RESUMEN
We previously showed increased steroid-resistant CD28null CD8+ senescent lymphocyte subsets in the peripheral blood from patients with chronic obstructive pulmonary disease (COPD). These cells expressed decreased levels of the glucocorticoid receptor (GCR), suggesting their contribution to the steroid-resistant property of these cells. COPD is a disease of the small airways (SA). We, therefore, hypothesized that there would be a further increase in these steroid-resistant lymphocytes in the lung, particularly in the SA. We further hypothesized that the pro-inflammatory/cytotoxic potential of these cells could be negated using prednisolone with low-dose cyclosporin A. Blood, bronchoalveolar lavage, large proximal, and small distal airway brushings were collected from 11 patients with COPD and 10 healthy aged-matched controls. The cytotoxic mediator granzyme b, pro-inflammatory cytokines IFNγ/TNFα, and GCR were determined in lymphocytes subsets before and after their exposure to 1µM prednisolone and/or 2.5 ng/mL cyclosporin A. Particularly in the SA, COPD subjects showed an increased percentage of CD28null CD8 T-cells and NKT-like cells, with increased expression of granzyme b, IFNγ and TNFα and a loss of GCR, compared with controls. Significant negative correlations between SA GCR expression and IFNγ/TNFα production by T and NKT-like cells (eg, T-cell IFNγ R = -0.834, P = 0.031) and with FEV1 (R = -0.890) were shown. Cyclosporine A and prednisolone synergistically increased GCR expression and inhibited pro-inflammatory cytokine production by CD28null CD8- T and NKT-like cells. COPD is associated with increased pro-inflammatory CD28null CD8+ T and NKT-like cells in the SA. Treatments that increase GCR in these lymphocyte subsets may improve the efficacy of clinical treatment.
Asunto(s)
Antígenos CD28 , Enfermedad Pulmonar Obstructiva Crónica , Anciano , Antígenos CD28/metabolismo , Linfocitos T CD8-positivos/metabolismo , Ciclosporina/farmacología , Ciclosporina/uso terapéutico , Granzimas/metabolismo , Humanos , Prednisolona/farmacología , Prednisolona/uso terapéutico , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Zinc homeostasis is vital to immune and other organ system functions, yet over a quarter of the world's population is zinc deficient. Abnormal zinc transport or storage protein expression has been linked to diseases, such as cancer and chronic obstructive pulmonary disorder. Although recent studies indicate a role for zinc regulation in vascular functions and diseases, detailed knowledge of the mechanisms involved remains unknown. This study aimed to assess protein expression and localization of zinc transporters of the SLC39A/ZIP family (ZIPs) and metallothioneins (MTs) in human subcutaneous microvessels and to relate them to morphological features and expression of function-related molecules in the microvasculature. Microvessels in paraffin biopsies of subcutaneous adipose tissues from 14 patients undergoing hernia reconstruction surgery were analysed for 9 ZIPs and 3 MT proteins by MQCM (multifluorescence quantitative confocal microscopy). Zinc regulation proteins detected in human microvasculature included ZIP1, ZIP2, ZIP8, ZIP10, ZIP12, ZIP14 and MT1-3, which showed differential localization among endothelial and smooth muscle cells. ZIP1, ZIP2, ZIP12 and MT3 showed significantly (p < 0.05) increased immunoreactivities, in association with increased microvascular muscularization, and upregulated ET-1, α-SMA and the active form of p38 MAPK (Thr180/Tyr182 phosphorylated, p38 MAPK-P). These findings support roles of the zinc regulation system in microvascular physiology and diseases.
Asunto(s)
Proteínas de Transporte de Catión , Humanos , Proteínas de Transporte de Catión/metabolismo , Zinc/metabolismo , Metalotioneína/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Recently identified molecular targets in pulmonary artery hypertension (PAH) include sphingosine-1-phosphate (S1P) and zinc transporter ZIP12 signaling. This study sought to determine linkages between these pathways, and with BMPR2 signaling. Lung tissues from a rat model of monocrotaline-induced PAH and therapeutic treatment with bone marrow-derived endothelial-like progenitor cells transduced to overexpress BMPR2 were studied. Multifluorescence quantitative confocal microscopy (MQCM) was applied for analysis of protein expression and localization of markers of vascular remodeling (αSMA and BMPR2), parameters of zinc homeostasis (zinc transporter SLC39A/ZIP family members 1, 10, 12 and 14; and metallothionein MT3) and S1P extracellular signaling (SPHK1, SPNS2, S1P receptor isoforms 1, 2, 3, 5) in 20-200 µm pulmonary microvessels. ZIP12 expression in whole lung tissue lysates was assessed by western blot. Spearman nonparametric correlations between MQCM readouts and hemodynamic parameters, Fulton index (FI), and right ventricular systolic pressure (RVSP) were measured. In line with PAH status, pulmonary microvessels in monocrotaline-treated animals demonstrated significant (p < .05, n = 6 per group) upregulation of αSMA (twofold) and downregulation of BMPR2 (20%). Upregulated ZIP12 (92%), MT3 (57.7%), S1PR2 (54.8%), and S1PR3 (30.3%) were also observed. Significant positive and negative correlations were demonstrated between parameters of zinc homeostasis (ZIP12, MT3), S1P signaling (S1PRs, SPNS2), and vascular remodeling (αSMA, FI, RVSP). MQCM and western blot analysis showed that monocrotaline-induced ZIP12 upregulation could be partially negated by BMPR2-targeted therapy. Our results indicate that altered zinc transport/storage and S1P signaling in the monocrotaline-induced PAH rat model are linked to each other, and could be alleviated by BMPR2-targeted therapy.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Proteínas de Transporte de Catión/metabolismo , Hipertensión Pulmonar/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Hipertensión Pulmonar/fisiopatología , Pulmón/metabolismo , Lisofosfolípidos/metabolismo , Masculino , Microvasos/metabolismo , Monocrotalina/farmacología , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Remodelación Vascular , Zinc/metabolismoRESUMEN
The environmental sustainability of megacities is a global problem, and megacities are experiencing increasing pressure and challenges with regard to providing a suitable living environment for people. Urban green space plays a crucial role in protecting urban ecological environments and in maintaining the physical and mental health of residents. In this study, a total of 94 soil samples from green spaces in Shanghai, including park green spaces and road green spaces in the eight central urban districts, were collected, and the contents of heavy metals and polycyclic aromatic hydrocarbons (PAHs) were analyzed to determine the distribution characteristics and influencing factors and to assess the associated health risks. The accumulation of heavy metals was greater in park green space soils than in road green space soils, although the variation coefficient of the former was lower than that of the latter. Conversely, the accumulation of PAHs was lower in park green space soils than that in road green space soils, although the variation coefficient of the former was higher than that of the latter. In particular, Cu, Zn, Pb, Cd, As, and PAHs have accumulated in Shanghai green space soils. With increasing soil depth (0-2 cm, 2-5 cm, 5-10 cm, and 10-30 cm), the PAH content increased in the park green space soils but decreased in the road green space soils. According to the "Technical guidelines for risk assessment of contaminated sites (MEP of China 2014)," the overall health risk posed by green space soils in urban areas in Shanghai can be considered safe, except at individual sampling sites. The PAH, Cu, and Zn contents of park green space soils might be related to the application of organic materials and to traffic and industry emissions. However, the soil pollutants in road green spaces are predominantly related to traffic and industrial emissions. Therefore, the monitoring and management of soil environmental quality must be strengthened.
Asunto(s)
Monitoreo del Ambiente/métodos , Metales Pesados/análisis , Hidrocarburos Policíclicos Aromáticos/análisis , Medición de Riesgo , Contaminantes del Suelo/análisis , Suelo/química , China , Humanos , Industrias , Hidrocarburos Policíclicos Aromáticos/toxicidad , Contaminantes del Suelo/toxicidadRESUMEN
As a fast-developing technique for in situ multi-element analysis method, laser induced breakdown spectroscopy - LIBS is, however, developing slowly on liquid analysis due to some technical difficulties. We propose a new method, namely capillary mode, to quantify the concentrations of the elements in solution using LIBS. A Nd:YAG laser with repetition of 10 Hz were used to analyze the solution of Na2CrO4 and no any sample preparation in measurements. The experimental results show that the splashing of liquid induced by laser pulses is decreased significantly and the pollution of mirrors is avoided effectively using liquid capillary mode. The results of quantitative analysis for liquid are also improved than other method. The calibration curves of Cr and Na are well characterized by straight lines and the regression coefficient values of the linear fit are better than 0.998. The limits of detection (LODs) of Cr and Na are determined to be 28.9 mg/L and 1.0 mg/L in this work, respectively. The experimental results show that the liquid capillary mode provides a more practical and very simple approach to improve accuracy of quantitative element analysis in liquids by LIBS technique.
RESUMEN
The proper regulation of zinc (Zn) trafficking proteins and the cellular distribution of Zn are critical for the maintenance of autophagic processes. However, there have been no studies that have examined Zn dyshomeostasis and the disease-related modulation of autophagy observed in the airways afflicted with chronic obstructive pulmonary disease (COPD). We hypothesized that dysregulated autophagy in airway epithelial cells (AECs) is related to Zn dysregulation in cigarette smoke (CS)-induced COPD. We applied a human ex vivo air-liquid interface model, a murine model of smoke exposure, and human lung tissues and investigated Zn, ZIP1, and ZIP2 Zn-influx proteins, autophagy [microtubule-associated 1A/1B-light chain-3 (LC3), Beclin-1], autophagic flux (Sequestosome), apoptosis [Bcl2; X-linked inhibitor of apoptosis (XIAP), poly (ADP)-ribose polymerase (PARP)], and inflammation [thymic stromal lymphopoietin (TSLP), regulated on activation, normal T cell expressed and secreted (RANTES), and IL-1ß]. Lung tissues from CS-exposed mice exhibit reduced free-Zn in AECs, with elevated ZIP1 and diminished ZIP2 expression. Interestingly, increased LC3 colocalized with ZIP1, suggesting an autophagic requirement for free-Zn to support its catabolic function. In human AECs, autophagy was initiated but was unable to efficiently degrade cellular debris, as evidenced by stable Beclin-1 and increased LC3-II, but with a concomitant elevation in Sequestosome. Autophagic dysfunction due to CS exposure coupled with Zn depletion also induced apoptosis, with the reduction of antiapoptotic and antiautophagic proteins Bcl2 and XIAP and PARP cleavage. This was accompanied by an increase in RANTES and TSLP, an activator of adaptive immunity. We conclude that the uncoupling of Zn trafficking and autophagy in AECs constitutes a fundamental disease-related mechanism for COPD pathogenesis and could provide a new therapeutic target.
Asunto(s)
Autofagia , Bronquios/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Homeostasis , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Zinc/metabolismo , Animales , Autofagosomas/metabolismo , Autofagosomas/ultraestructura , Proteínas de Transporte de Catión/metabolismo , Compartimento Celular , Diferenciación Celular , Células Cultivadas , Citocinas/metabolismo , Citosol/metabolismo , Células Epiteliales/ultraestructura , Fluorescencia , Humanos , Mediadores de Inflamación/metabolismo , Ratones , Fumar/efectos adversosRESUMEN
We reported defective efferocytosis associated with cigarette smoking and/or airway inflammation in chronic lung diseases, including chronic obstructive pulmonary disease, severe asthma, and childhood bronchiectasis. We also showed defects in phagocytosis of nontypeable Haemophilus influenzae (NTHi), a common colonizer of the lower airway in these diseases. These defects could be substantially overcome with low-dose azithromycin; however, chronic use may induce bacterial resistance. The aim of the present study was therefore to investigate two novel macrolides-2'-desoxy-9-(S)-erythromycylamine (GS-459755) and azithromycin-based 2'-desoxy molecule (GS-560660)-with significantly diminished antibiotic activity against Staphylococcus aureus, Streptococcus pneumonia, Moraxella catarrhalis, and H. influenzae We tested their effects on efferocytosis, phagocytosis of NTHi, cell viability, receptors involved in recognition of apoptotic cells and/or NTHi (flow cytometry), secreted and cleaved intracellular IL-1ß (cytometric bead array, immunofluorescence/confocal microscopy), and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) using primary alveolar macrophages and THP-1 macrophages ± 10% cigarette smoke extract. Dose-response experiments showed optimal prophagocytic effects of GS-459755 and GS-560660 at concentrations of 0.5-1 µg/ml compared with our findings with azithromycin. Both macrolides significantly improved phagocytosis of apoptotic cells and NTHi (e.g., increases in efferocytosis and phagocytosis of NTHi: GS-459755, 23 and 22.5%, P = 0.043; GS-560660, 23.5 and 22%, P = 0.043, respectively). Macrophage viability remained >85% following 24 h exposure to either macrolide at concentrations up to 20 µg/ml. Secreted and intracellular-cleaved IL-1ß was decreased with both macrolides with no significant changes in recognition molecules c-mer proto-oncogene tyrosine kinase; scavenger receptor class A, member 1; Toll-like receptor 2/4; or CD36. Particulate cytoplasmic immunofluorescence of NLRP3 inflammasome was also reduced significantly. We conclude that GS-459755 and GS-560660 may be useful for reducing airway inflammation in chronic lung diseases without inducing bacterial resistance.
Asunto(s)
Antibacterianos/uso terapéutico , Antiinflamatorios/uso terapéutico , Enfermedades Pulmonares/tratamiento farmacológico , Pulmón/patología , Macrólidos/uso terapéutico , Macrófagos Alveolares/patología , Fagocitosis/efectos de los fármacos , Adulto , Antibacterianos/farmacología , Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Enfermedad Crónica , Eritromicina/análogos & derivados , Eritromicina/farmacología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Haemophilus influenzae/efectos de los fármacos , Humanos , Interleucina-1beta/metabolismo , Enfermedades Pulmonares/microbiología , Enfermedades Pulmonares/patología , Macrólidos/farmacología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proto-Oncogenes Mas , Receptores de Superficie Celular/metabolismo , Fumar/efectos adversosRESUMEN
p-Cresyl sulfate (PCS) is a risk factor of cardiovascular disease in patients with chronic kidney disease. Here we tested whether serum PCS levels were related to the rate and evolution of carotid atherosclerosis in hemodialysis patients and identified a potential mechanism. A total of 200 hemodialysis patients were categorized as with or without carotid atherosclerotic plaque and followed for 5 years. Serum PCS levels were found to be higher in patients with than without carotid atherosclerotic plaque and positively correlated with increased total plaque area during follow-up. Multiple logistic regression and mixed effects model analyses showed that serum PCS levels were independently associated with the incidence and progression of carotid atherosclerotic plaque. PCS induced inflammatory factor and adhesion molecule expression in endothelial cells and macrophages. In addition, PCS triggered monocyte-endothelial cell interaction in vitro and in vivo through increased production of reactive oxygen species. Compared with controls, increase of PCS levels produced by gavage promoted atherogenesis in 5/6-nephrectomized apoE-/- mice; a process attenuated by NADPH oxidase inhibitors. Thus, increased serum PCS levels are associated with the occurrence and progression of carotid atherosclerosis in hemodialysis patients and promote atherogenesis through increased reactive oxygen species production.
Asunto(s)
Enfermedades de las Arterias Carótidas/sangre , Cresoles/sangre , Fallo Renal Crónico/complicaciones , Ésteres del Ácido Sulfúrico/sangre , Acetofenonas , Adulto , Anciano , Animales , Aorta/metabolismo , Apolipoproteínas E/deficiencia , Enfermedades de las Arterias Carótidas/etiología , Estudios de Casos y Controles , Progresión de la Enfermedad , Células Endoteliales/fisiología , Femenino , Humanos , Fallo Renal Crónico/sangre , Macrófagos/fisiología , Masculino , Ratones , Persona de Mediana Edad , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Corticosteroid resistance is a major barrier to effective treatment of COPD. We have shown that the resistance is associated with decreased expression of glucocorticoid receptor (GCR) by senescent CD28nullCD8+ pro-inflammatory lymphocytes in peripheral blood of COPD patients. GCR must be bound to molecular chaperones heat shock proteins (Hsp) 70 and Hsp90 to acquire a high-affinity steroid binding conformation, and traffic to the nucleus. We hypothesized a loss of Hsp70/90 from these lymphocytes may further contribute to steroid resistance in COPD. METHODS: Blood was collected from COPD (n = 10) and aged-matched controls (n = 10). To assess response to steroids, cytotoxic mediators, intracellular pro-inflammatory cytokines, CD28, GCR, Hsp70 and Hsp90 were determined in T and NKT-like cells in the presence of ± 10 µM prednisolone and 2.5 ng/mL cyclosporine A (binds to GCR-Hsp70/90 complex) using flow cytometry, western blot and fluorescence microscopy. RESULTS: A loss of expression of Hsp90 and GCR from CD28null CD8+ T and NKT-like cells in COPD was noted (Hsp70 unchanged). Loss of Hsp90 expression correlated with the percentage of CD28null CD8+ T and NKT-like cells producing IFNγ or TNFα in all subjects (eg, COPD: R = -0.763, p = 0.007 for T-cell IFNγ). Up-regulation of Hsp90 and associated decrease in pro-inflammatory cytokine production was found in CD28nullCD8+ T and NKT-like cells in the presence of 10 µM prednisolone and 2.5 ng/mL cyclosporine A. CONCLUSIONS: Loss of Hsp90 from cytotoxic/pro-inflammatory CD28nullCD8+ T and NKT-like cells could contribute to steroid resistance in COPD. Combination prednisolone and low-dose cyclosporine A therapy inhibits these pro-inflammatory cells and may reduce systemic inflammation in COPD.
Asunto(s)
Corticoesteroides/farmacología , Antiinflamatorios/farmacología , Resistencia a Medicamentos , Proteínas HSP90 de Choque Térmico/sangre , Células Asesinas Naturales/efectos de los fármacos , Prednisolona/farmacología , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Linfocitos T/efectos de los fármacos , Adulto , Antígenos CD28/sangre , Estudios de Casos y Controles , Ciclosporina/farmacología , Citocinas/sangre , Quimioterapia Combinada , Proteínas HSP70 de Choque Térmico/sangre , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Persona de Mediana Edad , Fenotipo , Enfermedad Pulmonar Obstructiva Crónica/sangre , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/sangre , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Oxidative stress, inflammation, increased bronchial epithelial cell apoptosis, and deficient phagocytic clearance of these cells (efferocytosis) by the alveolar macrophages are present in chronic obstructive pulmonary disease (COPD) and in response to cigarette smoke. We previously showed that the macrophage dysfunction is associated with changes to the sphingosine-1-phosphate (S1P) signalling system. We hypothesized that the antioxidant/anti-inflammatory agent, thymoquinone, would improve macrophage phagocytosis via modulation of the S1P system and protect bronchial epithelial cells from cigarette smoke or lipopolysaccharide (LPS)-induced apoptosis. Phagocytosis was assessed using flow cytometry, S1P mediators by Real-Time PCR, and apoptosis of 16HBE bronchial epithelial cells using flow cytometry and immunohistochemistry. Cigarette smoke and LPS decreased phagocytosis and increased S1P receptor (S1PR)-5 mRNA in THP-1 macrophages. Thymoquinone enhanced efferocytic/phagocytic ability, antagonized the effects of cigarette smoke extract and LPS on phagocytosis and S1PR5, and protected bronchial epithelial cells from cigarette smoke-induced apoptosis. Thymoquinone is worth further investigating as a potential therapeutic strategy for smoking-related lung diseases.
Asunto(s)
Antioxidantes/farmacología , Benzoquinonas/farmacología , Lisofosfolípidos/metabolismo , Macrófagos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Esfingosina/análogos & derivados , Apoptosis/efectos de los fármacos , Bronquios/citología , Línea Celular , Mezclas Complejas/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Humanos , Lipopolisacáridos/farmacología , Macrófagos/fisiología , ARN Mensajero/metabolismo , Receptores de Lisoesfingolípidos/genética , Humo/efectos adversos , Esfingosina/metabolismo , Productos de TabacoRESUMEN
The effects of inoculants on the composting of Sophora flavescens residues were evaluated based on several physical, chemical and biological parameters, as well as the infrared spectra. Compared to the control compost without inoculants, the treatment compost with inoculants (Bacillus subtilis strain G-13 and Chaetomium thermophilum strain GF-1) had a significantly longer thermophilic duration, higher cellulase activity and a higher degradation rate of cellulose, hemicellulose and lignin (P < 0.05). Thus, a higher maturity degree of compost with apparently lower C:N ratio (15.88 vs. 17.77) and NH4-N:NO3-N ratio (0.16 vs. 0.20) was obtained with the inoculation comparing with the control (P < 0.05). Besides, the inoculants could markedly accelerate the composting process and increase the maturity degree of compost as indicated by the germination index (GI) in which the treatment reached the highest GI of 133.2% at day 15 while the control achieved the highest GI of 125.7% at day 30 of the composting. Inoculation with B. subtilis and C. thermophilum is a useful method to enhance the S. flavescens residues composting according to this study.
RESUMEN
BACKGROUND: Histone acetyltransferases (HAT) and histone deacetylases (HDAC) are enzymes that upregulate and down-regulate pro-inflammatory gene transcription respectively. HDAC2 is required by corticosteroids to switch off activated inflammatory genes and is reduced in lung macrophages in COPD. We have shown that COPD patients have increased steroid resistant CD28null (senescent) pro-inflammatory T and NKT-like peripheral blood cells (particularly CD8+ subsets) and we hypothesized that these changes would be associated with a loss of HDAC2 from these senescent pro-inflammatory lymphocytes. METHODS: Blood was collected from 10 COPD and 10 aged-matched controls. Intracellular pro-inflammatory cytokines, IFNγ and TNFα, and expression of CD28, HDAC2 and HAT, were determined in lymphocyte subsets in the presence of ± 5 mg/ml theophylline (HDAC2 activator), 10 µM prednisolone and 2.5 ng/ml cyclosporine A (immunosuppressant), using flow cytometry. RESULTS: There was a loss of HDAC2 from CD28null CD8+ T and NKT-like cells in COPD. There was a significant negative correlation between HDAC2 expression and the percentage of CD28null CD8+ T and NKT-like cells producing IFNγ or TNFα in all subjects (eg, COPD: R = -.763, p < 0.001 for T-cell IFNγ). There was a synergistic upregulation of HDAC2 and associated decrease in pro-inflammatory cytokine production in CD28nullCD8+ T and NKT-like cells in the presence of 5 mg/L theophylline + 10(-6) M prednisolone or 2.5 ng/mL cyclosporine A (CsA). CONCLUSIONS: Lymphocyte senescence in COPD is associated with loss of HDAC2 in CD28nullCD8+ T and NKT-like cells. Alternative treatment options such as combined theophylline with low-dose CsA, that inhibit these pro-inflammatory cells, may reduce systemic inflammation in COPD.
Asunto(s)
Linfocitos T CD8-positivos/enzimología , Senescencia Celular , Histona Desacetilasa 2/sangre , Enfermedad Pulmonar Obstructiva Crónica/enzimología , Adulto , Anciano , Antígenos CD28/sangre , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Estudios de Casos y Controles , Senescencia Celular/efectos de los fármacos , Ciclosporina/farmacología , Regulación hacia Abajo , Activación Enzimática , Activadores de Enzimas/farmacología , Femenino , Histona Acetiltransferasas/sangre , Humanos , Inmunosupresores/farmacología , Interferón gamma/sangre , Masculino , Persona de Mediana Edad , Células T Asesinas Naturales/enzimología , Células T Asesinas Naturales/inmunología , Fenotipo , Prednisolona/farmacología , Enfermedad Pulmonar Obstructiva Crónica/sangre , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Teofilina/farmacología , Factor de Necrosis Tumoral alfa/sangreRESUMEN
BACKGROUND: Glucocorticoid (GC) resistance is a major barrier in COPD treatment. We have shown increased expression of the drug efflux pump, Pgp1 in cytotoxic/pro-inflammatory lymphocytes in COPD. Loss of lymphocyte co-stimulatory molecule CD28 (lymphocyte senescence) was associated with a further increase in their pro-inflammatory/cytotoxic potential and resistance to GC. We hypothesized that lymphocyte senescence and increased Pgp1 are also associated with down-regulation of the GC receptor (GCR). METHODS: Blood was collected from 10 COPD and 10 healthy aged-matched controls. Flow cytometry was applied to assess intracellular pro-inflammatory cytokines, CD28, Pgp1, GCR, steroid binding and relative cytoplasm/nuclear GCR by CD28+ and CD28null T, NKT-like cells. GCR localization was confirmed by fluorescent microscopy. RESULTS: COPD was associated with increased numbers of CD28nullCD8+ T and NKT-like cells. Loss of CD28 was associated with an increased percentage of T and NKT-like cells producing IFNγ or TNFα and associated with a loss of GCR and Dex-Fluor staining but unchanged Pgp1. There was a significant loss of GCR in CD8 + CD28null compared with CD8 + CD28+ T and NKT-like cells from both COPD and controls (eg, mean ± SEM 8 ± 3% GCR + CD8 + CD28null T-cells vs 49 ± 5% GCR + CD8 + CD28+ T-cells in COPD). There was a significant negative correlation between GCR expression and IFNγ and TNFα production by T and NKT-like cells(eg, COPD: T-cell IFNγ R = -.615; ) and with FEV1 in COPD (R = -.777). CONCLUSIONS: COPD is associated with loss of GCR in senescent CD28null and NKT-like cells suggesting alternative treatment options to GC are required to inhibit these pro-inflammatory/cytotoxic cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Senescencia Celular , Citocinas/inmunología , Mediadores de Inflamación/inmunología , Células T Asesinas Naturales/inmunología , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Receptores de Glucocorticoides/inmunología , Subfamilia B de Transportador de Casetes de Unión a ATP/sangre , Adulto , Anciano , Antígenos CD28/sangre , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Estudios de Casos y Controles , Citocinas/sangre , Regulación hacia Abajo , Resistencia a Medicamentos , Femenino , Citometría de Flujo , Glucocorticoides/uso terapéutico , Humanos , Mediadores de Inflamación/sangre , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Células T Asesinas Naturales/efectos de los fármacos , Células T Asesinas Naturales/metabolismo , Fenotipo , Enfermedad Pulmonar Obstructiva Crónica/sangre , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/sangreRESUMEN
Patients frequently experience a loss of salivary function following irradiation (IR) for the treatment of an oral cavity and oropharyngeal cancer. Herein, we tested if transfer of fibroblast growth factor-2 (FGF2) cDNA could limit salivary dysfunction after fractionated IR (7.5 or 9 Gy for 5 consecutive days to one parotid gland) in the miniature pig (minipig). Parotid salivary flow rates steadily decreased by 16 weeks post-IR, whereas blood flow in the targeted parotid gland began to decrease ~3 days after beginning IR. By 2 weeks, post-IR salivary blood flow was reduced by 50%, at which point it remained stable for the remainder of the study. The single preadministration of a hybrid serotype 5 adenoviral vector encoding FGF2 (AdLTR2EF1a-FGF2) resulted in the protection of parotid microvascular endothelial cells from IR damage and significantly limited the decline of parotid salivary flow. Our results suggest that a local treatment directed at protecting salivary gland endothelial cells may be beneficial for patients undergoing IR for oral cavity and oropharyngeal cancer.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Vectores Genéticos/administración & dosificación , Glándula Parótida/fisiopatología , Traumatismos Experimentales por Radiación/prevención & control , Animales , Dependovirus/genética , Células Endoteliales/metabolismo , Células Endoteliales/efectos de la radiación , Factor 2 de Crecimiento de Fibroblastos/genética , Terapia Genética , Glándula Parótida/citología , Glándula Parótida/efectos de la radiación , Traumatismos Experimentales por Radiación/patología , Saliva/citología , Saliva/efectos de la radiación , Porcinos , Porcinos EnanosRESUMEN
Animal venoms contain a fascinating array of divergent peptide toxins that have cross-activities on different types of voltage-gated ion channels. However, the underlying mechanism remains poorly understood. Jingzhaotoxin-III (JZTX-III), a 36-residue peptide from the tarantula Chilobrachys jingzhao, is specific for Nav1.5 and Kv2.1 channels over the majority of other ion channel subtypes. JZTX-III traps the Nav1.5 DII voltage sensor at closed state by binding to the DIIS3-S4 linker. In this study, electrophysiological experiments showed that JZTX-III had no effect on five voltage-gated potassium channel subtypes (Kv1.4, Kv3.1, and Kv4.1-4.3), whereas it significantly inhibited Kv2.1 with an IC50 of 0.71 ± 0.01 µM. Mutagenesis and modeling data suggested that JZTX-III docks at the Kv2.1 voltage-sensor paddle. Alanine replacement of Phe274, Lys280, Ser281, Leu283, Gln284, and Val288 could decrease JZTX-III affinity by 7-, 9-, 34-, 12-, 9-, and 7-fold, respectively. Among them, S281 is the most crucial determinant, and the substitution with Thr only slightly reduced toxin sensitivity. In contrast, a single conversion of Ser281 to Ala, Phe, Ile, Val, or Glu increased the IC50 value by >34-fold. Alanine-scanning mutagenesis experiments indicated that the functional surface of JZTX-III bound to the Kv2.1 channel is composed of four hydrophobic residues (Trp8, Trp28, Trp30, and Val33) and three charged residues (Arg13, Lys15, and Glu34). The bioactive surfaces of JZTX-III interacting with Kv2.1 and Nav1.5 are only partially overlapping. These results strongly supported the hypothesis that animal toxins might use partially overlapping bioactive surfaces to target the voltage-sensor paddles of two different types of ion channels. Increasing our understanding of the molecular mechanisms of toxins interacting with voltage-gated sodium and potassium channels may provide new molecular insights into the design of more potent ion channel inhibitors.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Oocitos/efectos de los fármacos , Péptidos/farmacología , Canales de Potasio Shab/metabolismo , Venenos de Araña/farmacología , Secuencia de Aminoácidos , Animales , Electrofisiología , Femenino , Activación del Canal Iónico/efectos de los fármacos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación/genética , Canal de Sodio Activado por Voltaje NAV1.5/química , Canal de Sodio Activado por Voltaje NAV1.5/genética , Oocitos/citología , Oocitos/metabolismo , Técnicas de Placa-Clamp , Conformación Proteica , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Canales de Potasio Shab/química , Canales de Potasio Shab/genética , Arañas/metabolismo , Xenopus laevisRESUMEN
BACKGROUND: A common feature of COPD is a defective lung macrophage phagocytic capacity that can contribute to chronic lung inflammation and infection. The precise mechanisms remain incompletely understood, although cigarette smoke is a known contributor. We previously showed deficiency of the LC3-associated phagocytosis (LAP) regulator, Rubicon, in macrophages from COPD subjects and in response to cigarette smoke. The current study investigated the molecular basis by which cigarette smoke extract (CSE) reduces Rubicon in THP-1, alveolar and blood monocyte-derived macrophages, and the relationship between Rubicon deficiency and CSE-impaired phagocytosis. METHODOLOGY: Phagocytic capacity of CSE-treated macrophages was measured by flow cytometry, Rubicon expression by Western blot and real time polymerase chain reaction, and autophagic-flux by LC3 and p62 levels. The effect of CSE on Rubicon degradation was determined using cycloheximide inhibition and Rubicon protein synthesis and half-life assessment. RESULTS: Phagocytosis was significantly impaired in CSE-exposed macrophages and strongly correlated with Rubicon expression. CSE-impaired autophagy, accelerated Rubicon degradation, and reduced its half-life. Lysosomal protease inhibitors, but not proteasome inhibitors, attenuated this effect. Autophagy induction did not significantly affect Rubicon expression. CONCLUSIONS: CSE decreases Rubicon through the lysosomal degradation pathway. Rubicon degradation and/or LAP impairment may contribute to dysregulated phagocytosis perpetuated by CSE.
Asunto(s)
Fumar Cigarrillos , Enfermedad Pulmonar Obstructiva Crónica , Humanos , Fumar Cigarrillos/efectos adversos , Fagocitosis , Macrófagos/metabolismo , Lisosomas/metabolismoRESUMEN
Previously (Shan et al, 2005), we reported that adenoviral vector-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to minipig parotid glands following irradiation (IR) transiently restored salivary flow to near normal levels. This study evaluated a serotype 2, adeno-associated viral (AAV2) vector for extended correction of IR (single dose; 20 Gy)-induced, parotid salivary hypofunction in minipigs. At 16 weeks following the IR parotid salivary flow decreased by 85-90%. AAV2hAQP1 administration at week 17 transduced only duct cells and resulted in a dose-dependent increase in salivary flow to approximately 35% of pre-IR levels (to approximately 1 ml per 10 min) after 8 weeks (peak response). Administration of a control AAV2 vector or saline was without effect. Little change was observed in clinical chemistry and hematology values after AAV2hAQP1 delivery. Vector-treated animals generated high anti-AAV2 neutralizing antibody titers by week 4 (approximately 1:1600) and significant elevations in salivary (approximately 15%), but not serum, granulocyte macrophage colony-stimulating factor levels. Following vector administration, salivary [Na(+)] was dramatically increased, from approximately 10 to approximately 55 mM (at 4 weeks) and finally to 39 mM (8 weeks). The findings demonstrate that localized delivery of AAV2hAQP1 to IR-damaged parotid glands leads to increased fluid secretion from surviving duct cells, and may be useful in providing extended relief of salivary hypofunction in previously irradiated patients.
Asunto(s)
Acuaporina 1/genética , Dependovirus/genética , Glándula Parótida/metabolismo , Glándula Parótida/efectos de la radiación , Animales , Acuaporina 1/administración & dosificación , ADN Complementario/metabolismo , Vectores Genéticos/genética , Humanos , Glándula Parótida/química , PorcinosRESUMEN
INTRODUCTION/RATIONALE: In chronic obstructive pulmonary disease (COPD), defective macrophage phagocytic clearance of cells undergoing apoptosis by efferocytosis may lead to secondary necrosis of the uncleared cells and contribute to airway inflammation. The precise mechanisms for this phenomenon remain unknown. LC3-associated phagocytosis (LAP) is indispensable for effective efferocytosis. We hypothesized that cigarette smoke inhibits the regulators of LAP pathway, potentially contributing to the chronic airways inflammation associated with COPD. METHODS: Bronchoalveolar (BAL)-derived alveolar macrophages, lung tissue macrophages obtained from lung resection surgery, and monocyte-derived macrophages (MDM) were prepared from COPD patients and control participants. Lung/airway samples from mice chronically exposed to cigarette smoke were also investigated. Differentiated THP-1 cells were exposed to cigarette smoke extract (CSE). The LAP pathway including Rubicon, as an essential regulator of LAP, efferocytosis and inflammation was examined using western blot, ELISA, flow cytometry, and/or immunofluorescence. RESULTS: Rubicon was significantly depleted in COPD alveolar macrophages compared with non-COPD control macrophages. Rubicon protein in alveolar macrophages of cigarette smoke-exposed mice and cigarette smoke-exposed MDM and THP-1 was decreased with a concomitant impairment of efferocytosis. We also noted increased expression of LC3 which is critical for LAP pathway in COPD and THP-1 macrophages. Furthermore, THP-1 macrophages exposed to cigarette smoke extract exhibited higher levels of other key components of LAP pathway including Atg5 and TIM-4. There was a strong positive correlation between Rubicon protein expression and efferocytosis. CONCLUSION: LAP is a requisite for effective efferocytosis and an appropriate inflammatory response, which is impaired by Rubicon deficiency. Our findings suggest dysregulated LAP due to reduced Rubicon as a result of CSE exposure. This phenomenon could lead to a failure of macrophages to effectively process phagosomes containing apoptotic cells during efferocytosis. Restoring Rubicon protein expression has unrecognized therapeutic potential in the context of disease-related modifications caused by exposure to cigarette smoke.
Asunto(s)
Fagocitosis , Enfermedad Pulmonar Obstructiva Crónica , Animales , Humanos , Pulmón/metabolismo , Macrófagos Alveolares/metabolismo , Ratones , Fagocitosis/fisiología , Enfermedad Pulmonar Obstructiva Crónica/etiología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Fumar/efectos adversosRESUMEN
INTRODUCTION: The role inflammasomes play in chronic obstructive pulmonary disease (COPD) is unclear. We hypothesised that the AIM2 inflammasome is activated in the airways of COPD patients, and in response to cigarette smoke. METHODS: Lung tissue, bronchoscopy-derived alveolar macrophages and bronchial epithelial cells from COPD patients and healthy donors; lungs from cigarette smoke-exposed mice; and cigarette smoke extract-stimulated alveolar macrophages from healthy controls and HBEC30KT cell line were investigated. AIM2 inflammasome activation was assessed by multi-fluorescence quantitative confocal microscopy of speck foci positive for AIM2, inflammasome component ASC and cleaved IL-1ß. Subcellular AIM2 localization was assessed by confocal microscopy, and immunoblot of fractionated cell lysates. Nuclear localization was supported by in-silico analysis of nuclear localization predicted scores of peptide sequences. Nuclear and cytoplasmic AIM2 was demonstrated by immunoblot in both cellular fractions from HBEC30KT cells. RESULTS: Increased cytoplasmic AIM2 speck foci, colocalized with cleaved IL-1ß, were demonstrated in COPD lungs (n = 9) vs. control (n = 5), showing significant positive correlations with GOLD stages. AIM2 nuclear-to-cytoplasmic redistribution was demonstrated in bronchiolar epithelium in cigarette-exposed mice and in HBEC30KT cells post 24 h stimulation with 5% cigarette smoke extract. Alveolar macrophages from 8 healthy non-smokers responded to cigarette smoke extract with an > 8-fold increase (p < 0.05) of cytoplasmic AIM2 and > 6-fold increase (p < 0.01) of colocalized cleaved IL-1ß speck foci, which were also localized with ASC. CONCLUSION: The AIM2 inflammasome is activated in the airway of COPD patients, and in response to cigarette smoke exposure, associated with a nuclear to cytoplasmic shift in the distribution of AIM2.
RESUMEN
BACKGROUND: No studies have reported the 3-kilometer running test (3KRT) intending to predict VO2max for water sports athletes. Therefore, the purpose of this study was to develop a new model to predict the maximal aerobic capacity (VO2max) for water sports athletes based on 3KRT. METHODS: One hundred and two water sports athletes completed two sessions of experiments consisting of a maximal graded exercise test (GXT) and a 3KRT. Multiple linear regression was applied to predict VO2max value based on the performance and physiological responses of 3KRT, along with participants' anthropometric and demographic variables. The predicted residual error sum of square (PRESS) and error terms (constant error and total error) were calculated to further evaluate the predictive accuracy. RESULTS: Two significant prediction models based on elapsed exercise time (T3KRT), post-exercise heart rate (PHR3KRT), body mass, and gender were proposed. One model including PHR3KRT was identified (VO2max=120.77-0.028×T3KRT [second]-0.11×PHR3KRT [bpm]-0.334×body mass [kg]+8.70×gender [1: male, 0: female]), with an adjusted R2 of 0.723. Another model excluding PHR3KRT was also identified (VO2max=103.65-0.034×T3KRT [second]-0.317×Body mass [kg] + 7.89×gender [1: male, 0: female]), with an adjusted R2 of 0.713. Both models were further validated by the result of PRESS statistics. CONCLUSIONS: This endurance 3-kilometer running test accurately predicted VO2max value for water sports athletes (rowers, canoeists, and kayakers), and the model excluding PHR3KRT would be easier to use.