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2.
Am J Physiol Lung Cell Mol Physiol ; 320(4): L600-L614, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33295836

RESUMO

The growing interest in regulating flavored E-liquids must incorporate understanding of the "flavoring profile" of each E-liquid-which flavorings (flavoring chemicals) are present and at what concentrations not just focusing on the flavor on the label. We investigated the flavoring profile of 10 different flavored E-liquids. We assessed bronchial epithelial cell viability and apoptosis, phagocytosis of bacteria and apoptotic cells by macrophages after exposure to E-cigarette vapor extract (EVE). We validated our data in normal human bronchial epithelial cells (NHBE) and alveolar macrophages (AM) from healthy donors. We also assessed cytokine release and validated in the saliva from E-cigarette users. Increased necrosis/apoptosis (16.1-64.5% apoptosis) in 16HBE cells was flavor dependent, and NHBEs showed an increased susceptibility to flavors. In THP-1 differentiated macrophages phagocytosis was also flavor dependent, with AM also showing increased susceptibility to flavors. Further, Banana and Chocolate were shown to reduce surface expression of phagocytic target recognition receptors on alveolar macrophages. Banana and Chocolate increased IL-8 secretion by NHBE, whereas all 4 flavors reduced AM IL-1ß secretion, which was also reduced in the saliva of E-cigarette users compared with healthy controls. Flavorant profiles of E-liquids varied from simple 2 compound mixtures to complex mixtures containing over a dozen flavorants. E-liquids with high benzene content, complex flavoring profiles, high chemical concentration had the greatest impacts. The Flavorant profile of E-liquids is key to disruption of the airway status quo by increasing bronchial epithelial cell apoptosis, causing alveolar macrophage phagocytic dysfunction, and altering airway cytokines.


Assuntos
Apoptose , Brônquios/patologia , Citocinas/metabolismo , Sistemas Eletrônicos de Liberação de Nicotina/estatística & dados numéricos , Aromatizantes/efeitos adversos , Macrófagos/patologia , Fagocitose , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Humanos , Macrófagos/efeitos dos fármacos , Fatores de Risco
3.
Clin Exp Allergy ; 48(6): 650-662, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29676834

RESUMO

BACKGROUND: Severe asthma affects quality of life; however, its impact on workplace productivity is poorly understood. OBJECTIVE: To compare workplace productivity-absenteeism and presenteeism-and impairment in daily activities in severe and non-severe asthma over time and identify characteristics associated with presenteeism in severe asthma. METHODS: The Severe Asthma Web-based Database is an ongoing observational registry from Australia, New Zealand and Singapore. At April 2017, 434 patients with severe asthma and 102 with non-severe asthma were enrolled (18-88 years; 59% female). Participants provided comprehensive clinical and questionnaire data at baseline and were followed-up every 6 months for 24 months. Absenteeism (percentage of time not at work), presenteeism (self-reported impairment at work) and impairment in daily activities outside work due to health problems in the last week were calculated. RESULTS: At baseline, 61.4% of participants with severe asthma and 66.2% with non-severe asthma under 65 years were employed. At younger ages (30-50 years), fewer severe asthma participants were employed (69% vs 100%). Presenteeism and impairment in daily activity were more frequently reported in severe asthma and in participants with poorer asthma control, poorer lung function and more past-year exacerbations (P < .01). Over time, deteriorating asthma control was associated with increasing presenteeism. Although absenteeism was not different between severe and non-severe asthma, worse asthma control was associated with absenteeism (P < .001). In participants with severe asthma, presenteeism was reported more frequently in those with poorer asthma control, poorer asthma-related quality of life and symptoms of depression or anxiety (P < .01). CONCLUSION AND CLINICAL RELEVANCE: Severe asthma was associated with impairment at work and outside the workplace. Improving asthma control and mental health may be important targets for optimizing workplace productivity in severe asthma. Presenteeism and absenteeism may represent key metrics for assessing intervention efficacy in people with severe asthma of working age.


Assuntos
Absenteísmo , Asma/epidemiologia , Eficiência , Qualidade de Vida , Local de Trabalho , Atividades Cotidianas , Adulto , Idoso , Asma/diagnóstico , Asma/etiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sistema de Registros , Índice de Gravidade de Doença , Inquéritos e Questionários
4.
Clin Exp Immunol ; 192(2): 242-250, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29352737

RESUMO

Immunosuppressive therapy fails to suppress the production of proinflammatory cytokines, particularly by CD8+ T cells, in stable lung transplant recipients and those undergoing chronic rejection, suggesting that some patients may become relatively resistant to immunosuppressants such as glucocorticoids (GC). We have shown loss of GC receptor (GCR) from the CD8+ cells, and we hypothesized that the drug membrane efflux pump, p-glycoprotein-1 (Pgp), may also be involved in lymphocyte steroid resistance following lung transplant. Pgp/GCR expression and interferon (IFN)-γ/tumour necrosis factor (TNF)-α proinflammatory cytokine production was measured in blood lymphocytes from 15 stable lung transplant patients, 10 patients with bronchiolitis obliterans syndrome (BOS) and 10 healthy aged-matched controls (± prednisolone ± Pgp inhibitor, cyclosporin A ± GCR activator, Compound A) using flow cytometry. Both Pgp+ and Pgp- lymphocyte subsets from all subjects produced IFN-γ/TNF-α proinflammatory cytokines. Pgp expression was increased in CD8+ Pgp+ T cells and correlated with IFN-γ/TNF-α expression and BOS grade. Reduced GCR was observed in CD8+ Pgp- T, natural killer (NK) T-like and NK cells from stable patients compared with controls, and reduced further in CD8+ Pgp- T cells in BOS. The addition of 2·5 ng/ml cyclosporin A and 1 µM prednisolone inhibit IFN-γ/TNF-α production significantly by CD8+ Pgp+ T cells from BOS patients. The addition of 10 µM Compound A and 1 µM prednisolone inhibit IFN-γ/TNF-α production significantly by CD8+ Pgp- T cells from BOS patients. BOS is associated with increased Pgp expression and loss of GCR from steroid-resistant proinflammatory CD8+ T cells. Treatments that inhibit Pgp and up-regulate GCR in CD8+ T cells may improve graft survival.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Bronquiolite Obliterante/imunologia , Linfócitos T CD8-Positivos/metabolismo , Transplante de Pulmão , Receptores de Glucocorticoides/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Adulto , Bronquiolite Obliterante/genética , Linfócitos T CD8-Positivos/efeitos dos fármacos , Resistência a Medicamentos , Citometria de Fluxo , Humanos , Interferon gama/sangue , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Pessoa de Meia-Idade , Receptores de Glucocorticoides/genética , Esteroides/administração & dosagem , Fator de Necrose Tumoral alfa/sangue , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATP
5.
Clin Exp Allergy ; 46(1): 60-70, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26767492

RESUMO

BACKGROUND: Non-eosinophilic asthma (NEA) is a distinct, often corticosteroid-resistant inflammatory asthma phenotype. NK and NKT-like cells are effector lymphocytes that we have shown, like CD28null T cells, to be relatively resistant to steroids and major sources of pro-inflammatory/cytotoxic mediators. We hypothesized that these cells and mediators would be increased in peripheral blood in NEA. METHODS: Adults with severe asthma and variable airflow obstruction, poorly controlled despite maintenance therapy with inhaled glucocorticosteroids and long-acting bronchodilators, were recruited. Blood was assessed in those with eosinophilic asthma (n = 12), NEA (n = 25) and healthy non-smoking controls (n = 30). We applied flow cytometry to measure T, CD28null, NK and NKT-like cells and their expression of granzyme B, perforin, and killer inhibitory/activating receptors CD94(Kp43), CD158b and CD107A. Intracellular pro-inflammatory cytokine production (IFN-γ and TNF-α) was assessed in 18 controls and 10 patients with asthma/group. RESULTS: In NEA, there was increased expression of granzyme B by CD8+ T cells vs. CONTROLS: There was increased expression of granzyme B and CD158 and decreased CD94 on NK cells, vs. healthy controls and those with eosinophilic asthma. IFN-γ production by NK cells and TNF-α production by NKT-like cells in NEA were significantly increased vs. CONTROLS: In both eosinophilic and NEA phenotypes, there were significant increases in CD4+28null T cells (72% and 81% increases, respectively, vs. controls) and their expression of pro-inflammatory cytokines. Significant correlations were noted between blood CD4+28null T cells and neutrophil numbers in induced sputum, and between corticosteroid dose and blood NKT-like cells, and their production of granzyme B and TNF-α and NK IFN-γ. CONCLUSION AND CLINICAL RELEVANCE: In poorly controlled asthma, altered expression of cytotoxic/pro-inflammatory mediators can be seen on a variety of lymphocyte subsets in the peripheral blood; these changes are most apparent in NEA. Whether this pattern of expression is a marker of treatment responsiveness and future risk of exacerbations remains to be determined.


Assuntos
Asma/sangue , Asma/imunologia , Citocinas/sangue , Mediadores da Inflamação/sangue , Idoso , Asma/diagnóstico , Biomarcadores , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Citocinas/metabolismo , Feminino , Granzimas/sangue , Humanos , Imunofenotipagem , Mediadores da Inflamação/metabolismo , Espaço Intracelular/metabolismo , Contagem de Leucócitos , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Perforina/sangue , Testes de Função Respiratória , Fatores de Risco , Escarro/citologia , Escarro/imunologia
6.
Clin Exp Immunol ; 178(1): 79-85, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24894428

RESUMO

There is a limited understanding how of lung cancer cells evade cytotoxic attack. Previously, we have shown reduced production of the cytotoxic mediator granzyme B by CD8(+) T cells in lung cancer tissue. We hypothesized that lung cancer would be further associated with decreased production of granzyme B, perforin and proinflammatory cytokines by other cytotoxic lymphocytes, natural killer (NK) T-like and NK cells, and that this would result from soluble mediators released by the cancer cells. Lung cancer and non-cancer tissue from five patients was identified by experienced pathologists. Tumour necrosis factor (TNF)-α, interferon (IFN)-γ, granzyme B and perforin were measured in CD4 and CD8(+) T, NK T-like cells and NK cells by flow cytometry. Correlation between cancer stage and granzyme B was analysed retrospectively for 21 patients. The effects of soluble factors released by lung cancer cells on production of cytotoxic mediators and cytokines was assessed, and the role of prostaglandin E2 (PGE)2 /COX investigated using indomethacin inhibition. There were significantly decreased percentages of T, NK T-like and NK cells expressing perforin, TNF-α and IFN-γ in cancer versus non-cancer tissue, and of CD8(+) T cells and CD8(+) NK T-like cells expressing granzyme B (e.g. NK T-like cells: non-cancer 30% ± 7 versus cancer 6% ± 2·5). Cancer cells released soluble factors that inhibited granzyme B, perforin and IFN-γ production that was partially associated with the PGE2 /COX2 pathway. Thus, lung cancer is associated with decreased expression of granzyme B, perforin and IFN-γ by infiltrating T cells, NK T-like and NK cells, possibly as a result of soluble factors produced by the cancer cells including PGE2 . This may be an important immune evasion mechanism.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Granzimas/biossíntese , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Neoplasias Pulmonares/metabolismo , Perforina/biossíntese , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Dinoprostona/metabolismo , Feminino , Granzimas/imunologia , Granzimas/metabolismo , Humanos , Interferon gama/imunologia , Células Matadoras Naturais/imunologia , Pulmão/metabolismo , Neoplasias Pulmonares/imunologia , Masculino , Pessoa de Meia-Idade , Perforina/imunologia , Estudos Retrospectivos , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
7.
Clin Exp Allergy ; 44(9): 1137-45, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24849076

RESUMO

BACKGROUND: In asthma, the airway inflammatory phenotype influences clinical characteristics and treatment response. Although induced sputum is the gold standard test for phenotyping asthma, a more accessible method is needed for clinical practice. OBJECTIVE: To investigate whether white blood cell counts and/or their derived ratios can predict sputum eosinophils or neutrophils in uncontrolled asthma. METHODS: This cross-sectional study evaluated 164 treated but uncontrolled asthmatic patients with sputum induction and blood collection. Receiver-operating characteristic (ROC) curves were used to assess the relationship between blood and sputum parameters. RESULTS: There was a significant positive relationship between blood eosinophil parameters and the percentage of sputum eosinophil count. A weak but significant correlation was found between sputum neutrophil percentage and blood neutrophil percentage (r = 0.219, P = 0.005). ROC curve analysis identified that blood eosinophil percentage count was the best predictor for eosinophilic asthma, with an area under the curve (AUC) of 0.907 (P < 0.001). The optimum cut-point for blood eosinophil percentage was 2.7%, and this yielded a sensitivity of 92.2% and a specificity of 75.8%. The absolute blood eosinophil count was also highly predictive with an AUC of 0.898 (P < 0.0001) at a blood eosinophil cut-off of 0.26 × 10(9) /L. The blood eosinophil/lymphocyte ratio (ELR) and eosinophil/neutrophil ratio (ENR) were increased in eosinophilic asthma, and the neutrophil/lymphocyte ratio (NLR) was increased in neutrophilic asthma. Neutrophilic asthma could also be detected by blood neutrophil percentages and NLR, but with less accuracy. CONCLUSIONS AND CLINICAL RELEVANCE: Blood eosinophil counts and derived ratios (ELR and ENR) can accurately predict eosinophilic asthma in patients with persistent uncontrolled asthma despite treatment. Blood neutrophil parameters are poor surrogates for the proportion of sputum neutrophils. Blood counts may be a useful aid in the monitoring of uncontrolled asthma.


Assuntos
Asma/sangue , Asma/diagnóstico , Contagem de Leucócitos , Fenótipo , Adolescente , Corticosteroides/administração & dosagem , Corticosteroides/uso terapêutico , Adulto , Idoso , Asma/tratamento farmacológico , Estudos Transversais , Eosinófilos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos , Curva ROC , Testes de Função Respiratória , Fatores de Risco , Escarro/citologia , Adulto Jovem
9.
Clin Exp Immunol ; 173(1): 150-60, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23607447

RESUMO

Bronchiolitis obliterans syndrome (BOS) is associated with lack of immunosuppression of T cell proinflammatory cytokines and increased T cell granzyme B. Repeated antigen-driven proliferation down-regulates T cell CD28. We hypothesized that down-regulation of CD28 and up-regulation of alternate co-stimulatory molecules (CD134, CD137, CD152 and CD154) on T cells may be associated with BOS. Co-stimulatory molecules, granzyme B, perforin and intracellular cytokines were measured by flow cytometry on T cells from stable lung transplant patients (n = 38), patients with BOS (n = 20) and healthy controls (n = 10). There was a significant increase in the percentage of CD4/28(null) and CD8/28(null) T cells producing granzyme B, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in BOS compared with stable patients. Down-regulation of CD28 was associated with steroid resistance and up-regulation of CD134, CD137, CD152 and CD154 on CD4(+) T cells and CD137 and CD152 on CD8(+) T cells. There was a significant correlation between increased CD28(null) /CD137 T cells producing IFN-γ, TNF-α with BOS grade (r = 0·861, P < 0·001 for CD28(null) /CD137 IFN-γ/CD8) and time post-transplant (r = 0·698, P < 0·001 for CD28(null) /CD137 IFN-γ/CD8). BOS is associated with down-regulation of CD28 and up-regulation of alternate co-stimulatory molecules on steroid-resistant peripheral blood proinflammatory CD4(+) and CD8(+) T cells. Therapeutic targeting of alternate co-stimulatory molecules on peripheral blood CD28(null) T cells and monitoring response using these assays may help in the management of patients with BOS.


Assuntos
Bronquiolite Obliterante/imunologia , Receptores Coestimuladores e Inibidores de Linfócitos T/biossíntese , Complicações Pós-Operatórias/imunologia , Subpopulações de Linfócitos T/metabolismo , Adulto , Bronquiolite Obliterante/etiologia , Antígenos CD28/análise , Ligante de CD40/biossíntese , Ligante de CD40/genética , Antígeno CTLA-4/biossíntese , Antígeno CTLA-4/genética , Estudos de Casos e Controles , Receptores Coestimuladores e Inibidores de Linfócitos T/genética , Ciclosporina/uso terapêutico , Feminino , Granzimas/análise , Humanos , Imunossupressores/uso terapêutico , Interferon gama/biossíntese , Interferon gama/genética , Transplante de Pulmão , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Perforina/análise , Complicações Pós-Operatórias/etiologia , Receptores OX40/biossíntese , Receptores OX40/genética , Subpopulações de Linfócitos T/imunologia , Tacrolimo/uso terapêutico , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Regulação para Cima
10.
Clin Exp Allergy ; 43(1): 29-35, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23278878

RESUMO

BACKGROUND: Many patients with non-eosinophilic asthma have increased numbers of neutrophils in the airways. The explanation for this chronic inflammation remains unclear, but may result from an impaired ability of alveolar macrophages to phagocytose apoptotic cells (a process termed 'efferocytosis'), as we have shown in chronic obstructive pulmonary disease (COPD). OBJECTIVES: To examine induced sputum as a non-invasive technique to characterize efferocytosis in chronic lung diseases and to compare efferocytosis in patients with non-eosinophilic asthma, eosinophilic asthma and COPD. METHODS: Participants with stable asthma (20 with eosinophilic and 30 with non-eosinophilic) and COPD (n = 11) underwent clinical assessment including allergy skin tests, saline challenge and sputum induction. Sputum cells were dispersed using dithiothreitol and resuspended in culture medium. Efferocytosis of apoptotic bronchial epithelial cells by sputum-derived macrophages was determined using flow cytometry. RESULTS: There were no significant differences in efferocytosis between paired sputum and bronchoalveolar lavage macrophages from three subjects. Efferocytosis was significantly impaired in patients with non-eosinophilic asthma [mean (SD) 0.95 (0.24)] compared with eosinophilic asthma [1.17 (0.19)] and to a similar degree as patients with COPD [1.04 (0.16)]. Sputum neutrophils were significantly higher in patients with COPD and non-eosinophilic asthma compared with eosinophilic asthma. CONCLUSION AND CLINICAL RELEVANCE: Induced sputum provides a reliable and non-invasive method for studying macrophage efferocytosis in chronic lung disease. Macrophage efferocytosis is impaired in non-eosinophilic asthma to a similar degree as that in COPD and may explain the persistent airway neutrophilia and chronic inflammation that characterizes this asthma subtype.


Assuntos
Asma/imunologia , Macrófagos Alveolares/imunologia , Fagocitose/imunologia , Idoso , Asma/patologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Feminino , Citometria de Fluxo , Humanos , Macrófagos Alveolares/patologia , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , Eosinofilia Pulmonar/imunologia , Eosinofilia Pulmonar/patologia , Escarro/citologia , Escarro/imunologia
11.
Eur Respir J ; 39(2): 329-43, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21737550

RESUMO

Pulmonary arterial hypertension (PAH) remains a fatal disease despite modern pharmacotherapy. Mutations in the gene for bone morphogenetic protein receptor type II (BMPR2) lead to reduced BMPR2 expression, which is causally linked to PAH. BMPR2 is predominantly expressed on pulmonary endothelium and has complex interactions with transforming growth factor (TGF)-ß signalling mechanisms. Our objectives were to assess the effect on PAH of upregulating BMPR2 by targeted adenoviral BMPR2 gene delivery to the pulmonary vascular endothelium. We used two established rat models of PAH: chronic hypoxia and monocrotaline (MCT). In both hypertensive models, those receiving BMPR2 had less right ventricular hypertrophy, less pulmonary vascular resistance, improved cardiac function and reduced vascular remodelling. In the MCT model, there was an increase in TGF-ß, which was prevented by BMPR2 treatment. In vitro, TGF-ß1-induced endothelial-mesenchymal transition (EndMT) in human pulmonary microvascular endothelial cells, which was associated with reduced BMPR2 expression. EndMT was partially ameliorated by stimulating BMPR2 signalling with appropriate ligands even in the ongoing presence of TGF-ß1. Collectively, these results indicate therapeutic potential for upregulation of the BMPR2 axis in PAH, which may be, in part, mediated by countering the remodelling effects of TGF-ß.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Terapia Genética/métodos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/terapia , Adenoviridae/genética , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Divisão Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/citologia , Transição Epitelial-Mesenquimal/genética , Técnicas de Transferência de Genes , Humanos , Hipertensão Pulmonar/metabolismo , Hipóxia/genética , Hipóxia/metabolismo , Hipóxia/terapia , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Transgenes/fisiologia , Regulação para Cima/genética
12.
Clin Exp Immunol ; 166(1): 94-102, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21910726

RESUMO

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease; it is a leading cause of death and existing treatments have no proven disease-modifying effect. The mechanisms underlying this resistance are largely unknown, but suggest the presence of some self-maintaining pathogenic process, possibly initiated by cigarette smoking, that prevents the normal resolution of inflammation. We have previously reported increased production of proinflammatory cytokines and granzyme b by CD8(+) T cells in COPD; costimulatory receptor/ligand interactions required include CD80:86/CD28, B7-1/CTLA4, 4-1BB/1BBL and OX40/OX40L. We hypothesized that a dysregulated expression/function of these molecules may play a role in inflammatory/autoimmune components of COPD. We analysed T cell co-stimulatory molecules in blood from 34 controls, 15 smokers and 48 COPD subjects. We assessed the potential functional relevance of CD8/CD28(null) cells in COPD by measuring their production of proinflammatory cytokines, co-stimulatory molecules, granzyme and perforin. A smoke-exposed murine model was applied to investigate the relative expression of CD8/CD28(null) T cells in blood, lung tissue and airway. CD8/CD28(null) cells were increased in both current- and ex-smoker COPD groups; these cells expressed significantly more interferon (IFN)-γ, OX40, 4-1BB, CTLA4, granzyme and perforin when stimulated than CD8/CD28(+) T cells. There were no changes in CD4/CD28(null) T cells. In mice exposed to cigarette smoke for 12 weeks, CD8/CD28(null) T cells were significantly increased in the airway with a trend for an increase in lung tissue and blood. Increased production of proinflammatory cytokines and expression of alternative co-stimulatory molecules by CD8/CD28(null) T cells may play a role in inflammatory or autoimmune responses in COPD and identify therapeutic targets.


Assuntos
Antígenos CD28/metabolismo , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Citocinas/metabolismo , Granzimas/metabolismo , Pulmão/imunologia , Linfócitos Nulos/metabolismo , Perforina/metabolismo , Doença Pulmonar Obstrutiva Crônica , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Antígenos CD28/imunologia , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Estudos de Casos e Controles , Citocinas/genética , Citometria de Fluxo , Expressão Gênica , Granzimas/genética , Humanos , Pulmão/patologia , Linfócitos Nulos/citologia , Linfócitos Nulos/imunologia , Camundongos , Pessoa de Meia-Idade , Perforina/genética , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , Fumar , Regulação para Cima
13.
Clin Exp Immunol ; 161(3): 584-90, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20528884

RESUMO

Immunosuppression therapy following lung transplant fails to prevent chronic rejection/bronchiolitis obliterans syndrome, which we have shown is associated with lack of suppression of peripheral blood T cell granzyme B, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. We hypothesized that these proinflammatory mediators may increase with time post-transplant in otherwise stable patients before clinical signs of declining lung function, and patients experiencing declining lung function would show a further increase in these mediators. Intracellular cytokine profiles and granzyme B were investigated in T cells in whole blood and airways from lung transplant patients using flow cytometry. There was a significant negative correlation between forced expiratory volume in 1 s (FEV(1) ), drug dose and time post-transplant. A significant correlation between increased granzyme B, IFN-γ, interleukin (IL)-2 and TNF-α and time post-transplant was noted in peripheral blood T cells but not lung T cells from stable patients. Patients with similar drug dose but experiencing declining FEV(1) showed a further increase in peripheral blood T cell IFN-γ, IL-2 and TNF-α. Time post-lung transplant correlates with increasing peripheral blood T cell granzyme B and proinflammatory cytokines. Declining FEV(1) is associated with a further increase in these proinflammatory mediators. Drugs that reduce these inflammatory mediators effectively may reduce the incidence of chronic graft rejection.


Assuntos
Citocinas/sangue , Granzimas/sangue , Transplante de Pulmão , Linfócitos T/metabolismo , Azatioprina/uso terapêutico , Líquido da Lavagem Broncoalveolar/química , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Ciclosporina/uso terapêutico , Citocinas/metabolismo , Quimioterapia Combinada , Citometria de Fluxo , Volume Expiratório Forçado , Rejeição de Enxerto/sangue , Rejeição de Enxerto/metabolismo , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Sobrevivência de Enxerto/imunologia , Granzimas/metabolismo , Humanos , Imunossupressores/uso terapêutico , Mediadores da Inflamação/sangue , Mediadores da Inflamação/metabolismo , Interferon gama/sangue , Interferon gama/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/fisiopatologia , Contagem de Linfócitos , Prednisolona/uso terapêutico , Linfócitos T/imunologia , Tacrolimo/uso terapêutico , Fatores de Tempo , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/metabolismo
14.
Clin Exp Immunol ; 158(2): 230-6, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19737140

RESUMO

Bronchiolitis obliterans syndrome (BOS) is characterized by persistent alloreactive, infective and non-specific epithelial injury, loss of epithelial integrity and dysregulated repair. We have reported increased apoptosis of epithelial cells collected from the large airway in lung transplant recipients. As part of the alloreactive response, T cells induce apoptosis of target epithelial cells by secreting granzyme b. We hypothesized that granzyme b would be increased in lung transplant patients with acute rejection and BOS and that commonly used immunosuppressive agents would fail to suppress this serine protease adequately. We investigated intracellular T cell granzyme b in blood, bronchoalveolar lavage (BAL) and large airway brushing (23 controls, 29 stable transplant, 23 BOS, 28 acute rejection, 31 infection) using flow cytometry and assessed the effect of clinically relevant concentrations of cyclosporin A, tacrolimus, methylprednisolone and a protease inhibitor, gabexate mesilate, on in vitro granzyme b production. Granzyme b was increased significantly in all compartments of all transplant groups compared to controls. Surprisingly, granzyme b was even higher in patients with BOS than in patients with acute rejection. In longitudinal analysis in three patients, blood granzyme b increased prior to or at the onset of BOS. In vitro, methylprednisolone and gabexate mesilate had no effect and cyclosporin A and tacrolimus only a moderate effect on production of granzyme b by CD8(+) T cells. Increased T cell granzyme b production may contribute to BOS pathogenesis and is not curtailed by current immunosuppressants. Longitudinal investigation of granzyme b in blood may provide an adjunctive non-invasive method for predicting BOS/OB.


Assuntos
Bronquiolite Obliterante/enzimologia , Granzimas/metabolismo , Imunossupressores/uso terapêutico , Transplante de Pulmão/imunologia , Subpopulações de Linfócitos T/enzimologia , Adulto , Idoso , Bronquiolite Obliterante/tratamento farmacológico , Bronquiolite Obliterante/etiologia , Bronquiolite Obliterante/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Broncoscopia/métodos , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo/métodos , Rejeição de Enxerto/enzimologia , Rejeição de Enxerto/imunologia , Granzimas/biossíntese , Humanos , Imunossupressores/farmacologia , Estudos Longitudinais , Transplante de Pulmão/efeitos adversos , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/efeitos dos fármacos
15.
Am J Transplant ; 9(4): 727-33, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19344464

RESUMO

Bronchiolitis obliterans syndrome (BOS) compromises lung transplant outcomes and is characterised by airway epithelial damage and fibrosis. The process whereby the normal epithelial configuration is replaced by fibroblastic scar tissue is poorly understood, but recent studies have implicated epithelial mesenchymal transition (EMT). The primary aim of this study was to assess the utility of flow cytometry in detecting and quantifying EMT in bronchial epithelial cells. Large airway brushings were obtained at 33 bronchoscopies in 16 BOS-free and 6 BOS grade 1-3 patients at 2-120 months posttransplant. Flow cytometry was used to assess expression of the mesenchymal markers alphaSMA, S100A4 and ED-A FN and HLA-DR. TGF beta 1 and HGF were measured in Bronchoalveolar lavage (BAL). Expression of all three mesenchymal markers was increased in BOS, as was HLA-DR. BAL HGF, but not TGF beta 1 was increased in BOS. Longitudinal investigation of one patient revealed a 100% increase in EMT markers concurrent with a 6-fold increase in BAL TGF beta 1 and the diagnosis of BOS at 17 months posttransplant. Flow cytometric evaluation of bronchial epithelium may provide a novel and rapid means to assess lung allografts at risk of BOS.


Assuntos
Bronquiolite Obliterante/epidemiologia , Células Epiteliais/citologia , Transplante de Pulmão/efeitos adversos , Mesoderma/citologia , Adulto , Idoso , Antígenos CD/análise , Brônquios/citologia , Brônquios/patologia , Brônquios/fisiologia , Brônquios/fisiopatologia , Líquido da Lavagem Broncoalveolar , Broncoscopia , Células Epiteliais/imunologia , Células Epiteliais/fisiologia , Feminino , Citometria de Fluxo , Antígenos HLA-DR/genética , Fator de Crescimento de Hepatócito/análise , Fator de Crescimento de Hepatócito/genética , Humanos , Imunoglobulina G/análise , Antígenos Comuns de Leucócito/análise , Transplante de Pulmão/estatística & dados numéricos , Masculino , Mesoderma/fisiologia , Pessoa de Meia-Idade , Medição de Risco , Fator de Crescimento Transformador beta1/análise , Fator de Crescimento Transformador beta1/genética , Transplante Homólogo/patologia , Transplante Homólogo/fisiologia
16.
J Pathol ; 214(1): 85-95, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17992660

RESUMO

The bone morphogenetic protein (BMP) type II receptor (BMPR-II) is predominantly expressed on the vascular endothelium in the adult lung. Although mutations in BMPR-II are known to underlie many cases of familial pulmonary arterial hypertension (FPAH), little is known regarding the expression of BMPs and their signalling pathways during normal lung development or the impact of BMPR-II mutations on endothelial cell function. We determined the cellular localization and expression levels of BMP4, BMP receptors, and activation of downstream signalling via phospho-Smad1 in a developmental series of human embryonic and fetal lungs by immunohistochemistry. The expression of BMP4 and BMP receptors was temporally and spatially regulated during lung development. BMPR-II expression correlated with phosphorylation of tissue Smad1 and was highest during the late pseudoglandular and early canalicular stage of lung development, when vasculogenesis is intense. Phospho-Smad1 expression was associated with markers of proliferation in endothelial cells. In vitro studies confirmed that BMPs 2 and 4 induced phosphorylation of Smad1/5 and pulmonary artery endothelial cell (PAEC) migration and proliferation. Adenoviral transfection of PAECs with mutant kinase-deficient BMPR-II, or siRNA knockdown of BMPR-II, inhibited Smad signalling and the proliferative response to BMP4. Our findings support a critical role for BMPs in lung vasculogenesis. Dysfunctional BMP signalling in PAECs during development may lead to abnormal pulmonary vascular development and contribute to the pathogenesis of FPAH.


Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Pulmão/embriologia , Proteína Morfogenética Óssea 4 , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Movimento Celular/fisiologia , Proliferação de Células , Sobrevivência Celular/fisiologia , Células Cultivadas , Endotélio Vascular/metabolismo , Desenvolvimento Fetal/fisiologia , Inativação Gênica , Humanos , Técnicas Imunoenzimáticas , Alvéolos Pulmonares/embriologia , Artéria Pulmonar/metabolismo , Circulação Pulmonar/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transdução de Sinais
17.
Transpl Infect Dis ; 10(2): 99-105, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17511818

RESUMO

Current immunosuppression protocols to prevent lung transplant rejection reduce pro-inflammatory and T-helper type 1 (Th1) cytokines. However, Th1 T-cell pro-inflammatory cytokine production is important in host defense against bacterial infection in the lungs. Excessive immunosuppression of Th1 T-cell pro-inflammatory cytokines leaves patients susceptible to infection. To investigate whether pulmonary infection in lung transplant recipients is associated with reduced Th1 T-cell pro-inflammatory cytokines, whole blood and bronchoalveolar lavage (BAL) fluid from 13 stable lung transplant patients with 'culture-negative' BAL and 13 patients with 'culture-positive' BAL was stimulated in vitro, and cytokine production by CD8+ and CD4+ T-cell subsets was determined using multiparameter flow cytometry. In BAL samples, there was a significant decrease in interleukin-2 (IL2) in CD3+ T cells and tumor necrosis factor-alpha (TNF-alpha) in CD8+ T cells (but not CD4+) in 'culture-positive' compared with 'culture-negative' transplant patients. There was no difference in blood Th1 T-cell cytokines between 'culture-positive' compared with 'culture-negative' transplant patients. A decrease in Th1 cytokines IL-2 and TNF-alpha in BAL T-cell subsets is associated with isolation of potentially pathogenic organisms in the lungs in stable lung transplant patients. Excessive immunosuppression of these Th1 T-cell pro-inflammatory cytokines in stable transplant patients may leave them susceptible to infection. Modifying immunosuppression by monitoring intracellular Th1 pro-inflammatory cytokines in BAL T cells may help to improve morbidity and infection rates in stable lung transplant patients.


Assuntos
Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/análise , Pneumopatias/imunologia , Transplante de Pulmão , Células Th1/imunologia , Adulto , Broncoscopia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Estudos de Casos e Controles , Citocinas/imunologia , Feminino , Citometria de Fluxo/métodos , Humanos , Hospedeiro Imunocomprometido , Interleucina-2/imunologia , Transplante de Pulmão/imunologia , Masculino , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Células Th1/citologia , Imunologia de Transplantes , Fator de Necrose Tumoral alfa
18.
Clin Exp Immunol ; 150(1): 22-9, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17614970

RESUMO

The role of T cells in the pathophysiology of chronic obstructive pulmonary disease (COPD) is not yet certain, although varying reports have shown increases in T helper 1 (Th1) and/or Th2 cytokines in peripheral blood and bronchoalveolar lavage (BAL). No studies have examined cytokine production by intraepithelial T cells obtained by bronchial brushing (BB). Intracellular cytokine analysis of T cell subsets from peripheral blood, BAL and BB from smoker and ex-smoker COPD patients, COPD patients receiving inhaled corticosteroids and smoker and non-smoker control subjects was studied using multi-parameter flow cytometry. CD4 : CD8 inversion was noted in the peripheral blood of smoker and ex-smoker COPD groups, in BAL and BB from smoker controls and BAL of COPD smokers. There was an increase in intracellular CD8(+) T cell Th1 proinflammatory cytokines in some COPD groups in the peripheral blood and in CD8(+) T cell tumour necrosis factor (TNF)-alpha in some COPD groups and smoker controls in BAL and BB. There was an increase in proinflammatory cytokines in COPD smokers compared with ex-smokers and a decrease in COPD smokers receiving inhaled corticosteroids in the airways. There was a negative correlation between forced expiratory volume in 1 s (FEV(1)) and the percentage of BAL and intraepithelial CD8(+) T cells producing TNF-alpha. COPD patients exhibit systemic inflammation as evidenced by increased intracellular Th1 proinflammatory cytokines in blood, BAL and intraepithelial CD8(+) T cells, whereas smoker controls showed localized Th1 response in the lung only. Systemic therapeutic targeting of TNF-alpha production by CD8(+) T cells may improve morbidity in COPD patients while targeting of TNF-alpha in the lung may prevent smokers progressing to COPD.


Assuntos
Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/biossíntese , Doença Pulmonar Obstrutiva Crônica/imunologia , Células Th1/imunologia , Adulto , Idoso , Brônquios/imunologia , Broncoscopia , Contagem de Linfócito CD4 , Linfócitos T CD8-Positivos/imunologia , Feminino , Volume Expiratório Forçado/imunologia , Humanos , Mediadores da Inflamação/metabolismo , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Mucosa Respiratória/imunologia
19.
Cancer Gene Ther ; 14(1): 105-16, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17024232

RESUMO

Treatment of advanced lung cancer is one of the major challenges in current medicine because of the high morbidity and mortality of the disease. Advanced stage lung cancer is refractory to conventional therapies and has an extremely poor prognosis. Thus, new therapeutic approaches are needed. Lung tumor formation depends on angiogenesis in which the vascular endothelial growth factor (VEGF) produced by cancer cells plays a pivotal role. Neutralizing VEGF with a soluble VEGF receptor suppresses tumor growth; however, the anticancer effect with this therapy is weakened after the intratumoral vascular network is completed. In this study, we turned the expression of VEGF by tumors to therapeutic advantage using a conditionally replication-competent adenovirus (CRAd) in which the expression of E1 is controlled by the human VEGF promoter. This virus achieved good levels of viral replication in lung cancer cells and induced a substantial anticancer effect in vitro and in vivo. As a further enhancement, the cancer cell killing effect was improved with tropism modification of the virus to express the knob domain of Ad3, which improved infectivity for cancer cells. These VEGF promoter-based CRAds also showed a significant cell killing effect for various types of cancer lines other than lung cancer. Conversely, the VEGF promoter has low activity in normal tissues, and the CRAd caused no damage to normal bronchial epithelial cells. Since tumor-associated angiogenesis via VEGF signalling is common in many types of cancers, these CRAds may be applicable to a wide range of tumors. We concluded that VEGF promoter-based CRAds have the potential to be an effective strategy for cancer treatment.


Assuntos
Adenoviridae/genética , Terapia Genética , Neoplasias/terapia , Regiões Promotoras Genéticas , Fator A de Crescimento do Endotélio Vascular/genética , Replicação Viral , Adenoviridae/fisiologia , Sequência de Bases , Primers do DNA , Humanos , Transgenes
20.
Clin Exp Immunol ; 145(3): 413-9, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16907908

RESUMO

Allograft rejection remains a major cause of morbidity and mortality following lung transplantation and is associated with an increased expression of T cell proinflammatory cytokines. We have shown that CD4(+) T cell proinflammatory cytokine production was significantly reduced in peripheral blood and bronchoalveolar lavage (BAL) of stable lung transplant patients, consistent with immunosuppression therapy. However, analysis of inflammatory cytokine profiles of intraepithelial T cells in bronchial brushing (BB) may be more relevant than peripheral blood or BAL T cells for assessing immune graft status. To investigate the immunomodulatory effects of currently used immunosuppressive regimens on bronchial intraepithelial T cell cytokine production, whole blood, BAL and BB from stable lung transplant patients and control volunteers were stimulated in vitro and cytokine production by CD8(+) and CD4(+) T cell subsets determined using multi-parameter flow cytometry. In bronchial intraepithelial T cell subsets in control subjects and transplant patients there was compartmentalization of interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha production, a decrease in interleukin (IL)-2 production by CD4(+) T cells and CD4 : CD8 inversion compared with blood and BAL. Although there was a decrease in T cell proinflammatory cytokine production in blood of transplant patients, this was not found in BAL or bronchial intraepithelial CD8 T cell subsets, suggesting that the same level of immunosuppression may not occur in the lung of transplant recipients. Drugs that effectively reduce CD8 T cell proinflammatory cytokine production in the lung compartment may improve current protocols for reducing graft rejection in these patients.


Assuntos
Brônquios , Citocinas/análise , Transplante de Pulmão/imunologia , Mucosa Respiratória/imunologia , Linfócitos T/imunologia , Adulto , Líquido da Lavagem Broncoalveolar/imunologia , Broncoscopia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Estudos de Casos e Controles , Citocinas/imunologia , Citometria de Fluxo/métodos , Humanos , Hospedeiro Imunocomprometido , Pneumopatias/imunologia , Pneumopatias/cirurgia , Contagem de Linfócitos , Pessoa de Meia-Idade , Estatísticas não Paramétricas , Transplante Homólogo
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