07.17 The mutated rna splicing protein hnrnp-a3 is a novel autoantigen in systemic rheumatic diseases a link to warburg effect in ra

Objective Novel mutated hnRNP-A3 (MA3)was cloned out of RA synovial tissue involved in alternative splicing of PK2 linking it directly to Warburg effect and lactate production in RA. Methods After immunoblotting and 2D-gel-eletrophoresis out of a semipurified hnRNP fraction two protein spots were sequenced and identified to be highly similar to hnRN-A3. hnRNP-A3 variants were cloned from RA synovial-tissue. 3700 RA sera were screened for the presence of mutated anti-hnRNP-A3 autoantibodies using recombinant proteins and mutated citrullinated A3 peptides (MCA3) thereof. Identification of RNA and antibody binding sites to hnRNP A3 (MA3). Expression of hnRNP-A3 in synovial tissue was analysed by immunohistochemistry. Results Autoantibodies to MA3 protein were detected in 13% of RA (n=215) patients, in 9% SLE(n=154), in 27% of MCTD patients (n=44/10) and in less than 5% of 129 patients with other rheumatic disorders but not at all in healthy controls on immunoblot. When using native MA3-ELISA 22% of early RA patients (n=130) were detected and 87% of these patients had erosive arthritis. Identical modification on MA3 as in cancer cells were identified in synovial tissue and verified by MS and DNA sequencing. Using 2–3 citrullinated MCA3 peptides up to 81% of patients (n=150) with established and 67% (n=2926) of patients with an early RA with a specificity of 97% were detected. In early RA 27% and 25% in established RA of CCP2 negative and 93% of CCP2 positive patients were identified. By combining with the already established CCP2 and the new MCA3, 72% of early patients are positive. MCA3 autoantibodies predominantly occur (p<0.001) in an erosive, severe course of disease. MRL Lpr/lpr sera were hnRNP-A3 reactive and the antibody generation is Toll 7 and 9 dependent. Anti-hnRNP-A3-antibodies are directed to conformational RNA binding epitopes. Expression of hnRNP-A3 revealed the antigen is overexpressed in RA synovial tissue. Conclusion Mutated hnRNP-A3 is as a novel Toll7/9 dependent autoantigen in systemic rheumatic diseases. These mutated proteins are components of RNA and DNA containing alternative splicing complexes leading to the Warburg effect and predominantly occurring in an erosive and severe courses of RA.

Background Inflammation is a vital and complex process in response to diverse tissue damaging stimuli such as trauma, injury and pathogen. NLRP1, NLRP3 and NLRC4 belonging to the intracellular proteins Nod like receptor family, are capable of sensing the inflammatory inducers and trigger the assembly of a large complex called the inflammasome. By inducing the caspase-1 activation, inflammasome plays a crucial role in the release of IL-1b and IL-18, two critical cytokines of the initial steps of inflammatory responses.
Whereas mutations in NLRP3 and NLRC4 have been linked to two rare monogenic systemic autoinflammatory diseases (SAIDs), several polymorphisms in the NLRP1 gene have been associated extensively to an increased risk of autoimmune disorders (e.g. vitiligo, psoriasis, type 1 diabetes, and rheumatoid arthritis). We identified for the first time two distinct NLRP1 mutations in patients displaying a novel SAID combining autoinflammation and autoimmunity. We named this disease NAIAD, for NLRP1-associated autoinflammation arthritis and dyskeratosis.
The aim of our study was to unravel how mutation in NLRP1 impaired its function and triggered autoinflammation. Materials and methods Peripheral blood mononuclear cells from patients were analysed to identify the immunologic components involved in these novel diseases, using flow cytometry. The pathogenic effect of the NLRP1 mutations in inflammation was investigated using in vitro functional assays in transfected HEK293T. Results The level of caspase-1, IL-18 and IL-1b in serum samples from patients was increased as compared to controls and asymptomatic parents. Moreover, patient's cells displayed constitutive production of IL-1b. Functional studies in HEK293T revealed that the NLRP1 mutations resulted in a constitutive activation of the NLRP1 inflammasome.
Conclusions We demonstrated that two mutations in the NLRP1 gene are involved in autoinflammation in human. This novel disease could be a novel inflammasomopathy combining autoinflammatory and autoimmune features. Our data, combined with that in the literature, highlight the pleomorphic role of NLRP1 in inflammation and immunity. Objective Novel mutated hnRNP-A3 (MA3)was cloned out of RA synovial tissue involved in alternative splicing of PK2 linking it directly to Warburg effect and lactate production in RA. Methods After immunoblotting and 2D-gel-eletrophoresis out of a semipurified hnRNP fraction two protein spots were sequenced and identified to be highly similar to hnRN-A3. hnRNP-A3 variants were cloned from RA synovial-tissue. 3700 RA sera were screened for the presence of mutated anti-hnRNP-A3 autoantibodies using recombinant proteins and mutated citrullinated A3 peptides (MCA3) thereof. Identification of RNA and antibody binding sites to hnRNP A3 (MA3). Expression of hnRNP-A3 in synovial tissue was analysed by immunohistochemistry. Results Autoantibodies to MA3 protein were detected in 13% of RA (n=215) patients, in 9% SLE(n=154), in 27% of MCTD patients (n=44/10) and in less than 5% of 129 patients with other rheumatic disorders but not at all in healthy controls on immunoblot. When using native MA3-ELISA 22% of early RA patients (n=130) were detected and 87% of these patients had erosive arthritis. Identical modification on MA3 as in cancer cells were identified in synovial tissue and verified by MS and DNA sequencing. Using 2-3 citrullinated MCA3 peptides up to 81% of patients (n=150) with established and 67% (n=2926) of patients with an early RA with a specificity of 97% were detected. In early RA 27% and 25% in established RA of CCP2 negative and 93% of CCP2 positive patients were identified.

07.17
By combining with the already established CCP2 and the new MCA3, 72% of early patients are positive. MCA3 autoantibodies predominantly occur (p<0.001) in an erosive, severe course of disease. MRL Lpr/lpr sera were hnRNP-A3 reactive and the antibody generation is Toll 7 and 9 dependent. Anti-hnRNP-A3-antibodies are directed to conformational RNA binding epitopes. Expression of hnRNP-A3 revealed the antigen is overexpressed in RA synovial tissue. Conclusion Mutated hnRNP-A3 is as a novel Toll7/9 dependent autoantigen in systemic rheumatic diseases. These mutated proteins are components of RNA and DNA containing alternative splicing complexes leading to the Warburg effect and predominantly occurring in an erosive and severe courses of RA. Background Heat shock proteins (Hsps) are chaperones playing important roles in skeletal muscle physiology, adaptation to exercise or stress, and activation of inflammatory cells.
The aim of our study was to assess Hsp90 expression in muscle biopsies and plasma of patients with idiopathic inflammatory myopathies (IIM) and to characterise its association with IIM-related features. Methods Total of 277 patients with IIM (198 females, 79 males; mean age 54.8; disease duration 4.1 years; dermatomyositis (DM, 104)/polymyositis (PM, 104)/cancer associated myositis (CAM, 42)/necrotizing myopathy (IMNM, 27)) and 100 age-/sex-matched healthy individuals were included in plasma analysis and 50 muscle biopsy samples were stained for Hsp90 (in PM, DM, IMNM, myodystrophy, myasthenia gravis, 10 each). Plasma Hsp90 was measured by ELISA (eBioscience, Vienna). CK, LD, ALT, AST and CRP were analysed by routine techniques and IIM-specific autoantibodies by inline blot/immunoprecipitation. Data are presented as median.