RESUMEN
COVID-19 is causing a major once-in-a-century global pandemic. The scientific and clinical community is in a race to define and develop effective preventions and treatments. The major features of disease are described but clinical trials have been hampered by competing interests, small scale, lack of defined patient cohorts and defined readouts. What is needed now is head-to-head comparison of existing drugs, testing of safety including in the background of predisposing chronic diseases, and the development of new and targeted preventions and treatments. This is most efficiently achieved using representative animal models of primary infection including in the background of chronic disease with validation of findings in primary human cells and tissues. We explore and discuss the diverse animal, cell and tissue models that are being used and developed and collectively recapitulate many critical aspects of disease manifestation in humans to develop and test new preventions and treatments.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , Antivirales/farmacología , Betacoronavirus/patogenicidad , Infecciones por Coronavirus/inmunología , Modelos Animales de Enfermedad , Neumonía Viral/inmunología , Vacunas Virales/biosíntesis , Enzima Convertidora de Angiotensina 2 , Animales , Animales Modificados Genéticamente , Antivirales/síntesis química , Betacoronavirus/efectos de los fármacos , Betacoronavirus/genética , Betacoronavirus/fisiología , COVID-19 , Vacunas contra la COVID-19 , Gatos , Quirópteros , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/virología , Cricetulus , Femenino , Hurones , Haplorrinos , Humanos , Masculino , Ratones , Organoides/efectos de los fármacos , Organoides/inmunología , Organoides/virología , Pandemias , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/inmunología , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/genética , Neumonía Viral/virología , SARS-CoV-2 , Índice de Severidad de la Enfermedad , Especificidad de la Especie , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Virales/administración & dosificaciónRESUMEN
There is limited evidence linking airway inflammation and lung function impairment in older non-smoking asthmatics with fixed airflow obstruction (FAO), which can develop despite treatment with inhaled corticosteroids (ICS). We assessed lung function (spirometry, forced oscillation technique (FOT)), lung elastic recoil and airway inflammation using bronchoalveolar lavage (BAL) in non-smoking adult asthmatics with FAO, following 2 months treatment with high-dose ICS/long-acting beta-agonist. Subjects demonstrated moderate FAO, abnormal FOT indices and loss of lung elastic recoil. This cross-sectional study showed a lack of a relationship between BAL neutrophils, eosinophils, inflammatory cytokines and lung function impairment. Other inflammatory pathways or the effect of inflammation on lung function over time may explain FAO development.
Asunto(s)
Corticoesteroides/uso terapéutico , Asma/tratamiento farmacológico , Asma/fisiopatología , No Fumadores , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Corticoesteroides/farmacología , Anciano , Asma/diagnóstico , Líquido del Lavado Bronquioalveolar , Estudios de Cohortes , Estudios Transversales , Femenino , Volumen Espiratorio Forzado/efectos de los fármacos , Volumen Espiratorio Forzado/fisiología , Humanos , Inflamación/diagnóstico , Inflamación/tratamiento farmacológico , Inflamación/fisiopatología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Pruebas de Función Respiratoria/métodos , Espirometría/métodosRESUMEN
Asthma is an obstructive respiratory disease characterised by chronic inflammation with airway hyperresponsiveness. In asthmatic airways, there is an increase in airway smooth muscle (ASM) cell bulk, which differs from non-asthmatic ASM in characteristics. This study aimed to assess the usefulness of hTERT immortalisation of human ASM cells as a research tool. Specifically we compared proliferative capacity, inflammatory mediator release and extracellular matrix (ECM) production in hTERT immortalised and parent primary ASM cells from asthmatic and non-asthmatic donors. Our studies revealed no significant differences in proliferation, IL-6 and eotaxin-1 production, or CTGF synthesis between donor-matched parent and hTERT immortalised ASM cell lines. However, deposition of ECM proteins fibronectin and fibulin-1 was significantly lower in immortalised ASM cells compared to corresponding primary cells. Notably, previously reported differences in proliferation and inflammatory mediator release between asthmatic and non-asthmatic ASM cells were retained, but excessive ECM protein deposition in asthmatic ASM cells was lost in hTERT ASM cells. This study shows that hTERT immortalised ASM cells mirror primary ASM cells in proliferation and inflammatory profile characteristics. Moreover, we demonstrate both strengths and weaknesses of this immortalised cell model as a representation of primary ASM cells for future asthma pathophysiological research.
Asunto(s)
Miocitos del Músculo Liso/fisiología , Fenotipo , Sistema Respiratorio/citología , Telomerasa/metabolismo , Asma/patología , Proliferación Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Humanos , Factores Inmunológicos/metabolismo , Telomerasa/genéticaRESUMEN
Sex differences in disease susceptibility due to maternal programming have been reported. We previously observed that maternal smoking induced renal disease and neurological changes are restricted to males, while both male and female offspring develop metabolic disorders. We have also found that maternal L-carnitine supplementation during gestation and lactation can significantly improve glucose intolerance and hyperlipidaemia in male offspring. This study aimed to determine whether such beneficial effects can also occur in female offspring. Balb/c female mice were exposed to cigarette smoke (SE) 6 weeks prior to gestation, during gestation and lactation. A subgroup of the SE dams was given L-carnitine (1.5 mmol/L in drinking water) during gestation and lactation. Female offspring were studied at 20 days (weaning) and 13 weeks (adulthood). Maternal smoking increased liver weight (%) and blood glucose levels at 20 days, as well as glucose intolerance and plasma triglycerides levels at adulthood (P < .05). The hepatic lipid metabolic marker adipose triglyceride lipase was downregulated in the SE offspring at 20 days (P < .05). At 13 weeks, the hepatic pro-inflammatory markers IL-1ß and TNF-α mRNA expression were upregulated, while the anti-inflammatory marker IL-10 mRNA expression was downregulated in the SE offspring (P < .05). Liver fibrosis was apparent at 20 days and 13 weeks. Maternal L-carnitine supplementation either normalised or suppressed the detrimental effects induced by maternal smoke exposure (P < .05). We conclude that maternal L-carnitine supplementation improves metabolic parameters in the female offspring of SE dams.
Asunto(s)
Carnitina/farmacología , Fumar Cigarrillos/efectos adversos , Suplementos Dietéticos , Glucosa/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Exposición Materna/efectos adversos , Animales , Biomarcadores/metabolismo , Femenino , Fibrosis , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos BALB C , EmbarazoRESUMEN
BACKGROUND: Although extracellular matrix (ECM) proteins are associated with irreversible lower airway changes, the relationship with upper airway remodelling which occurs during chronic rhinosinusitis (CRS) is poorly understood. This study assessed the expression of ECM proteins periostin, fibulin-1, fibronectin and collagenIV in nasal mucosa of patients with and without histologic features of remodelling. METHODS: A cross-sectional study of sinonasal mucosal biopsies taken from patients, undergoing surgery for CRS was performed, where patients were grouped according to remodelling, defined by basement membrane thickening (BMT over 7.5 micrometer) and subepithelial fibrosis. An overall view and three random fields of immunostained tissue sections that included epithelium, basement membrane and submucosa, were imaged using Zeiss Zen software. The area and intensity of positive staining were scored by two blinded observers, using a 12-point ordinal scale of weak to strong. RESULTS: 65 patients (47.6 +/- 13.4years, 44.6% female) were assessed. Patients were grouped as controls 26.2%, BMT/no fibrosis 38.5% or BMT and fibrosis 33.8%. Stronger grade of periostin expression was associated with remodelling changes and tissue eosinophilia over 10/HPF. Fibulin-1, fibronectin and collagenIV did not differ. CONCLUSION: Periostin expression was associated with the presence of BMT, fibrosis and tissue eosinophilia and may identify patients undergoing remodelling changes.
Asunto(s)
Biomarcadores/metabolismo , Moléculas de Adhesión Celular/metabolismo , Eosinófilos/metabolismo , Fibronectinas/metabolismo , Mucosa Nasal/metabolismo , Sinusitis/complicaciones , Remodelación de las Vías Aéreas (Respiratorias) , Moléculas de Adhesión Celular/química , Enfermedad Crónica , Estudios Transversales , Fibronectinas/química , HumanosRESUMEN
BACKGROUND: Asthma affects 300 million people worldwide. In asthma, the major cause of morbidity and mortality is acute airway narrowing, due to airway smooth muscle (ASM) hypercontraction, associated with airway remodelling. However, little is known about the transcriptional differences between healthy and asthmatic ASM cells. OBJECTIVES: To investigate the transcriptional differences between asthmatic and healthy airway smooth muscle cells (ASMC) in culture and investigate the identified targets using in vitro and ex vivo techniques. METHODS: Human asthmatic and healthy ASMC grown in culture were run on Affymetrix_Hugene_1.0_ST microarrays. Identified candidates were confirmed by PCR, and immunohistochemistry. Functional analysis was conducted using in vitro ASMC proliferation, attachment and contraction assays and ex vivo contraction of mouse airways. RESULTS: We suggest a novel role for latrophilin (LPHN) receptors, finding increased expression on ASMC from asthmatics, compared with non-asthmatics in vivo and in vitro, suggesting a role in mediating airway function. A single nucleotide polymorphism in LPHN1 was associated with asthma and with increased LPHN1 expression in lung tissue. When activated, LPHNs regulated ASMC adhesion and proliferation in vitro, and promoted contraction of mouse airways and ASMC. CONCLUSIONS: Given the need for novel inhibitors of airway remodelling and bronchodilators in asthma, the LPHN family may represent promising novel targets for future dual therapeutic intervention.
Asunto(s)
Asma/genética , Asma/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de Péptidos/genética , Acetilcolina/farmacología , Animales , Estudios de Casos y Controles , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Masculino , Glicoproteínas de Membrana , Proteínas de la Membrana/farmacología , Ratones , Ratones Endogámicos BALB C , Contracción Muscular/efectos de los fármacos , Miocitos del Músculo Liso/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Sistema Respiratorio/citología , Venenos de Araña/farmacología , Transcripción GenéticaRESUMEN
BACKGROUND: IL-17A plays an important role in respiratory disease and is a known regulator of pulmonary inflammation and immunity. Recent studies have linked IL-17A with exacerbation in asthma and COPD. We have shown that the enzyme cyclooxygenase-2 (COX-2) and its prostanoid products, prostaglandin E2 (PGE2 ) in particular, are key contributors in in vitro models of infectious exacerbation; however, the impact of IL-17A was not known. METHODS AND RESULTS: We address this herein and show that IL-17A induces a robust and sustained upregulation of COX-2 protein and PGE2 secretion from airway smooth muscle (ASM) cells. COX-2 can be regulated at transcriptional, post-transcriptional and/or post-translational levels. We have elucidated the underlying molecular mechanisms responsible for the sustained upregulation of TNF-α-induced COX-2 by IL-17A in ASM cells and show that is not via increased COX-2 gene expression. Instead, TNF-α-induced COX-2 upregulation is subject to regulation by the proteasome, and IL-17A acts to increase TNF-α-induced COX-2 protein stability as confirmed by cycloheximide chase experiments. In this way, IL-17A acts to amplify the COX-2-mediated effects of TNF-α and greatly enhances PGE2 secretion from ASM cells. CONCLUSION: As PGE2 is a multifunctional prostanoid with diverse roles in respiratory disease, our studies demonstrate a novel function for IL-17A in airway inflammation by showing for the first time that IL-17A impacts on the COX-2/PGE2 pathway, molecules known to contribute to disease exacerbation.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Interleucina-17/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Sistema Respiratorio/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Bronquios/metabolismo , AMP Cíclico/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-17/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Estabilidad Proteica , ARN Mensajero/genética , Receptores Adrenérgicos beta 2/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND: Asthma is a widespread chronic health problem exacerbated by common viral and bacterial infections. Further research is required to understand how infection worsens asthma control in order to advance therapeutic options in the future. Recent research has revealed that ß2 -adrenergic receptor (ß2 -AR) agonists lose bronchodilatory efficacy because the receptor-mediated molecular pathways responsible for their beneficial actions are desensitized by infection. To date, most studies have focussed on viral infection, leaving the impact of bacterial infection on ß2 -AR desensitization relatively under-investigated. We address this in this study. METHODS AND RESULTS: Utilizing an in vitro model of bacterial exacerbation in airway smooth muscle (ASM) cells, we show that activation of toll-like receptor 2 (TLR2; mimicking bacterial infection) in the presence of an inflammatory stimulus leads to ß2 -AR desensitization. This occurs via TLR2-dependent upregulation of cyclooxygenase 2 (COX-2) mRNA expression and increased secretion of PGE2 . Importantly, PGE2 causes heterologous ß2 -AR desensitization and reduces cAMP production in response to short-acting (salbutamol) and long-acting (formoterol) ß2 -agonists. Thus, bacterial infectious stimuli act in a PGE2 -dependent manner to severely curtail the beneficial actions of ß2 -agonists. The impact of ß2 -AR desensitization is demonstrated by reduced gene expression of the critical anti-inflammatory molecule MKP-1 in response to ß2 -agonists, as well as impaired bronchodilation in a mouse lung slices. CONCLUSIONS: Taken together, our results show that, like viruses, bacteria induce prostanoid-dependent ß2 -AR desensitization on ASM cells. Notably, COX-2 inhibition with the specific inhibitor celecoxib represses PGE2 secretion, presenting a feasible pharmacological option for treatment of infectious exacerbation in asthma in the future.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/farmacología , Asma/microbiología , Infecciones Bacterianas/complicaciones , Receptores Adrenérgicos beta 2/inmunología , Taquifilaxis/inmunología , Receptor Toll-Like 2/inmunología , Animales , Antiasmáticos/farmacología , Asma/inmunología , Asma/metabolismo , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/metabolismo , Bronquios/efectos de los fármacos , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/inmunología , Dinoprostona/biosíntesis , Dinoprostona/inmunología , Fosfatasa 1 de Especificidad Dual/biosíntesis , Fosfatasa 1 de Especificidad Dual/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Técnicas In Vitro , Ratones , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/inmunología , Miocitos del Músculo Liso/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Adrenérgicos beta 2/efectos de los fármacos , Receptores Adrenérgicos beta 2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 2/efectos de los fármacos , Receptor Toll-Like 2/metabolismoRESUMEN
In asthma, airway smooth muscle (ASM) chemokine (C-X-C motif) receptor 3 (CXCR3) ligand production may attract mast cells or T lymphocytes to the ASM, where they can modulate ASM functions. In ASM cells (ASMCs) from people with or without asthma, we aimed to investigate JAK-STAT1, JNK, and Ca²âº involvement in chemokine (C-X-C motif) ligand (CXCL)10 and CXCL11 production stimulated by interferon-γ, IL-1ß, and TNF-α combined (cytomix). Confluent, growth-arrested ASMC were treated with inhibitors for pan-JAK (pyridone-6), JAK2 (AG-490), JNK (SP-600125), or the sarco(endo)plasmic reticulum Ca²âºATPase (SERCA) pump (thapsigargin), Ca²âº chelator (BAPTA-AM), or vehicle before and during cytomix stimulation for up to 24 h. Signaling protein activation as well as CXCL10/CXCL11 mRNA and protein production were examined using immunoblot analysis, real-time PCR, and ELISA, respectively. Cytomix-induced STAT1 activation was lower and CXCR3 ligand mRNA production was more sensitive to pyridone-6 and AG-490 in asthmatic than nonasthmatic ASMCs, but CXCL10/CXCL11 release was inhibited by the same proportion. Neither agent caused additional inhibition of release when used in combination with the JNK inhibitor SP-600125. Conversely, p65 NF-κB activation was higher in asthmatic than nonasthmatic ASMCs. BAPTA-AM abolished early CXCL10/CXCL11 mRNA production, whereas thapsigargin reduced it in asthmatic cells and inhibited CXCL10/CXCL11 release by both ASMC types. Despite these inhibitory effects, neither Ca²âº agent affected early activation of STAT1, JNK, or p65 NF-κB. In conclusion, intracellular Ca²âº regulated CXCL10/CXCL11 production but not early activation of the signaling molecules involved. In asthma, reduced ASM STAT1-JNK activation, increased NF-κB activation, and altered Ca²âº handling may contribute to rapid CXCR3 ligand production and enhanced inflammatory cell recruitment.
Asunto(s)
Calcio/fisiología , Quimiocina CXCL10/biosíntesis , Quimiocina CXCL11/biosíntesis , Pulmón/metabolismo , Miocitos del Músculo Liso/metabolismo , Adolescente , Adulto , Anciano , Asma/metabolismo , Quimiocinas CXC/metabolismo , Femenino , Humanos , Interferón gamma , Interleucina-1beta/farmacología , Enfermedades Pulmonares/metabolismo , Neoplasias Pulmonares/metabolismo , Masculino , Persona de Mediana Edad , FN-kappa B/metabolismo , Receptores CXCR3/metabolismo , Factor de Transcripción STAT1 , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: Lymphangioleiomyomatosis (LAM) is characterized by the abnormal growth of smooth muscle-like cells (LAM cells) and cystic destruction of the lung parenchyma. LAM cell-derived matrix metalloproteinases (MMPs) are thought to play a prominent role in the tissue destruction. The aim of this study was to determine whether doxycycline, a known MMP inhibitor, can inhibit LAM cell proliferation or mitochondrial function and/or modulate MMPs and their tissue inhibitors (TIMPs). EXPERIMENTAL APPROACH: Wild-type and tuberous sclerosis complex-2 (TSC2)-null mouse embryonic fibroblasts (MEFs) were cultured in DMEM containing 10% fetal bovine serum (FBS). Human LAM cells were derived from the lungs of LAM patients and airway smooth muscle cells from control subjects. Cells were stimulated with FBS with or without doxycycline for up to 9 days. Proliferation was assessed by manual cell counts and MTT assay, MMP production by zymography and ELISA, and TIMP production using elisa. KEY RESULTS: Doxycycline did not change FBS-induced proliferation in MEFs or human cells. However, doxycycline did reduce metabolic activity of both wild-type and TSC2-null MEFs and LAM cells, but had no effect on control cells. Furthermore, doxycycline reduced MMP-2 from MEFs and decreased active-MMP-2 from LAM cells but had no effect on TIMP-1 and TIMP-2 from human LAM cells. CONCLUSIONS AND IMPLICATIONS: Doxycycline decreased MMP levels and cell metabolic activity, which raises the possibility of therapeutic efficacy in LAM.
Asunto(s)
Doxiciclina/farmacología , Linfangioleiomiomatosis/tratamiento farmacológico , Linfangioleiomiomatosis/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Proteínas Supresoras de Tumor/deficiencia , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/metabolismo , Humanos , Linfangioleiomiomatosis/patología , Metaloproteinasa 2 de la Matriz/biosíntesis , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterised by chronic bronchitis, largely irreversible remodelling of the small airways, and emphysematous destruction of the alveoli. COPD is projected to be the third leading cause of death worldwide by 2020. COPD often results from prolonged exposure to irritants such as cigarette smoke or inhaled particulates. Current pharmacotherapies for COPD are unable to reverse the pathological changes of this disease, and this is partially due to a limited understanding of the intricate mechanisms by which chronic exposure lead to the different pathological components of COPD. This review examines how the mechanisms that underlie various components of COPD can be modelled in vitro, specifically using cigarette smoke extract with cells cultured from primary human lung tissue, and how the effectiveness of current and novel pharmacotherapies on successfully attenuating these pathological changes can also be examined in vitro.
Asunto(s)
Modelos Biológicos , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Fumar/efectos adversos , Remodelación de las Vías Aéreas (Respiratorias) , Animales , Células Cultivadas , Humanos , Pulmón/citología , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/etiologíaRESUMEN
The antipsoriatic dimethylfumarate (DMF) has been anecdotically reported to reduce asthma symptoms and to improve quality of life of asthma patients. DMF decreases the expression of proinflammatory mediators by inhibiting the transcription factor NF-kappaB and might therefore be of interest for the therapy of inflammatory lung diseases. In this study, we determined the effect of DMF on platelet-derived growth factor (PDGF)-BB- and TNFalpha-induced asthma-relevant cytokines and NF-kappaB activation by primary human asthmatic and nonasthmatic airway smooth muscle cells (ASMC). Confluent nonasthmatic and asthmatic ASMC were incubated with DMF (0.1-100 microM) and/or dexamethasone (0.0001-0.1 microM), NF-kappaB p65 siRNA (100 nM), the NF-kappaB inhibitor helenalin (1 microM) before stimulation with PDGF-BB or TNFalpha (10 ng/ml). Cytokine release was measured by ELISA. NF-kappaB, mitogen and stress-activated kinase (MSK-1), and CREB activation was determined by immunoblotting and EMSA. TNFalpha-induced eotaxin, RANTES, and IL-6 as well as PDGF-BB-induced IL-6 expression was inhibited by DMF and by dexamethasone from asthmatic and nonasthmatic ASMC, but the combination of both drugs showed no glucocorticoid sparing effect in either of the two groups. NF-kappaB p65 siRNA and/or the NF-kappaB inhibitor helenalin reduced PDGF-BB- and TNFalpha-induced cytokine expression, suggesting the involvement of NF-kappaB signaling. DMF inhibited TNFalpha-induced NF-kappaB p65 phosphorylation, NF-kappaB nuclear entry, and NF-kappaB-DNA complex formation, whereas PDGF-BB appeared not to activate NF-kappaB within 60 min. Both stimuli induced the phosphorylation of MSK-1, NF-kappaB p65 at Ser276, and CREB, and all were inhibited by DMF. These data suggest that DMF downregulates cytokine secretion not only by inhibiting NF-kappaB but a wider range of NF-kappaB-linked signaling proteins, which may explain its potential beneficial effect in asthma.
Asunto(s)
Bronquios/citología , Citocinas/metabolismo , Fumaratos/farmacología , Inmunosupresores/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Neumonía/tratamiento farmacológico , Factor de Transcripción ReIA/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Becaplermina , Bronquios/inmunología , Células Cultivadas , Quimiocina CCL11/metabolismo , Quimiocina CCL5/metabolismo , Enfermedad Crónica , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Dexametasona/farmacología , Dimetilfumarato , Inhibidores Enzimáticos/farmacología , Glucocorticoides/farmacología , Humanos , Proteínas I-kappa B/metabolismo , Interleucina-6/metabolismo , Isoquinolinas/farmacología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/inmunología , Inhibidor NF-kappaB alfa , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Neumonía/inmunología , Neumonía/metabolismo , Proteínas Proto-Oncogénicas c-sis , ARN Interferente Pequeño , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Sesquiterpenos/farmacología , Sesquiterpenos de Guayano , Sulfonamidas/farmacología , Factor de Transcripción ReIA/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: CD40 and OX40 Ligand (OX40L) are cell-surface molecules expressed on airway smooth muscle (ASM) that can enhance inflammatory cell activation and survival. The aim of this study was to examine the effect of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on ASM CD40 and OX40L expression. METHODS: CD40 and OX40L expression on human ASM cells from asthmatic and nonasthmatic donors following stimulation with TNF-alpha and/or IFN-gamma was measured using cell-surface enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Involvement of signalling pathway was investigated with pharmacological inhibitors. Soluble TNF receptor levels were quantified by ELISA. RESULTS: Interferon-gamma and TNF-alpha synergistically increased CD40 expression to a greater extent on asthmatic than on nonasthmatic ASM. In contrast, IFN-gamma reduced TNF-alpha-induced OX40L expression to a similar extent in both cell types. TNF-alpha and IFN-gamma induced CD40 via nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription-3 in both cell types and modulated OX40L via NF-kappaB and c-Jun N terminal kinase in nonasthmatic cells. Similar effects on the induction of OX40L in asthmatic cells were seen with NF-kappaB, but these were not statistically significant. The reduced OX40L expression with TNF-alpha and IFN-gamma involved extracellular regulated kinase 1/2 activation. CONCLUSION: Asthmatic ASM may modulate airway inflammation locally by increasing CD40 and OX40L expression in response to cytokines. IFN-gamma may regulate ASM pro-inflammatory actions by differentially modulating ASM CD40 and OX40L expression.
Asunto(s)
Asma/inmunología , Antígenos CD40/metabolismo , Interferón gamma/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Ligando OX40/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Adulto , Anciano , Asma/metabolismo , Antígenos CD40/agonistas , Antígenos CD40/inmunología , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , MAP Quinasa Quinasa 4/efectos de los fármacos , MAP Quinasa Quinasa 4/inmunología , MAP Quinasa Quinasa 4/metabolismo , Persona de Mediana Edad , Miocitos del Músculo Liso/inmunología , FN-kappa B/efectos de los fármacos , FN-kappa B/inmunología , FN-kappa B/metabolismo , Ligando OX40/antagonistas & inhibidores , Ligando OX40/inmunología , Factor de Transcripción STAT3/efectos de los fármacos , Factor de Transcripción STAT3/inmunología , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunologíaRESUMEN
BACKGROUND: Rhinovirus infection is responsible for considerable morbidity and mortality as the major cause of exacerbations of asthma, and is also known to induce exacerbations of cystic fibrosis and chronic obstructive pulmonary disease. Exacerbations of these diseases are also frequently associated with bacterial and atypical bacterial infection. Alveolar macrophages are the major immune cells in the airways and are important in defence against bacterial infections. METHODS: The authors investigated whether rhinovirus modifies cytokine release, the pattern recognition receptor expression and phagocytosis by human alveolar macrophages in response to bacterial products. RESULTS: Viable rhinovirus was detected in macrophages up to 3 days after exposure and viral RNA expression persisted for 10 days. Infectious but not UV inactivated rhinovirus increased tumour necrosis factor alpha (TNFalpha) and interleukin (IL)8 release by macrophages. In contrast, infectious rhinovirus impaired lipopolysaccharide and lipoteichoic acid induced TNFalpha and IL8 secretion by macrophages. Rhinovirus induced impairment of macrophage antibacterial immune responses did not involve IL10, prostaglandin E(2) or downregulation of Toll-like receptor 2. Furthermore, the macrophage phagocytic response to labelled bacterial particles, but not to latex beads, was impaired. CONCLUSION: The authors have identified impairment of cytokine responses to bacterial lipopolysaccharide and lipoteichoic acid by alveolar macrophages in response to infectious rhinovirus. Virus induced impairment of antibacterial host defence has important implications in the pathogenesis of exacerbations of respiratory diseases.
Asunto(s)
Macrófagos Alveolares/inmunología , Infecciones por Picornaviridae/inmunología , Rhinovirus/inmunología , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Femenino , Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Bacterias Grampositivas/inmunología , Infecciones por Bacterias Grampositivas/inmunología , Humanos , Inmunidad Celular , Interleucina-8/metabolismo , Lipopolisacáridos/farmacología , Macrófagos Alveolares/virología , Masculino , Persona de Mediana Edad , Fagocitosis/inmunología , Infecciones por Picornaviridae/virología , Ácidos Teicoicos/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Lymphangioleiomyomatosis (LAM) is associated with abnormal airway smooth muscle that leads to the characteristic pathology of lung nodule formation and destruction of lung tissue. The current authors have previously identified abnormal behaviour of airway smooth muscle cells from patients with asthma. In this study, cells and tissue sections derived from patients with LAM (n=7), asthma (n=8), and nonasthmatic controls (n=9) were compared. The presence of the antigen human melanosome (HM)B-45 was investigated, along with the proliferation and release of extracellular matrix proteins, release of endogenous prostaglandin E2 (PGE2), vascular endothelial growth factor and connective tissue growth factor, and the expression of integrins. Positive HMB-45 staining was found in all LAM patients and no controls. Proliferation of LAM cells was not different from control cells nor was its inhibition by beta-agonists, corticosteroids, rapamycin or PGE2. However, endogenous PGE2 levels were markedly decreased in LAM cells, and this was associated with decreased expression of the inducible form of cyclooxygenase (COX-2). The increased levels of connective tissue growth factor seen in asthma cells were not observed in LAM. Elastin mRNA in response to transforming growth factor-beta stimulation was markedly lower in LAM cells than either asthma or control cells. In conclusion, lymphangioleiomyomatosis cells exhibit abnormal properties in vitro that may contribute to pathophysiology and symptomatology in patients with lymphangioleiomyomatosis.
Asunto(s)
Dinoprostona/biosíntesis , Proteínas de la Matriz Extracelular/biosíntesis , Sustancias de Crecimiento/biosíntesis , Integrinas/biosíntesis , Linfangioleiomiomatosis/metabolismo , Proteínas de Neoplasias/biosíntesis , Adolescente , Adulto , Anciano , Antígenos de Neoplasias , Asma/metabolismo , Asma/fisiopatología , Células Cultivadas , Factor de Crecimiento del Tejido Conjuntivo , Ciclooxigenasa 2/biosíntesis , Femenino , Humanos , Proteínas Inmediatas-Precoces/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Pulmón , Linfangioleiomiomatosis/fisiopatología , Masculino , Antígenos Específicos del Melanoma , Persona de Mediana Edad , Miocitos del Músculo Liso , Factor de Crecimiento Transformador beta , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
Neutrophils and mononuclear cells are implicated in the pathogenesis of several inflammatory conditions including chronic obstructive pulmonary disease (COPD). Neutrophil-derived serine proteases, such as cathepsin G (CG) and neutrophil elastase (NE), may interact with mononuclear cells via protease-activated receptors (PARs) which are seven-transmembrane G protein-coupled receptors activated by proteolytic cleavage of the extracellular N-terminus, and which, on activation, induce the release of several mediators and cytokines. We determined whether CG and NE could affect PAR-1 expression and function in mononuclear cells. Human blood mononuclear cells were isolated from 20 healthy donors. Surface and intracellular receptor expression and calcium mobilisation (using the calcium chelator, FLUO3-AM) were studied by fluorescence-assisted cell sorting (FACS analysis). Positive controls, i.e. thrombin (0.1-100 mU/ml) and the PAR-1-activating peptide SFLLRN (100 microM) induced a rapid and transient intemalisation of PAR-1 in monocytes and lymphocytes. CG but not NE had a similar effect. By contrast, in monocytes intracellular calcium mobilisation was induced by thrombin and SFLLRN but not by CG and NE. Thus, CG can induce intracellular PAR-1 sequestration without activation of the receptor, and may act as an antagonist and prevent subsequent activation of PAR-1 in mononuclear cells. These findings may be of relevance to the pathogenesis of COPD.
Asunto(s)
Catepsinas/farmacología , Elastasa de Leucocito/farmacología , Monocitos/enzimología , Neutrófilos/enzimología , Receptores de Trombina/metabolismo , Calcio/metabolismo , Catepsina G , Células Cultivadas , Humanos , Receptor PAR-1 , Serina EndopeptidasasRESUMEN
Aspergillus fumigatus is an important opportunistic fungal pathogen that can cause acute invasive disease in neutropenic hosts. Invasive aspergillosis is being diagnosed with increasing frequency, and morbidity and mortality remain high despite prompt antifungal therapy. Because little is known about the virulence factors used by A. fumigatus, a tissue culture model was developed to mimic the interaction of the fungus with the endothelium. Differential display was used to compare gene expression in fungal cells grown on endothelial cells with that of cells grown in the absence of endothelial cell contact, and genes that were up-regulated were selected for analysis as putatively virulence-related genes. Two of these up-regulated genes were chosen for further study and were identified as genes encoding the regulatory subunit of cyclic adenosine monophosphate (cAMP)-dependent protein kinase and a member of the ras gene family, both of which are involved in cAMP-mediated signaling in fungi. This model system provides a new approach to the identification of potentially virulence-related genes induced in A. fumigatus by the interaction with host cells.
Asunto(s)
Aspergillus fumigatus/crecimiento & desarrollo , Aspergillus fumigatus/genética , Endotelio Vascular/microbiología , Regulación Fúngica de la Expresión Génica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Secuencia de Aminoácidos , Aspergilosis/microbiología , Aspergillus fumigatus/patogenicidad , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Endotelio Vascular/citología , Genes Fúngicos , Genes Reguladores , Genes ras , Humanos , Datos de Secuencia Molecular , Venas Umbilicales , Regulación hacia Arriba , VirulenciaRESUMEN
An imbalance between proteases and antiproteases may play a role in emphysema, which is characterized by increased degradation of extracellular matrix, and in airway remodeling in chronic bronchitis and asthma, in which there is increased collagen deposition. We assessed the effect of smoking on release of matrix metalloprotease-9 (MMP-9) and of its inhibitor, tissue inhibitor of metalloprotease-1 (TIMP-1), from alveolar macrophages, and determined the effects of proinflammatory (interleukin [IL]-1beta and lipopolysaccharide [LPS]) and antiinflammatory (IL-10) stimuli on the release of MMP-9 and TIMP-1. We performed bronchoalveolar lavage in 11 smokers and 11 nonsmokers, and cultured airway macrophages in the presence of control medium, IL-1beta, and LPS. Airway macrophages from smokers released greater amounts of MMP-9 and TIMP-1 at baseline and in response to IL-1beta and LPS than did those of nonsmokers. Airway macrophages from smokers produced more TNF-alpha and IL-10. IL-10 increased TIMP-1 release without modifying that of MMP-9, leading to a decrease in the MMP-9 to TIMP-1 ratio. Anti-IL-10 antibody had no effect on MMP-9 production induced by LPS. We conclude that the release of proteases and antiproteases by airway macrophages is increased in cigarette smokers, and can be regulated by exogenous IL-10.
Asunto(s)
Interleucina-10/fisiología , Macrófagos Alveolares/inmunología , Metaloproteinasa 9 de la Matriz/metabolismo , Fumar/efectos adversos , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Adulto , Líquido del Lavado Bronquioalveolar/inmunología , Broncoscopía , Femenino , Humanos , Lipopolisacáridos/inmunología , Masculino , Enfisema Pulmonar/etiología , Enfisema Pulmonar/inmunología , Fumar/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Expression of platelet thromboxane receptors is transcriptionally increased during megakaryocytic differentiation stimulated by phorbol 12-myristate 13-acetate (PMA). We previously cloned and characterized the promoter region of the human thromboxane receptor gene and localized PMA-responsive elements to a region between 1.84 and 1.95 kilobase pairs (kb) 5' of the transcription initiation site (D'Angelo, D. D., Davis, M. G., Houser, W. A., Eubank, J. J., Ritchie, M. E., and Dorn, G. W., II (1995) Circ. Res. 77, 466-474). Herein we report the localization of the PMA response element to a 14-nucleotide C-rich sequence, flanked by an octanucleotide inverted repeat, located -1.938 to -1.925 kb 5' of the transcription start site of this gene. We further identify the PMA-responsive enhancer factor that binds to this C-rich sequence as Sp1. Heterologous thromboxane receptor gene promoter/thymidilate kinase reporter constructs transfected into K562 cells exhibited PMA responsiveness when the C-rich element was included with additional 3' sequence from -1.924 to -1.84 kb. However, mutations of the C-rich element that disrupted a GC box located on the inverse strand eliminated PMA responsiveness and, in gel mobility shift assays, eliminated binding of Sp1. PMA treatment of K562 cells significantly increased, by 5-fold, Sp1 binding to the C-rich element and increased both phosphorylated and nonphosphorylated Sp1 protein levels by 2-fold. Furthermore, PMA treatment transiently increased Sp1 mRNA levels prior to increasing thromboxane receptor mRNA, suggesting that up-regulation of Sp1 contributes to up-regulation of thromboxane receptors. Finally, we have detected an unidentified K562 nuclear protein that binds specifically to the sense strand of the C-rich sequence overlapping the Sp1 binding site and that, by stabilizing a double stem-loop conformation of this DNA segment, may also play a role in Sp1 regulation of this gene. These studies are the first to describe regulatory and regulated roles for Sp1 in PMA-responsive gene expression and suggest that modulation of Sp1 levels controls thromboxane receptor expression during megakaryocytic differentiation.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Proteína Quinasa C/fisiología , Receptores de Tromboxanos/genética , Factor de Transcripción Sp1/fisiología , Acetato de Tetradecanoilforbol/farmacología , Composición de Base , Secuencia de Bases , Sitios de Unión , Cartilla de ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Urinary tract infection (UTI) continues to be a common nosocomial infection. From a 2-year city-county hospital experience, 212 nosocomial UTI were identified in 153 patients from 3747 admissions. Mean age was 54 years; 102 were men. Foley catheterization was an associated factor in 129 patients (84%). UTI was caused by 40 different species of bacteria. In 28 infections (13%), the UTI was polymicrobial. Only nine patients had bacteremia. The bacteriology of the UTI depended on whether the patient had received systemic antibiotics previously during the hospitalization. Prior antibiotic administration increased the probability of Pseudomonas and Serratia as pathogens. Thus, patients that have had antibiotic therapy demonstrate a distribution of pathogens that are different from patients not receiving antibiotics, and a distribution different from the community-acquired UTI. Continued emphasis on the shorter duration and more judicious use of systemic antibiotics for both prophylaxis and therapy is warranted.