-diffusion technique employing human spleen extract as a source of the Ro(SSA) antigen is, at present, the most cost-effective test to detect anti-Ro(SSA) antibodies.     

Calreticulin binds preferentially with B cell linear epitopes of Ro60 kD autoantigen,enhancing recognition by anti-Ro60 kD autoantibodies

Calreticulin is a molecular chaperone to newly synthesized polypeptides. Previous studies suggested that calreticulin is probably a protein member of the Ro/La RNP complex. The aims of this study were (a) to investigate whether linear B cell epitopes of the Ro/La RNP complex are bound to calreticulin and (b) if the complex peptide–calreticulin is recognized specifically by anti‐Ro autoantibodies. Calreticulin was isolated from either human or pig spleen using a multi‐step purification method and found to interact preferentially with biotinylated peptides derived from the sequence of the Ro60 kD 175–184aa(10p) and 216–232aa(17p). The interaction of the peptide–calreticulin complex was favoured by the combination of heat treatment, divalent cations and ATP. La/SSB epitopes did not react with calreticulin. Peptides corresponding to La/SSB epitopes as well as the common epitope of Sm did not interact with calreticulin. Thirty‐eight anti‐Ro60 KD positive and 23 anti‐Ro60 kD negative sera of patients with systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS) were tested. All anti‐Ro60 kD positive sera bound the complex calreticulin‐17p, while 95% of the same sera had activity against the complex calreticulin ? 10p. Tested individually, calreticulin, pep10p and pep17p presented very low reactivity (8%, 11% and 29%, respectively) against anti‐Ro60 kD positive sera. Anti‐Ro60 KD negative sera did not exhibit significant reactivity either with calreticulin, 10ρ and 17ρ or with the complexes calreticulin ? 10p and calreticulin‐17p (<5%). These results suggest that calreticulin can induce conformation‐dependent recognition of the Ro60 kD epitopes, leading eventually to their recognition by autoantibodies. This is the first time that such a relationship is shown between a chaperone protein and fragments of an intracellular autoantigen. This work also provides insights into the understanding of mechanisms for autoantibody production. Furthermore, this association can be proved useful for the development of new sensitive assays for autoantibody detection.     

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.     

Post-translational modifications of the major linear epitope 169-190aa of Ro60 kDa autoantigen alter the autoantibody binding

Ro60 kDa is a member of the Ro/LaRNP ribonucleoprotein complex and its major linear B cell epitope, corresponding to the region 169–190aa, has been found to be the initial target of the autoimmune response in patients with systemic lupus erythematosus. This sequence contains one serine and two arginine amino acid residues, which can potentially be modified post‐translationally by phosphorylation or citrullination, respectively. The aim of this study was to develop an immunoassay for anti‐Ro60 kDa epitope antibody detection and to investigate the changes in the antigenicity of the Ro60 kDa epitope when it is post‐translationally modified, by either citrullination or phosphorylation. Peptide analogues corresponding to the unmodified form of the epitope, its phosphorylated form, and a form with both arginine residues citrullinated were synthesized. The peptide coating conditions were investigated and it was found that the use of highly hydrophilic surfaces increase the efficiency of the coating, as well as the sensitivity of the method for anti‐peptide antibody detection. All peptides were tested by the optimized enzyme‐linked immunosorbent assay (ELISA) against 119 sera from patients with primary Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis with anti‐Ro/SSA reactivity, 20 sera from patients with systemic diseases without anti‐Ro/SSA immune reactivity, as well as against 65 sera from normal individuals. A large proportion of the tested sera reacted against all three peptide analogues, although with a preference for the unmodified form of the epitope. In conclusion, post‐translational modifications of the major Ro60 kDa B cell epitope can alter the autoantibody binding.     

Cross-recognition between histones and La/SSB may account for anti-DNA reactivity in SLE patients

Antibodies to La/SSB are detected in sera of patients with primary Sjogren's syndrome (pSS) and systemic lupus erythematosus (SLE). The vast majority of anti-La/SSB positive sera contain antibodies directed towards a linear B-cell epitope of La/SSB spanning the sequence 349-364aa (pep349-364). The aim of this study was to evaluate the fluctuation of antibody levels to major B-cell epitopes of La/SSB over time and investigate for their possible crossreactions. Sequential sera from 15 SLE and 15 pSS patients, followed from 3 to 10 years were obtained. All patients with SLE were positive for anti-Ro/SSA, anti-La/SSB and anti-dsDNA antibodies and patients with pSS were positive for anti-Ro/SSA and anti-La/SSB antibodies. Sera from 30 patients with SLE without anti-La/SSB antibodies and 30 healthy individuals served as disease and negative control respectivelly. All sera tested for the presence of anti-pep349-364 antibodies, using a specific ELISA. Specific anti-pep349-364 IgG was purified from sera of SLE patients and evaluated for cross reactivity against dsDNA and histones. In all SLE sera the levels of anti-pep349-364 antibodies varied in time and fluctuated in parallel with anti dsDNA antibodies. Anti-pep349-364 IgG purified from 7 SLE patients. Five out of 7 were found to react with calf thymus DNA in ELISA. All purified (7/7) anti-pep349-364 IgG preparations reacted with histone H1 and failed to produce a positive immunofluorescence pattern in Crithidia luciliae anti-dsDNA assay which lacks histones. Competative inhibition experiments demonstrated that histone H1 could inhibit completely the binding of anti-pep349-364 IgG to pep349-364 while pep349-364 inhibited by 70% the binding of anti-pep349-364 IgG to histone H1. These findings indicate that a subgroup of SLE patients possess cross-reacting anti-histone H1 antibodies and anti-pep349-364 antibodies, which can be faulty considered as anti-dsDNA reactivity in regular ELISA techniques.     

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.     

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.     

Anti-Ro fine specificity defined by multiple antigenic peptides identifies components of tertiary epitopes

Anti-Ro (or SSA) is a clinically important autoantibody that is found in 25–40% of patients with systemic lupus erythematosus as well as an even greater proportion of patients with Sjögren's syndrome or subacute cutaneous lupus. We have studied the binding of anti-Ro sera to multiple antigenic peptides constructed from the sequence of the 60-kD Ro molecule. The results demonstrate that sera bind these peptides in solid-phase assay. Surprisingly, some of these peptides also form a precipitin line in double immunodiffusion with anti-Ro sera. Formation of lines of identity in double immunodiffusion as well as absorption studies indicate that peptides distant in the primary amino acid sequence and without shared sequence are bound by the same antibody. In addition, data from surface plasmon resonance demonstrate that peptides identified in this manner have protein–protein interactions. Thus, these techniques may identify the components of conformational epitopes.     

Immunization with peptides from 60 kDa Ro in diverse mouse strains

Antibodies binding the Ro (or SSA) and La (or SBB) proteins are commonly found in a high proportion of sera from patients with systemic lupus erythematosus or Sjögren's syndrome. The mechanism by which these autoantibodies arise is not known. Others and we have shown that immunization of nonautoimmune‐prone mice with short peptides from the Ro ribonucleoprotein particle can induce autoimmunity to 60 kDa Ro and 52 kDa Ro as well as to the 48 kDa La protein after epitope spreading. We have explored the differences in the epitope spreading after 60 kDa Ro peptide immunization in several strains of mice. There is intra‐ and intermolecular diversification of the immune response after immunization of DBA/2J animals with a monomer peptide representing the residues 480–494 of the 60 kDa Ro protein, but this peptide does not induce epitope spreading when used as the immunogen in either C57Bl/6J or PL/J mice. Similar to previously studied BALB/c mice, DBA/2J mice have antibodies binding many epitopes of 60 kDa Ro, and some sera bind 52 kDa Ro as well as La. These mice have antinuclear antibody in their sera. These data demonstrate that Ro peptide immunization results in different outcomes depending upon the strain of mouse used. Furthermore, these data suggest that genetic variation is important with regard to responding towards short peptide immunization by epitope spreading.     

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-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.