title: Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis.
abstract: BACKGROUND
  Kveim-reagent (Kv) skin testing was a historical method of diagnosing sarcoidosis. Intradermal injection of treated sarcoidosis spleen tissue resulted in a granuloma response at injection site by 4-6 weeks. Previous work indicates proteins as the possible trigger of this reaction. We aimed to identify Kv-specific proteins and characterise the ex vivo response of Peripheral Blood Mononuclear Cells (PBMCs) from sarcoidosis, tuberculosis and healthy control patients when stimulated with both Kv and selected Kv-specific proteins.
  
  
  METHODS
  Kv extracts were separated by 1D-SDS-PAGE and 2D-DIGE and then underwent mass spectrometric analysis for protein identification. Sarcoidosis and control PBMCs were first stimulated with Kv and then with three selected recombinant protein candidates which were identified from the proteomic analysis. PBMC secreted cytokines were subsequently measured by Multiplex Cytokine Assay.
  
  
  RESULTS
  We observed significantly increased IFN-γ and TNF-α secretion from Kv-stimulated PBMCs of sarcoidosis patients vs. PBMCs from healthy volunteers (IFN-γ: 207.2 pg/mL vs. 3.86 pg/mL, p = 0.0018; TNF-α: 2375 pg/mL vs. 42.82 pg/mL, p = 0.0003). Through proteomic approaches we then identified 74 sarcoidosis tissue-specific proteins. Of these, 3 proteins (vimentin, tubulin and alpha-actinin-4) were identified using both 1D-SDS-PAGE and 2D-DIGE. Data are available via ProteomeXchange with identifier PXD005150. Increased cytokine secretion was subsequently observed with vimentin stimulation of sarcoidosis PBMCs vs. tuberculosis PBMCs (IFN-γ: 396.6 pg/mL vs 0.1 pg/mL, p = 0.0009; TNF-α: 1139 pg/mL vs 0.1 pg/mL, p<0.0001). This finding was also observed in vimentin stimulation of sarcoidosis PBMCs compared to PBMCs from healthy controls (IFN-γ: 396.6 pg/mL vs. 0.1 pg/mL, p = 0.014; TNF-α: 1139 pg/mL vs 42.29 pg/mL, p = 0.027). No difference was found in cytokine secretion between sarcoidosis and control PBMCs when stimulated with either tubulin or alpha-actinin-4.
  
  
  CONCLUSIONS
  Stimulation with both Kveim reagent and vimentin induces a specific pro-inflammatory cytokine secretion from sarcoidosis PBMCs. Further investigation of cellular immune responses to Kveim-specific proteins may identify novel biomarkers to assist the diagnosis of sarcoidosis.
authors.affiliation: Tuberculosis Research Unit, National Heart and Lung Institute, Imperial College London, London, United Kingdom.
authors.forename: Christian
authors.initials: C
authors.lastname: Eberhardt
authors.affiliation: Cambridge Interstitial Lung Disease Group, Papworth NHS Trust, Cambridge, United Kingdom.
authors.forename: Muhunthan
authors.initials: M
authors.lastname: Thillai
authors.affiliation: Tuberculosis Research Unit, National Heart and Lung Institute, Imperial College London, London, United Kingdom.
authors.forename: Robert
authors.initials: R
authors.lastname: Parker
authors.affiliation: Tuberculosis Research Unit, National Heart and Lung Institute, Imperial College London, London, United Kingdom.
authors.forename: Nazneen
authors.initials: N
authors.lastname: Siddiqui
authors.affiliation: Tuberculosis Research Unit, National Heart and Lung Institute, Imperial College London, London, United Kingdom.
authors.forename: Lee
authors.initials: L
authors.lastname: Potiphar
authors.affiliation: Centre for Pathology, Department of Medicine, Imperial College London, London, United Kingdom.
authors.forename: Rob
authors.initials: R
authors.lastname: Goldin
authors.affiliation: Institute for Women's Health, Faculty of Population Health Sciences, University College London, London, United Kingdom.
authors.forename: John F
authors.initials: JF
authors.lastname: Timms
authors.affiliation: Interstitial Lung Unit, Royal Brompton Hospital, Imperial College London NHS Healthcare Trust, London, United Kingdom.
authors.forename: Athol U
authors.initials: AU
authors.lastname: Wells
authors.affiliation: Department of Respiratory Medicine, St. Mary's Hospital London, Imperial College London NHS Healthcare Trust, London, United Kingdom.
authors.forename: Onn M
authors.initials: OM
authors.lastname: Kon
authors.affiliation: Department of Respiratory Medicine, St. Mary's Hospital London, Imperial College London NHS Healthcare Trust, London, United Kingdom.
authors.forename: Melissa
authors.initials: M
authors.lastname: Wickremasinghe
authors.affiliation: Tuberculosis Research Unit, National Heart and Lung Institute, Imperial College London, London, United Kingdom.
authors.forename: Donald
authors.initials: D
authors.lastname: Mitchell
authors.affiliation: Barts Cancer Institute- a Cancer Research UK Centre of Excellence, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, United Kingdom.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Weeks
authors.affiliation: Tuberculosis Research Unit, National Heart and Lung Institute, Imperial College London, London, United Kingdom.
authors.forename: Ajit
authors.initials: A
authors.lastname: Lalvani
mesh_terms: D000328:Adult; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007111:Immunity, Cellular; D007202:Indicators and Reagents / Q000737:chemistry*; D008297:Male; D008875:Middle Aged; D040901:Proteomics; D012507:Sarcoidosis / Q000276:immunology*; D053719:Tandem Mass Spectrometry
chemical_list: D007202:Indicators and Reagents