Purification and partial sequencing of the nuclear autoantigen RA33 shows that it is indistinguishable from the A2 protein of the heterogeneous nuclear ribonucleoprotein complex.

RA33 is a nuclear autoantigen with an apparent molecular mass of 33 kD. Autoantibodies against RA33 are found in about 30% of sera from RA patients, but only occasionally in sera from patients with other connective tissue diseases. To characterize RA33, the antigen was purified from HeLa cell nuclear extracts to more than 90% homogeneity by affinity chromatography on heparin-Sepharose and by chromatofocusing. Sequence analysis of five tryptic peptides revealed that their sequences matched corresponding sequences of the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Furthermore, RA33 was shown to be present in the 40S hnRNP complex and to behave indistinguishably from A2 in binding to single stranded DNA. In summary, these data strongly indicate that RA33 and A2 are the same protein, and thus identify on a molecular level a new autoantigen.

Vascular endothelial growth factor in patients with psoriatic arthritis.

OBJECTIVE: To determine the levels of vascular endothelial growth factor (VEGF) in patients with active psoriatic arthritis, patients with inactive psoriatic arthritis, and healthy controls. Serum VEGF levels were correlated with clinical and laboratory features in patients with active psoriasis arthritis. METHODS: Serum samples from 14 patients with active psoriatic arthritis, 14 patients with inactive psoriatic arthritis, and 9 healthy controls were investigated. VEGF levels in the serum were measured using a sensitive sandwich ELISA. RESULTS: The mean serum VEGF concentration in patients with active PA was 394.4 pg/ml (394 +/- 171.8), in patients with inactive PA 200.4 pg/ml (200.4 +/- 115.7), and in healthy subjects 214.3 pg/ml (214.3 +/- 162.1). Patients with active psoriasis arthritis had significantly higher levels of VEGF compared to patients with inactive psoriasis arthritis and healthy individuals (p > 0.001). In contrast, VEGF levels were comparable in patients with inactive psoriatic arthritis and controls (p =0.659). Furthermore, in patients with psoriatic arthritis, VEGF levels were positively correlated with ESR, HAQ, PASI and VAS. CONCLUSION: VEGF levels may be regarded as a good indicator of active psoriasis arthritis.

Antibodies to antinuclear subsets in systemic lupus erythematosus and rheumatoid arthritis.

In this study, technical, clinical and genetic aspects of anti-Ro/SSA, anti-Sm, and anti-RNP are discussed; different techniques may reveal differences in autoantibody frequencies and associations. Finally, the importance of anti-RA33, a new autoantibody characteristic of rheumatoid arthritis, is evidenced.

New treatment possibilities in rheumatoid arthritis.

It is very difficult to predict future treatment modalities especially in diseases like rheumatoid arthritis (RA) with unknown etiology and pathogenesis. In the near future, traditional disease-modifying antirheumatic drugs (DMARD) alone, in combination with each other, or together with cyclosporine, FK506, Rapamycin, or Leflunomide, will probably be the main treatment for RA. Currently biological anti-TNFalpha agents like humanized MAb and recombinant TNF-receptor constructs are now launched in the market. This therapy alone, or in combination with methotrexate is very effective in RA patients. There are, however, concerns over increase in serious infections. Autologous stem cell transplantation will probably be used in certain patient with serious autoimmune diseases.

Clinical and immunological aspects of autoantibodies to RA33/hnRNP-A/B proteins--a link between RA, SLE and MCTD.

Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are major constituents of the spliceosome. They are composed of approximately 30 different proteins which can bind to nascent pre-mRNA. Among these, the hnRNP-A/B proteins form a subgroup of highly related proteins consisting of two adjacent RNA binding domains (RBD) within the N-terminal parts, whereas the C-terminal halves contain almost 50% glycine residues. These proteins, in particular A2/RA33, are targeted by autoantibodies from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD). In SLE anti-hnRNP antibodies frequently occur together with antibodies to U1 small nuclear RNP (U1-snRNP) and Sm, other proteins of the spliceosome. Preliminary epitope mapping studies have revealed major antibody binding sites in the RNA binding regions for all three diseases. Nevertheless, there is some indication of disease specific epitope recognition. Studies in animal models have demonstrated anti-RA33/hnRNP-A/B antibodies in lupus-prone mouse strains. Thus, autoantibodies to the spliceosomal hnRNP-A/B proteins are a common feature of RA, SLE, and MCTD. However, these diseases differ in their reactivities to other spliceosomal proteins, especially anti-U1 snRNP and Sm. Therefore, anti-RA33/hnRNP-A/B autoantibodies are not only valuable diagnostic markers but may also allow additional insights into the pathogenesis of rheumatic autoimmune diseases.

Sensitivity and specificity of anti-Sa autoantibodies for rheumatoid arthritis.

OBJECTIVE: To assess the sensitivity and specificity of the recently described anti-Sa autoantibodies in order to determine their potential usefulness for the diagnosis of rheumatoid arthritis (RA). METHODS: Sera from 67 patients with RA (including 31 patients with early RA of <3 months duration), from 180 patients with other rheumatic diseases and from 30 healthy control subjects were investigated by immunoblotting employing partially purified Sa antigen. Additionally, all sera were tested for rheumatoid factor (RF), anti-A2/RA33, antinuclear antibodies (ANA) and ANA subsets. RESULTS: Twenty-one (31%) of the 67 patients with RA, but only four of the 180 control patients, were anti-Sa positive; therefore, anti-Sa was approximately 98% specific for RA. Anti-Sa was not associated with either RF or with anti-A2/RA33. The latter antibody was present in 21 RA sera, only eight of which also contained anti-Sa. Thus, 34 RA sera (51%) showed at least one of these two autoantibodies and, importantly, 18 of these sera were RF negative. Furthermore, of the 31 patients with early RA, 12 (40%) were anti-Sa and/or anti-A2/RA33 positive. CONCLUSION: Although the numbers studied remain small, taken together, these data suggest that anti-Sa may represent a promising novel serological marker with high specificity for RA.

Autoantibody to the nuclear antigen RA33: a marker for early rheumatoid arthritis.

Sera from 47 patients with early (< 3 months) arthritis of any type were investigated for anti-RA33, a new anti-nuclear autoantibody characteristic of RA, and the diagnoses determined within the following 8-14 months. In addition, seven patients with unclassified arthritis of > 4 months duration, who were all anti-RA33 positive, were followed for up to 2 years to establish their final rheumatologic diagnoses. Four of 47 early arthritis patients' sera were anti-RA33 positive at the initial evaluation; 14 of these 47 patients (30%) could be classified as RA (according to established criteria) at the final evaluation. All four anti-RA33 positive patients belonged to the RA group (27% of RA patients); of the 33 non-RA patients none had anti-RA33 (P = 0.005). Rheumatoid factor was found in four RA (none of whom had anti-RA33), but also in two non-RA patients (P = 0.05). Finally, the study involved seven additional patients with longer standing, initially unclassified, anti-RA33 positive arthritis: in all of them a diagnosis of RA could be established within 3 years of disease onset. These results suggest that anti-RA33 helps to discriminate early RA from other forms of early arthritis and, in the absence of an established diagnoses, it is predictive of RA. Its discriminative capacity appears to be better than and complementary to that of RF.

The concurrence of rheumatoid arthritis and limited systemic sclerosis: clinical and serologic characteristics of an overlap syndrome.

OBJECTIVE: The characteristics of 3 patients with longstanding rheumatoid arthritis (RA) and consecutive evolution of limited cutaneous systemic sclerosis (IcSSc) were evaluated and compared with those of patients with IcSSc alone (n = 20) or with RA alone (n = 120). METHODS: Clinical features of the different patient populations were compared. Serologic analyses included tests for antinuclear antibodies (ANA) and ANA subsets, in particular anticentromere antibodies (ACA) and anti-heterogeneous nuclear RNP (hnRNP)-A2/RA33 (anti-A2/RA33). RESULTS: The 3 patients with RA developed IcSSc 11, 29, or 50 years after the onset of RA. Features of IcSSc were Raynaud's phenomenon, sclerodactyly, and telangiactasias in all 3 patients, and esophageal dysmotility in 1 patient. Rheumatoid factor (RF) and anti-A2/ RA33 were each found in 2 patients, and 1 of these patients was seropositive for both RF and anti-A2/RA33. ACA titers were positive in all cases. However, similar to the development of RA prior to IcSSc, the occurrence of autoantibodies typical of RA preceded the occurrence of ACA, at least in 2 of the patients. Using affinity-purified antibodies, cross-reactivities between anti-centromere protein A (CENP-A) and anti-CENP-B antibodies with anti-A2/RA33 antigens were seen in the 2 anti-A2/RA33-positive patients. Such cross-reactivities were not found in IcSSc patients without concomitant RA. Epitope mapping revealed that both autoantibody specificities recognized the known major epitopes: anti-CENP-B reacted with the C-terminal region and anti-A2/RA33 with the second RNA binding domain in the N-terminal region of hnRNP-A2. CONCLUSION: The RA-lcSSc overlap syndrome in these 3 patients with longstanding RA was characterized by an incomplete CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasias) syndrome. The study demonstrated the presence of autoantibodies typical of both diseases and cross-reactivity of ACA with hnRNP-A2/RA33 in the sera of these patients.

Isolation and characterization of messenger RNA from male inflorescences and pollen of the white birch (Betula verrucosa).

A glycoprotein with a molecular weight (MW) of 17 kilodaltons (kD), Bet v I, represents the major allergen of the white birch (Betula verrucosa, BV) and plays an important role in tree-pollen-induced type I allergic reactions. In order to characterize the major and also some minor allergens of BV, we investigated the IgE-binding properties of these allergens using immunoblot techniques. Normal and patients' sera were employed for this study. Furthermore, RNA from male inflorescences and from pollen of BV were isolated and purified by affinity chromatography on oligo-dT-cellulose. Poly(A)+-mRNA thus obtained was translated in vitro in a cell-free wheat germ system and the proteins synthesized were separated by SDS-PAGE and transferred to nitrocellulose. The blots were incubated with normal human sera and with sera from patients allergic to birch pollen. Bound IgE antibodies were detected with 125I-labeled anti-IgE. We observed major IgE binding to a protein of an MW of 12.5 kD, and little IgE binding to a 17-kD protein, presumably Bet v I. Comparing the products of in vitro translation from mRNA preparations of mature pollen and of male inflorescences collected in June, October and February, little seasonal variations could be observed. As the in vitro translation system does not glycosylate proteins, our results show that the majority of IgE in patients' sera is not directed against the carbohydrate moieties of these allergens.

Molecular definition of heterogeneous nuclear ribonucleoprotein R (hnRNP R) using autoimmune antibody: immunological relationship with hnRNP P.

Serum from a patient showing symptoms related to autoimmunity was found to contain autoantibodies to the nuclear mitotic apparatus (NuMA) protein and to several novel nuclear antigens with estimated molecular weights of 40, 43, 72, 74 and 82 kDa. Using this serum for screening a human cDNA expression library a 2.5 kb cDNA clone was isolated which encoded the complete sequence of a protein of 633 amino acids. Sequence analysis revealed a modular structure of the protein: an acidic N-terminal region of approximately 150 amino acids was followed by three adjacent consensus sequence RNA binding domains located in the central part of the protein. In the C-terminal portion a nuclear localization signal and an octapeptide (PPPRMPPP) with similarity to a major B cell epitope of the snRNP core protein B were identified. This was followed by a glycine- and arginine-rich section of approximately 120 amino acids forming another type of RNA binding motif, a RGG box. Interestingly, three copies of a tyrosine-rich decapeptide were found interspersed in the RGG box region. The major in vitro translation product of the cDNA co-migrated in SDS-PAGE with the 82 kDa polypeptide that was recognized by autoantibodies. The structural motifs as well as the immunofluorescence pattern generated by anti-82 kDa antibodies suggested that the antigen was one of the proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Subsequently the 82 kDa antigen was identified as hnRNP R protein by its presence in immunoprecipitated hnRNP complexes and co-migration of the recombinant protein with this hitherto uncharacterized hnRNP constituent in two-dimensional gel electrophoresis. The concomitant autoimmune response to a hnRNP component of the pre-mRNA processing machinery and to NuMA, a protein engaged in mitotic events and reported to be associated with mRNA splicing complexes in interphase, may indicate physical and functional association of these antigens. Support for this notion comes from observations that concomitant or coupling of autoantibody responses to proteins which are associated with each other as components of subcellular particles are often found in autoimmune diseases.

Fine specificity of anti-Ro(SSA) autoantibodies and clinical manifestations in patients with systemic lupus erythematosus.

OBJECTIVE: To determine the fine specificity of the anti-Ro(SSA) autoimmune response in patients with systemic lupus erythematosus (SLE), and to correlate it with clinical and serological manifestations. METHODS: The frequency of anti-Ro and anti-La autoantibodies was determined by double immunodiffusion (DID), ELISA, and immunoblotting (IB) in 69 patients with SLE and 39 controls. Protein and RNA immunoprecipitation were used to further characterize anti-Ro positive sera. RESULTS: Anti-Ro antibodies were detected in 37 (54%) patients: 33 (48%) were positive by DID, 35 (51%) by ELISA, and 25 (35%) by IB; 32 sera were reactive in at least 2 of these 3 assay systems. By IB, 12 patients had antibodies to both the 60 kDa Ro (Ro60) and the 52 kDa Ro (Ro52), 11 patients were anti-Ro60 positive, 2 patients were anti-Ro52 positive, and 12 patients were not reactive with blotted Ro antigens. However, in immunoprecipitation assays all but one anti-Ro positive sera precipitated both Ro proteins. Anti-La reactivities were found in 15 anti-Ro positive patients: 13 sera were positive by IB, 11 by ELISA, and 9 by DID. Significant associations of anti-Ro antibodies with clinical symptoms were found for sicca syndrome, which was increased in anti-Ro positive patients (p < 0.05 vs anti-Ro negative patients), and for nephritis, for which an inverse correlation was found, since it was less frequently diagnosed in anti-Ro positive patients (p < 0.01). However, this association was seen only for those anti-Ro positive patients who were not reactive with Ro52 by IB. No difference was observed between anti-Ro/La and anti-Ro positive patients. CONCLUSION: DID and ELISA were of comparable sensitivity for detection of anti-Ro, IB was the most sensitive method for detection of anti-La. Moreover, our data indicate that IB may help to characterize clinically distinct subgroups of anti-Ro positive patients with SLE. Thus, determination of anti-Ro by IB may increase the prognostic value of this autoantibody.

Demonstration of a new antinuclear antibody (anti-RA33) that is highly specific for rheumatoid arthritis.

Using immunoblot analysis with soluble nuclear extracts from HeLa cells, we identified autoantibodies to an antigen with a molecular weight of approximately 33,000 in 36% of 95 sera from rheumatoid arthritis patients, but in only 1 of 170 controls. The antigen, termed RA33, was resistant to DNase and RNase digestion but sensitive to proteinase K treatment. There was no discernible relation to other autoantibodies. Thus, this newly described autoantibody appears to be highly specific for rheumatoid arthritis.

Induction of systemic lupus erythematosus by interferon-gamma in a patient with rheumatoid arthritis.

The development of systemic lupus erythematosus (SLE) after 38 months of therapy with recombinant human interferon gamma (rIFN-gamma) was observed in a patient with rheumatoid arthritis. In addition to glomerulonephritis and a butterfly rash, previously negative tests for antinuclear, anti-dsDNA and anti-Sm antibodies became positive. We assume that rIFN-gamma induced the de novo development of SLE in our patient.

Autoimmune response to the spliceosome. An immunologic link between rheumatoid arthritis, mixed connective tissue disease, and systemic lupus erythematosus.

OBJECTIVE: To assess the significance of autoantibodies to RA33, the A2 protein of the heterogeneous nuclear ribonucleoproteins (hnRNP), and to the related hnRNP proteins A1, B1, and B2 in rheumatic diseases. METHODS: Using a partially purified preparation of hnRNP-A and hnRNP-B proteins, sera from 303 patients with various rheumatic diseases were investigated by immunoblotting. For the analysis of cross-reactivities, autoantibodies were affinity purified by blot elution. RESULTS: Anti-A2/RA33 was found in 35% of rheumatoid arthritis (RA) patients, 38% of mixed connective tissue disease (MCTD) patients, 23% of systemic lupus erythematosus (SLE) patients, and, apart from single exceptions, not in patients with other rheumatic diseases. All anti-A2/RA33-positive sera were also reactive with B1 and B2, and anti-A2/RA33 antibodies cross-reacted with both proteins. Antibodies to hnRNP-A1 were found less frequently; moreover, the majority of anti-A1-positive sera also contained anti-A2/RA33 antibodies. In anti-A1, anti-A2/RA33 double-positive sera, cross-reactivity between the 2 antibodies was generally observed. In SLE patients, the presence of anti-A2/RA33 was correlated with the presence of anti-(U1) small nuclear RNP (snRNP) and anti-Sm (P < 0.0001 and P < 0.005, respectively), but there was no evidence for cross-reactivity between antibodies to hnRNP and antibodies to snRNP antigens. CONCLUSION: Since both hnRNPs and snRNPs are essential components of the spliceosome, the data show that the immune systems of patients with RA, SLE, and MCTD react to this functional complex. However, compared with MCTD and SLE patients, RA patients have a more restricted immune response to the spliceosome: they react to hnRNP proteins, particularly to hnRNP-A2/RA33, but not to snRNPs.