The myositis clinical phenotype associated with anti-Zo autoantibodies: a case series of nine UK patients.

OBJECTIVES: It has been over 10 years since the first report of autoantibodies directed against phenylalanyl tRNA synthetase (anti-Zo) in a patient with features of the anti-synthetase syndrome. In that time no further cases have been published. Here we aim to characterize more fully the clinical phenotype of anti-Zo-associated myositis by describing the clinical features of nine patients. METHODS: Anti-Zo was identified by protein-immunoprecipitation in patients referred for extended spectrum myositis autoantibody testing at our laboratory. Results were confirmed by immunodepletion using a reference serum. Medical records were retrospectively reviewed to provide detailed information of the associated clinical phenotype for all identified patients. Where possible, HLA genotype was imputed using Illumina protocols. RESULTS: Nine patients with anti-Zo were identified. The median age at disease onset was 51 years, and six patients were female. Seven patients had evidence of inflammatory muscle disease, seven of interstitial lung disease and six of arthritis. The reported pattern of interstitial lung disease varied with usual interstitial pneumonia, non-specific interstitial pneumonia and organizing pneumonia all described. Other features of the anti-synthetase syndrome such as RP and mechanics hands were common. HLA data was available for three patients, all of whom had at least one copy of the HLA 8.1 ancestral haplotype. CONCLUSION: Patients with anti-Zo presenting with features of the anti-synthetase syndrome and interstitial lung disease is a common finding. Like other myositis autoantibodies, there is likely to be a genetic association with the HLA 8.1 ancestral haplotype.

Immunoproteomic analysis of the protective response obtained with subunit and commercial vaccines against Glässer's disease in pigs.

An immunoproteomic analysis of the protective response of subunit and commercial vaccines in colostrum-deprived pigs against Glässer's disease was carried out. A mixture of proteins with affinity to porcine transferrin (PAPT) from Haemophilus parasuis Nagasaki strain (serovar 5) was inoculated intramuscularly (PAPT(M)) and intratracheally (PAPT(Cp)), along with a commercial bacterin. PAPT were separated using 2 dimensional electrophoresis (2DE) gels and with them, 2DE Western blots were carried out. A total of 17 spots were identified as positive with sera of pigs from any of the three vaccinated groups, the highest number of immunoreactive proteins being detected in those having received PAPT(Cp). Among them, six proteins (FKBP-type peptidyl-prolyl cis-trans isomerase, neuraminidase exo-α-sialidase, xanthine-guanine phosphoribosyl transferase, CMP-N-acetylneuraminic acid synthetase, phenylalanyl-tRNA synthetase and glyceraldehyde 3-phosphate dehydrogenase) were found to be novel immunogens in H. parasuis. These proteins showed a high potential as candidates in future subunit vaccines against Glässer's disease. The three experimental groups developed specific systemic total IgG (IgGt), IgG1, IgG2 and IgM antibodies after immunizations. In addition, those receiving PAPT(Cp) yielded a serum IgA response.

Anti-synthetase syndrome: a new autoantibody to phenylalanyl transfer RNA synthetase (anti-Zo) associated with polymyositis and interstitial pneumonia.

OBJECTIVE: Autoantibodies directed against the aminoacyl tRNA synthetases are associated with myositis, arthritis, Raynaud's phenomenon, mechanic's hands, fever and interstitial pneumonia, clinically referred to as the anti-synthetase syndrome (ASS). The aim of this study was to characterize the autoantibody profile in a patient with clinical features of ASS whose routine diagnostic testing was negative for the previously identified anti-synthetase autoantibodies. METHODS: Serum from a patient presenting with interstitial pneumonia followed by proximal myopathy, Raynaud's phenomenon and arthrlagia was analysed for autoantigen specificity by routine methods including indirect immunofluorescence, immunodiffusion, ELISA and immunoblotting. The autoantibody specificity was further analysed by RNA and protein immunoprecipitation. Novel autoantigens found on protein immunoprecipitation were further characterized using a proteomic approach, combining immunoprecipitation, SDS-PAGE and MALDI-TOF mass spectrometry. RESULTS: Diagnostic testing on the patient's serum was negative by ELISA and immunodiffusion. Indirect immunofluorescence using Hep-2 cells was ANA negative, although a strong cytoplasmic speckle was seen. Immunoblotting with the patient serum displayed an unknown positive band at approximately 60 kDa. Protein immunoprecipitation revealed the presence of two proteins with molecular weights of approximately 60 and 70 kDa, and RNA immunoprecipitation revealed the presence of a band corresponding to a tRNA synthetase. Using a combination of immunoprecipitation and mass spectrometry, the novel immunoprecipitation targets were identified as phenylalanyl tRNA synthetase alpha and beta chains. CONCLUSIONS: We report the identification of previously uncharacterized autoantibodies to phenylalanyl tRNA synthetase, entitled anti-Zo. This is the eighth anti-synthetase autoantibody in a patient with anti-synthetase syndrome.

[Detection of autoantibodies against phenylalanyl-, tyrosyl-, and tryptophanyl-tRNA-synthetase and anti-idiotypic antibodies to it in serum from patients with autoimmune diseases]. / Vyiavlenie v syvorotkakh bol'nykh autoimmunnymi zabolevaniiami autoantitel protiv fenilalanil-, tirozil- i triptofanil-tRNK-sintetaz i antiidiotipicheskikh antitel k nim.

Sera of patients bearing autoimmune diseases (rheumatoid arthritis and systemic lupus erythematosus) and sera of clinically healthy donors were examined by ELISA for the presence of autoantibodies against tryptophanyl-, tyrosyl- and phenylalanyl-tRNA synthetases. Pure bovine synthetases served as antigens. It was shown that in patients with both autoimmune diseases all three enzyme autoantibodies were revealed at serum dilution 1/1600-1/3200. Moreover, by means of monoclonal antibodies against the same enzymes used for immunoaffinity sorption, antiidiotypic antibodies of IgG type against autoantibodies were detected. A conclusion has been made that autoimmune diseases are characterized by autoimmune response for many aminoacyl-tRNA synthetases irrespectively of their quaternary structure, intracellular location etc both at the level of primary and secondary antibodies.

[Express method of isolation of mammalian phenylalanine-tRNA-synthetase and preparation of monoclonal antibodies against this enzyme]. / Bystryi metod vydeleniia fenilalanil-tRNK-sintetazy iz mlekopitaiushchikh i poluchenie monoklonal'nykh antitel k étomu fermentu.

A rapid and efficient procedure for isolating homogeneous beef liver phenylalanyl-tRNA synthetase (EC.6.1.1) was developed that enables to purify the enzyme 5000 fold and to achieve the activity of 8 e.a.u. per mg of protein. The molecular mass of the native enzyme was estimated to be 260 kDa, for alpha subunit - 59 kDa, and for beta - 72 kDa. Two cellular clones were derived by means of hybridization of immunised splenocytes with myeloma cells. They secrete monoclonal antibodies, designated P6 and P1 2, that bind to human placental and bovine liver phenylalanyl-tRNA synthetases but not to the same enzymes from E. coli and T. thermophilus. P6 and P1 2 antibodies do not affect the aminoacylation capacity of human or bovine phenylalanyl-tRNA synthetases. By immunoblotting, it was shown that P6 antibodies recognize the alpha subunit of the enzyme.

Glycyl-tRNA synthetase of Escherichia coli: immunological homology with phenylalanyl-tRNA synthetase.

Antibodies to Escherichia coli glycyl-tRNA synthetase (GlyRS) cross-react extensively with E. coli phenylalanyl-tRNA synthetase (PheRS). These data indicate that structural homology exists between these two enzymes, the only two aminoacyl-tRNA synthetases in E. coli having an alpha 2 beta 2 subunit structure. Although only limited similarities are found in the protein sequences deduced from their known gene sequences, the presence of common epitopes in GlyRS and PheRS adds to a rather long list of physical and chemical similarities between those proteins. In addition, antibodies directed at the alpha- and beta-subunits of GlyRS inhibit both GlyRS and PheRS in the same relative manner, indicating that the function as well as the structure of subunits is similar in each enzyme. In contrast, GlyRS antibodies did not cross-react with a number of other aminoacyl-tRNA synthetase activities from E. coli, yeast, or Bacillus.

Cross-reactivity of phenylalanyl-transfer ribonucleic acid ligases from different microorganisms.

The cross-reaction of phenylalanyl-transfer ribonucleic acid (tRNA) ligases from different microorganisms with antibodies raised against the purified enzyme from Escherichia coli has been investigated. The results of immunotitration and immunodiffusion experiments and of the sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of immunoprecipitates revealed: (i) a high degree of immunochemical identity of this enzyme only within the family Enterobacteriaceae; (ii) intermediate-to-weak cross-reaction with the phenylalanyl-tRNA ligases from Pseudomonadaceae, Rhodopseudomonas spheroides, and Bacillus stearothermophilus; (iii) no detectable cross-reaction (with the methods employed) with the enzymes from several gram-positive organisms, Euglena gracilis, and several fungi. As revealed by immunochemical analysis, a merodiploid strain of E. coli carrying an episome (F148) that covers the aroD region of the E. coli chromosome possesses at least twice the amount of phenylalanyl-tRNA ligase in comparison with its haploid parent strain. This suggests that the cistrons for both the alpha and beta polypeptides of this enzyme are mapping in this area.

Altered alpha subunits in phenylalanyl-tRNA synthetases from p-fluorophenylalanine-resistant strains of Escherichis coli.

Three different phenylalanyl-tRNA synthetases have been purified to near homogeneity, one from a wild-type strain of Escherichia coli and the others from two independently isolated p-fluorophenyalanine-resistant strains. The mutant enzymes were not able to use p-fluorophenylalanine as a substrate for activation and attachment to tRNA. They proved to be indistinguishable from the wild-type enzyme by several electrophoretic and immunological criteria. The alpha and beta subunits of all three enzymes have been prepared by a method described in this paper. The isolated subunits per se did not reveal any significant enzyme activity, but combined they were able to form active phenylalanyl tRNA synthetase after a defined reconstitution process. Mixed reconstitution experiments between wild-type and mutant subunits indicate that the mutant alpha subunit is responsible for p-fluorophenylalanine resistance and therefore seems to carry the phenylalanine-binding site or to participate in its formation.