RESUMEN
Viral-induced exacerbation of asthma remains a major cause of hospitalization and mortality. New human-relevant models of the airways are urgently needed to understand how respiratory infections may trigger asthma attacks and to advance treatment development. Here, we describe a new human-relevant model of rhinovirus-induced asthma exacerbation that recapitulates viral infection of asthmatic airway epithelium and neutrophil transepithelial migration, and enables evaluation of immunomodulatory therapy. Specifically, a microengineered model of fully differentiated human mucociliary airway epithelium was stimulated with IL-13 to induce a T-helper cell type 2 asthmatic phenotype and infected with live human rhinovirus 16 (HRV16) to reproduce key features of viral-induced asthma exacerbation. We observed that the infection with HRV16 replicated key hallmarks of the cytopathology and inflammatory responses observed in human airways. Generation of a T-helper cell type 2 microenvironment through exogenous IL-13 stimulation induced features of asthmatic airways, including goblet cell hyperplasia, reduction of cilia beating frequency, and endothelial activation, but did not alter rhinovirus infectivity or replication. High-resolution kinetic analysis of secreted inflammatory markers revealed that IL-13 treatment altered IL-6, IFN-λ1, and CXCL10 secretion in response to HRV16. Neutrophil transepithelial migration was greatest when viral infection was combined with IL-13 treatment, whereas treatment with MK-7123, a CXCR2 antagonist, reduced neutrophil diapedesis in all conditions. In conclusion, our microengineered Airway Lung-Chip provides a novel human-relevant platform for exploring the complex mechanisms underlying viral-induced asthma exacerbation. Our data suggest that IL-13 may impair the hosts' ability to mount an appropriate and coordinated immune response to rhinovirus infection. We also show that the Airway Lung-Chip can be used to assess the efficacy of modulators of the immune response.
Asunto(s)
Asma/virología , Bioingeniería , Progresión de la Enfermedad , Dispositivos Laboratorio en un Chip , Pulmón/patología , Pulmón/virología , Microtecnología , Modelos Biológicos , Movimiento Celular , Células Cultivadas , Efecto Citopatogénico Viral , Humanos , Infiltración Neutrófila , Receptores de Interleucina-8B/antagonistas & inhibidores , Receptores de Interleucina-8B/metabolismo , RhinovirusRESUMEN
Here we describe the development of a human lung 'small airway-on-a-chip' containing a differentiated, mucociliary bronchiolar epithelium and an underlying microvascular endothelium that experiences fluid flow, which allows for analysis of organ-level lung pathophysiology in vitro. Exposure of the epithelium to interleukin-13 (IL-13) reconstituted the goblet cell hyperplasia, cytokine hypersecretion and decreased ciliary function of asthmatics. Small airway chips lined with epithelial cells from individuals with chronic obstructive pulmonary disease recapitulated features of the disease such as selective cytokine hypersecretion, increased neutrophil recruitment and clinical exacerbation by exposure to viral and bacterial infections. With this robust in vitro method for modeling human lung inflammatory disorders, it is possible to detect synergistic effects of lung endothelium and epithelium on cytokine secretion, identify new biomarkers of disease exacerbation and measure responses to anti-inflammatory compounds that inhibit cytokine-induced recruitment of circulating neutrophils under flow.
Asunto(s)
Epitelio/efectos de los fármacos , Inflamación/metabolismo , Interleucina-13/farmacología , Dispositivos Laboratorio en un Chip , Enfermedades Pulmonares/tratamiento farmacológico , Enfermedades Pulmonares/metabolismo , Humanos , Inflamación/patología , Técnicas de Cultivo de TejidosRESUMEN
Hatchling marine turtles emerge at night from underground nests on oceanic beaches and then use visual cues to crawl from the nest site to the sea ("seafinding"). However, the light wavelengths (λ's) used to accomplish this orientation have not been thoroughly documented, nor do we understand why some λ's are favored over others. We measured nocturnal radiance on the horizon at 20 nm intervals between 340 and 600 nm at two nesting beach sites and then, under laboratory conditions, determined the lowest intensities of those λ's that induced green turtle and loggerhead hatchlings to crawl toward each light source (a low positive "phototaxis threshold"). Both species were similarly sensitive and were attracted to all λ's. Radiance measures at all λ's were greater toward the seaward horizon than toward the landward horizon, providing an important orientation cue regardless of variation in lunar illumination. Previous studies document that both species detect λ's longer than those that are most attractive. We hypothesize that seafinding is a specialized response mediated by cones that are sensitive to the shorter λ's (to minimize the effects of dark noise) but such as rods, are especially sensitive to low levels of nocturnal illumination.
Asunto(s)
Luz , Fototaxis , Navegación Espacial , Tortugas , Animales , Señales (Psicología) , Iluminación , Actividad Motora , Comportamiento de Nidificación , Océanos y Mares , Especificidad de la EspecieRESUMEN
BACKGROUND: Airway smooth muscle (ASM) contributes to local inflammation and plays an immunomodulatory role in airway diseases. This is partially regulated by p38 mitogen-activated protein kinase (MAPK), which further activates two closely related isoforms of the MAPK-activated protein kinases (MKs), MK2 and MK3. The MKs have similar substrate specificities but less is known about differences in their functional responses. This study was undertaken to identify differential downstream inflammatory targets of MK2 and MK3 signaling and assess cross-talk between the MAPK pathway and NF-κB signaling relevant to ASM function. METHODS: Wild-type and kinase-deficient MK2 (MK2WT, MK2KR) and MK3 (MK3WT, MK33A) were expressed in human ASM cells stimulated for 20â¯h with 10â¯ng/ml each interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and interferon (IFN)-γ. Inflammatory mediator secretion was assessed by Luminex assays and ELISA. Signaling pathway activation was monitored by Western blotting. RESULTS: Expression of these MKs and stimulation with 10â¯ng/ml IL-1ß, TNFα and IFNγ for 20â¯h did not affect secretion of multiple cytokines including IL-4, IL-5, IL-13 and monocyte chemotactic protein (MCP)-1/CCL2 but did differentially affect the secretion of regulated upon activation, normal T cell expressed and secreted (RANTES)/CCL5, IL-6 and granulocyte macrophage-colony stimulating factor (GM-CSF). RANTES/CCL5 secretion was decreased by MK2WT or MK3WT and stimulated by inhibition of MK2 or MK3 activity with expression of the kinase-deficient enzymes MK2KR or MK33A. IL-6 and GM-CSF secretion was decreased by inhibition of MK2 activity with MK2KR and while MK3WT had no effect, the kinase-deficient MK33A further decreased secretion of these mediators. Cross-talk of the MKs with other signaling pathways was investigated by examining NF-κB activation, which was inhibited by expression of MK3 but not affected by MK2. CONCLUSIONS: These results suggest an inhibitory role for MK2 and MK3 activity in RANTES/CCL5 secretion and cross-talk of MK3 with NF-κB to regulate IL-6 and GM-CSF. These findings differentiate MK2 and MK3 function in ASM cells and provide insight that may enable selective targeting of MKs in ASM to modulate local inflammation in airway disease.
Asunto(s)
Bronquios/citología , Péptidos y Proteínas de Señalización Intracelular/genética , Miocitos del Músculo Liso/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Western Blotting , Células Cultivadas , Quimiocina CCL5/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Inflamación/patología , Mediadores de Inflamación/metabolismo , Interleucina-6/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Miocitos del Músculo Liso/patología , FN-kappa B/metabolismo , Transducción de SeñalRESUMEN
(R)-6-[(3-{[4-(5-{[2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl]amino}pent-1-yn-1-yl)phenyl] carbamoyl}phenyl)sulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide trifluoroacetic acid (GS-5759) is a bifunctional ligand composed of a quinolinone-containing pharmacophore [ß2-adrenoceptor agonist orthostere (ß2A)] found in several ß2-adrenoceptor agonists, including indacaterol, linked covalently to a phosphodiesterase 4 (PDE4) inhibitor related to 6-[3-(dimethylcarbamoyl)benzenesulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GSK 256066) by a pent-1-yn-1-ylbenzene spacer. GS-5759 had a similar affinity for PDE4B1 and the native ß2-adrenoceptor expressed on BEAS-2B human airway epithelial cells. However, compared with the monofunctional parent compound, ß2A, the KA of GS-5759 for the ß2-adrenoceptor was 35-fold lower. Schild analysis determined that the affinities of the ß-adrenoceptor antagonists, (2R,3R)-1-[(2,3-dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl) amino]-2-butanol (ICI 118551) and propranolol, were agonist-dependent, being significantly lower for GS-5759 than ß2A. Collectively, these data can be explained by "forced proximity," bivalent binding where the pharmacophore in GS-5759 responsible for PDE4 inhibition also interacts with a nonallosteric domain within the ß2-adrenoceptor that enhances the affinity of ß2A for the orthosteric site. Microarray analyses revealed that, after 2-hour exposure, GS-5759 increased the expression of >3500 genes in BEAS-2B cells that were highly rank-order correlated with gene expression changes produced by indacaterol and GSK 256066 in combination (Ind/GSK). Moreover, the line of regression began close to the origin with a slope of 0.88, indicating that the magnitude of most gene expression changes produced by Ind/GSK was quantitatively replicated by GS-5759. Thus, GS-5759 is a novel compound exhibiting dual ß2-adrenoceptor agonism and PDE4 inhibition with potential to interact on target tissues in a synergistic manner. Such polypharmacological behavior may be particularly effective in chronic obstructive pulmonary disease and other complex disorders where multiple processes interact to promote disease pathogenesis and progression.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Enfermedad Pulmonar Obstructiva Crónica/genética , Quinolonas/farmacología , Receptores Adrenérgicos beta 2/metabolismo , Sistema Respiratorio/patología , Sulfonas/farmacología , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Agonistas de Receptores Adrenérgicos beta 2/uso terapéutico , Aminoquinolinas/farmacología , Línea Celular , Interacciones Farmacológicas , Células Epiteliales/metabolismo , Humanos , Indanos/farmacología , Inhibidores de Fosfodiesterasa 4/farmacología , Inhibidores de Fosfodiesterasa 4/uso terapéutico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/patología , Quinolonas/uso terapéutico , Sulfonas/uso terapéuticoRESUMEN
CXC chemokine receptor 2 (CXCR2) is a key receptor in the chemotaxis of neutrophils to sites of inflammation. The studies reported here describe the pharmacological characterization of danirixin, a CXCR2 antagonist in the diaryl urea chemical class. Danirixin has high affinity for CXCR2, with a negative log of the 50% inhibitory concentration (pIC50) of 7.9 for binding to Chinese hamster ovary cell (CHO)-expressed human CXCR2, and 78-fold selectivity over binding to CHO-expressed CXCR1. Danirixin is a competitive antagonist against CXCL8 in Ca2+-mobilization assays, with a KB (the concentration of antagonist that binds 50% of the receptor population) of 6.5 nM and antagonist potency (pA2) of 8.44, and is fully reversible in washout experiments over 180 minutes. In rat and human whole-blood studies assessing neutrophil activation by surface CD11b expression following CXCL2 (rat) or CXCL1 (human) challenge, danirixin blocks the CD11b upregulation with pIC50s of 6.05 and 6.3, respectively. Danirixin dosed orally also blocked the influx of neutrophils into the lung in vivo in rats following aerosol lipopolysaccharide or ozone challenge, with median effective doses (ED50s) of 1.4 and 16 mg/kg respectively. Thus, danirixin would be expected to block chemotaxis in disease states in which neutrophils are increased in response to inflammation, such as pulmonary diseases. In comparison with navarixin, a CXCR2 antagonist from a different chemical class, the binding characterization of danirixin is distinct. These observations may offer insight into the previously observed clinical differences in induction of neutropenia between these compounds.
Asunto(s)
Piperidinas/farmacología , Receptores de Interleucina-8B/antagonistas & inhibidores , Receptores de Interleucina-8B/metabolismo , Sulfonas/farmacología , Animales , Células CHO , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Humanos , Interleucina-8/farmacología , Masculino , Ratas , Ratas Endogámicas LewRESUMEN
Inhaled long-acting ß(2)-adrenoceptor agonists (LABA) that act as bronchodilators and the oral anti-inflammatory phosphodiesterase 4 (PDE4) inhibitor roflumilast are both approved therapies for chronic obstructive pulmonary disease (COPD). Here we describe the activity of a novel, inhaled, bifunctional, small molecule (R)-6-[(3-{[4-(5-{[2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl]amino}pent-1-yn-1-yl)phenyl]carbamoyl}phenyl)sulfonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GS-5759), which has specific ß(2) agonist and PDE4 inhibitory activity. GS-5759 demonstrated potent and full agonist activity at ß(2) adrenoceptors (EC(50) = 8 ± 4 nM) and is a potent inhibitor of the PDE4 enzyme (IC(50) = 5 ± 3 nM). In cell assays, GS-5759 inhibited lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) production in human peripheral mononuclear cells (PBMC) with an IC(50) = 0.3 nM [confidence interval (CI) 0.1-0.6] and in human neutrophils formyl-methionyl-leucyl-phenylalanine (fMLP)-induced super oxide anion production with an IC(50) = 3 nM (CI 0.8-8). The addition of the ß(2) antagonist ICI 118551 shifted the IC(50) in these cell assays to 4 and 38 nM, respectively, demonstrating the contribution of both ß(2) agonist and PDE4 inhibitory activity to GS-5759. GS-5759 was also a potent inhibitor of profibrotic and proinflammatory mediator release from human lung fibroblasts. GS-5759 relaxed guinea pig airway smooth muscle strips precontracted with carbachol in a concentration-dependent manner with an EC(50) = 0.5 µM (CI 0.2-2) and had slow dissociation kinetics with an Off T(1/2) > 720 minutes at an EC(80) concentration of 3 µM. GS-5759 is a novel bifunctional molecule with both potent ß(2) agonist and PDE4 inhibitor activity that could provide inhaled bronchodilator and anti-inflammatory therapy for COPD.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/farmacología , Fibroblastos/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Inhibidores de Fosfodiesterasa 4/farmacología , Quinolonas/farmacología , Sistema Respiratorio/efectos de los fármacos , Sulfonas/farmacología , Agonistas de Receptores Adrenérgicos beta 2/síntesis química , Agonistas de Receptores Adrenérgicos beta 2/química , Animales , Técnicas de Cultivo de Célula , Citocinas/antagonistas & inhibidores , Citocinas/inmunología , Fibroblastos/enzimología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Cobayas , Humanos , Leucocitos Mononucleares/enzimología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Masculino , Estructura Molecular , Músculo Liso/enzimología , Músculo Liso/inmunología , Músculo Liso/metabolismo , Inhibidores de Fosfodiesterasa 4/síntesis química , Inhibidores de Fosfodiesterasa 4/química , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Quinolonas/síntesis química , Quinolonas/química , Sistema Respiratorio/enzimología , Sistema Respiratorio/inmunología , Sistema Respiratorio/metabolismo , Sulfonas/síntesis química , Sulfonas/química , Factores de TiempoRESUMEN
Activation of muscarinic subtype 3 (M3) muscarinic cholinergic receptors (mAChRs) increases airway tone, whereas its blockade improves lung function and quality of life in patients with pulmonary diseases. The present study evaluated the pharmacological properties of a novel mAChR antagonist, GSK573719 (4-[hydroxy(diphenyl)methyl]-1-{2-[(phenylmethyl)oxy]ethyl}-1-azoniabicyclo[2.2.2]octane; umeclidinium). The affinity (Ki) of GSK573719 for the cloned human M1-M5 mAChRs ranged from 0.05 to 0.16 nM. Dissociation of [(3)H]GSK573719 from the M3 mAChR was slower than that for the M2 mAChR [half-life (t1/2) values: 82 and 9 minutes, respectively]. In Chinese hamster ovary cells transfected with recombinant human M3 mAChRs, GSK573719 demonstrated picomolar potency (-log pA2 = 23.9 pM) in an acetylcholine (Ach)-mediated Ca(2+) mobilization assay. Concentration-response curves indicate competitive antagonism with partial reversibility after drug washout. Using isolated human bronchial strips, GSK573719 was also potent and showed competitive antagonism (-log pA2 = 316 pM) versus carbachol, and was slowly reversible in a concentration-dependent manner (1-100 nM). The time to 50% restoration of contraction at 10 nM was about 381 minutes (versus 413 minutes for tiotropium bromide). In mice, the ED50 value was 0.02 µg/mouse intranasally. In conscious guinea pigs, intratracheal administration of GSK573719 dose dependently blocked Ach-induced bronchoconstriction with long duration of action, and was comparable to tiotropium; 2.5 µg elicited 50% bronchoprotection for >24 hours. Thus, GSK573719 is a potent anticholinergic agent that demonstrates slow functional reversibility at the human M3 mAChR and long duration of action in animal models. This pharmacological profile translated into a 24-hour duration of bronchodilation in vivo, which suggested umeclidinium will be a once-daily inhaled treatment of pulmonary diseases.
Asunto(s)
Enfermedades Pulmonares/tratamiento farmacológico , Antagonistas Muscarínicos/uso terapéutico , Quinuclidinas/uso terapéutico , Administración por Inhalación , Animales , Células CHO , Calcio/metabolismo , Carbacol/farmacología , Antagonistas Colinérgicos/farmacología , Cricetinae , Cricetulus , Cobayas , Cinética , Pulmón/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/administración & dosificación , Pletismografía , Quinuclidinas/administración & dosificación , Receptor Muscarínico M3/efectos de los fármacos , Receptores Muscarínicos , Derivados de Escopolamina/farmacología , Bromuro de TiotropioRESUMEN
Neutrophils are the most abundant leukocyte and have a short lifespan, dying by apoptosis approximately five days after leaving the bone marrow. Their apoptosis can be delayed at sites of inflammation to extend their functional lifespan, but inappropriate inhibition of apoptosis contributes to chronic inflammatory disease. Levels of the physiological iron chelator lactoferrin are raised at sites of inflammation and we have shown previously that iron-unsaturated lactoferrin inhibited human neutrophil apoptosis, but the mechanisms involved were not determined. Here we report that the anti-apoptotic effect of lactoferrin is dependent upon its iron saturation status as iron-saturated lactoferrin did not affect neutrophil apoptosis. We also show that the effect of lactoferrin is mediated at an early stage in apoptosis as it inhibited activation of sphingomyelinase, generation of ceramide, activation of caspase 8 and Bax and cleavage of Bid. Lactoferrin did not inhibit apoptosis induced by exogenous ceramide, supporting the proposal that it acts upstream of ceramide generation. We therefore conclude that raised lactoferrin levels are likely to contribute to chronic inflammation by delaying neutrophil apoptosis and that this is achieved by inhibiting proximal apoptotic signaling events.
Asunto(s)
Apoptosis/efectos de los fármacos , Lactoferrina/farmacología , Neutrófilos/efectos de los fármacos , Apoproteínas/farmacología , Apoptosis/fisiología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ceramidas/farmacología , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Hierro/metabolismo , Neutrófilos/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
BACKGROUND: Small airway narrowing is an important pathology which impacts lung function in chronic obstructive pulmonary disease (COPD). The accumulation of fibroblasts and myofibroblasts contribute to inflammation, remodeling and fibrosis by production and release of mediators such as cytokines, profibrotic factors and extracellular matrix proteins. This study investigated the effects of the phosphodiesterase 4 inhibitor roflumilast, combined with the long acting ß2 adrenergic agonist indacaterol, both approved therapeutics for COPD, on fibroblast functions that contribute to inflammation and airway fibrosis. METHODS: The effects of roflumilast and indacaterol treatment were characterized on transforming growth factor ß1 (TGFß1)-treated normal human lung fibroblasts (NHLF). NHLF were evaluated for expression of the profibrotic mediators endothelin-1 (ET-1) and connective tissue growth factor (CTGF), expression of the myofibroblast marker alpha smooth muscle actin, and fibronectin (FN) secretion. Tumor necrosis factor-α (TNF-α) was used to induce secretion of chemokine C-X-C motif ligand 10 (CXCL10), chemokine C-C motif ligand 5 (CCL5) and granulocyte macrophage colony-stimulating factor (GM-CSF) from NHLF and drug inhibition was assessed. RESULTS: Evaluation of roflumilast (1-10 µM) showed no significant inhibition alone on TGFß1-induced ET-1 and CTGF mRNA transcripts, ET-1 and FN protein production, alpha smooth muscle expression, or TNF-α-induced secretion of CXCL10, CCL5 and GM-CSF. A concentration-dependent inhibition of ET-1 and CTGF was shown with indacaterol treatment, and a submaximal concentration was chosen for combination studies. When indacaterol (0.1 nM) was added to roflumilast, significant inhibition was seen on all inflammatory and fibrotic mediators evaluated, which was superior to the inhibition seen with either drug alone. Roflumilast plus indacaterol combination treatment resulted in significantly elevated phosphorylation of the transcription factor cAMP response element-binding protein (CREB), an effect that was protein kinase A-dependent. Inhibition of protein kinase A was also found to reverse the inhibition of indacaterol and roflumilast on CTGF. CONCLUSIONS: These results demonstrate that addition of roflumilast to a LABA inhibits primary fibroblast/myofibroblast function and therapeutically this may impact lung fibroblast proinflammatory and profibrotic mediator release which contributes to small airway remodeling and airway obstruction in COPD.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/farmacología , Aminopiridinas/farmacología , Benzamidas/farmacología , Fibroblastos/metabolismo , Indanos/farmacología , Inflamación/prevención & control , Pulmón/metabolismo , Inhibidores de Fosfodiesterasa 4/farmacología , Fibrosis Pulmonar/prevención & control , Quinolonas/farmacología , Actinas/metabolismo , Células Cultivadas , Quimiocina CCL5/metabolismo , Quimiocina CXCL10/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Ciclopropanos/farmacología , Endotelina-1/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibronectinas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Inflamación/metabolismo , Pulmón/citología , Pulmón/efectos de los fármacos , Fibrosis Pulmonar/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The phosphodiesterase 4 inhibitor (PDE4i) roflumilast has been approved in the US and EU for treatment of GOLD stage 3 and 4 chronic obstructive pulmonary disease (COPD). Inhaled ß2 adrenoceptor agonist bronchodilators and anti-inflammatory glucocorticosteroids are also used as standard of care in COPD. We investigated the anti-inflammatory interaction of roflumilast in combination with long-acting ß2 agonists (LABA), salmeterol or formoterol, or a glucocorticosteroid, dexamethasone, on cytokine production from LPS-stimulated human primary peripheral blood mononuclear cells (PBMC). Salmeterol or formoterol caused a concentration-dependent inhibition of tumor necrosis factor-α (TNFα) secretion with an IC50 of 0.33 pM (C.I. 0.006-19) and 34 pM (C.I. 13-87), respectively. When roflumilast was evaluated, the addition of salmeterol (1 nM) to roflumilast caused the IC50 for roflumilast to shift from 1.8 nM (C.I. 0.8-4) to 4.1 pM (C.I.0.3-69) (p < 0.01), and maximal inhibition increased from 72.5 ± 3.2% to 90.9 ± 3.1%. Addition of formoterol to roflumilast also produced an increased TNFα inhibition more than either drug alone (p < 0.05). The inhibition of TNFα production with salmeterol was both ß2 adrenoceptor- and protein kinase A-dependent. Addition of roflumilast (10 nM) in the presence of dexamethasone increased the inhibition of LPS-induced TNFα and CCL3. Roflumilast in combination with salmeterol, formoterol, or dexamethasone increased the inhibition of LPS-induced TNFα from human PBMC, in an additive manner. Addition of roflumilast to either a ß2 adrenoceptor agonist or a glucocorticosteroid may provide superior anti-inflammatory activity and greater efficacy in COPD patients and be dose sparing.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/farmacología , Aminopiridinas/farmacología , Benzamidas/farmacología , Dexametasona/farmacología , Inflamación/tratamiento farmacológico , Agonistas de Receptores Adrenérgicos beta 2/administración & dosificación , Albuterol/administración & dosificación , Albuterol/análogos & derivados , Albuterol/farmacología , Aminopiridinas/administración & dosificación , Antiinflamatorios/administración & dosificación , Antiinflamatorios/farmacología , Benzamidas/administración & dosificación , Ciclopropanos/administración & dosificación , Ciclopropanos/farmacología , Dexametasona/administración & dosificación , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Etanolaminas/administración & dosificación , Etanolaminas/farmacología , Fumarato de Formoterol , Glucocorticoides/administración & dosificación , Glucocorticoides/farmacología , Humanos , Inflamación/fisiopatología , Concentración 50 Inhibidora , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos , Masculino , Inhibidores de Fosfodiesterasa 4/administración & dosificación , Inhibidores de Fosfodiesterasa 4/farmacología , Xinafoato de Salmeterol , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Tyrosine ureas had been identified as potent muscarinic receptor antagonists with promising in vivo activity. Controlling the stereochemistry of the chiral quaternary ammonium center had proved to be a serious issue for this series, however. Herein we describe the preparation and SAR of tyrosine urea antagonists containing achiral quaternary ammonium centers. The most successful such moiety was the 2-methylimidazo[2,1-b][1,3]thiazol-7-ium group which yielded highly potent antagonists with long duration of action in an inhaled animal model of bronchoconstriction.
Asunto(s)
Antagonistas Muscarínicos/química , Compuestos de Amonio Cuaternario/química , Receptores Muscarínicos/química , Tirosina/química , Urea/análogos & derivados , Animales , Bronquios/efectos de los fármacos , Ratones , Antagonistas Muscarínicos/síntesis química , Antagonistas Muscarínicos/farmacología , Receptores Muscarínicos/metabolismo , Relación Estructura-Actividad , Urea/síntesis química , Urea/farmacologíaRESUMEN
A novel series of N-substituted tropane derivatives was characterized as potent muscarinic acetylcholine receptor antagonists (mAChRs). Kinetic washout studies showed that the N-endosubstituted analog 24 displayed much slower reversibility at mAChRs than the methyl-substituted parent molecule darotropium. In addition, it was shown that this characteristic appeared to translate into enhanced which duration of action in a mouse model of bronchonstriction.
Asunto(s)
Antagonistas Muscarínicos/síntesis química , Tropanos/síntesis química , Animales , Enfermedades Bronquiales/tratamiento farmacológico , Diseño de Fármacos , Ratones , Antagonistas Muscarínicos/farmacología , Receptores Muscarínicos/efectos de los fármacos , Relación Estructura-Actividad , Tropanos/farmacologíaRESUMEN
OBJECTIVE: Interleukin-6 (IL-6) is a proinflammatory cytokine with regulatory effects on the survival and differentiation of T cells. It exerts its biologic function in 2 ways: by directly binding to the IL-6 receptor (IL-6R; CD126) or via trans-signaling, in which soluble IL-6R/IL-6 complexes bind to the signaling component CD130. This study was undertaken to assess the expression and regulation of CD126 and CD130 and determine how these affect the response of CD4+ T cells to IL-6 in the joints of patients with rheumatoid arthritis (RA). METHODS: Flow cytometry and immunofluorescence microscopy were used to determine the expression, function, and regulation of CD126 and CD130 in CD4+ T cells from the peripheral blood (PB), synovial fluid (SF), and synovial tissue of RA patients. RESULTS: Compared to the findings in RA PB, CD4+ T cells in the SF and synovial tissue expressed low levels of CD126. In contrast, whereas CD4+ T cell expression of CD130 was minimal in the SF, its level in the synovial tissue was high. Consistent with this phenotype, synovial tissue T cells responded to trans-signaling by soluble IL-6R/IL-6 complexes, whereas no response was evident in CD4+ T cells from the SF. Down-regulation of both receptor components in SF T cells could be explained by exposure to high levels of IL-6. Increased levels of CD130 messenger RNA and protein in synovial tissue CD4+ T cells suggested that CD130 is up-regulated locally. Among a range of cytokines tested, only IL-10 induced CD130 expression in T cells. CONCLUSION: The inflamed microenvironment in the synovial tissue maintains responsiveness to IL-6 trans-signaling through the up-regulation of CD130 expression in CD4+ T cells, and this process may be driven by IL-10.
Asunto(s)
Artritis Reumatoide/inmunología , Linfocitos T CD4-Positivos/inmunología , Interleucina-6/metabolismo , Líquido Sinovial/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Receptor gp130 de Citocinas/metabolismo , Humanos , Subunidad alfa del Receptor de Interleucina-6/metabolismo , Articulaciones/inmunología , Masculino , Persona de Mediana Edad , Membrana Sinovial/inmunologíaRESUMEN
We examined the hypothesis that stromal fibroblasts modulate the ability of endothelial cells (EC) to recruit lymphocytes in a site-specific manner. PBL were perfused over HUVEC that had been cultured with fibroblasts isolated from the inflamed synovium or the skin of patients with rheumatoid arthritis or osteoarthritis, or from normal synovium, with or without exposure to the inflammatory cytokines TNF-alpha+IFN-gamma. Fibroblasts from inflamed synovium, but no others, caused unstimulated HUVEC to bind flowing lymphocytes. This adhesion was supported by alpha(4)beta(1)-VCAM-1 interaction and stabilised by activation of PBL through CXCR4-CXCL12. Antibody neutralisation of IL-6 during co-culture effectively abolished the ability of EC to bind lymphocytes. Cytokine-stimulated EC supported high levels of lymphocyte adhesion, through the presentation of VCAM-1, E-selectin and chemokine(s) acting through CXCR3. Interestingly, co-culture with dermal fibroblasts caused a marked reduction in cytokine-induced adhesion, while synovial fibroblasts had variable effects depending on their source. In the dermal co-cultures, neutralisation of IL-6 or TGF-beta caused partial recovery of cytokine-induced lymphocyte adhesion; this was complete when both were neutralised. Exogenous IL-6 was also found to inhibit response to TNF-alpha+IFN-gamma. Normal stromal fibroblasts appear to regulate the cytokine-sensitivity of vascular endothelium, while fibroblasts associated with chronic inflammation bypass this and develop a directly inflammatory phenotype. Actions of IL-6 might be pro-inflammatory or anti-inflammatory, depending on the local milieu.
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Movimiento Celular/inmunología , Células Endoteliales/inmunología , Fibroblastos/inmunología , Inflamación/inmunología , Linfocitos/inmunología , Antivirales/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Células Cultivadas , Quimiocina CXCL12/inmunología , Quimiocina CXCL12/metabolismo , Técnicas de Cocultivo , Selectina E/inmunología , Selectina E/metabolismo , Células Endoteliales/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Humanos , Interferón gamma/farmacología , Interleucina-6/antagonistas & inhibidores , Interleucina-6/inmunología , Interleucina-6/metabolismo , Interleucina-6/farmacología , Linfocitos/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores CXCR3/efectos de los fármacos , Receptores CXCR3/inmunología , Receptores CXCR3/metabolismo , Receptores CXCR4/inmunología , Receptores CXCR4/metabolismo , Membrana Sinovial/inmunología , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/inmunología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Ca(2+) and Sr(2+) entry pathways activated by pro-inflammatory agonists FMLP, LTB(4) and PAF have been compared to thapsigargin in human neutrophils. 2-APB (10microM) increased Ca(2+) influx and to a greater extent in agonist than in thapsigargin stimulated neutrophils. This action of 2-APB was specific to Ca(2+) because 2-APB did not augment Sr(2+) entry in agonist and thapsigargin stimulated neutrophils. This suggests that Ca(2+) and Sr(2+) entry can be used to discriminate between receptor and non-receptor (store)-operated Ca(2+) influx. Our data show for the first time that Pyr3 whilst partially inhibiting agonist induced Ca(2+) influx almost completely abolished Ca(2+) influx after thapsigargin stimulation.
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Calcio/inmunología , Neutrófilos/inmunología , Receptores Sensibles al Calcio/inmunología , Compuestos de Boro/farmacología , Calcio/metabolismo , Humanos , Factores Inmunológicos/farmacología , Leucotrieno B4/inmunología , N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Factor de Activación Plaquetaria/inmunología , Receptores Sensibles al Calcio/metabolismo , Espectrometría de Fluorescencia , Estroncio/inmunología , Estroncio/metabolismoRESUMEN
The action of two potent store operated Ca2+ entry (SOCE) inhibitors, ML-9 and GdCl3 on Ca2+ fluxes induced by the pro-inflammatory agonists FMLP, PAF, LTB(4) as well as the receptor-independent stimulus thapsigargin has not been documented in human neutrophils. In this study, ML-9 enhanced both release and subsequent Ca2+ influx in response to agonists whereas it enhanced Ca2+ release by thapsigargin, but inhibited Ca2+ influx. In contrast, 1muM GdCl3 completely inhibited Ca2+ influx in response to thapsigargin, but only partially blocked Ca2+ influx after agonist stimulation. These results strongly suggest a major role for receptor-operated Ca2+ influx in human neutrophils.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Calcio/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Receptores Sensibles al Calcio/metabolismo , HumanosRESUMEN
Here we describe examples of studies that have contributed both to a basic understanding of the biology of imperiled marine turtles, and to their management and conservation. Key elements include, first and foremost, correctly identifying species that differ strikingly in their morphology at different life stages because with growth, they change size by several orders of magnitude and have accompanying shape changes. We also review comprehensive field studies documenting the need for management actions to correct abnormal shifts in sex ratios caused by climate change. We highlight the need to describe those perturbations in terms that are clear to regulators and personnel responsible for management and conservation policies. Finally, we review several basic studies that enhance our understanding of how selection has shaped morphological, functional, and performance attributes, and describe how that knowledge can be applied to the tasks required for enhancing species recovery.
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Conservación de los Recursos Naturales , Rasgos de la Historia de Vida , Comportamiento de Nidificación , Tortugas/anatomía & histología , Tortugas/fisiología , Animales , Cambio Climático , Femenino , MasculinoRESUMEN
AIMS: There is very little published literature on Enhanced Recovery Principles (ERP) used in primary joint replacements applied to revision hip and knee arthroplasty (rTHA, rTKA). METHODS: Retrospective series of 268 rTHA and rTKA surgeries from 2010 -2018, treated with ERP, focusing on multimodal pain management, blood management and early functional recovery. RESULTS: No patients from the latest cohort required readmission within 6 weeks. Only 20 patients (7.5%) required a blood transfusion. Surgical site local anaesthetic infiltration was associated with lower PCA use in aseptic rTHA and rTKA (p<0.001; p<0.001). Revisions for infection had a longer length of stay (LOS) and increased PCA usage in both rTHA (6.5 vs. 5.2 days) and rTKA (10.1 vs. 5.3 days), similar to our previous study.1 Use of an intra-articular catheter for analgesia in rTKA showed reduced PCA use. Tourniquets were not beneficial for blood loss in rTKA and had greater PCA use post-operatively (p<0.001). CONCLUSION: The application of ERP to revision THA and TKA surgery is safe and effective.
RESUMEN
Non-excitable cells such as neutrophil granulocytes are the archetypal inflammatory immune cell involved in critical functions of the innate immune system. The electron current generated (I(e)) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential. For continuous function of the NADPH oxidase, I(e) has to be balanced to preserve electroneutrality, if not; sufficient depolarisation would prevent electrons from leaving the cell and neutrophil function would be abrogated. Subsequently, the depolarisation generated by the neutrophil NADPH oxidase I(e) must be counteracted by ion transport. The finding that depolarisation required counter-ions to compensate electron transport was followed by the observation that chloride channels activated by swell can counteract the NADPH oxidase membrane depolarisation. In this mini review, we discuss the research findings that revealed the essential role of swell activated chloride channels in human neutrophil function.