Imperial College London



JAK/STAT pathway activation in COPD1Liang Yew-Booth, 1Mark A. Birrell, 1Ming Sum Lau, 1Katie Baker, 1Victoria Jones, 2Iain Kilty and 1Maria G. Belvisi1Respiratory Pharmacology, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, Exhibition Road, London, SW7 2AZ, UK. 2Pfizer Inc, 610 Main Street, Cambridge MA 02139, USACorrespondence: Prof Maria Belvisi, Tel: +44 (0)207 594 7828 Fax: +44 (0)207 594 3100, E-mail: m.belvisi@imperial.ac.ukTake home message: A comprehensive investigation into the activation status of the JAK-STATs pathways in the lungs of COPD patients. Sources of support: Post doctorial funding for L Y-B from PfizerWord count: 2553AbstractThe Janus Kinase/ Signal Transducers and Activators of Transcription (JAK/STAT) pathways represent critical nodes in inflammatory signalling and inhibitors of these pathways may represent treatments for chronic obstructive pulmonary disease (COPD). There is, however, limited target validation data demonstrating an increase in the activation status of the various JAK/STAT proteins in tissue from COPD patients. Our aim was to determine the extent of JAK/STAT pathway activation in diseased lung tissue.We determined the expression of phosphorylated STAT proteins as a marker of JAK/STAT pathway activation in human parenchymal samples from non-smokers, smokers and COPD patients by western blotting. Also, to determine the localisation of STAT phosphorylation, Immunohistochemical (IHC) staining of phospho-STAT proteins was assessed in epithelial cells, macrophages and the overall parenchymal section.Western blotting revealed a significant increase in the phosphorylation of tyrosine, but not serine sites, on STAT1 and 3 in lungs from COPD patients compared to non-smokers. Analysis of the IHC staining revealed a statistically significant increase in phospho-STAT2 in the parenchymal macrophages of smokers compared to non-smokers.These data suggest that the JAK/STAT pathway is activated in COPD patients and provides encouraging target validation data which supports drug discovery efforts in this area.IntroductionChronic obstructive pulmonary disease (COPD) is an inflammatory disease of the lung, most commonly resulting from cigarette smoke (CS) exposure, characterised by a largely irreversible and progressive airflow limitation ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1513/pats.200603-065MS", "ISSN" : "1546-3222", "PMID" : "16921123", "author" : [ { "dropping-particle" : "", "family" : "Hogg", "given" : "James C", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Proceedings of the American Thoracic Society", "id" : "ITEM-1", "issue" : "6", "issued" : { "date-parts" : [ [ "2006", "8" ] ] }, "page" : "489-93", "title" : "State of the art. Bronchiolitis in chronic obstructive pulmonary disease.", "type" : "article-journal", "volume" : "3" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1164/rccm.201204-0596PP", "abstract" : "Chronic obstructive pulmonary disease (COPD) is a global health problem and since 2001 the Global Initiative for Chronic Obstructive Lung Disease (GOLD) has published its strategy document for the diagnosis and management of COPD. This executive summary presents the main contents of the second 5-year revision of the GOLD document that has implemented some of the vast knowledge about COPD accumulated over the last years. Today, GOLD recommends that spirometry is required for the clinical diagnosis of COPD in order to avoid misdiagnosis and to ensure proper evaluation of severity of airflow limitation. The document highlights that the assessment of the COPD patient should always include assessment of 1) symptoms, 2) severity of airflow limitation, 3) history of exacerbations, and 4) comorbidities. The first three points can be used to evaluate level of symptoms and risk of future exacerbations and this is done in a way that split COPD patients into 4 categories - A, B, C and D. Non-pharmacologic and pharmacologic management of COPD match this assessment in an evidence-based attempt to relieve symptoms and reduce risk of exacerbations. Identification and treatment of comorbidities must have high priority and a separate chapter in the document addresses management of comorbidities as well as COPD in the presence of comorbidities. The revised document also contains a new chapter on exacerbations of COPD. The GOLD initiative will continue to bring COPD to the attention of all relevant shareholders and will hopefully inspire future national and local guidelines on the management of COPD.", "author" : [ { "dropping-particle" : "", "family" : "Vestbo", "given" : "J\u00f8rgen", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hurd", "given" : "Suzanne S", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Agusti", "given" : "Alvar G", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jones", "given" : "Paul W", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Vogelmeier", "given" : "Claus", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Anzueto", "given" : "Antonio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barnes", "given" : "Peter J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fabbri", "given" : "Leonardo M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Martinez", "given" : "Fernando J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nishimura", "given" : "Masaharu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Stockley", "given" : "Robert A", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sin", "given" : "Don D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rodriguez-Roisin", "given" : "Roberto", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Agust\u00ed", "given" : "Alvar G", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nishimura", "given" : "Junji", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "American journal of respiratory and critical care medicine", "id" : "ITEM-2", "issue" : "4", "issued" : { "date-parts" : [ [ "2013", "8" ] ] }, "page" : "347-365", "title" : "Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease", "type" : "article-journal", "volume" : "187" }, "uris" : [ "" ] } ], "mendeley" : { "previouslyFormattedCitation" : "[1, 2]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[1, 2]. Currently there are no disease-modifying therapies to prevent the relentless disease progression in patients with COPD. As a consequence, COPD is associated with high mortality and increasing prevalence worldwide ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1164/rccm.201204-0596PP", "abstract" : "Chronic obstructive pulmonary disease (COPD) is a global health problem and since 2001 the Global Initiative for Chronic Obstructive Lung Disease (GOLD) has published its strategy document for the diagnosis and management of COPD. This executive summary presents the main contents of the second 5-year revision of the GOLD document that has implemented some of the vast knowledge about COPD accumulated over the last years. Today, GOLD recommends that spirometry is required for the clinical diagnosis of COPD in order to avoid misdiagnosis and to ensure proper evaluation of severity of airflow limitation. The document highlights that the assessment of the COPD patient should always include assessment of 1) symptoms, 2) severity of airflow limitation, 3) history of exacerbations, and 4) comorbidities. The first three points can be used to evaluate level of symptoms and risk of future exacerbations and this is done in a way that split COPD patients into 4 categories - A, B, C and D. Non-pharmacologic and pharmacologic management of COPD match this assessment in an evidence-based attempt to relieve symptoms and reduce risk of exacerbations. Identification and treatment of comorbidities must have high priority and a separate chapter in the document addresses management of comorbidities as well as COPD in the presence of comorbidities. The revised document also contains a new chapter on exacerbations of COPD. The GOLD initiative will continue to bring COPD to the attention of all relevant shareholders and will hopefully inspire future national and local guidelines on the management of COPD.", "author" : [ { "dropping-particle" : "", "family" : "Vestbo", "given" : "J\u00f8rgen", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hurd", "given" : "Suzanne S", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Agusti", "given" : "Alvar G", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jones", "given" : "Paul W", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Vogelmeier", "given" : "Claus", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Anzueto", "given" : "Antonio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barnes", "given" : "Peter J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Fabbri", "given" : "Leonardo M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Martinez", "given" : "Fernando J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nishimura", "given" : "Masaharu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Stockley", "given" : "Robert A", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Sin", "given" : "Don D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rodriguez-Roisin", "given" : "Roberto", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Agust\u00ed", "given" : "Alvar G", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nishimura", "given" : "Junji", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "American journal of respiratory and critical care medicine", "id" : "ITEM-1", "issue" : "4", "issued" : { "date-parts" : [ [ "2013", "8" ] ] }, "page" : "347-365", "title" : "Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease", "type" : "article-journal", "volume" : "187" }, "uris" : [ "" ] } ], "mendeley" : { "previouslyFormattedCitation" : "[2]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[2]. Therefore, significant research effort has been directed to understanding the mechanism by which CS exposure leads to the inflammation seen in COPD in the hope of discovering novel, effective anti-inflammatory therapies to tackle underlying pathogenic disease mechanisms.Cytokines are thought to be critical orchestrators of the persistent inflammation seen in several inflammatory diseases including COPD ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1183/09031936.01.00229701", "author" : [ { "dropping-particle" : "", "family" : "Chung", "given" : "K F", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "European Respiratory Journal", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2001", "7" ] ] }, "page" : "50-59", "title" : "Cytokines in chronic obstructive pulmonary disease", "type" : "article-journal", "volume" : "18" }, "uris" : [ "" ] } ], "mendeley" : { "previouslyFormattedCitation" : "[3]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[3]. Many of these cytokines signal through the Janus Kinase/ Signal Transducers and Activators of Transcription (JAK/STAT) pathway and/or are produced as a result of JAK/STAT pathway signalling ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "ISSN" : "1054-3589", "PMID" : "10582086", "author" : [ { "dropping-particle" : "", "family" : "Schindler", "given" : "C", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Strehlow", "given" : "I", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Advances in pharmacology (San Diego, Calif.)", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2000", "1" ] ] }, "page" : "113-74", "title" : "Cytokines and STAT signaling.", "type" : "article-journal", "volume" : "47" }, "uris" : [ "" ] } ], "mendeley" : { "previouslyFormattedCitation" : "[4]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[4]. In mammals, there are 4 JAK family members: JAK1, JAK2, JAK3 and TYK2 and 7 STAT family members: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. Binding of a cytokine to its receptor activates the appropriate pair of JAK subunits which phosphorylate the receptor cytoplasmic domain creating binding sites for STAT proteins. Once bound the STAT proteins are phosphorylated at the tyrosine residue and then form homo- or hetero-dimers, which rapidly migrate to the nucleus and act as transcription factors to mediate gene expression ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1126/science.277.5332.1630", "ISSN" : "00368075", "author" : [ { "dropping-particle" : "", "family" : "Darnell Jr.", "given" : "J. E.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Science", "id" : "ITEM-1", "issue" : "5332", "issued" : { "date-parts" : [ [ "1997", "9", "12" ] ] }, "page" : "1630-1635", "title" : "STATs and Gene Regulation", "type" : "article-journal", "volume" : "277" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "ISSN" : "0014-2956", "PMID" : "9342212", "abstract" : "Since the discovery of their physiological roles in cytokine signalling, the Janus kinases (JAKs) and the signal transducers and activators of transcription (STATs) have attracted considerable attention, to the point that the concept of a intracellular signalling pathway, named JAK/STAT, has emerged. As originally defined, this pathway involves ligand-dependent activation of a particular class of receptor-associated tyrosine kinases, the JAK proteins, which phosphorylate themselves and receptor components, creating recruitment sites for STAT transcription factors. The STATs are phosphorylated, they dissociate from the receptor x JAK complex and translocate to the nucleus where they participate in transcriptional gene activation. Although this pathway was found initially to be activated by interferons, it is now known that a large number of cytokines, growth factors and hormonal factors activate JAK and/or STAT proteins. Recent findings have suggested that the interdependence of JAKs and STATs might not be absolute as originally thought.", "author" : [ { "dropping-particle" : "", "family" : "Pellegrini", "given" : "S", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Dusanter-Fourt", "given" : "I", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "European journal of biochemistry / FEBS", "id" : "ITEM-2", "issue" : "3", "issued" : { "date-parts" : [ [ "1997", "9", "15" ] ] }, "page" : "615-33", "title" : "The structure, regulation and function of the Janus kinases (JAKs) and the signal transducers and activators of transcription (STATs).", "type" : "article-journal", "volume" : "248" }, "uris" : [ "" ] }, { "id" : "ITEM-3", "itemData" : { "ISSN" : "0022-1767", "PMID" : "17312100", "abstract" : "Universal and essential to cytokine receptor signaling, the JAK-STAT pathway is one of the best understood signal transduction cascades. Almost 40 cytokine receptors signal through combinations of four JAK and seven STAT family members, suggesting commonality across the JAK-STAT signaling system. Despite intense study, there remain substantial gaps in understanding how the cascades are activated and regulated. Using the examples of the IL-6 and IL-10 receptors, I will discuss how diverse outcomes in gene expression result from regulatory events that effect the JAK1-STAT3 pathway, common to both receptors. I also consider receptor preferences by different STATs and interpretive problems in the use of STAT-deficient cells and mice. Finally, I consider how the suppressor of cytokine signaling (SOCS) proteins regulate the quality and quantity of STAT signals from cytokine receptors. New data suggests that SOCS proteins introduce additional diversity into the JAK-STAT pathway by adjusting the output of activated STATs that alters downstream gene activation.", "author" : [ { "dropping-particle" : "", "family" : "Murray", "given" : "Peter J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of immunology (Baltimore, Md. : 1950)", "id" : "ITEM-3", "issue" : "5", "issued" : { "date-parts" : [ [ "2007", "3", "1" ] ] }, "page" : "2623-9", "title" : "The JAK-STAT signaling pathway: input and output integration.", "type" : "article-journal", "volume" : "178" }, "uris" : [ "" ] } ], "mendeley" : { "previouslyFormattedCitation" : "[5\u20137]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[5–7].The JAK/STAT pathway has been targeted by several companies for treatments for a wide range of inflammatory diseases. For example, inhibitors of the pathway have been approved for the treatment of rheumatoid arthritis ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/nbt0113-3", "ISSN" : "1546-1696", "PMID" : "23302910", "author" : [ { "dropping-particle" : "", "family" : "Garber", "given" : "Ken", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature biotechnology", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2013", "1" ] ] }, "page" : "3-4", "publisher" : "Nature Publishing Group", "title" : "Pfizer's first-in-class JAK inhibitor pricey for rheumatoid arthritis market.", "type" : "article-journal", "volume" : "31" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1038/nbt0611-467", "ISSN" : "1546-1696", "PMID" : "21654650", "author" : [ { "dropping-particle" : "", "family" : "Garber", "given" : "Ken", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature biotechnology", "id" : "ITEM-2", "issue" : "6", "issued" : { "date-parts" : [ [ "2011", "6" ] ] }, "page" : "467-8", "publisher" : "Nature Publishing Group", "title" : "Pfizer's JAK inhibitor sails through phase 3 in rheumatoid arthritis.", "type" : "article-journal", "volume" : "29" }, "uris" : [ "" ] } ], "mendeley" : { "previouslyFormattedCitation" : "[8, 9]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[8, 9]. In the respiratory field, the JAK/STAT pathway has been implicated in the pathogenesis of asthma ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.intimp.2010.04.014", "ISSN" : "1878-1705", "PMID" : "20430118", "abstract" : "Allergic asthma is a chronic inflammatory condition of the lung characterized by reversible airway obstruction, high serum immunoglobulin (Ig) E levels, and chronic airway inflammation. A number of cells including mast cells, T cells, macrophages and dendritic cells play a role in the pathogenesis of the disease. Janus kinase (JAK)-3, a non-receptor protein tyrosine kinase, traditionally known to mediate cytokine signaling, also regulates functional responses of these cells. In this review the role of JAK-3 in regulating various pathogenic processes in allergic asthma is discussed. We propose that targeting JAK-3 is a rationale approach to control the inflammatory responses of multiple cell types responsible for the pathogenesis of allergic asthma.", "author" : [ { "dropping-particle" : "", "family" : "Malaviya", "given" : "Rama", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Laskin", "given" : "Debra L", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Malaviya", "given" : "Ravi", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "International immunopharmacology", "id" : "ITEM-1", "issue" : "8", "issued" : { "date-parts" : [ [ "2010", "8" ] ] }, "page" : "829-36", "publisher" : "Elsevier B.V.", "title" : "Janus kinase-3 dependent inflammatory responses in allergic asthma.", "type" : "article-journal", "volume" : "10" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1172/JCI15786", "author" : [ { "dropping-particle" : "", "family" : "Pernis", "given" : "Alessandra B", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rothman", "given" : "Paul B", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "The Journal of clinical investigation", "id" : "ITEM-2", "issue" : "10", "issued" : { "date-parts" : [ [ "2002", "5" ] ] }, "page" : "1279-1283", "title" : "JAK-STAT signaling in asthma.", "type" : "article-journal", "volume" : "109" }, "uris" : [ "" ] } ], "mendeley" : { "previouslyFormattedCitation" : "[10, 11]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[10, 11]. There is currently considerable interest in developing JAK/STAT inhibitors as a potential treatment for COPD. However, there are no published studies which have comprehensively investigated the activation of all the STAT family members in tissue from COPD patients. Therefore the aim of this study was to determine the extent of JAK/STAT pathway activation by detecting the presence of phosphorylated STAT proteins 1-6 in lung tissue from non-smokers, smokers (with no apparent lung disease) and COPD patients by western blot and immunohistochemistry (IHC). This will provide important target validation data regarding the role of the JAK/STAT pathway in COPD which may be useful in the development of more specific novel treatments for COPD.Materials and MethodsThe current study used human parenchymal samples from non-smokers, smokers (with no apparent lung disease) and COPD patients assessing phosphorylation of STAT proteins as a marker of JAK/STAT pathway activation by western blot and IHC.Generation of human samplesHuman donor and recipient lung tissue samples, surplus to clinical requirements, were obtained from a transplant programme supported by the NIHR Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London. Informed written consent and ethical approval was obtained. Samples were divided into 3 groups: donor (non-smokers), donor (smokers, with no apparent lung disease) and COPD patients. Lung function data was available for 6 of the COPD patients: average FEV1 of 0.54 (0.38-0.68) L, or 18.6 (15-26) % predicted, FVC 2.09 (1.11 – 2.62) L, all subjects were GOLD stage III. Limited information on smoking history was available, the average pack history for the smoking donor group was 27.8 (14-37) years (n = 4) and COPD group was 47.6 (30-80) years (n = 5). None of the COPD patients were active smokers at the time of the lung transplant.Table 1. Subject demographicsDonor (non-smoker)Donor (smoker)COPDn121023Age50.1 ± 13.539.8 ± 13.155.3 ± 4.6Gender3M:9F7M:3F7M:16FInvestigating STAT phosphorylation by western blotProtein was extracted from frozen human lung parenchymal samples in the presence of protease and phosphatase inhibitors and standard western blotting techniques were used for the investigation of STAT phosphorylation. For an explanation of the full methods please see the online supplement. Primary antibodies were purchased from New England Biolabs, Hitchin, UK and used at a 1:500 dilution unless stated: phospho-STAT-1 (9167), phospho-STAT-2 (4441), phospho-STAT -3 (9131), phospho-STAT -4 (5267), phospho-STAT -5 (9351), phospho-STAT -6 (9364), STAT-1 (9172), STAT-2 (4594), STAT-3 (9132), STAT-4 (2653), STAT-5 (9363), STAT-6 (9362), β-actin (A5060; Sigma-Aldrich Ltd., Poole, UK; 1:1000 dilution). Antibodies for phosphorylated ser 727 on STAT1 (9177S) and STAT3 (9134S) were purchased from New England Biolabs. A HRP-conjugated goat anti-rabbit secondary antibody was used (P0448; Dako, Ely, UK; 1:5000 dilution). Band density of the phospho-STAT proteins were analysed relative to density of the relevant STAT protein or the loading control, actin, using ImageJ software.Investigating STAT phosphorylation by IHCStandard IHC techniques were used for the investigation of STAT phosphorylation. For an explanation of the full methods please see the online supplement. Primary antibodies for IHC were purchased from New England Biolabs unless stated: phospho-STAT-1 (9167), phospho-STAT-2 (4441), phospho-STAT-3 (9131), phospho-STAT-4 (71-7900; Invitrogen) phospho-STAT-5 (9359), phospho-STAT-6 (9364). A donkey anti-rabbit secondary antibody was purchased from Jackson Immunoresearch, Newmarket, Suffolk, UK (1:200 dilution).The intensity of the phospho-STAT staining in each sample was scored by eye using a light microscope. Two independent observers, blinded to the sample group to avoid bias, scored the slides. For each slide, the staining intensity was graded relatively based on the following scale: 1, 2, 3 and 4 from no staining to strong brown staining. A score was recorded for the staining confined to the macrophages, epithelium and for the overall slide. Ten different fields on each slide were scored, and the average score was calculated for each slide. An average score of the scores for each of the two observers was then calculated.ResultsIn order to determine whether the JAK-STAT pathway is activated in smokers and COPD patients compared to non-smokers, the tyrosine phosphorylation of STATs 1-6, as markers of JAK-STAT pathway activation, were assessed by western blot and IHC. We were also able to investigate whether the phosphorylation was localised to epithelial cells or macrophages, by assessing the intensity of immunohistochemical staining in these particular cell types.Determining phosphorylation of STAT1 by western blot and IHC in human parenchymal samples from non-smokers, smokers and COPD patientsWestern blotting revealed that tyrosine phosphorylation of STAT1 appears to be increased in smokers and COPD patients compared to non-smokers although this only reached significance in COPD patients indicating that the JAK-STAT1 pathway is activated in COPD patients (Figure 1). We could not detect any difference in levels of phosphorylation at ser 727 in the three groups. This suggests that the STAT 1 activation was via JAK (data not shown). Immunohistochemical staining for tyrosine phospho-STAT1 displayed a similar trend towards a small increase in smokers and COPD patients compared to non-smokers but this did not reach significance (Figure 2). Staining was stronger in macrophages than epithelial cells across all three groups.Determining phosphorylation of STAT2 by western blot and IHC in human parenchymal samples from non-smokers, smokers and COPD patientsWe could not detect any phosphorylated tyrosine in STAT2 in any of the groups by western blot. However a significant increase in phospho- tyrosine-STAT2 staining was seen in the macrophages of smokers compared to non-smokers by IHC (Figure 3). This increase did not reach significance in epithelial cells or the overall section and no such increase was seen in COPD patients. Determining phosphorylation of STAT3 by western blot and IHC in human parenchymal samples from non-smokers, smokers and COPD patientsSimilar to the results for phospho-STAT1, there was a significant increase in the amount of phospho-tyrosine-STAT3 detected by western blot in COPD patients compared to non-smokers (Figure 4). A trend towards an increase in smokers compared to non-smokers did not reach significance. We could not detect any difference in levels of phosphorylation at ser 727 in the three groups. This suggests that the STAT 3 activation was via JAK (data not shown). Despite these findings by western blot no increase in tyrosine phospho-STAT3 staining was seen in COPD patients compared to non-smokers by IHC (Figure 5). There appeared to be a small increase in phospho-STAT3 in macrophages specifically from smokers compared to non-smokers but this did not reach significance.Determining phosphorylation of STAT4 by western blot and IHC in human parenchymal samples from non-smokers, smokers and COPD patientsPhosphorylated tyrosine in STAT4 could not be detected in any of the samples by western blot and no difference in phospho-STAT4 staining by IHC could be detected across the groups in either cell type (epithelial cells or macrophages) evaluated or the overall section (Figure 6).Determining phosphorylation of STAT5 by western blot and IHC in human parenchymal samples from non-smokers, smokers and COPD patientsPhosphorylated tyrosine in STAT5 could not be measured in any of the samples by western blot. Differences in phospho-tyrosine-STAT5 staining by IHC between the groups were also not detected (Figure 7).Determining phosphorylation of STAT6 by western blot and IHC in human parenchymal samples from non-smokers, smokers and COPD patientsAlthough we could measure phosphorylated tyrosine in STAT6 by western blot, there was no difference between the groups (Figure 8). Very little phospho-tyrosine-STAT6 was observed by IHC and there were no significant differences between the groups (Figure 9).Discussion There are currently no effective treatments to stop the progression of COPD. Currently there is interest in developing inhibitors of the JAK/STAT pathway as a novel treatment for COPD as this pathway plays a key role in the signalling of several cytokines thought to drive the persistent inflammation seen in COPD ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1183/09031936.01.00229701", "author" : [ { "dropping-particle" : "", "family" : "Chung", "given" : "K F", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "European Respiratory Journal", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2001", "7" ] ] }, "page" : "50-59", "title" : "Cytokines in chronic obstructive pulmonary disease", "type" : "article-journal", "volume" : "18" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "ISSN" : "1054-3589", "PMID" : "10582086", "author" : [ { "dropping-particle" : "", "family" : "Schindler", "given" : "C", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Strehlow", "given" : "I", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Advances in pharmacology (San Diego, Calif.)", "id" : "ITEM-2", "issued" : { "date-parts" : [ [ "2000", "1" ] ] }, "page" : "113-74", "title" : "Cytokines and STAT signaling.", "type" : "article-journal", "volume" : "47" }, "uris" : [ "" ] } ], "mendeley" : { "previouslyFormattedCitation" : "[3, 4]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[3, 4]. Moreover, inhibitors of this pathway have been approved for other inflammatory diseases such as rheumatoid arthritis ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/nbt0113-3", "ISSN" : "1546-1696", "PMID" : "23302910", "author" : [ { "dropping-particle" : "", "family" : "Garber", "given" : "Ken", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature biotechnology", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2013", "1" ] ] }, "page" : "3-4", "publisher" : "Nature Publishing Group", "title" : "Pfizer's first-in-class JAK inhibitor pricey for rheumatoid arthritis market.", "type" : "article-journal", "volume" : "31" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1038/nbt0611-467", "ISSN" : "1546-1696", "PMID" : "21654650", "author" : [ { "dropping-particle" : "", "family" : "Garber", "given" : "Ken", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature biotechnology", "id" : "ITEM-2", "issue" : "6", "issued" : { "date-parts" : [ [ "2011", "6" ] ] }, "page" : "467-8", "publisher" : "Nature Publishing Group", "title" : "Pfizer's JAK inhibitor sails through phase 3 in rheumatoid arthritis.", "type" : "article-journal", "volume" : "29" }, "uris" : [ "" ] } ], "mendeley" : { "previouslyFormattedCitation" : "[8, 9]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[8, 9]. We wanted to investigate which STAT proteins are activated in COPD by both western blot and IHC as surprisingly there are no published studies showing the activation status of all STATs1-6 in lung samples as compared to non-smoker and healthy smoker controls. While prospective patient selection enables the collection of data from carefully phenotyped patients, BAL, sputum samples, biopsies data can often be compromised by the small sample sizes, limited area sampled and in some cases artefacts induced by the sampling techniques. For these reasons it is important to carry out work in lung tissue in which larger areas can be sampled and tissues can be preserved using methodologies which have less impact on assay protocols. Importantly, the use of transplant tissue also allows us to use tissue from end stage COPD patients who would not normally be consented for invasive procedures as part of a clinical trial.To our knowledge, this is the first study to show increased phosphorylation of tyrosine on STAT1 in lung parenchymal samples taken from COPD patients compared to smokers without COPD and non-smokers. This is particularly interesting as a recent genome wide association study using a Norwegian cohort suggests that a polymorphism in the STAT1 gene may confer susceptibility to developing COPD ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1183/09031936.00091709", "abstract" : "Lack of reproducibility of findings has been a criticism of genetic association studies on complex diseases, such as chronic obstructive pulmonary disease (COPD). We selected 257 polymorphisms of 16 genes with reported or potential relationships to COPD and genotyped these variants in a case-control study that included 953 COPD cases and 956 control subjects. We explored the association of these polymorphisms to three COPD phenotypes: a COPD binary phenotype and two quantitative traits (post-bronchodilator forced expiratory volume in 1 s (FEV(1)) % predicted and FEV(1)/forced vital capacity (FVC)). The polymorphisms significantly associated to these phenotypes in this first study were tested in a second, family-based study that included 635 pedigrees with 1,910 individuals. Significant associations to the binary COPD phenotype in both populations were seen for STAT1 (rs13010343) and NFKBIB/SIRT2 (rs2241704) (p<0.05). Single-nucleotide polymorphisms rs17467825 and rs1155563 of the GC gene were significantly associated with FEV(1) % predicted and FEV(1)/FVC, respectively, in both populations (p<0.05). This study has replicated associations to COPD phenotypes in the STAT1, NFKBIB/SIRT2 and GC genes in two independent populations, the associations of the former two genes representing novel findings.", "author" : [ { "dropping-particle" : "", "family" : "Bakke", "given" : "P S", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Zhu", "given" : "G", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gulsvik", "given" : "a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kong", "given" : "X", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Agusti", "given" : "a G N", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Calverley", "given" : "P M a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Donner", "given" : "C F", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Levy", "given" : "R D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Make", "given" : "B J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Par\u00e9", "given" : "P D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rennard", "given" : "S I", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Vestbo", "given" : "J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wouters", "given" : "E F M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Anderson", "given" : "W", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lomas", "given" : "D a", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Silverman", "given" : "E K", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pillai", "given" : "S G", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "The European respiratory journal : official journal of the European Society for Clinical Respiratory Physiology", "id" : "ITEM-1", "issue" : "2", "issued" : { "date-parts" : [ [ "2011", "2" ] ] }, "page" : "255-263", "title" : "Candidate genes for COPD in two large data sets.", "type" : "article-journal", "volume" : "37" }, "uris" : [ "" ] } ], "mendeley" : { "previouslyFormattedCitation" : "[12]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[12]. Equally, we were able to show increased phosphorylation of STAT3 by western in COPD patients compared to non-smokers. This finding is consistent with elevated levels of IL-6 that are seen in the induced sputum and lung tissue of COPD patients ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.cyto.2010.02.004", "ISSN" : "1096-0023", "PMID" : "20181491", "abstract" : "Lung diseases like cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are associated with chronic airway inflammation. The aim of our study was to compare a complex biomarker profile in order to characterize specific inflammatory patterns in sputum of patients with CF and COPD. Induced sputum samples of 19 CF-, 26 COPD patients and 21 healthy controls were analyzed for concentrations of IL-1beta, IL-2, IL-6, IL-8, IL-13, IP-10, MCP-1, IFN-gamma and TNF-alpha using the new cytometric bead array (CBA) technology. Significant differences in airway biomarker profiles of CF and COPD were detected. Patients with CF showed a significant increase in IL-1beta, IL-6, IL-8, IL-13, TNF-alpha, IFN-gamma and MCP-1. COPD patients showed an increase in IL-6, IL-8, IL-13 and MCP-1 compared to healthy controls. CF and COPD compared to each other exhibited differences in IL-1beta, IL-2, IL-8, TNF-alpha, IFN-gamma and MCP-1 levels. Significant correlations between the parameters of lung function and sputum biomarker levels were found. Analyzing induced sputum allows characterization of specific airway biomarker profiles in CF and COPD and can be related to the clinical status of the patient. CBA of induced sputum seems to be a pivotal tool to characterize pulmonary inflammation.", "author" : [ { "dropping-particle" : "", "family" : "Eickmeier", "given" : "Olaf", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Huebner", "given" : "Marisa", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Herrmann", "given" : "Eva", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Zissler", "given" : "Ulrich", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Rosewich", "given" : "Martin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Baer", "given" : "Patrick C", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Buhl", "given" : "Roland", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Schmitt-Groh\u00e9", "given" : "Sabina", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Zielen", "given" : "Stefan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Schubert", "given" : "Ralf", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cytokine", "id" : "ITEM-1", "issue" : "2", "issued" : { "date-parts" : [ [ "2010", "5" ] ] }, "page" : "152-7", "publisher" : "Elsevier Ltd", "title" : "Sputum biomarker profiles in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) and association between pulmonary function.", "type" : "article-journal", "volume" : "50" }, "uris" : [ "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1152/ajplung.00285.2011", "ISSN" : "1522-1504", "PMID" : "22268122", "abstract" : "Interleukin (IL)-6 is a potent immunomodulatory cytokine that is associated with emphysema, a major component of chronic obstructive pulmonary disease (COPD). IL-6 signaling via the gp130 coreceptor is coupled to multiple signaling pathways, especially the latent transcription factor signal transducer and activator of transcription (Stat)3. However, the pathological role of endogenous gp130-dependent Stat3 activation in emphysema is ill defined. To elucidate the role of the IL-6/gp130/Stat3 signaling axis in the cellular and molecular pathogenesis of emphysema, we employed a genetic complementation strategy using emphysematous gp130(F/F) mice displaying hyperactivation of endogenous Stat3 that were interbred with mice to impede Stat3 activity. Resected human lung tissue from patients with COPD and COPD-free individuals was also evaluated by immunohistochemistry. Genetic reduction of Stat3 hyperactivity in gp130(F/F):Stat3(-/+) mice prevented lung inflammation and excessive protease activity; however, emphysema still developed. In support of these findings, Stat3 activation levels in human lung tissue correlated with the extent of pulmonary inflammation but not airflow obstruction in COPD. Furthermore, COPD lung tissue displayed increased levels of IL-6 and apoptotic alveolar cells, supporting our previous observation that increased endogenous IL-6 expression in the lungs of gp130(F/F) mice contributes to emphysema by promoting alveolar cell apoptosis. Collectively, our data suggest that IL-6 promotes emphysema via upregulation of Stat3-independent apoptosis, whereas IL-6 induction of lung inflammation occurs via Stat3. We propose that while discrete targeting of Stat3 may alleviate pulmonary inflammation, global targeting of IL-6 potentially represents a therapeutically advantageous approach to combat COPD phenotypes where emphysema predominates.", "author" : [ { "dropping-particle" : "", "family" : "Ruwanpura", "given" : "Saleela M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "McLeod", "given" : "Louise", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Miller", "given" : "Alistair", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jones", "given" : "Jessica", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Vlahos", "given" : "Ross", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ramm", "given" : "Georg", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Longano", "given" : "Anthony", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bardin", "given" : "Philip G", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bozinovski", "given" : "Steven", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Anderson", "given" : "Gary P", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jenkins", "given" : "Brendan J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "American journal of physiology. Lung cellular and molecular physiology", "id" : "ITEM-2", "issue" : "7", "issued" : { "date-parts" : [ [ "2012", "4", "1" ] ] }, "page" : "L627-39", "title" : "Deregulated Stat3 signaling dissociates pulmonary inflammation from emphysema in gp130 mutant mice.", "type" : "article-journal", "volume" : "302" }, "uris" : [ "" ] } ], "mendeley" : { "previouslyFormattedCitation" : "[13, 14]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[13, 14] and the fact that IL-6 is recognised to activate STAT3 ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "ISSN" : "0022-1767", "PMID" : "17312100", "abstract" : "Universal and essential to cytokine receptor signaling, the JAK-STAT pathway is one of the best understood signal transduction cascades. Almost 40 cytokine receptors signal through combinations of four JAK and seven STAT family members, suggesting commonality across the JAK-STAT signaling system. Despite intense study, there remain substantial gaps in understanding how the cascades are activated and regulated. Using the examples of the IL-6 and IL-10 receptors, I will discuss how diverse outcomes in gene expression result from regulatory events that effect the JAK1-STAT3 pathway, common to both receptors. I also consider receptor preferences by different STATs and interpretive problems in the use of STAT-deficient cells and mice. Finally, I consider how the suppressor of cytokine signaling (SOCS) proteins regulate the quality and quantity of STAT signals from cytokine receptors. New data suggests that SOCS proteins introduce additional diversity into the JAK-STAT pathway by adjusting the output of activated STATs that alters downstream gene activation.", "author" : [ { "dropping-particle" : "", "family" : "Murray", "given" : "Peter J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Journal of immunology (Baltimore, Md. : 1950)", "id" : "ITEM-1", "issue" : "5", "issued" : { "date-parts" : [ [ "2007", "3", "1" ] ] }, "page" : "2623-9", "title" : "The JAK-STAT signaling pathway: input and output integration.", "type" : "article-journal", "volume" : "178" }, "uris" : [ "" ] } ], "mendeley" : { "previouslyFormattedCitation" : "[7]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[7]. Interestingly, we could not detect any phosphorylation of Ser 727 on either STAT1 or STAT3. As these sites are not thought to be phosphorylated by JAK it may imply that the activation of STATs we observes is specific to JAK signalling. These data may suggest that whilst the JAK/STAT pathway is involved in the pathogenesis of COPD only certain STATs are involved. Therefore the possibility exists to target specific STAT proteins rather than broad-spectrum inhibition which may increase the side effect liability.Whilst the results obtained by IHC did not entirely match the results obtained by western blot, the differences in results may simply be explained by the different protocols employed. For example, fixing the lung tissue in formalin before IHC can mask epitopes of the target proteins by crosslinking proteins or/and alter their tertiary structure ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "ISSN" : "0046-8177", "PMID" : "7512074", "abstract" : "Different variations of the antigen retrieval technique using different retrieval solutions have been evaluated for their effectiveness in restoring the antigenicity of six intranuclear antigens, each of which is a potentially valuable prognostic indicator in formalin-fixed, paraffin-embedded tissue sections. The results of immunohistochemical staining for estrogen receptor, progesterone receptor, androgen receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen were compared following the different antigen retrieval approaches. The strongest immunostaining signal with the clearest background was obtained by microwave heating of dewaxed paraffin sections for 10 minutes in 0.05 mol/L glycine HCl (pH 3.5) or in citrate buffer solution (pH 6). Urea solution, distilled water, and lead thiocyanate solution yielded improvements with some antigens, but less consistently and less impressively than glycine HCl buffer or citrate buffer. Following antigen retrieval nuclear staining was sharply defined and could be achieved consistently in a variety of tissues after formalin fixation for as long as 7 days. The duration of fixation, however, was an important variable; generally, the longer the fixation time the more vigorous the retrieval procedure required. This study demonstrates the ability to stain a variety of intranuclear antigens, which are not readily demonstrable otherwise, in formalin-paraffin sections with a high degree of consistency and reproducibility. The availability of methods that are effective in paraffin sections may facilitate studies of the possible value of these markers as prognostic indicators for predicting the response of major tumors to different forms of therapy. This study also provided insight into the basic principles of the antigen retrieval method, which may be helpful in attempts to develop a more uniformly standardized technique applicable to many different antigen systems.", "author" : [ { "dropping-particle" : "", "family" : "Taylor", "given" : "C R", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Shi", "given" : "S R", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chaiwun", "given" : "B", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Young", "given" : "L", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Imam", "given" : "S A", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Cote", "given" : "R J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Human pathology", "id" : "ITEM-1", "issue" : "3", "issued" : { "date-parts" : [ [ "1994", "3" ] ] }, "page" : "263-70", "title" : "Strategies for improving the immunohistochemical staining of various intranuclear prognostic markers in formalin-paraffin sections: androgen receptor, estrogen receptor, progesterone receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 ant", "type" : "article-journal", "volume" : "25" }, "uris" : [ "" ] } ], "mendeley" : { "previouslyFormattedCitation" : "[15]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[15], although steps were taken to minimise this and unmask the epitopes. Alternatively the discrepancy in the results may be due to differences in antibody affinity when utilised for each technique. Indeed, phosphorylated STAT2 was found to be increased by IHC in macrophages from smokers compared to non-smokers but we could not detect any phosphorylated tyrosine STAT2 in any of the samples by western. Apart from the increase in STAT2 activation in macrophages, no significant differences in STAT activation between the groups were detected by IHC. Whilst we were able to show an increase in STAT3 phosphorylation by western in COPD patients compared to non-smokers, our IHC results did not illustrate this. Intriguingly, Ruwanpura and colleagues were only able to demonstrate an association between activation of STAT3 and inflammation but not COPD status when comparing COPD patients to ‘healthy’ smokers using IHC ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1152/ajplung.00285.2011", "ISSN" : "1522-1504", "PMID" : "22268122", "abstract" : "Interleukin (IL)-6 is a potent immunomodulatory cytokine that is associated with emphysema, a major component of chronic obstructive pulmonary disease (COPD). IL-6 signaling via the gp130 coreceptor is coupled to multiple signaling pathways, especially the latent transcription factor signal transducer and activator of transcription (Stat)3. However, the pathological role of endogenous gp130-dependent Stat3 activation in emphysema is ill defined. To elucidate the role of the IL-6/gp130/Stat3 signaling axis in the cellular and molecular pathogenesis of emphysema, we employed a genetic complementation strategy using emphysematous gp130(F/F) mice displaying hyperactivation of endogenous Stat3 that were interbred with mice to impede Stat3 activity. Resected human lung tissue from patients with COPD and COPD-free individuals was also evaluated by immunohistochemistry. Genetic reduction of Stat3 hyperactivity in gp130(F/F):Stat3(-/+) mice prevented lung inflammation and excessive protease activity; however, emphysema still developed. In support of these findings, Stat3 activation levels in human lung tissue correlated with the extent of pulmonary inflammation but not airflow obstruction in COPD. Furthermore, COPD lung tissue displayed increased levels of IL-6 and apoptotic alveolar cells, supporting our previous observation that increased endogenous IL-6 expression in the lungs of gp130(F/F) mice contributes to emphysema by promoting alveolar cell apoptosis. Collectively, our data suggest that IL-6 promotes emphysema via upregulation of Stat3-independent apoptosis, whereas IL-6 induction of lung inflammation occurs via Stat3. We propose that while discrete targeting of Stat3 may alleviate pulmonary inflammation, global targeting of IL-6 potentially represents a therapeutically advantageous approach to combat COPD phenotypes where emphysema predominates.", "author" : [ { "dropping-particle" : "", "family" : "Ruwanpura", "given" : "Saleela M", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "McLeod", "given" : "Louise", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Miller", "given" : "Alistair", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jones", "given" : "Jessica", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Vlahos", "given" : "Ross", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ramm", "given" : "Georg", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Longano", "given" : "Anthony", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bardin", "given" : "Philip G", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bozinovski", "given" : "Steven", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Anderson", "given" : "Gary P", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jenkins", "given" : "Brendan J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "American journal of physiology. Lung cellular and molecular physiology", "id" : "ITEM-1", "issue" : "7", "issued" : { "date-parts" : [ [ "2012", "4", "1" ] ] }, "page" : "L627-39", "title" : "Deregulated Stat3 signaling dissociates pulmonary inflammation from emphysema in gp130 mutant mice.", "type" : "article-journal", "volume" : "302" }, "uris" : [ "" ] } ], "mendeley" : { "previouslyFormattedCitation" : "[14]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[14]. Whilst this appears to support our data showing no difference in phosphorylated STAT3 staining between COPD patients and smokers, it suggests that as COPD patients display airway inflammation but non-smokers should not, we should have observed a difference between these two groups. This suggests that the antibody in this case was more suitable for Western blotting rather than IHC, or that the dynamic range for two different assays are different.Increased phospho-STAT4 staining by IHC in smokers and COPD patients compared to non-smokers has been reported previously by Di Stefano and colleagues ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1183/09031936.04.00080303", "ISSN" : "0903-1936", "author" : [ { "dropping-particle" : "", "family" : "Stefano", "given" : "a.", "non-dropping-particle" : "Di", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Caramori", "given" : "G.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Capelli", "given" : "a.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gnemmi", "given" : "I.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ricciardolo", "given" : "F.L.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Oates", "given" : "T.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Donner", "given" : "C.F.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chung", "given" : "K.F.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barnes", "given" : "P.J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Adcock", "given" : "I.M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "European Respiratory Journal", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2004", "7", "1" ] ] }, "page" : "78-85", "title" : "STAT4 activation in smokers and patients with chronic obstructive pulmonary disease", "type" : "article-journal", "volume" : "24" }, "uris" : [ "" ] } ], "mendeley" : { "previouslyFormattedCitation" : "[16]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[16]. Whilst we used the same antibody we were unable to replicate their finding. This could be due to differences in the general protocol or the samples employed. The previous paper used samples from patients with mild or moderate COPD ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1183/09031936.04.00080303", "ISSN" : "0903-1936", "author" : [ { "dropping-particle" : "", "family" : "Stefano", "given" : "a.", "non-dropping-particle" : "Di", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Caramori", "given" : "G.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Capelli", "given" : "a.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gnemmi", "given" : "I.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ricciardolo", "given" : "F.L.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Oates", "given" : "T.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Donner", "given" : "C.F.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chung", "given" : "K.F.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Barnes", "given" : "P.J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Adcock", "given" : "I.M.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "European Respiratory Journal", "id" : "ITEM-1", "issue" : "1", "issued" : { "date-parts" : [ [ "2004", "7", "1" ] ] }, "page" : "78-85", "title" : "STAT4 activation in smokers and patients with chronic obstructive pulmonary disease", "type" : "article-journal", "volume" : "24" }, "uris" : [ "" ] } ], "mendeley" : { "previouslyFormattedCitation" : "[16]" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }[16] whereas the samples in our study were from a transplant programme and therefore the patients had end stage disease. It may be that STAT4 is only activated during the early stages of COPD rather than the later stages. There are limitations associated with the samples utilised for this study, which although unavoidable here, should be addressed in future investigations. Firstly, equal numbers of samples were not utilised in each group with a maximum of 12 non-smokers or 10 smokers samples compared to 23 COPD patients samples. As mentioned previously the COPD patients are at the end stage of the disease (the FEV1 data available from 6 of the COPD patients indicated an FEV1 of 18.6 ± 1.7% predicted) and they have stopped smoking which may affect the inflammatory status of their lungs. This is may explain why an increase in STAT2 activation in macrophages was only observed in smokers but not COPD patients compared to donor non-smokers. The COPD patients in our study would also have been taking a range of differing medications depending on their exact disease phenotype. Although the efficacy of these medications is controversial, they may affect the JAK STAT pathway via effects on cytokine production. A larger study with equal groups would be needed to validate these initial findings. It would also be interesting to assess STAT phosphorylation at the different GOLD stages of COPD to investigate how the STAT proteins are involved in disease progression and if different STATs are involved at different stages. In summary, the results of this study suggest a role for the JAK/STAT pathway in COPD and STAT1 and 3 in particular warrant further investigation as targets for novel COPD therapeutics.Figure LegendsFigure 1: STAT1 phosphorylation in human parenchymal samples from non-smokers, smokers and COPD patients by western blotTop panel: Representative image of western blot for phospho-STAT1, STAT1 and β-actin as a loading control. D, donor; S, smoker; C, COPD.Bottom panels: The level of phospho-STAT1 was measured by western blot relative to the loading control, actin (left panel) and total level of STAT1 (right panel) in human parenchymal samples from donor non-smokers, smokers and COPD patients. Data is presented as mean ± SEM (AU) of densitometry readings. Statistical significance was assessed using a Kruskal-Wallis test and Dunn’s multiple comparison post-test, *p<0.05 vs. donor.Results are representative of 12 non-smokers, 10 healthy smokers and 22 subjects with COPD.Figure 2: STAT1 phosphorylation in human parenchymal samples from non-smokers, smokers and COPD patients by IHCA] Parenchymal sections from donor non-smokers, smokers and COPD patients were stained for phospho-STAT1 by IHC. The level of staining in epithelial cells, macrophages and the overall section was assessed on a scale from 1 to 4 by two independent observers. Ten fields per section were assessed and an average was calculated. Data is presented as the mean ± SEM. Epi, epithelial cells; Macs, macrophages.Representative photomicrographs of a parenchymal section immunostained for identification of phospho‐STAT1 from a donor (B), smoker (C) and COPD (D) patients. Scale bar = 100 ?m. The arrow indicates positive staining.Results are representative of 12 non-smokers, 10 healthy smokers and 23 subjects with COPD.Figure 3: STAT2 phosphorylation in human parenchymal samples from non-smokers, smokers and COPD patients by IHCA] Parenchymal sections from donor non-smokers, smokers and COPD patients were stained for phospho-STAT2 by IHC. The level of staining in epithelial cells, macrophages and the overall section was assessed on a scale from 1 to 4 by two independent observers. Ten fields per section were assessed and an average was calculated. Data is presented as the mean ± SEM. Statistical significance was assessed using a Kruskal-Wallis test and Dunn’s multiple comparison post-test, *p<0.05 vs. donor. Epi, epithelial cells; Macs, macrophages.Representative photomicrographs of a parenchymal section immunostained for identification of phospho‐STAT2 from a donor (B), smoker (C) and COPD (D) patients. Scale bar = 100 ?m.Results are representative of 12 non-smokers, 10 healthy smokers and 23 subjects with COPD.Figure 4: STAT3 phosphorylation in human parenchymal samples from non-smokers, smokers and COPD patients by western blotTop panel: Representative image of western blot for phospho-STAT3, STAT3 and β-actin as a loading control. D, donor; S, smoker; C, COPD.Bottom panels: The level of phospho-STAT3 was measured by western blot relative to the loading control, actin (left panel) and total level of STAT3 (right panel) in human parenchymal samples from donor non-smokers, smokers and COPD patients. Data is presented as mean ± SEM (AU) of densitometry readings. Statistical significance was assessed using a Kruskal-Wallis test and Dunn’s multiple comparison post-test, *p<0.05 vs. donor.Results are representative of 12 non-smokers, 10 healthy smokers and 22 subjects with COPD.Figure 5: STAT3 phosphorylation in human parenchymal samples from non-smokers, smokers and COPD patients by IHCA] Parenchymal sections from donor non-smokers, smokers and COPD patients were stained for phospho-STAT3 by IHC. The level of staining in epithelial cells, macrophages and the overall section was assessed on a scale from 1 to 4 by two independent observers. Ten fields per section were assessed and an average was calculated. Data is presented as the mean ± SEM. Epi, epithelial cells; Macs, macrophages.Representative photomicrographs of a parenchymal section immunostained for identification of phospho‐STAT3 from a donor (B), smoker (C) and COPD (D) patients. Scale bar = 100 ?m.Results are representative of 12 non-smokers, 10 healthy smokers and 23 subjects with COPD.Figure 6: STAT4 phosphorylation in human parenchymal samples from non-smokers, smokers and COPD patients by IHCA] Parenchymal sections from donor non-smokers, smokers and COPD patients were stained for phospho-STAT4 by IHC. The level of staining in epithelial cells, macrophages and the overall section was assessed on a scale from 1 to 4 by two independent observers. Ten fields per section were assessed and an average was calculated. Data is presented as the mean ± SEM. Epi, epithelial cells; Macs, macrophages.Representative photomicrographs of a parenchymal section immunostained for identification of phospho‐STAT4 from a donor (B), smoker (C) and COPD (D) patients. Scale bar = 100 ?m.Results are representative of 12 non-smokers, 10 healthy smokers and 23 subjects with COPD.Figure 7: STAT5 phosphorylation in human parenchymal samples from non-smokers, smokers and COPD patients by IHCA] Parenchymal sections from donor non-smokers, smokers and COPD patients were stained for phospho-STAT5 by IHC. The level of staining in epithelial cells, macrophages and the overall section was assessed on a scale from 1 to 4 by two independent observers. Ten fields per section were assessed and an average was calculated. Data is presented as the mean ± SEM. Epi, epithelial cells; Macs, macrophages.Representative photomicrographs of a parenchymal section immunostained for identification of phospho‐STAT5 from a donor (B), smoker (C) and COPD (D) patients. Scale bar = 100 ?m.Results are representative of 12 non-smokers, 10 healthy smokers and 23 subjects with COPD.Figure 8: STAT6 phosphorylation in human parenchymal samples from non-smokers, smokers and COPD patients by western blotTop panel: Representative image of western blot for phospho-STAT6, STAT6 and β-actin as a loading control. D, donor; S, smoker; C, COPD.Bottom panels: The level of phospho-STAT3 was measured by western blot relative to the loading control, actin (left panel) and total level of STAT6 (right panel) in human parenchymal samples from donor non-smokers, smokers and COPD patients. Data is presented as mean ± SEM (AU) of densitometric readings. Statistical significance was assessed using a Kruskal-Wallis test and Dunn’s multiple comparison post-test, *p<0.05 vs. donor.Results are representative of 12 non-smokers, 10 healthy smokers and 22 subjects with COPD.Figure 9: STAT6 phosphorylation in human parenchymal samples from non-smokers, smokers and COPD patients by IHCA] Parenchymal sections from donor non-smokers, smokers and COPD patients were stained for phospho-STAT6 by IHC. The level of staining in epithelial cells, macrophages and the overall section was assessed on a scale from 1 to 4 by two independent observers. Ten fields per section were assessed and an average was calculated. Data is presented as the mean ± SEM. Epi, epithelial cells; Macs, macrophages.Representative photomicrographs of a parenchymal section immunostained for identification of phospho‐STAT6 from a donor (B), smoker (C) and COPD (D) patients. Scale bar = 100 ?m.Results are representative of 12 non-smokers, 10 healthy smokers and 23 subjects with COPD.ReferencesADDIN Mendeley Bibliography CSL_BIBLIOGRAPHY 1. Hogg JC. State of the art. Bronchiolitis in chronic obstructive pulmonary disease. Proc. Am. Thorac. Soc. 2006; 3: 489–493.2. Vestbo J, Hurd SS, Agusti AG, Jones PW, Vogelmeier C, Anzueto A, Barnes PJ, Fabbri LM, Martinez FJ, Nishimura M, Stockley RA, Sin DD, Rodriguez-Roisin R, Agustí AG, Nishimura J. Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease. Am. J. Respir. Crit. Care Med. 2013; 187: 347–365.3. Chung KF. Cytokines in chronic obstructive pulmonary disease. Eur. Respir. J. 2001; 18: 50–59.4. Schindler C, Strehlow I. Cytokines and STAT signaling. Adv. Pharmacol. 2000; 47: 113–174.5. Darnell Jr. JE. STATs and Gene Regulation. Science (80-. ). 1997; 277: 1630–1635.6. Pellegrini S, Dusanter-Fourt I. The structure, regulation and function of the Janus kinases (JAKs) and the signal transducers and activators of transcription (STATs). Eur. J. Biochem. 1997; 248: 615–633.7. Murray PJ. The JAK-STAT signaling pathway: input and output integration. J. Immunol. 2007; 178: 2623–2629.8. Garber K. Pfizer’s first-in-class JAK inhibitor pricey for rheumatoid arthritis market. Nat. Biotechnol. Nature Publishing Group; 2013; 31: 3–4.9. Garber K. Pfizer’s JAK inhibitor sails through phase 3 in rheumatoid arthritis. Nat. Biotechnol. Nature Publishing Group; 2011; 29: 467–468.10. Malaviya R, Laskin DL, Malaviya R. Janus kinase-3 dependent inflammatory responses in allergic asthma. Int. Immunopharmacol. Elsevier B.V.; 2010; 10: 829–836.11. Pernis AB, Rothman PB. JAK-STAT signaling in asthma. J. Clin. Invest. 2002; 109: 1279–1283.12. Bakke PS, Zhu G, Gulsvik a, Kong X, Agusti a GN, Calverley PM a, Donner CF, Levy RD, Make BJ, Paré PD, Rennard SI, Vestbo J, Wouters EFM, Anderson W, Lomas D a, Silverman EK, Pillai SG. Candidate genes for COPD in two large data sets. Eur. Respir. J.? Off. J. Eur. Soc. Clin. Respir. Physiol. 2011; 37: 255–263.13. Eickmeier O, Huebner M, Herrmann E, Zissler U, Rosewich M, Baer PC, Buhl R, Schmitt-Grohé S, Zielen S, Schubert R. Sputum biomarker profiles in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) and association between pulmonary function. Cytokine Elsevier Ltd; 2010; 50: 152–157.14. Ruwanpura SM, McLeod L, Miller A, Jones J, Vlahos R, Ramm G, Longano A, Bardin PG, Bozinovski S, Anderson GP, Jenkins BJ. Deregulated Stat3 signaling dissociates pulmonary inflammation from emphysema in gp130 mutant mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 2012; 302: L627–39.15. Taylor CR, Shi SR, Chaiwun B, Young L, Imam SA, Cote RJ. Strategies for improving the immunohistochemical staining of various intranuclear prognostic markers in formalin-paraffin sections: androgen receptor, estrogen receptor, progesterone receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 ant. Hum. Pathol. 1994; 25: 263–270.16. Di Stefano a., Caramori G, Capelli a., Gnemmi I, Ricciardolo FL, Oates T, Donner CF, Chung KF, Barnes PJ, Adcock IM. STAT4 activation in smokers and patients with chronic obstructive pulmonary disease. Eur. Respir. J. 2004; 24: 78–85. ................
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