Yasuo Ohosone

Spectrum and clinical significance of autoantibodies against transfer RNA

OBJECTIVE To characterize the clinical features of patients who have autoantibodies against transfer RNA (tRNA) or tRNA-associated proteins. METHODS Sera from 1,472 patients with suspected systemic rheumatic disease were screened by RNA immunoprecipitation of HeLa cell extracts. The specificities of the antibodies that precipitated tRNAs were further analyzed by immunoprecipitation using deproteinized RNAs and 35S-methionine-labeled HeLa cell extracts, followed by immunoblotting. RESULTS Forty-one serum samples (2.8%) were found to immunoprecipitate tRNAs. Thirteen patients were identified as having previously defined anti-aminoacyl-tRNA synthetase antibodies (anti-histidyl-tRNA synthetase in 4 patients, anti-threonyl-tRNA synthetase in 1, anti-alanyl-tRNA synthetase in 3, anti-glycyl-tRNA synthetase in 4, and anti-isoleucyl-tRNA synthetase in 1). All 13 patients had myositis and/or interstitial pneumonitis. Sera from the remaining 28 patients immunoprecipitated previously unidentified tRNAs, including 13 serum samples that bound deproteinized cognate tRNA; 24 of the 28 patients met criteria for either systemic lupus erythematosus (SLE) or Sjögrens syndrome (SS). In addition, nonerosive polyarthritis, leukocytopenia, rheumatoid factor, and characteristic annular or papulosquamous recurrent erythema were noted in these patients; however, renal involvement was rare. Sera from 16 of these 28 patients also contained anti-Ro/SSA and/or anti-La/SSB antibodies. While 189 patient sera precipitated Ro/SSA and/or La/SSB-associated RNAs but not tRNA, only 12 of the patients (6.3%) developed skin lesions (P=0.0009, odds ratio 8.85). CONCLUSION Novel autoantibodies against tRNAs or tRNA-associated proteins were identified in 28 sera. These autoantibodies appear to be distinct from anti-aminoacyl-tRNA synthetase antibodies and are associated with SLE and SS. The presence of anti-Ro/SSA and/or anti-La/SSB along with anti-tRNA antibodies is more strongly associated with recurrent erythema than is the presence of anti-Ro/SSA or anti-La/SSB alone.

snRNPs and scRNPs as autoantigens: clues to the etiology of the connective tissue diseases

A N T I R O / S S A A U T O A N T I B O D I E S RECOGNIZE S E V E R A L P O L Y P E P T I D E S J.F. Meilof, I. Bantjes, A.P. van Dam, R. J. Smeenk. Centr . Lab. Netherl . Red Cross Blood Transfus ion Service, Ams te rdam, The Netherlands. The presence of antibodies to the Ro /SS-A autoant igen (a-SS-A-Ab) is associated in SLE with various clinical manifes ta t ions , amongst which cutaneous and cardiological complicat ions (Congenitial Heart Block). A-SS-A-Ab are classically identified in Ouchter lony or Counter immuno-elect rophoresis (CIE) assays using established reference sera. To characterize the Ro / SS-A autoantigen we Clinical rheumatology 19 developed an immunoblottingtest (IBT) using a Ro/SS-A preparation biochemically purified from bovine spleen, and an ELISA using a commercially obtained affinity purified Ro/SS-A preparation. Of 21 sera of patients containing a-SS-A-Ab (according to the CIE method) 60~ recognized a 56 kD doublet in the IBT, the remainder weakly stained a 48 kD band or no bands at all. All sera reactive with the 56 kD doublet were also found positive with the ELISA and reacted with a single 56 kD band in an IBT using the commercial Ro/SS-A preparation, suggesting that the ELISA mainly detects antibodies against the 56 kD doublet. The observed discrepancy between CIE and ELISA was not based on lack of (a certain) polypeptide in the commercial preparation, as discrepant sera did react with this antigen if used in a CIE assay. From these results we conclude that Ro/SS-A comprises at least two distinct antigenic polypeptides.

Anti-transfer RNA Antibodies in Two Patients with Pulmonary Fibrosis, Raynaud's Phenomenon and Polyarthritis

Patient W.S. (a 61-year-old woman) and patient T.M. (a 41-year-old man) developed recurrent fevers, polyarthritis, Raynauds phenomenon and interstitial pulmonary fibrosis without apparent polymyositis. From HeLa cell extracts, sera from both patients immunoprecipitated all species of intact and deproteinised tRNAs. To identify the antibody binding site more precisely, tRNAs transcribed in vitro from clonedEscherichia coli tRNA genes and various mutants were prepared and used as antigens for immunoprecipitation. When the TψC loop, or the D loop were deleted, such mutants were not bound by both sera, suggesting that the D and TψC loops were required for antibody binding. Abrogation of tRNA binding occurred when18G of tRNATrp was replaced with18A to break the tertiary L-shape structure of tRNA. These results strongly suggest that sera from W.S. and T.M. recognise the tertiary conformation of L-shaped tRNA which is constructed with both D and TψC loops. These autoantibodies may also serve as a marker for a new subset of patients with connective tissue diseases that is distinct from anti-aminoacyl-tRNA synthetase syndrome.

Domain reactivity of autoantibodies to calpastatin in patients with systemic rheumatic diseases

Abstract Autoantibodies to calpastatin (endogenous inhibitor of calpain, a calcium-dependent neutral proteinase) have been detected in sera of patients with rheumatoid arthritis (RA) and other diseases. We investigated the epitope reactivity of anticalpastatin autoantibodies in patients with rheumatic diseases. cDNAs encoding each calpastatin domain (L, I, II, III, and IV) were amplified by PCR and ligated into an expression vector. The fusion proteins were expressed in E. coli. The presence of autoantibodies specific for each calpastatin domain was assayed in sera of patients with various rheumatic diseases by immunoblotting the fusion proteins with these sera. Of the RA patient sera, 81% reacted with at least one calpastatin domain. This reaction was significantly greater than with sera from patients with systemic lupus erythematosus (46%), scleroderma (32%), polymyositis/dermatomyositis (43%), and normal controls (13%). Domains I and II were recognized by RA patient sera significantly more than by other patient sera, whereas domains III and IV reacted almost equally among all patient sera. Although, collectively, sera from RA and lupus patients reacted equally with all domains, scleroderma sera tended to react with only domains I and IV and myositis sera tended to recognize only domains III and IV. Patients with RA positive for anticalpastatin antibodies exhibited more active disease (i.e., a higher erythrocyte sedimentation rate and C-reative protein level) than antibody-negative patients. Our results suggest that anticalpastatin antibodies were detected in RA with the highest frequency and that different domain reactivity was shown among different diseases. The presence of these antibodies in sera may be related to the type of disease and, in RA, with disease activity, suggesting their importance in rheumatic disorders.

The Association of Serum Hepatitis C Virus RNA with Serum Aminotransferase Activities

Serum samples from 71 patients with hepatitis C virus infection at various stages were studied to determine the clinical significance of the detection of hepatitis C virus RNA by the polymerase chain reaction. There was a significant correlation of serum HCV RNA with elevation of the serum aminotransferase levels. These data suggest that the detection of HCV RNA in the serum might be useful for evaluation of the extent of ongoing liver damage.