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Objective: To describe the clinical characteristics and treatment of severe community-acquired pneumonia(SCAP) caused by Legionella pneumophila with acute respiratory failure and to analyze the risk factors for mortality. Methods: From October 2011 to October 2019, 34 patients were diagnosed with SCAP caused by Legionella pneumophila with acute respiratory failure.There were 25 males and 9 females, aged from 17 to 82 years, with a median age of 61 (48, 69) years. According to the prognosis, the patients were divided into a survival group and a death group for comparative analysis.The survival group included 24 patients, 17 males and 7 females, with a median age of 65 (55, 70) years. There were 10 cases in the death group, 8 males and 2 females, with a median age of 53 (50, 58) years. Multivariable logistic regression analysis was used for risk factors of ICU mortality. Results: The median time of admission to ICU was 7 (5, 11) days, the median time of stay in RICU was 12 (7, 22) days, and the PaO(2)/FiO(2) was 134 (91, 216) mmHg(1 mmHg=0.133 kPa). Ten patients died during ICU hospitalization, with a mortality of 29%. Sequential organ failure assessment (SOFA) of death group was 9 (7, 12), which was significantly higher than that of the survival group [4 (3, 8)], P=0.018. The time from onset of pneumonia symptoms to initiation of targeted treatment of the death group was 10 (7, 14) d, which was significantly longer than that of the survival group of [4 (3, 7) d], P=0.019. Multivariable logistic regression analysis showed that SOFA score (OR=1.461, 95%CI 1.041-2.051, P=0.028) and the time from onset of pneumonia symptoms to initiation of targeted treatment (OR=1.293, 95%CI 1.029-1.625, P=0.027) were independent risk factors for hospital mortality. Conclusions: The ICU mortality of severe legionella pneumonia was high. Critical organ dysfunctions and delayed initial targeted treatment were related with the increase of ICU mortality.
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Infecciones Comunitarias Adquiridas/diagnóstico , Legionella pneumophila/aislamiento & purificación , Enfermedad de los Legionarios/diagnóstico , Neumonía Bacteriana/diagnóstico , Insuficiencia Respiratoria , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Infecciones Comunitarias Adquiridas/microbiología , Infecciones Comunitarias Adquiridas/mortalidad , Infecciones Comunitarias Adquiridas/terapia , Femenino , Humanos , Unidades de Cuidados Intensivos , Enfermedad de los Legionarios/mortalidad , Enfermedad de los Legionarios/terapia , Masculino , Persona de Mediana Edad , Neumonía Bacteriana/mortalidad , Pronóstico , Síndrome de Dificultad Respiratoria , Estudios Retrospectivos , Tasa de Supervivencia , Adulto JovenRESUMEN
Superparamagnetic iron oxide (Fe3O4) and highly anisotropic barium hexaferrite (BaFe12O19) nanoparticles were coated with an anti-inflammatory drug and magnetically transported through mucus produced by primary human airway epithelial cells. Using wet planetary ball milling, dl-2-amino-3-phosphonopropionic acid-coated BaFe12O19 nano-particles (BaNPs) of 1-100 nm in diameter were prepared in water. BaNPs and conventional 20-30-nm Fe3O4 nanoparticles (FeNPs) were then encased in a polymer (PLGA) loaded with dexamethasone (Dex) and tagged for imaging. PLGA-Dex-coated BaNPs and FeNPs were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), and superconducting quantum interference device (SQUID) magnetometry. Both PLGA-Dex-coated BaNPs and FeNPs were transferred to the surface of a ~100-µm thick mucus layer of air-liquid interface cultured primary normal human tracheobronchial epithelial (NHTE) cells. Within 30 min, the nanoparticles were pulled successfully through the mucus layer by a permanent neodymium magnet. The penetration time of the nanomedicine was monitored using confocal microscopy and tailored by varying the thickness of the PLGA-Dex coating around the particles.
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BACKGROUND: Rhinovirus (RV) infection in asthma induces varying degrees of airway inflammation (e.g. neutrophils), but the underlying mechanisms remain unclear. OBJECTIVE: The major goal was to determine the role of genetic variation [e.g. single nucleotide polymorphisms (SNPs)] of Toll-interacting protein (Tollip) in airway epithelial responses to RV in a type 2 cytokine milieu. METHODS: DNA from blood of asthmatic and normal subjects was genotyped for Tollip SNP rs5743899 AA, AG and GG genotypes. Human tracheobronchial epithelial (HTBE) cells from donors without lung disease were cultured to determine pro-inflammatory and antiviral responses to IL-13 and RV16. Tollip knockout and wild-type mice were challenged with house dust mite (HDM) and infected with RV1B to determine lung inflammation and antiviral response. RESULTS: Asthmatic subjects carrying the AG or GG genotype (AG/GG) compared with the AA genotype demonstrated greater airflow limitation. HTBE cells with AG/GG expressed less Tollip. Upon IL-13 and RV16 treatment, cells with AG/GG (vs. AA) produced more IL-8 and expressed less antiviral genes, which was coupled with increased NF-κB activity and decreased expression of LC3, a hallmark of the autophagic pathway. Tollip co-localized and interacted with LC3. Inhibition of autophagy decreased antiviral genes in IL-13- and RV16-treated cells. Upon HDM and RV1B, Tollip knockout (vs. wild-type) mice demonstrated higher levels of lung neutrophilic inflammation and viral load, but lower levels of antiviral gene expression. CONCLUSIONS AND CLINICAL RELEVANCE: Our data suggest that Tollip SNP rs5743899 may predict varying airway response to RV infection in asthma.
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Alelos , Péptidos y Proteínas de Señalización Intracelular/genética , Infecciones por Picornaviridae/genética , Infecciones por Picornaviridae/virología , Polimorfismo de Nucleótido Simple , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/virología , Rhinovirus/inmunología , Adulto , Anciano , Animales , Autofagia , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Predisposición Genética a la Enfermedad , Genotipo , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , FN-kappa B/metabolismo , Infecciones por Picornaviridae/inmunología , Infecciones por Picornaviridae/patología , Interferencia de ARN , Pruebas de Función Respiratoria , Carga ViralRESUMEN
Targeted knockout of genes in primary human cells using CRISPR-Cas9-mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly, we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2, 3 and 4 agonists revealed that IL-8 (a proinflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9-mediated gene knockouts in AEC culture (both submerged and polarized), and suggest a proinflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli.
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Vectores Genéticos , Lentivirus , Mucosa Respiratoria/metabolismo , Antígeno CD146/genética , Antígeno CD146/inmunología , Antígeno CD146/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Inflamación/genética , Cultivo Primario de Células , Mucosa Respiratoria/inmunologíaRESUMEN
Cigarette smoke (CS) is a main risk factor for chronic obstructive pulmonary disease (COPD). Oxidative stress induced by CS causes DNA and lung damage. Oxidant/antioxidant imbalance occurs in the distal air spaces of smokers and in patients with COPD. We studied the effect of oxidative stress generated by CS both in vivo and in vitro on murine primary alveolar type II (ATII) cells isolated from nuclear erythroid 2-related factor-2 (Nrf2)(-/-) mice. We determined human primary ATII cell injury by CS in vitro and analyzed ATII cells isolated from smoker and non-smoker lung donors ex vivo. We also studied whether trolox (water-soluble derivative of vitamin E) could protect murine and human ATII cells against CS-induced DNA damage and/or decrease injury. We analyzed oxidative stress by 4-hydroxynonenal expression, reactive oxygen species (ROS) generation by Amplex Red Hydrogen Peroxide Assay, Nrf2, heme oxygenase 1, p53 and P53-binding protein 1 (53BP1) expression by immonoblotting, Nrf2 nuclear translocation, Nrf2 and p53 DNA-binding activities, apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and cytokine production by ELISA. We found that ATII cells isolated from Nrf2(-/-) mice are more susceptible to CS-induced oxidative DNA damage mediated by p53/53BP1 both in vivo and in vitro compared with wild-type mice. Therefore, Nrf2 activation is a key factor to protect ATII cells against injury by CS. Moreover, trolox abolished human ATII cell injury and decreased DNA damage induced by CS in vitro. Furthermore, we found higher inflammation and p53 mRNA expression by RT-PCR in ATII cells isolated from smoker lung donors in comparison with non-smokers ex vivo. Our results indicate that the Nrf2 and p53 cross talk in ATII cells affect the susceptibility of these cells to injury by CS. Trolox can protect against oxidative stress, genotoxicity and inflammation induced by CS through ROS scavenging mechanism, and serve as a potential antioxidant prevention strategy against oxidative injury of ATII cells in CS-related lung diseases.
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Células Epiteliales Alveolares/efectos de los fármacos , Antioxidantes/farmacología , Cromanos/farmacología , Factor 2 Relacionado con NF-E2/agonistas , Nicotiana/toxicidad , Alveolos Pulmonares/efectos de los fármacos , Proteína p53 Supresora de Tumor/agonistas , Aldehídos/metabolismo , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/metabolismo , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Noqueados , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Alveolos Pulmonares/citología , Alveolos Pulmonares/metabolismo , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Humo/efectos adversos , Fumar/efectos adversos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
BACKGROUND: Caveolin-1 has emerged as a critical regulator of signaling pathways involved in lung fibrosis and inflammation. METHODS: Therefore, we investigated whether caveolin-1 is deficient in asthmatic patients and in a murine model of asthma. RESULTS: Immunohistochemical analyses of endobronchial biopsies showed a remarkable loss of caveolin-1 in the lungs of asthmatic patients compared with controls. This loss was most evident in bronchial epithelial cells and associated with an increase in the expression of extracellular matrix proteins: collagen I, tenascin, and periostin. Cultured primary bronchial epithelial cells of asthmatics had lower caveolin-1 expression compared with control cells. In addition, caveolin-1 expression was significantly decreased in peripheral blood monocytes from asthma patients. The loss of caveolin-1 was also observed in a mouse model for asthma (mice sensitized and challenged with aspergillus fumigatus). CONCLUSIONS: To our knowledge, this is the first demonstration that the regulatory protein caveolin-1 is reduced in patients with asthma.
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Asma/metabolismo , Bronquios/metabolismo , Caveolina 1/metabolismo , Células Epiteliales/metabolismo , Monocitos/metabolismo , Animales , Caveolina 1/deficiencia , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Humanos , Pulmón/metabolismo , Pulmón/patología , Ratones , Transducción de SeñalRESUMEN
BACKGROUND: Chronic rhinosinusitis (CRS) is a disease characterized by inflammation of the nasal mucosa and paranasal sinuses. This inflammation may result in part from decreased epithelial barrier and innate immune responses, leading to frequent bacterial and fungal colonization. The objectives of this study were to investigate the expression of innate immune proteins of the palate lung and nasal epithelium clone (PLUNC) family in patients with CRS. METHODS: Nasal tissue samples were collected from control subjects and CRS patients with and without nasal polyps. Expression of the members of the PLUNC family was analyzed by real-time PCR. Expression of SPLUNC1 and LPLUNC2 proteins was analyzed by ELISA, immunoblot, and immunohistochemical analysis. RESULTS: Levels of mRNA for most of the members of the PLUNC family were profoundly reduced in nasal polyps (NPs) compared to uncinate tissue from control subjects or patients with CRS. LPLUNC2 and SPLUNC1 proteins were decreased in NPs of patients with CRS compared to uncinate tissue from control subjects. Immunohistochemical data revealed that within submucosal glands of sinonasal tissues, SPLUNC1 and LPLUNC2 were differentially expressed, in serous and mucous cells, respectively. The decrease in the expression of these molecules is probably explained by a decrease in the number of glands in NPs as revealed by correlations with levels of the glandular marker lactoferrin. CONCLUSIONS: Decreased SPLUNC1 and LPLUNC2 in NPs reflect a profound decrease in the number of submucosal glands. Decreased glands may lead to a localized defect in the production and release of glandular innate defense molecules.
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Expresión Génica , Glicoproteínas/genética , Pólipos Nasales/genética , Fosfoproteínas/genética , Rinitis/genética , Sinusitis/genética , Adolescente , Adulto , Anciano , Enfermedad Crónica , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Glicoproteínas/inmunología , Humanos , Lactoferrina/genética , Lactoferrina/inmunología , Masculino , Persona de Mediana Edad , Mucosa Nasal/inmunología , Mucosa Nasal/metabolismo , Pólipos Nasales/inmunología , Fosfoproteínas/inmunología , Rinitis/inmunología , Sinusitis/inmunología , Adulto JovenRESUMEN
In 2009 the journal published in the region of 200 papers including reviews, editorials, opinion pieces and original papers that ran the full gamut of allergic disease. It is instructive to take stock of this output to determine patterns of interest and where the cutting edge lies. We have surveyed the field of allergic disease as seen through the pages of Clinical and Experimental Allergy (CEA) highlighting trends, emphasizing notable observations and placing discoveries in the context of other key papers published during the year. The review is divided into similar sections as the journal. In the field of Asthma and Rhinitis CEA has contributed significantly to the debate about asthma phenotypes and expressed opinions about the cause of intrinsic asthma. It has also added its halfpennyworth to the hunt for meaningful biomarkers. In Mechanisms the considerable interest in T cell subsets including Th17 and T regulatory cells continues apace and the discipline of Epidemiology continues to invoke a steady stream of papers on risk factors for asthma with investigators still trying to explain the post-second world war epidemic of allergic disease. Experimental Models continue to make important contributions to our understanding of pathogenesis of allergic disease and in the Clinical Allergy section various angles on immunotherapy are explored. New allergens continue to be described in the allergens section to make those allergen chips even more complicated. A rich and vibrant year helpfully summarized by some of our associate editors.
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Alergia e Inmunología/tendencias , Hipersensibilidad/inmunología , Publicaciones Periódicas como Asunto/tendencias , Animales , Asma/inmunología , Bibliometría , Modelos Animales de Enfermedad , Humanos , Hipersensibilidad/epidemiología , Hipersensibilidad/terapia , Rinitis/inmunología , Medición de Riesgo , Factores de Riesgo , Subgrupos de Linfocitos T/inmunología , Factores de TiempoRESUMEN
Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the United States and the incidence is increasing as the population ages. Cigarette smoking is the primary risk factor; however, only a minority of smokers develop the disease. Inhalation of cigarette smoke introduces an abundance of free radicals into the lungs, causing oxidative stress and inflammation. We hypothesized that after the initial burst of oxidative stress associated with cigarette smoke exposure, a sustained source of endogenous free radical production is modulated by the antioxidant enzyme extracellular superoxide dismutase (ECSOD) and the superoxide-generating complex NADPH oxidase (NOX). Primary mouse macrophages exposed to cigarette smoke extract exhibited increased oxidative stress as indicated by fluorogenic dyes and isoprostane concentration, which was suppressed in the presence of both a superoxide dismutase mimetic and a NOX inhibitor. Similarly, primary macrophages isolated from ECSOD-overexpressing mice or NOX-deficient mice showed reduced oxidative stress in response to cigarette smoke treatment. In addition, both reduced glutathione and cytokines (MIP2 and IFNγ) were increased in bronchoalveolar lavage fluid of wild-type mice exposed to cigarette smoke but not in ECSOD-overexpressing or NOX-deficient mice. These data suggest that the mechanisms underlying the host defense against cigarette smoke-induced oxidative damage and subsequent development of COPD may include endogenous oxidases and antioxidant enzymes.
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NADPH Oxidasas/metabolismo , Nicotiana/efectos adversos , Estrés Oxidativo , Proteínas Recombinantes/metabolismo , Humo/efectos adversos , Superóxido Dismutasa/metabolismo , Animales , Quimiocina CXCL2/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Pulmón/patología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , NADPH Oxidasas/antagonistas & inhibidores , Compuestos Onio/farmacología , Enfermedad Pulmonar Obstructiva Crónica/patología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/genética , Superóxido Dismutasa/genética , Regulación hacia ArribaRESUMEN
BACKGROUND: Infection with Mycoplasma pneumoniae (Mp) in asthma can occur both acutely and chronically with an associated Th2 inflammatory response and/or increased numbers of bronchial mast cells. Mast cells have previously been shown to promote mycoplasma clearance in mice; however, it is unknown whether mast cells would aid Mp clearance under allergic conditions. OBJECTIVE: Our aim was to determine the impact of allergic inflammation on mast cell-mediated lung Mp clearance. Furthermore, as we have previously demonstrated an essential role for IL-6 in lung Mp clearance we also investigated the role of mast cell-derived IL-6. METHODS: Mast cell-deficient (WBB6F1/J-Kit(W)/Kit(W-v)) mice were challenged with ovalbumin to induce airway inflammation before Mp infection. The role of mast cell-derived IL-6 in bacterial clearance was further investigated by reconstitution of mast cell-deficient mice with IL-6(-/-) mast cells. RESULTS: Allergic mast cell-deficient mice exhibited increased lung Mp burden compared with control littermates. Intravenous adoptive transfer of wild-type and IL-6(-/-) mast cells significantly improved Mp clearance in mast cell-deficient mice. Acutely after Mp infection, allergen-challenged mast cell-deficient mice had increased levels of the pro-inflammatory cytokines IL-6 and TNF-alpha in the bronchoalveolar lavage (BAL) fluid. The total number of neutrophils was also increased in mast cell-deficient mice. CONCLUSIONS: Our results establish that mast cells aid host defense against Mp in an allergic setting and that while IL-6 is necessary for lung Mp clearance, mast cell-derived IL-6 is not required.
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Asma/complicaciones , Interacciones Huésped-Patógeno/inmunología , Hipersensibilidad/microbiología , Inmunidad Innata , Pulmón/inmunología , Mastocitos/inmunología , Mycoplasma pneumoniae , Neumonía por Mycoplasma/inmunología , Animales , Hipersensibilidad/inmunología , Interleucina-6/inmunología , Pulmón/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neumonía por Mycoplasma/complicacionesRESUMEN
BACKGROUND: Over 40% of chronic stable asthma patients have evidence of respiratory Mycoplasma pneumoniae (Mp) infection as detected by PCR, but not by serology and culture, suggesting that a low-level Mp is involved in chronic asthma. However, the role of such a low-level Mp infection in the regulation of allergic inflammation remains unknown. OBJECTIVE: To determine the impact of a low-level Mp infection in mice with established airway allergic inflammation on allergic responses such as eosinophilia and chemokine eotaxin-2, and the underlying mechanisms [i.e. the prostaglandin E(2) (PGE(2)) pathway] since PGE(2) inhalation before an allergen challenge suppressed the eosinophil infiltration in human airways. METHODS: BALB/c mouse models of ovalbumin (OVA)-induced allergic asthma with an ensuing low- or high-dose Mp were used to assess IL-4 expression, bronchoalveolar lavage (BAL) eosinophil, eotaxin-2 and PGE(2) levels, and lung mRNA levels of microsomal prostaglandin E synthase-1 (mPGES-1). Primary alveolar macrophages (pAMs) from naïve BALB/c mice were cultured to determine whether Mp-induced PGE(2) or exogenous PGE(2) down-regulates IL-4/IL-13-induced eotaxin-2. RESULTS: Low-dose Mp in allergic mice significantly enhanced IL-4 and eotaxin-2, and moderately promoted lung eosinophilia, whereas high-dose Mp significantly reduced lung eosinophilia and tended to decrease IL-4 and eotaxin-2. Moreover, in both OVA-naïve and allergic mice, lung mPGES-1 mRNA and BAL PGE(2) levels were elevated in mice infected with high-dose, but not low-dose Mp. In pAMs, IL-4/IL-13 significantly increased eotaxin-2, which was reduced by Mp infection accompanied by dose-dependent PGE(2) induction. Exogenous PGE(2) inhibited IL-4/IL-13-induced eotaxin-2 in a dose-dependent manner. CONCLUSIONS: This study highlights a novel concept on how different bacterial loads in the lung modify the established allergic airway inflammation and thus interact with an allergen to further induce Th2 responses. That is, unlike high-level Mp, low-level Mp fails to effectively induce PGE(2) to down-regulate allergic responses (e.g. eotaxin-2), thus maintaining or even worsening allergic inflammation in asthmatic airways.
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Asma/inmunología , Dinoprostona/inmunología , Mycoplasma pneumoniae/inmunología , Neumonía por Mycoplasma/inmunología , Animales , Asma/microbiología , Lavado Broncoalveolar , Quimiocina CCL24/inmunología , Relación Dosis-Respuesta Inmunológica , Eosinofilia/inmunología , Eosinofilia/microbiología , Femenino , Humanos , Inflamación/inmunología , Inflamación/microbiología , Interleucina-13/inmunología , Interleucina-4/inmunología , Oxidorreductasas Intramoleculares/inmunología , Ratones , Ratones Endogámicos BALB C , Prostaglandina-E Sintasas , Células Th2/inmunologíaRESUMEN
Reduced vascular endothelial growth factor (VEGF) has been reported in bronchoalveolar lavage fluid and lungs of severe emphysema patients. Airway epithelial cells (AEC) are exposed to various environmental insults like cigarette smoke and bacterial infections, but their direct effect on VEGF production in well-differentiated primary human AEC remains unclear. The current authors determined the effect of cigarette smoke extract (CSE) alone and in combination with Mycoplasma pneumoniae (Mp) on VEGF production in well-differentiated primary normal human bronchial epithelial (NHBE) and small airway epithelial cells (SAEC) in air-liquid interface cultures. Secretion and expression of VEGF were determined by ELISA and real-time RT-PCR, respectively. Cell growth, apoptosis, extracellular signal-regulated kinase (ERK)1/2 and protein kinase (PK)C signalling pathways were evaluated to further dissect VEGF regulation under CSE treatment. CSE significantly reduced VEGF secretion in NHBE and SAEC. In SAEC, Mp alone significantly increased the VEGF, while the presence of CSE attenuated Mp-induced VEGF production. While ERK inhibitor reduced VEGF secretion only in NHBE, a PKC inhibitor significantly decreased VEGF secretion in both NHBE and SAEC. In conclusion, direct cigarette smoke extract exposure significantly reduced vascular endothelial growth factor production in well-differentiated primary human airway epithelial cells, in part through modifying extracellular signal-regulated kinase 1/2 and protein kinase C signalling pathways.
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Células Epiteliales/efectos de los fármacos , Pulmón/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Nicotiana , Humo , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Análisis de Varianza , Apoptosis/efectos de los fármacos , Western Blotting , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/metabolismo , Humanos , Pulmón/metabolismo , Mycoplasma pneumoniae , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Exhaled nitric oxide is increased in asthma, but the mechanisms controlling its production, including the effects of T-helper type 2 (Th2) cytokines, are poorly understood. In mouse and submerged human epithelial cells, Th2 cytokines inhibit expression of inducible nitric oxide synthase (iNOS). Arginases have been proposed to contribute to asthma pathogenesis by limiting the arginine substrate available to NOS enzymes, but expression of any of these enzymes has not been extensively studied in primary human cells. OBJECTIVES: We hypothesized that primary human airway epithelial cells in air-liquid interface (ALI) culture would increase iNOS expression and activity in response to IL-13, while decreasing arginase expression. METHODS: iNOS and arginase mRNA (real-time PCR) and protein expression (Western blot and immunofluorescence) as well as iNOS activity (nitrite levels) were measured in ALI epithelial cells cultured from bronchial brushings of normal and asthmatic subjects following IL-13 stimulation. RESULTS: IL-13 up-regulated iNOS mRNA primarily at a transcriptional level in epithelial cells. iNOS protein and activity also increased, arginase1 protein expression decreased while arginase 2 expression did not change. The changes in iNOS protein correlated strongly with changes in nitrites, and inclusion of arginase (1 or 2) did not substantially change the relationship. Interestingly, iNOS mRNA and protein were not correlated. CONCLUSIONS: These results contrast with many previous results to confirm that Th2 stimuli enhance iNOS expression and activity. While arginase 1 protein decreases in response to IL-13, neither arginase appears to substantially impact nitrite levels in this system.
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Arginasa/metabolismo , Bronquios/efectos de los fármacos , Epitelio/efectos de los fármacos , Interleucina-13/farmacología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa/biosíntesis , Nitritos/metabolismo , Adulto , Arginasa/genética , Asma/enzimología , Asma/patología , Western Blotting , Bronquios/enzimología , Bronquios/patología , Broncoscopía , Células Cultivadas , Relación Dosis-Respuesta a Droga , Epitelio/enzimología , Epitelio/patología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interferón gamma/farmacología , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo II/genética , Reacción en Cadena de la Polimerasa , Estabilidad del ARN , ARN Mensajero/metabolismo , Estadísticas no ParamétricasRESUMEN
As excess mucin expression can contribute to the exacerbation of asthma, the present authors hypothesised that Mycoplasma pneumoniae significantly induces MUC5AC (the major airway mucin) expression in airway epithelial cells isolated directly from asthmatic subjects. A total of 11 subjects with asthma and six normal controls underwent bronchoscopy with airway brushing. Epithelial cells were cultured at an air-liquid interface and incubated with and without M. pneumoniae for 48 h, and in the presence and absence of nuclear factor (NF)-kappaB and a toll-like receptor (TLR)2 inhibitor. Quantitative PCR was performed for MUC5AC and TLR2 mRNA. MUC5AC protein and total protein were determined by ELISA. M. pneumoniae exposure significantly increased MUC5AC mRNA and protein expression after 48 h in epithelial cells isolated from asthmatic, but not from normal control subjects, at all concentrations as compared to unexposed cells. TLR2 mRNA expression was significantly increased in asthmatic epithelial cells at 4 h compared with unexposed cells. NF-kappaB and TLR2 inhibition reduced MUC5AC expression to the level of the unexposed control in both groups. Mycoplasma pneumoniae exposure significantly increased MUC5AC mRNA and protein expression preferentially in airway epithelial cells isolated from asthmatic subjects. The toll-like receptor 2 pathway may be involved in this process.
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Asma/metabolismo , Células Epiteliales/microbiología , Regulación de la Expresión Génica , Mucinas/biosíntesis , Mycoplasma pneumoniae/metabolismo , Adulto , Asma/inmunología , Asma/microbiología , Broncoscopía/métodos , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/citología , Femenino , Humanos , Masculino , Modelos Biológicos , Mucina 5AC , Mucinas/metabolismo , Mycoplasma pneumoniae/fisiología , FN-kappa B/metabolismo , Factores de Tiempo , Receptor Toll-Like 2/metabolismoRESUMEN
INTRODUCTION: The multifocal visual-evoked potential (mfVEP) has been widely used in the study of diseases of the visual system. However, the sensitivity of the mfVEP in the objective detection of relative field defects has not been determined. This study investigates variations in mfVEP responses while simulating relative field defects by using different luminous transmission masks [neutral density (ND) filters] on the stimulus pattern. METHODS: Simulated relative field defects with four different luminous transmissions were obtained by using 0.2, 0.4, 0.6, and 0.8 ND filters, 5 degrees in size, at two different retinal eccentricities (10 and 16 degrees) on a standard mfVEP dartboard stimulus. Eleven normal subjects were recruited for mfVEP measurements. The response amplitudes and latencies of the N1 and P1 of the mfVEP, with and without small simulated relative field defects, were compared. RESULTS: The mfVEP amplitudes of N1 and P1 decreased substantially when 0.6 and 0.8 ND filters were introduced. The effects were similar at both the 10- and 16-degree eccentricities but there was no change in latency with simulated field defects at either location. CONCLUSIONS: The mfVEP can detect a simulated relative field defect 5 degrees in size starting with 0.6 log unit reduction in luminance at both 10-degree and 16-degree eccentricities. This illustrates that the sensitivity of the mfVEP measurement is nearly comparable with that of the Humphrey Visual Field Analyser.
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Potenciales Evocados Visuales/fisiología , Trastornos de la Visión/fisiopatología , Campos Visuales/fisiología , Adulto , Femenino , Humanos , Masculino , Estimulación Luminosa/métodos , Retina/fisiopatología , Percepción Visual/fisiologíaRESUMEN
Small airway (SA) inflammation in asthmatics is poorly understood. Surgical biopsies to obtain peripheral lung tissue are seldom justified in asthmatics. Therefore, the authors hypothesised that transbronchial biopsy could be an alternative approach to evaluate SA in asthma. Transbronchial and endobronchial biopsy tissue samples (TBBX and EBBX) from 12 severe asthmatics were evaluated for airway and parenchymal total inflammatory cell count expressed as the sum of immunostained T-cells (CD3), macrophages (CD68), mast cells (tryptase AAI), neutrophils (neutrophil elastase) and eosinophils (EG2) per mm2. The large airways (LA) were evaluated in EBBXs, while SA, medium airways (MA) and alveolar tissue (AT) were evaluated in TBBXs. When cell counts from SA, MA, LA and AT were compared, SA had a significantly higher cell count than MA or LA (SA 1011 x mm(-2) (539-1,290), MA 346 x mm(-2) (223-415), LA 332 x mm(-2) (189-416), AT 464 x mm(-2) (298-834)). The cell density and pattern of the inflammatory cell distribution in subjects with TBBXs appeared similar to those in three severe asthmatics whose inflammatory cells were analysed in surgical tissue samples. This study suggests that small airway may be identified and analysed in transbronchial biopsy tissue samples and therefore transbronchial biopsy tissue samples could expand the analysis of inflammation and tissue remodelling in asthma.
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Obstrucción de las Vías Aéreas/etiología , Obstrucción de las Vías Aéreas/patología , Asma/complicaciones , Asma/patología , Biopsia , Bronquios/patología , Adulto , Broncoscopía , Femenino , Humanos , Recuento de Leucocitos , Masculino , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Índice de Severidad de la EnfermedadRESUMEN
BACKGROUND: Although 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), a product of 15-lipoxygenase (15-LO), may be involved in mild to moderate asthma, little is known about its potential roles in severe asthma. OBJECTIVES: This study was performed to evaluate 15(S)-HETE levels in bronchoalveolar lavage fluid (BALF) from severe asthmatics with and without airway eosinophils and from the control groups. In addition, 15-LO protein expression was examined in endobronchial biopsy, while its expression and activation were evaluated in BAL cells. RESULTS: While 15(S)-HETE levels in BALF were significantly higher in all severe asthmatics than normal subjects, severe asthmatics with airway eosinophils had the highest levels compared with mild, moderate asthmatics and normal subjects. 15(S)-HETE levels were associated with tissue eosinophil numbers, sub-basement membrane thickness and BALF tissue inhibitor of metalloproteinase-1 levels, and were accompanied by increased 15-LO expression in bronchial epithelium. In addition, activation of 15-LO was suggested by the increased proportion of 15-LO in the cytoplasmic membrane of alveolar macrophages from severe asthmatics. CONCLUSION: The data suggest that severe asthmatics with persistent airway eosinophils manifest high levels of 15(S)-HETE in BALF, which may be associated with airway fibrosis. It is likely that 15-LO expression and activation by airway cells explain the increased 15(S)-HETE levels.
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Araquidonato 15-Lipooxigenasa/metabolismo , Asma/metabolismo , Líquido del Lavado Bronquioalveolar/química , Eosinófilos/inmunología , Ácidos Hidroxieicosatetraenoicos/metabolismo , Transducción de Señal/fisiología , Adulto , Asma/inmunología , Bronquios/metabolismo , Colágeno/metabolismo , Femenino , Fibrosis , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Although airway angiogenesis and edema have been proposed to contribute to the airway remodeling process in patients with asthma, there are few studies looking at these structural components in the airway tissue of asthma patients. Mycoplasma infection may be associated with chronic asthma and has been shown to induce angiogenesis and edema in a murine model. PARTICIPANTS AND MEASUREMENTS: We evaluated blood vessels and edema by immunohistochemistry in endobronchial biopsy samples from 10 normal control subjects and 15 patients with mild-to-moderate asthma before and after a 6-week treatment with clarithromycin (n = 8) or placebo (n = 7). Type IV collagen and alpha(2)-macroglobulin were used to identify blood vessels and edema in the tissue, respectively. Mycoplasma pneumoniae was evaluated by polymerase chain reaction. SETTING: National Jewish Medical and Research Center. RESULTS: At baseline, the vascularity, the number of blood vessels, and the edematous area in the airway tissue were not significantly different between asthmatic patients and normal control subjects. However, asthmatic patients demonstrated increased blood vessel size compared with normal control subjects (p = 0.03). After clarithromycin treatment in asthmatic patients, the number of blood vessels was increased (p = 0.02), while edema decreased (p = 0.049). Asthmatic patients who tested positive for M pneumoniae showed a significant increase in vascularity than asthmatic patients who tested negative for M pneumoniae (p = 0.02). CONCLUSION: Our data suggest that angiogenesis and edema may not be significant features of airway remodeling in patients with chronic, mild-to-moderate asthma. Clarithromycin treatment in asthmatic patients could reduce the edematous area as identified by alpha(2)-macroglobulin staining, which may lead to airway tissue shrinkage and cause an artificial increase in the number of blood vessels.
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Antibacterianos/uso terapéutico , Asma/patología , Arterias Bronquiales/patología , Claritromicina/uso terapéutico , Edema/patología , Adulto , Asma/tratamiento farmacológico , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/patología , Bronquios/patología , Colágeno/análisis , Edema/tratamiento farmacológico , Femenino , Humanos , Inmunohistoquímica , Masculino , Mycoplasma pneumoniae/aislamiento & purificación , Neovascularización Fisiológica/efectos de los fármacos , Neumonía por Mycoplasma/patología , alfa-Macroglobulinas/análisisRESUMEN
Despite advances in understanding the pathophysiology of asthma, morbidity and mortality in pediatrics continue to rise. Little is known about the initiation and chronicity of inflammation resulting in asthma in this young population. We evaluated 20 "wheezing" children (WC) (median age 14.9 mo) with a minimum of two episodes of wheezing or prolonged wheezing > or = 2 mo in a 6-mo period with bronchoscopy and bronchoalveolar lavage (BAL). Comparisons were made with six normal controls (NC) (median age 23.3 mo) undergoing general anesthesia for elective surgery. BAL fluid cell counts and differentials were determined. The eicosanoids, leukotriene (LT) B(4), LTE(4), prostaglandin (PG)E(2), and 15-hydroxyeicosatetraenoic acid (HETE) and the mast cell mediators, beta-tryptase and PGD(2), were evaluated by enzyme immunoassay (EIA). WC had significant elevations in total BAL cells/ml (p = 0.01), as well as, lymphocytes (LYMPH, p = 0.007), macrophages/monocytes (M&M, p = 0.02), polymorphonuclear cells (PMN, p = 0.02), epithelial cells (EPI, p = 0.03), and eosinophils (EOS, p = 0.04) compared with NC. Levels of PGE(2) (p = 0.0005), 15-HETE (p = 0.002), LTE(4) (p = 0.04), and LTB(4) (p = 0.05) were also increased in WC compared with NC, whereas PGD(2) and beta-tryptase were not. This study confirms that inflammation is present in the airways of very young WC and may differ from patterns seen in adults with asthma.