Fine specificity of the autoimmune response to the Ro/SSA and La/SSB ribonucleoproteins.
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The fine specificity of the Ro and La proteins has been studied by several techniques. In general, there is agreement in a qualitative sense that autoantibodies bind multiple epitopes. For some specific antibody binding, different studies agree quantitatively, for instance, the binding of the carboxyl terminus of 60-kd Ro as described by 2 studies using different techniques and the presence of an epitope within the leucine zipper of 52-kd Ro. In addition, there is general agreement about the location of a prominent epitope at the RRM motif region of the La molecule. On the other hand, the many specific epitope regions of the molecules differ among these studies. These discrepancies are likely the result of using different techniques, sera, and peptide constructs as well as a result of inherent advantages and disadvantages in the individual approaches. Several theories concerning the origin of not only the antibodies, but also the diseases themselves, have been generated from studies of the fine specificity of antibody binding. These include a theory of a primordial foreign antigen for anti-Ro autoimmunity, molecular mimicry with regard to La and CCHB, as well as the association of anti-Ro with HLA. These remain unproven, but are of continuing interest. An explanation for the association of anti-60-kd Ro and anti-52-kd Ro in the sera of patients has sprung from evaluating antibody binding. Data demonstrating multiple epitopes are part of a large body of evidence that strongly suggests an antigen-driven immune response. This means that the autoantigens are directly implicated in initiating and sustaining autoimmunity in their associated diseases. A number of studies have investigated the possibility of differences in the immune response to these antigens in SS and SLE sera. While several differences have been reported, none have been reproduced in a second cohort of patients. Furthermore, none of the reported differences may be sufficiently robust for clinical purposes, such as distinguishing between SS with systemic features and mild SLE, although some might be promising. For instance, in at least 3 groups of SLE patients, no binding of residues spanning amino acids 21-41 of 60-kd Ro has been found. Meanwhile, 1 of those studies found that 41% of sera from patients with primary SS bound the 60-kd Ro peptide 21-41. Perhaps future studies will elaborate a clinical role of such a difference among SS and SLE patients. Study of the epitopes of these autoantigens has, in part, led to a new animal model of anti-Ro and anti-La. Non-autoimmune-prone animals are immunized with proteins or peptides that make up the Ro/La RNP. Such animals develop an autoimmune response to the entire particle, not just the immunogen. This response has been hypothesized to arise from autoreactive B cells. In another, older animal model of disease, the MRL-lpr/lpr mouse, B cells have recently been shown to be required for the generation of abnormal, autoreactive T cells. Thus, there are now powerful data indicating that B cells that produce autoantibodies are directly involved in the pathogenesis of disease above and beyond the formation of immune complexes. Given that the autoreactive B cell is potentially critical to the underlying pathogenesis of disease, then studying these cells will be crucial to further understanding the origin of diseases associated with Ro and La autoimmunity. Hopefully, an increased understanding will eventually lead to improved treatment of patients. Progress in the area of treatment will almost surely be incremental, and studies of the fine specificity of autoantibody binding will be a part of the body of basic knowledge contributing to ultimate advancement. In the future, the animal models will need to be examined with regard to immunology and immunochemistry as well as genetics. The development of these autoantibodies has not been studied extensively because upon presentation to medical care, virtually all patients have a full- (+info)
A critical evaluation of enzyme immunoassays for detection of antinuclear autoantibodies of defined specificities. I. Precision, sensitivity, and specificity.
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OBJECTIVE: To determine the performance characteristics of enzyme-based immunoassay (EIA) kits for the detection of antinuclear and other autoantibodies of defined specificities. METHODS: Nine manufacturers of EIA kits to detect antibodies of defined specificities participated in a study in which they received coded sera from the Centers for Disease Control and Prevention. These coded sera contained different dilutions of antibody of one specificity mixed with sera containing antibodies of other specificities. The manufacturers were asked to use their standard technology to determine antibody content and send the data to a committee of the International Union of Immunological Societies for analysis. The data were analyzed for sensitivity and specificity in the detection of anti-double-stranded DNA (anti-dsDNA), anti-single-stranded DNA, antihistone, anti-Sm, anti-U1 RNP, anti-SSA/Ro, anti-SSB/La, anti-Scl-70 (DNA topoisomerase I), anticentromere, and anti-Jo-1 antibodies. In addition, replicate samples were included in the coded sera to evaluate the precision of each EIA method. RESULTS: Lack of sensitivity and specificity was most evident in the anti-dsDNA and anti-Sm kits, although 2 kits for anti-dsDNA achieved acceptable sensitivity and specificity. Generally, anti-SSA/Ro, anti-SSB/La, anti-Scl-70, anticentromere, and anti-Jo-1 kits performed well. Many false-positive results were obtained with a multiple myeloma serum containing cryoprecipitates, but multiple myeloma sera without cryoprecipitates presented no problem in the EIA system. Precision, based on evaluation of replicate samples, varied from very good to poor. CONCLUSION: No single manufacturer was clearly superior to others in terms of their products' overall sensitivity, specificity, and precision. Areas that needed improvement were in kits for the detection of antibodies to dsDNA and to Sm antigen. Some EIA kits achieved good sensitivity and specificity. Individual manufacturers were informed of the performance of their respective kits so they could take measures to correct perceived deficiencies and thus improve the reliability of a group of important diagnostic assays used in the evaluation of systemic rheumatic diseases. (+info)
Bacillus subtilis histone-like protein, HBsu, is an integral component of a SRP-like particle that can bind the Alu domain of small cytoplasmic RNA.
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Small cytoplasmic RNA (scRNA) is metabolically stable and abundant in Bacillus subtilis cells. Consisting of 271 nucleotides, it is structurally homologous to mammalian signal recognition particle RNA. In contrast to 4.5 S RNA of Escherichia coli, B. subtilis scRNA contains an Alu domain in addition to the evolutionarily conserved S domain. In this study, we show that a 10-kDa protein in B. subtilis cell extracts has scRNA binding activity at the Alu domain. The in vitro binding selectivity of the 10-kDa protein shows that it recognizes the higher structure of the Alu domain of scRNA caused by five consecutive complementary sequences in the two loops. Purification and subsequent analyses demonstrated that the 10-kDa protein is HBsu, which was originally identified as a member of the histone-like protein family. By constructing a HBsu-deficient B. subtilis mutant, we showed that HBsu is essential for normal growth. Immunoprecipitating cell lysates using anti-HBsu antibody yielded scRNA. Moreover, the co-precipitation of HBsu with (His)6-tagged Ffh depended on the presence of scRNA, suggesting that HBsu, Ffh, and scRNA make a ternary complex and that scRNA serves as a functional unit for binding. These results demonstrated that HBsu is the third component of a signal recognition particle-like particle in B. subtilis that can bind the Alu domain of scRNA. (+info)
HTLV-I associated Sjogren's syndrome is aetiologically distinct from anti-centromere antibodies positive Sjogren's syndrome.
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OBJECTIVE: To investigate whether Sjogren's syndrome (SS) with anti-HTLV-I antibodies is aetiopathologically distinguishable from SS without these antibodies, the study compared prevalence of autoantibodies in serum samples of SS patients with or without anti-HTLV-I antibodies. METHODS: The test group included 135 patients with primary SS and 97 patients with secondary SS. Serum samples of the patients were examined for the presence of anti-nuclear antibodies (ANA), anti-SS-A/Ro antibodies, anti-SS-B/La antibodies, anti-centromere antibodies (ACA), and anti-HTLV-I antibodies. RESULTS: Anti-HTLV-I antibodies were detected in 25.0% of primary SS patients and in 29.2% of secondary SS patients. There were no significant differences in the mean age, sex, values of asparate aminotransferase, alanine aminotransferase, alkaline phosphatase, serum complements and IgG between HTLV-I seropositive and seronegative SS patients. The rheumatoid factor, ANA, anti-SS-A/Ro, and anti-SS-B/La antibodies in serum samples of SS patients were detected in 60.0%, 84.0%, 51.9%, and 12.0%, respectively. There was no significant difference in the prevalence of these antibodies between HTLV-I seropositive and seronegative SS patients. Using the indirect immunofluorescence test, 14.2% showed a discrete speckled staining pattern. All serum samples contained significant amounts of ACA determined by enzyme linked immunosorbent assay. These antibodies were detected in only 4% of HTLV-I seropositive SS patients but were present in 19.9% of HTLV-I seronegative SS patients. Furthermore, the prevalences of anti-SS-A/Ro and anti-SS-B/La antibodies in serum samples of ACA positive patients were significantly lower than those in ACA negative SS patients. CONCLUSION: These results suggest that SS patients with anti-SS-A/Ro or anti-SS-B/La antibodies, or both, might be aetiopathologically distinct from SS patients with ACA. HTLV-I might be involved in the pathogenesis of SS in a subset of patients with anti-SS-A/Ro or anti-SS-B/La antibodies, or both, but not SS patients with ACA. (+info)
Immunization of mice with human 60-kd Ro peptides results in epitope spreading if the peptides are highly homologous between human and mouse.
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OBJECTIVE: Immunization with peptide fragments of autoantigens may lead to an immune response at both the T and B cell level that is directed not only at the immunogen, but also at the autoantigen from which the peptide came. In addition, a complex multicomponent particle may become the target of this expanded immune response. The purpose of this study was to determine the ability of several different peptides from 60-kd Ro to induce expansion of the immune response to the Ro/La RNP particle. METHODS: We immunized BALB/c mice with 3 different oligopeptides from human 60-kd Ro (or, SSA). RESULTS: Animals immunized with peptides either identical to or differing by only 1 amino acid developed autoimmunity to the entire Ro RNP particle. Animals immunized with a human peptide highly divergent from the corresponding mouse sequence developed an immune response to the immunogen only and showed little evidence of epitope spreading. Furthermore, these mice did not have antibodies that bound the poorly conserved mouse homolog peptide, and the antibody response to this peptide did not include IgG1. CONCLUSION: These data indicate that B lymphocytes specific for the self-peptide that is homologous to the immunogen are a critical determinant for spreading of the immune response to other components of self. (+info)
Distribution and antigen specificity of anti-U1RNP antibodies in patients with systemic sclerosis.
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Systemic sclerosis (SSc) is a generalized connective tissue disease which is characterized by the presence of several autoantibodies. To determine the prevalence and antigen specificity of anti-U1RNP antibodies (anti-U1RNP) in patients with SSc, serum samples from 223 patients with SSc, 117 patients with systemic lupus erythematosus (SLE), 18 patients with mixed connective tissue disease (MCTD) and 40 healthy control subjects were examined by indirect immunofluorescent analysis (IIF), double immunodiffusion, and immunoblotting using nuclear extract of HeLa cells. Eighteen of the 223 (8%) serum samples from patients with SSc were shown to be positive for anti-U1RNP. The frequency of anti-U1RNP positivity in limited cutaneous SSc (14%) was significantly higher than that in those with diffuse cutaneous SSc (3%). Anti-Sm antibodies were detected in patients with SLE positive for anti-U1RNP, but not in those with SSc positive for anti-U1RNP or those with MCTD. Immunoblotting demonstrated that anti-70-kD antibodies were detected more often in patients with SSc positive for anti-U1RNP and in those with MCTD than in those with SLE. Furthermore, anti-U1RNP was closely correlated with pulmonary fibrosis and joint involvement in patients with SSc. These results suggest that anti-70-kD antibodies are useful in the classification of patients with anti-U1RNP. (+info)
Rapid nucleolytic degradation of the small cytoplasmic Y RNAs during apoptosis.
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We have investigated the fate of the RNA components of small ribonucleoprotein particles in apoptotic cells. We show that the cytoplasmic Ro ribonucleoprotein-associated Y RNAs are specifically and rapidly degraded during apoptosis via a caspase-dependent mechanism. This is the first study describing the selective degradation of a specific class of small structural RNA molecules in apoptotic cells. Cleavage and subsequent truncation of Y RNAs was observed upon exposure of cells to a variety of apoptotic stimuli and were found to be inhibited by Bcl-2, zinc, and several caspase inhibitors. These results indicate that apoptotic degradation of Y RNAs is dependent on caspase activation, which suggests that the nucleolytic activity responsible for hY RNA degradation is activated downstream of the caspase cascade. The Y RNA degradation products remain bound by the Ro60 protein and in part also by the La protein, the only two proteins known to be stably associated with intact Ro ribonucleoprotein particles. The size of the Y RNA degradation products is consistent with the protection from degradation of the most highly conserved region of the Y RNAs by the bound Ro60 and La proteins. Our results indicate that the rapid abrogation of the yet unknown function of Y RNAs might be an early step in the systemic deactivation of the dying cell. (+info)
RNA editing associated with the generation of two distinct conformations of the trypanosomatid Leptomonas collosoma 7SL RNA.
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Analysis of the trypanosomatid Leptomonas collosoma 7SL RNA revealed the existence of two distinct stable 7SL RNA conformers (7SL I and II). Sequence analysis of the RNAs indicated a single base difference between the conformers at position 133 (C in 7SL II and U in 7SL I) located in domain III. This change appears to be the result of a post-transcriptional editing event, since the single-copy 7SL RNA gene codes exclusively for a C at this position. The edited form (7SL I) was found preferentially in the cytoplasm, and the pre-edited form in the nucleus. 7SL I is mainly bound to ribosomes, whereas 7SL II is more abundant in ribosome-free particles. Mutations introduced in regions outside the editing site were found to occur in a single conformation, suggesting that the editing event is not the only factor that determines the conformation of the molecule. This study is the first description of an editing event on a small RNA other than tRNA and is the first report of C --> U editing in trypanosomes. We propose a novel role for RNA editing in controlling the conformation of the 7SL RNA in vivo. (+info)