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Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis
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 aim...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5256960/ https://www.ncbi.nlm.nih.gov/pubmed/28114394 http://dx.doi.org/10.1371/journal.pone.0170285 |
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author | Eberhardt, Christian Thillai, Muhunthan Parker, Robert Siddiqui, Nazneen Potiphar, Lee Goldin, Rob Timms, John F. Wells, Athol U. Kon, Onn M. Wickremasinghe, Melissa Mitchell, Donald Weeks, Mark E. Lalvani, Ajit |
author_facet | Eberhardt, Christian Thillai, Muhunthan Parker, Robert Siddiqui, Nazneen Potiphar, Lee Goldin, Rob Timms, John F. Wells, Athol U. Kon, Onn M. Wickremasinghe, Melissa Mitchell, Donald Weeks, Mark E. Lalvani, Ajit |
author_sort | Eberhardt, Christian |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-5256960 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-52569602017-02-06 Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis Eberhardt, Christian Thillai, Muhunthan Parker, Robert Siddiqui, Nazneen Potiphar, Lee Goldin, Rob Timms, John F. Wells, Athol U. Kon, Onn M. Wickremasinghe, Melissa Mitchell, Donald Weeks, Mark E. Lalvani, Ajit PLoS One Research Article 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. Public Library of Science 2017-01-23 /pmc/articles/PMC5256960/ /pubmed/28114394 http://dx.doi.org/10.1371/journal.pone.0170285 Text en © 2017 Eberhardt et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Eberhardt, Christian Thillai, Muhunthan Parker, Robert Siddiqui, Nazneen Potiphar, Lee Goldin, Rob Timms, John F. Wells, Athol U. Kon, Onn M. Wickremasinghe, Melissa Mitchell, Donald Weeks, Mark E. Lalvani, Ajit Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis |
title | Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis |
title_full | Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis |
title_fullStr | Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis |
title_full_unstemmed | Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis |
title_short | Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis |
title_sort | proteomic analysis of kveim reagent identifies targets of cellular immunity in sarcoidosis |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5256960/ https://www.ncbi.nlm.nih.gov/pubmed/28114394 http://dx.doi.org/10.1371/journal.pone.0170285 |
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