title: Identification of new autoantibody specificities directed at proteins involved in the transforming growth factor β pathway in patients with systemic sclerosis.
abstract: BACKGROUND
  Antinuclear antibodies (ANAs), usually detected by indirect immunofluorescence on HEp-2 cells, are identified in 90% of patients with systemic sclerosis (SSc). Thus, approximately 10% of SSc patients have no routinely detectable autoantibodies, and for 20% to 40% of those with detectable ANAs, the ANAs do not have identified specificity (unidentified ANAs). In this work, we aimed to identify new target autoantigens in SSc patients.
  
  
  METHODS
  Using a proteomic approach combining two-dimensional electrophoresis and immunoblotting with HEp-2 cell total and enriched nuclear protein extracts as sources of autoantigens, we systematically analysed autoantibodies in SSc patients. Sera from 45 SSc patients were tested in 15 pools from groups of three patients with the same phenotype. A sera pool from 12 healthy individuals was used as a control. Proteins of interest were identified by mass spectrometry and analysed using Pathway Studio software.
  
  
  RESULTS
  We identified 974 and 832 protein spots in HEp-2 cell total and enriched nuclear protein extracts, respectively. Interestingly, α-enolase was recognised by immunoglobulin G (IgG) from all pools of patients in both extracts. Fourteen and four proteins were recognised by IgG from at least 75% of the 15 pools in total and enriched nuclear protein extracts, respectively, whereas 15 protein spots were specifically recognised by IgG from at least four of the ten pools from patients with unidentified ANAs. The IgG intensity for a number of antigens was higher in sera from patients than in sera from healthy controls. These antigens included triosephosphate isomerase, superoxide dismutase mitochondrial precursor, heterogeneous nuclear ribonucleoprotein L and lamin A/C. In addition, peroxiredoxin 2, cofilin 1 and calreticulin were specifically recognised by sera from phenotypic subsets of patients with unidentified ANAs. Interestingly, several identified target antigens were involved in the transforming growth factor β pathway.
  
  
  CONCLUSIONS
  We identified several new target antigens shared among patients with SSc or specific to a given phenotype. The specification of new autoantibodies could help in understanding the pathophysiology of SSc. Moreover, these autoantibodies could represent new diagnostic and/or prognostic markers for SSc.
authors.affiliation: Institut Cochin, Université Paris Descartes, CNRS UMR 8104, 8 rue Méchain, F-75014 Paris, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Bussone
authors.forename: Hanadi
authors.initials: H
authors.lastname: Dib
authors.forename: Mathieu C
authors.initials: MC
authors.lastname: Tamby
authors.forename: Cedric
authors.initials: C
authors.lastname: Broussard
authors.forename: Christian
authors.initials: C
authors.lastname: Federici
authors.forename: Geneviève
authors.initials: G
authors.lastname: Woimant
authors.forename: Luc
authors.initials: L
authors.lastname: Camoin
authors.forename: Loïc
authors.initials: L
authors.lastname: Guillevin
authors.forename: Luc
authors.initials: L
authors.lastname: Mouthon
mesh_terms: D000974:Antibodies, Antinuclear / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity / Q000276:immunology; D001324:Autoantigens / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification; D016130:Immunophenotyping; D007822:Laryngeal Neoplasms; D009687:Nuclear Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D011379:Prognosis; D020543:Proteome / Q000276:immunology; D040901:Proteomics / Q000379:methods*; D012595:Scleroderma, Systemic / Q000175:diagnosis / Q000276:immunology* / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D016212:Transforming Growth Factor beta / Q000276:immunology* / Q000378:metabolism
chemical_list: D000974:Antibodies, Antinuclear; D001324:Autoantigens; D015415:Biomarkers; D007074:Immunoglobulin G; D009687:Nuclear Proteins; D020543:Proteome; D016212:Transforming Growth Factor beta