Autoantibody and metalloproteinase activity in early arthritis.

OBJECTIVES: The aim of the study was to evaluate the frequency of anti-mutated citrullinated vimentin antibodies (a-Sa), anti-citrullinated α-enolase peptide 1 antibodies (a-CEP-1), anti-filaggrin antibodies (AFAs), heterogeneous nuclear ribonucleoprotein compies/anti-RA33-antibodies (a-hnRNP/RA33), anti-carbamylated protein antibodies (a-CarP), and metalloproteinase (MMPs) activity in patients with early inflammatory arthritis (EIA). METHODS: Seventy-four patients with EIA: 51 diagnosed with RA (rheumatoid arthritis) and 23 with UA (undifferentiated arthritis), and 20 healthy volunteers were enrolled to the study. Inflammatory markers, rheumatoid factor (RF), and antibodies mentioned above were assessed in all patients. RESULTS: In the EIA group, we observed significantly higher concentration of a-CEP-1 (65.8 ± 111.6 RU/mL) than in controls (2.0 ± 0.0 RU/mL). In RF(+) RA patients, we observed higher concentration of a-Sa and a-CEP-1 than in other groups. A-Sa were positive in 69% of RF(+) RA, 37% of RF(-) RA, 26% of UA patients and in 10% of controls. A-CEP-1 were positive in 77% of RF(+) RA patients, in 56% of RF(-) RA patients, in 8.7% of UA patients, but they were negative in controls. In patients with RF(+) RA, positive a-CarP were present statistically significantly more often than in RF (-) RA patients. No statistically significant difference in frequency of a-hnRNP/RA33 and AFA between RF(+) RA, RF(-) RA, and UA was observed. CONCLUSIONS: Our results suggest that a-CEP-1 may help in differentiation between RF(-) RA and UA. a-CEP-1 and a-Sa may be useful while diagnosing EIA. a-CarP may be used in differentiation of RA RF(-) and UA. However, a follow-up study is needed to evaluate the prognostic value of analyzed antibodies.

RNA-binding protein Ptbp1 is essential for BCR-mediated antibody production.

The RNA-binding protein polypyrimidine tract-binding protein-1 (Ptbp1) binds to the pyrimidine-rich sequence of target RNA and controls gene expression via post-transcriptional regulation such as alternative splicing. Although Ptbp1 is highly expressed in B lymphocytes, its role to date is largely unknown. To clarify the role of Ptbp1 in B-cell development and function, we generated B-cell-specific Ptbp1-deficient (P1BKO) mice. B-cell development in the bone marrow, spleen and peritoneal cavity of the P1BKO mice was nearly normal. However, the P1BKO mice had significantly lower levels of natural antibodies in serum compared with those of the control mice. To investigate the effect of Ptbp1 deficiency on the immune response in vivo, we immunized the P1BKO mice with T-cell-independent type-2 (TI-2) antigen NP-Ficoll and T-cell-dependent (TD) antigen NP-CGG. We found that B-cell-specific Ptbp1 deficiency causes an immunodeficiency phenotype due to defective production of antibody against both TI-2 and TD antigen. This immunodeficiency was accompanied by impaired B-cell receptor (BCR)-mediated B-cell activation and plasmablast generation. These findings demonstrate that Ptbp1 is essential for the humoral immune response.

The RNA-binding protein PTBP1 is necessary for B cell selection in germinal centers.

Antibody affinity maturation occurs in germinal centers (GCs), where B cells cycle between the light zone (LZ) and the dark zone. In the LZ, GC B cells bearing immunoglobulins with the highest affinity for antigen receive positive selection signals from helper T cells, which promotes their rapid proliferation. Here we found that the RNA-binding protein PTBP1 was needed for the progression of GC B cells through late S phase of the cell cycle and for affinity maturation. PTBP1 was required for proper expression of the c-MYC-dependent gene program induced in GC B cells receiving T cell help and directly regulated the alternative splicing and abundance of transcripts that are increased during positive selection to promote proliferation.

Automated fluorescent miscroscopic image analysis of PTBP1 expression in glioma.

Multiplexed immunofluorescent testing has not entered into diagnostic neuropathology due to the presence of several technical barriers, amongst which includes autofluorescence. This study presents the implementation of a methodology capable of overcoming the visual challenges of fluorescent microscopy for diagnostic neuropathology by using automated digital image analysis, with long term goal of providing unbiased quantitative analyses of multiplexed biomarkers for solid tissue neuropathology. In this study, we validated PTBP1, a putative biomarker for glioma, and tested the extent to which immunofluorescent microscopy combined with automated and unbiased image analysis would permit the utility of PTBP1 as a biomarker to distinguish diagnostically challenging surgical biopsies. As a paradigm, we utilized second resections from patients diagnosed either with reactive brain changes (pseudoprogression) and recurrent glioblastoma (true progression). Our image analysis workflow was capable of removing background autofluorescence and permitted quantification of DAPI-PTBP1 positive cells. PTBP1-positive nuclei, and the mean intensity value of PTBP1 signal in cells. Traditional pathological interpretation was unable to distinguish between groups due to unacceptably high discordance rates amongst expert neuropathologists. Our data demonstrated that recurrent glioblastoma showed more DAPI-PTBP1 positive cells and a higher mean intensity value of PTBP1 signal compared to resections from second surgeries that showed only reactive gliosis. Our work demonstrates the potential of utilizing automated image analysis to overcome the challenges of implementing fluorescent microscopy in diagnostic neuropathology.

Quantitative validation of anti-PTBP1 antibody for diagnostic neuropathology use: Image analysis approach.

Traditional diagnostic neuropathology relies on subjective interpretation of visual data obtained from a brightfield microscopy. This approach causes high variability, unsatisfactory reproducibility, and inability for multiplexing even among experts. These problems may affect patient outcomes and confound clinical decision-making. Also, standard histological processing of pathological specimens leads to auto-fluorescence and other artifacts, a reason why fluorescent microscopy is not routinely implemented in diagnostic pathology. To overcome these problems, objective and quantitative methods are required to help neuropathologists in their clinical decision-making. Therefore, we propose a computerized image analysis method to validate anti-PTBP1 antibody for its potential use in diagnostic neuropathology. Images were obtained from standard neuropathological specimens stained with anti-PTBP1 antibody. First, the noise characteristics of the images were modeled and images are de-noised according to the noise model. Next, images are filtered with sigma-adaptive Gaussian filtering for normalization, and cell nuclei are detected and segmented with a k-means-based deterministic approach. Experiments on 29 data sets from 3 cases of brain tumor and reactive gliosis show statistically significant differences between the number of positively stained nuclei in images stained with and without anti-PTBP1 antibody. The experimental analysis of specimens from 3 different brain tumor groups and 1 reactive gliosis group indicates the feasibility of using anti-PTBP1 antibody in diagnostic neuropathology, and computerized image analysis provides a systematic and quantitative approach to explore feasibility.

MicroRNA-555 has potent antiviral properties against poliovirus.

Vaccination with live-attenuated polio vaccine has been the primary reason for the drastic reduction of poliomyelitis worldwide. However, reversion of this attenuated poliovirus vaccine occasionally results in the emergence of vaccine-derived polioviruses that may cause poliomyelitis. Thus, the development of anti-poliovirus agents remains a priority for control and eradication of the disease. MicroRNAs (miRNAs) have been shown to regulate viral infection through targeting the viral genome or reducing host factors required for virus replication. However, the roles of miRNAs in poliovirus (PV) replication have not been fully elucidated. In this study, a library of 1200 miRNA mimics was used to identify miRNAs that govern PV replication. High-throughput screening revealed 29 miRNAs with antiviral properties against Sabin-2, which is one of the oral polio vaccine strains. In particular, miR-555 was found to have the most potent antiviral activity against three different oral polio attenuated vaccine strains tested. The results show that miR-555 reduced the level of heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C) required for PV replication in the infected cells, which in turn resulted in reduction of PV positive-strand RNA synthesis and production of infectious progeny. These findings provide the first evidence for the role of miR-555 in PV replication and reveal that miR-555 could contribute to the development of antiviral therapeutic strategies against PV.

Heterogeneous nuclear RNPs as targets of autoantibodies in systemic rheumatic diseases.

OBJECTIVE: To investigate the abundance of autoantibodies to heterogeneous nuclear RNPs (hnRNPs) in systemic rheumatic diseases. METHODS: Recombinant human hnRNPs A1, B1, C1, E1, F, Gi, H1, I, K, and P2 were prepared. Antibodies to these antigens were determined by Western blotting and by enzyme-linked immunosorbent assay (ELISA) (for hnRNPs B1, E1, F, and H1) in serum samples obtained from patients with chronic fatigue syndrome (control subjects) and from patients with various connective tissue diseases. RESULTS: Western blotting analysis in 106 control subjects and 298 patients with a connective tissue disease revealed that antibodies to all tested hnRNP antigens, except hnRNP Gi, were significantly more prevalent in patients with Sjögren's syndrome (SS) than in control subjects. The highest reactivity was observed for hnRNPs B1, E1, F, and H1 (reactivity in >45% of patients with SS and in 2.8% of control subjects). Reactivity with hnRNPs B1, E1, F, and H1 was also evaluated by ELISA in 89 control subjects and 228 patients with a connective tissue disease. Reactivity with at least 2 of the 4 tested antigens was observed in 1.1% of control subjects, 16% of patients with systemic lupus erythematosus (SLE), and 18% of patients with SS. Reactivity with at least 3 of the 4 antigens was observed in 0% of the control subjects, 3.2% of patients with SLE, and 15% of patients with SS. CONCLUSION: Several hnRNPs are target antigens in SS. The combined presence of antibodies to several hnRNPs was strongly associated with connective tissue disease in general and with SS in particular.

Antagonistic effects of cellular poly(C) binding proteins on vesicular stomatitis virus gene expression.

Immunoprecipitation and subsequent mass spectrometry analysis of the cellular proteins from cells expressing the vesicular stomatitis virus (VSV) P protein identified the poly(C) binding protein 2 (PCBP2) as one of the P protein-interacting proteins. To investigate the role of PCBP2 in the viral life cycle, we examined the effects of depletion or overexpression of this protein on VSV growth. Small interfering RNA-mediated silencing of PCBP2 promoted VSV replication. Conversely, overexpression of PCBP2 in transfected cells suppressed VSV growth. Further studies revealed that PCBP2 negatively regulates overall viral mRNA accumulation and subsequent genome replication. Coimmunoprecipitation and immunofluorescence microscopic studies showed that PCBP2 interacts and colocalizes with VSV P protein in virus-infected cells. The P-PCBP2 interaction did not result in reduced levels of protein complex formation with the viral N and L proteins, nor did it induce degradation of the P protein. In addition, PCBP1, another member of the poly(C) binding protein family with homology to PCBP2, was also found to interact with the P protein and inhibit the viral mRNA synthesis at the level of primary transcription without affecting secondary transcription or genome replication. The inhibitory effects of PCBP1 on VSV replication were less pronounced than those of PCBP2. Overall, the results presented here suggest that cellular PCBP2 and PCBP1 antagonize VSV growth by affecting viral gene expression and highlight the importance of these two cellular proteins in restricting virus infections.

Anti-hnRNP and other autoantibodies in systemic sclerosis with joint involvement.

OBJECTIVES: To investigate joint involvement in SSc and its relationship with autoantibody to the hnRNP and to anti-cyclic citrullinated peptide (anti-CCP). METHODS: Sera from 55 SSc patients were investigated. Joint involvement was determined by clinical, radiological and ultrasonographical evaluation. Anti-hnRNP proteins A1 and A2 (anti-hnRNP-A1/A2) antibodies were determined by immunoblotting. Anti-CCP, ACA, anti-topo I (ATA), Sm, U1-RNP, ribosomal RNP, Ro/SSA, La/SSB autoantibody and RF were determined. RESULTS: Six patients were positive for anti-hnRNP-A2 autoantibody and two were anti-A1 positive. Eight patients had joint erosions: seven of the eight patients positive for anti-hnRNP-A2 or A1 presented articular involvement (P < 0.04) and five of the eight erosive patients were positive for either of the two autoantibodies (P < 0.02). Of the four patients positive for anti-CCP, none had anti-hnRNP but three had erosive aspects. ATAs were found in 10 patients, six of which were also positive for anti-hnRNP (P < 0.05). RF was positive in 16 patients and in seven among those with articular involvement (P < 0.04). RF was significantly associated with anti-hnRNP in patients with erosive arthritis (P < 0.02), but not with the presence of anti-hnRNP alone. Epitope mapping of the three strongest anti-hnRNP-A2-positive sera recognized the same major epitope as patients with RA. SSc patients have higher incidence of erosions and anti-hnRNP-A2/A1 positivity. RF test and anti-hnRNP had a statistically significant diagnostic value for articular involvement. CONCLUSIONS: These parameters might suggest that autoantibody to both hnRNP antigens might become a non-specific but useful marker for joint involvement in SSc patients and identify SSc patients prone to develop joint damage.

Impaired translation of CCAAT/enhancer binding protein alpha mRNA in bronchial smooth muscle cells of asthmatic patients.

BACKGROUND: Bronchial smooth muscle (BSM) cells of asthmatic patients have an impaired expression of CCAAT/enhancer binding protein (C/EBP) alpha, which is associated with increased proliferation. OBJECTIVE: We sought to assess the translational regulation of CEBPA mRNA in cultured BSM cells of healthy control subjects (n = 11) and asthmatic patients (n = 12). METHODS: Translation efficiency was studied by using a translation control reporter system driven by the control elements present in the CEBPA mRNA. Translation efficiency was determined by the ratio of 2 artificial hemagglutinin (HA.11) proteins: p23 and p12. We also analyzed levels of proteins that control translation of CEBPA mRNA, namely heterogeneous nuclear ribonucleoprotein E2, calreticulin, eukaryotic translation initiation factor (eIF4E), and 4E binding protein. RESULTS: Compared with healthy control subjects, BSM cells of asthmatic patients proliferate faster (2.1-fold) and are primed for IL-6 secretion. Real-time RT-PCR showed that BSM cells of asthmatic patients express normal levels of CEBPA mRNA, whereas they express lower levels of C/EBPalpha (p42). Transient transfections with the translation control reporter system construct showed a disturbed p12/p23 ratio in BSM cells of asthmatic patients relative to healthy control subjects, which coincided with lower levels of eIF4E. CONCLUSION: BSM cells of asthmatic patients have normal levels of CEBPA mRNA but inadequately reinitiate the translation into C/EBPalpha. Impaired translation control upstream of eIF4E might underlie the observed increased proliferation and priming of BSM cells of asthmatic patients.

Memory T cell RNA rearrangement programmed by heterogeneous nuclear ribonucleoprotein hnRNPLL.

Differentiation of memory cells involves DNA-sequence changes in B lymphocytes but is less clearly defined in T cells. RNA rearrangement is identified here as a key event in memory T cell differentiation by analysis of a mouse mutation that altered the proportions of naive and memory T cells and crippled the process of Ptprc exon silencing needed to generate CD45RO in memory T cells. A single substitution in a memory-induced RNA-binding protein, hnRNPLL, destabilized an RNA-recognition domain that bound with micromolar affinity to RNA containing the Ptprc exon-silencing sequence. Hnrpll mutation selectively diminished T cell accumulation in peripheral lymphoid tissues but not proliferation. Exon-array analysis of Hnrpll mutant naive and memory T cells revealed an extensive program of alternative mRNA splicing in memory T cells, coordinated by hnRNPLL. A remarkable overlap with alternative splicing in neural tissues may reflect a co-opted strategy for diversifying memory T cells.

Significantly increased antibody response to heterogeneous nuclear ribonucleoproteins in cerebrospinal fluid of multiple sclerosis patients but not in patients with human T-lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis.

It has been reported that antibodies (Abs) against heterogeneous nuclear ribonucleoproteins (hnRNPs) are associated with human T-lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and multiple sclerosis (MS). However, these studies were done under nonmasked conditions. In order to determine whether Abs against hnRNPs associate with HAM/TSP and MS, the authors assayed Abs against two major hnRNPs, hnRNP A1 and A2/B1, in 105 cerebrospinal fluid (CSF) samples under fully masked conditions. Samples included 40 cases of HAM/TSP, 28 of MS, and 37 of other neurological diseases. Anti-hnRNP A1 Abs, and especially anti-hnRNP A2/B1 Abs, were found significantly more often in the CSF of MS patients than in other groups. However, there was no difference in the incidence of anti-hnRNP A1 Abs between HAM/TSP and other disease groups.

A specific SR protein binds preferentially to the secretory protein gene transcripts in salivary glands of Chironomus tentans.

The members of the serine-arginine (SR) family of proteins play multiple roles in posttranscriptional gene expression. Initially considered as essential splicing factors confined to the nucleus and regulating constitutive and alternative splicing, SR proteins are now known to shuttle between the nucleus and the cytoplasm and to be involved in mRNA biogenesis, transport, and translation. In Chironomus tentans, hrp45 is an SR protein structurally similar to the Drosophila SRp55/B52 SR protein. We have studied how hrp45, hrp36 [a heterogenous nuclear ribonucleoprotein (hnRNP) protein], and small nuclear RNP (snRNP) proteins are distributed in the transcriptionally active loci of polytene chromosomes in C. tentans. Immunofluorescence visualization of the proteins in double-labeling experiments revealed that hrp45 preferentially associates with a small number of puffs. On the other hand, hrp36 and snRNP proteins were found distributed in a large number of loci with little quantitative difference. Remarkably, hrp45-labeled loci coincide with the sites of transcription of premessenger RNPs of secretory protein (sp) genes. Because the labeling was found sensitive to RNase A treatment, we conclude that the SR protein hrp45 preferentially binds to sp gene transcripts in salivary gland cells. The preferential association of a specific SR protein with a particular type of gene transcripts reflects substrate-specific function(s) of an SR protein, in vivo. The possible roles that hrp45 might be playing in speedy and efficient processing of sp gene transcripts are discussed.

Use of a recombinant protein containing major epitopes of hnRNP G to detect anti-hnRNP G antibodies in dogs with systemic lupus erythematosus.

The objective of this study was to express major epitopes of heterogeneous nuclear ribonucleoprotein G (hnRNP G) for detecting anti-hnRNP G antibodies in dogs with systemic lupus erythematosus (SLE). HnRNP G cDNA clone was isolated from HEp-2 cells, and a DNA fragment encoding immunodominant region (residues 189-272) of hnRNP G (hnRNP Gi) was subcloned into pET32 vector to construct a prokaryotic expression plasmid named pEThnRNPGi. After induction, Escherichia coli carrying pEThnRNPGi expressed a recombinant protein of 28 kDa, comprising recombinant hnRNP Gi and fusion tag. Purified recombinant hnRNP Gi protein was further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and its identity was confirmed. Western blot analysis showed that recombinant hnRNP Gi was specifically recognized by anti-hnRNP G positive sera of SLE dogs, and not by negative control sera. In conclusion, recombinant hnRNP Gi protein expressed in this study may serve as a useful reagent to assist in the immunological diagnosis of canine SLE.

Heterogeneous nuclear ribonucleoprotein P2 is an autoantibody target in mice deficient for Mer, Axl, and Tyro3 receptor tyrosine kinases.

Deficiencies in clearance of apoptotic cells predispose to the development of autoimmune disease. This is evident in mice lacking the receptor tyrosine kinases Tyro3, Axl, and Mer. Deficient mice exhibit an increased abundance of apoptotic cells in tissues and manifest diverse autoimmune conditions. To test these mice for the presence of autoantibodies to apoptotic cells, we generated spontaneous splenic B cell hybridomas and used a novel microscopy screen to detect Ab binding to apoptotic Jurkat cells. From hybridomas secreting IgG Abs reactive with apoptotic cells, we selected one that recreated the major serum specificity for apoptotic cells. The Ab LHC7.15 bound to an Ag that is differentially distributed between the nucleus and the cytoplasm in live and apoptotic cells. In late apoptotic cells, the Ag coalesces into aggregates that bleb from the cell surface. Immunopurification of the Ag, followed by mass spectrometry, identified a protein of 69 kDa whose partial sequence matched heterogeneous nuclear ribonucleoprotein P2. This multifunctional protein binds DNA, RNA, and several known ribonucleoprotein autoantigens. Our observations indicate that a ribonucleoprotein complex, formed and translocated to the cell surface in apoptosis, represents a potent stimulus for breaking tolerance and inducing systemic autoimmunity in mice with defective clearance of cell remnants.

Autoantibodies to heterogeneous nuclear ribonucleoproteins.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are among the most abundant proteins in the eukaryotic cell nucleus and play a direct role in several aspects of the RNA life including splicing, export of the mature RNAs and translation. To date, approximately 30 proteins have been identified. A growing body of evidence points to hnRNPs as an important target of the autoimmune response in rheumatic diseases. Autoantibodies to A and B proteins of the hnRNP complex have been detected in late 1980s in rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Beyond their role as diagnostic test in clinical practice, these autoantibodies are starting to be regarded as important tools to obtain deeper insight into the pathogenesis of autoimmune rheumatic diseases. Furthermore, new anti-hnRNP antibodies have been recognized in the last ten years extending the spectrum of anti-hnRNP reactivity in different autoimmune disorders.

[Advances in neuroimmunological laboratory studies on neuromuscular diseases].

The recent methodological advances in molecular biology, immunology, and genetics have clarified neuroimmunological problems in axonal Guillain-Barré syndrome, seronegative myasthenia gravis, paraneoplastic neurologic syndromes and many others. In addition to clinical and serological studies in peripheral neuropathies, the origins and measurement of anti-ganglioside antibodies and relationships to similar carbohydrate structures on infectious organisms are discussed in the context of molecular mimicry hypothesis, especially related with both the localization of relevant gangliosides in the nerve and clinical phenotypes. Major advances have been made in animal modeling of anti-ganglioside antibody-associated disease. An explanation for muscular weakness in 10-15% of patients with seronegative myasthenia gravis who lack autoantibodies to acetylcholine receptors(AchRs) appears to be the autoantibodies to muscle-specific receptor tyrosine kinase (MuSK). MuSK mediates agrin-induced clustering of AchRs during synapse formation. These autoantibodies to the extracellular domain of MuSK inhibit its function in tissue culture. Isoelectric focusing (IEF) and agar gel electrophoresis (AGE) are used to examine cerebrospinal fluid (CSF) and sera from patients with multiple sclerosis (MS). The CSF oligoclonal IgG bands (OB) are less frequently observed in Japanese MS patients compared with Caucasian patients. Few optic-spinal form of MS (OS-MS) was positive for OB by agarose gel electrophoresis, but IEF is more sensitive than AGE. Recent IEF data revealed some OS-MS patients were positive for OB. The neuroimmunological advances revealed the relationship between the neuroimmunological diseases and the putative autoantibodies as diagnostic markers, for example, HAM and hnRNP-A1, MSand anti-hnRNP-B1 antibody, opsoclonus-myoclonus syndrome and anti-GluRdelta2 antibody, Rasmussen encephalitis and anti-GluR3 antibody, paraneoplastic brainstem encephalitis and anti-Ma2 antibody, and so on.

Autoimmune antibodies to hnRNPG protein in dogs with systemic lupus erythematosus: epitope mapping of the antigen.

We previously reported the presence of circulating autoantibodies to hnRNPG protein in dogs with systemic lupus erythematosus (Soulard et al. 1993, 1994). These antibodies appeared to be specifically limited to German shepherd dog species. In the present report, we have analysed the nature of the hnRNPG epitopes responsible for autoantibody specificity. By using a set of 11 dog sera selected for their strong reactivity to hnRNPG protein, we have found that these sera had the ability to recognize two epitopes: (1) within a stretch of 33 amino acids located around the central part of protein, that is readily detected by immunoblotting; (2) a N-terminal conformation of the protein close to the RNA binding domain (RBD), that is revealed by immunoprecipitation. Our data strongly support the notion that the dog autoimmune response against hnRNPG protein is antigen-driven.

[Autoantibodies to the heterogeneous nuclear ribonucleoproteins and the autoimmune diseases].

Autoantibodies to the heterogeneous nuclear ribonucleoproteins (hnRNP) have a great role in the diagnosis of rheumatic diseases. hnRNP-A/B proteins are the main autoantigens in Rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCTD). Anti-RA33 autoantibodies which target the hnRNP proteins A2, B1, B2 (the 'RA33 complex') help to the diagnosis of RA at early stage. Therefore, anti-hnRNP autoantibodies are not only valuable diagnostic markers but may also allow deep insights into the pathogenetic mechanisms of rheumatic diseases.