RESUMO
INTRODUCTION: Rubinstein-Taybi syndrome (RSTS) is a rare genetic syndrome caused primarily by a mutation in the CREBBP gene found on chromosome 16. Patients with RSTS are at greater risk for a variety of medical problems, including upper airway obstruction and aspiration. Childhood interstitial lung disease (ILD) thus far has not been definitively linked to RSTS. Here we present three patients with RSTS who developed ILD and discuss possible mechanisms by which a mutation in CREBBP may be involved in the development of ILD. METHODS: Routine hematoxylin and eosin staining was performed on lung biopsy tissue for histological analysis. Immunofluorescent staining was performed on lung biopsy tissue for markers of fibrosis, surfactant deficiency and histone acetylation. Cases 1 and 2 had standard clinical microarray analysis. Case 3 had whole exome sequencing. Bioinformatics analyses were performed to identify possible causative genes using ToppGene. RESULTS: Computed tomography images in all cases showed consolidated densities overlying ground glass opacities. Lung histopathology revealed accumulation of proteinaceous material within alveolar spaces, evidence of fibrosis, and increased alveolar macrophages. Immunofluorescent staining showed increase in surfactant protein C staining, patchy areas of increased anti-smooth muscle antibody staining, and increased staining for acetylated histone 2 and histone 3 lysine 9. DISCUSSION: Clinical characteristics, radiographic imaging, lung histopathology, and immunofluorescent staining results shared by all cases demonstrated findings consistent with ILD. Immunofluorescent staining suggests two possible mechanisms for the development of ILD: abnormal surfactant metabolism and/or persistent activation of myofibroblasts. These two pathways could be related to dysfunctional CREBBP protein.
Assuntos
Doenças Pulmonares Intersticiais , Síndrome de Rubinstein-Taybi , Proteína de Ligação a CREB/genética , Criança , Humanos , Doenças Pulmonares Intersticiais/etiologia , Doenças Pulmonares Intersticiais/genética , Mutação , Síndrome de Rubinstein-Taybi/complicações , Síndrome de Rubinstein-Taybi/diagnóstico , Síndrome de Rubinstein-Taybi/genética , Sequenciamento do ExomaRESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with limited treatment options. Despite endothelial cells (ECs) comprising 30% of the lung cellular composition, the role of EC dysfunction in pulmonary fibrosis (PF) remains unclear. We hypothesize that sterol regulatory element-binding protein 2 (SREBP2) plays a critical role in the pathogenesis of PF via EC phenotypic modifications. Transcriptome data demonstrate that SREBP2 overexpression in ECs led to the induction of the TGF, Wnt, and cytoskeleton remodeling gene ontology pathways and the increased expression of mesenchymal genes, such as snail family transcriptional repressor 1 (snai1), α-smooth muscle actin, vimentin, and neural cadherin. Furthermore, SREBP2 directly bound to the promoter regions and transactivated these mesenchymal genes. This transcriptomic change was associated with an epigenetic and phenotypic switch in ECs, leading to increased proliferation, stress fiber formation, and ECM deposition. Mice with endothelial-specific transgenic overexpression of SREBP2 (EC-SREBP2[N]-Tg mice) that were administered bleomycin to induce PF demonstrated exacerbated vascular remodeling and increased mesenchymal transition in the lung. SREBP2 was also found to be markedly increased in lung specimens from patients with IPF. These results suggest that SREBP2, induced by lung injury, can exacerbate PF in rodent models and in human patients with IPF.
Assuntos
Células Endoteliais/metabolismo , Fibrose Pulmonar/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Animais , Humanos , CamundongosRESUMO
BACKGROUND: Lung regeneration plays an important role in lung repair after injury. It is reliant upon proliferation of multiple cell types in the lung, including endothelium, epithelium, and fibroblasts, as well as remodeling of the extracellular matrix. METHODS: Lung regeneration following injury progresses via an initial inflammatory response during which macrophages clear the tissue of cellular debris. This process continues through cellular proliferation when existing cells and progenitors act to repopulate cells lost during injury, followed by tissue maturation in which newly formed cells achieve a differentiated phenotype. RESULTS: Signaling pathways critical for lung regeneration include FGF, EGF, WNT, and NOTCH. In addition, HDACs, miRNAs, ELASTIN, and MMP14 have been shown to regulate lung regeneration. Partial pneumonectomy (PNX) has been used as a therapeutic and investigational tool for several decades. Following PNX the remaining lung increases in size to compensate for loss of volume and respiratory capacity. CONCLUSIONS: Much has been learned about the triggers and mechanisms regulating pulmonary regeneration. However, the role of thymocyte differentiation antigen-1 (Thy-1) in post-PNX lung growth remains incompletely characterized. Thy-1 is a phosphatidylinositol glycoprotein with a relative molecular weight of 25000~37000 Da, which is expressed in almost all types of fibroblasts and regulates many biological functions. It not only supports the structure of fibroblasts, but also can balance cell proliferation, migration and regulate the synthesis of immune inflammatory mediators.
Assuntos
Lesão Pulmonar/fisiopatologia , Pulmão/fisiologia , Pneumonectomia , Regeneração/fisiologia , Antígenos Thy-1/deficiência , Animais , Diferenciação Celular , Movimento Celular , Proliferação de Células , Pulmão/metabolismo , Pulmão/cirurgia , Camundongos , Camundongos Endogâmicos C57BL , Pneumonectomia/métodos , Transdução de Sinais , Células-Tronco/fisiologiaRESUMO
Loss of Thy-1 expression in fibroblasts correlates with lung fibrogenesis; however, the clinical relevance of therapeutic targeting of myofibroblasts via Thy-1-associated pathways remains to be explored. Using single (self-resolving) or repetitive (nonresolving) intratracheal administration of bleomycin in type 1 collagen-GFP reporter mice, we report that Thy-1 surface expression, but not mRNA, is reversibly diminished in activated fibroblasts and myofibroblasts in self-resolving fibrosis. However, Thy-1 mRNA expression is silenced in lung with nonresolving fibrosis following repetitive bleomycin administration, associated with persistent activation of αv integrin. Thy1-null mice showed progressive αv integrin activation and myofibroblast accumulation after a single dose of bleomycin. In vitro, targeting of αv integrin by soluble Thy-1-Fc (sThy-1), but not RLE-mutated Thy-1 or IgG, reversed TGF-ß1-induced myofibroblast differentiation in a dose-dependent manner, suggesting that Thy-1's integrin-binding RGD motif is required for the reversibility of myofibroblast differentiation. In vivo, treatment of established fibrosis induced either by single-dose bleomycin in WT mice or by induction of active TGF-ß1 by doxycycline in Cc10-rtTA-tTS-Tgfb1 mice with sThy-1 (1000 ng/kg, i.v.) promoted resolution of fibrosis. Collectively, these findings demonstrate that sThy-1 therapeutically inhibits the αv integrin-driven feedback loop that amplifies and sustains fibrosis.
Assuntos
Integrinas/metabolismo , Fibrose Pulmonar/metabolismo , Antígenos Thy-1/metabolismo , Animais , Sítios de Ligação , Bleomicina/administração & dosagem , Camundongos , Camundongos Knockout , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Fibrose Pulmonar/induzido quimicamente , Antígenos Thy-1/genéticaRESUMO
Fibrosis is characterized by persistent deposition of extracellular matrix (ECM) by fibroblasts. Fibroblast mechanosensing of a stiffened ECM is hypothesized to drive the fibrotic program; however, the spatial distribution of ECM mechanics and their derangements in progressive fibrosis are poorly characterized. Importantly, fibrosis presents with significant histopathological heterogeneity at the microscale. Here, we report that fibroblastic foci (FF), the regions of active fibrogenesis in idiopathic pulmonary fibrosis (IPF), are surprisingly of similar modulus as normal lung parenchyma and are nonlinearly elastic. In vitro, provisional ECMs with mechanical properties similar to those of FF activate both normal and IPF patient-derived fibroblasts, whereas type I collagen ECMs with similar mechanical properties do not. This is mediated, in part, by αvß3 integrin engagement and is augmented by loss of expression of Thy-1, which regulates αvß3 integrin avidity for ECM. Thy-1 loss potentiates cell contractility-driven strain stiffening of provisional ECM in vitro and causes elevated αvß3 integrin activation, increased fibrosis, and greater mortality following fibrotic lung injury in vivo. These data suggest a central role for αvß3 integrin and provisional ECM in overriding mechanical cues that normally impose quiescent phenotypes, driving progressive fibrosis through physical stiffening of the fibrotic niche.
Assuntos
Fibroblastos/patologia , Fibrose Pulmonar Idiopática/patologia , Integrina alfaVbeta3/metabolismo , Pulmão/patologia , Animais , Bleomicina/toxicidade , Células Cultivadas , Modelos Animais de Doenças , Progressão da Doença , Matriz Extracelular/patologia , Feminino , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Pulmão/citologia , Masculino , Camundongos , Camundongos Knockout , Cultura Primária de Células , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismoRESUMO
BACKGROUND: Angioplasty and stenting is a recognized treatment option for patients with intracranial atherosclerosis. OBJECTIVE: To evaluate the long-term evolutionary luminal changes of intracranial atherosclerosis after angioplasty and stenting. METHODS: This was a retrospective study with patient consent. Eighty-two patients presenting with acute and minor cerebral ischemia due to stenosis ≥70%, who had received medical therapy with or without stenting (Wingspan), were invited. Luminal imaging was provided using 3-dimensional rotational angiography (3-DRA) at baseline and 12 mo, and cone-beam computed tomography angiography with intravenous contrast (CBCT) was provided at follow-up (median 82.4 mo [interquartile range 61.9-96.9 mo]). RESULTS: Thirty-six patients in the stenting group and 26 patients in the medical group were recruited and completed the study. There was no statistically significant difference in demographics between the 2 patient groups. The luminal gain at 12 or 80 mo as compared to baseline in the stenting group was significantly greater than that in the medical group (12 mo: median gain 30% vs 7.2%, P < .001; 80 mo: median gain 42.9% vs 7.2%, P < .0001). Luminal loss or unchanged lumen was correlated with recurrent ischemic event. The differences in the stenosis degree assessment between CBCT and 3-DRA in the same 10 patients with or without stenting were 1.2 ± 0.6% or 0.2 ± 0.06%, respectively. There was a correlation between recurrent ischemic events and luminal loss. CONCLUSION: Arterial lumen after angioplasty and stenting can probably be well maintained and delayed luminal gain does occur, long-term luminal loss is associated with recurrent ischemic events, CBCT might be useful as a less-invasive means for long-term assessment.
Assuntos
Angioplastia , Arteriosclerose Intracraniana/diagnóstico por imagem , Arteriosclerose Intracraniana/cirurgia , Stents , Idoso , Vasos Sanguíneos/diagnóstico por imagem , Isquemia Encefálica/diagnóstico por imagem , Isquemia Encefálica/cirurgia , Angiografia Cerebral , Tomografia Computadorizada de Feixe Cônico , Constrição Patológica/diagnóstico por imagem , Constrição Patológica/cirurgia , Feminino , Seguimentos , Humanos , Imageamento Tridimensional , Masculino , Pessoa de Meia-Idade , Recidiva , Estudos Retrospectivos , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/cirurgia , Fatores de Tempo , Resultado do TratamentoRESUMO
Bone marrow-derived mesenchymal stem cells (MSC) have been promoted for multiple therapeutic applications. Many beneficial effects of MSCs are paracrine, dependent on extracellular vesicles (EVs). Although MSC-derived EVs (mEVs) are beneficial for acute lung injury and pulmonary fibrosis, mechanisms of mEV uptake by lung fibroblasts and their effects on myofibroblastic differentiation have not been established. We demonstrate that mEVs, but not fibroblast EVs (fEVs), suppress TGFß1-induced myofibroblastic differentiation of normal and idiopathic pulmonary fibrosis (IPF) lung fibroblasts. MEVs display increased time- and dose-dependent cellular uptake compared to fEVs. Removal or blocking of Thy-1, or blocking Thy-1-beta integrin interactions, decreased mEV uptake and prevented suppression of myofibroblastic differentiation. MicroRNAs (miRs) 199a/b-3p, 21-5p, 630, 22-3p, 196a-5p, 199b-5p, 34a-5p and 148a-3p are selectively packaged in mEVs. In silico analyses indicated that IPF lung fibroblasts have increased expression of genes that are targets of mEV-enriched miRs. MiR-630 mimics blocked TGFß1 induction of CDH2 in normal and IPF fibroblasts, and antagomiR-630 abrogated the effect of mEV on CDH2 expression. These data suggest that the interaction of Thy-1 with beta integrins mediates mEV uptake by lung fibroblasts, which blocks myofibroblastic differentiation, and that mEVs are enriched for miRs that target profibrotic genes up-regulated in IPF fibroblasts.
Assuntos
Diferenciação Celular/fisiologia , Vesículas Extracelulares/metabolismo , Fibroblastos/citologia , Células-Tronco Mesenquimais/citologia , Miofibroblastos/citologia , Antígenos Thy-1/metabolismo , Fibroblastos/metabolismo , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Células-Tronco Mesenquimais/metabolismo , Miofibroblastos/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by cellular phenotype alterations and deposition of extracellular matrix proteins. The alternative activation of macrophages in the lungs has been associated as a major factor promoting pulmonary fibrosis, however the mechanisms underlying this phenomenon are poorly understood. In the present study, we have defined a molecular mechanism by which signals transmitted from the extracellular matrix via the α4ß1 integrin lead to the activation of Rac2 which regulates alternative macrophage differentiation, a signaling axis within the pulmonary macrophage compartment required for bleomycin induced pulmonary fibrosis. Mice deficient in Rac2 were protected against bleomycin-induced fibrosis and displayed diminished collagen deposition in association with lower expression of alternatively activated profibrotic macrophage markers. We have demonstrated a macrophage autonomous process by which the injection of M2 and not M1 macrophages restored the bleomycin induced pulmonary fibrosis susceptibility in Rac2-/- mice, establishing a critical role for a macrophage Rac2 signaling axis in the regulation of macrophage differentiation and lung fibrosis in vivo. We also demonstrate that markers of alternative macrophage activation are increased in patients with IPF. Taken together, these studies define an important role for an integrin-driven Rac2 signaling axis in macrophages, and reveal that Rac2 activation is required for polarization of macrophages towards a profibrotic phenotype and progression of pulmonary fibrosis in vivo.
Assuntos
Fibrose Pulmonar Idiopática/imunologia , Ativação de Macrófagos , Macrófagos/imunologia , Proteínas rac de Ligação ao GTP/genética , Animais , Bleomicina/toxicidade , Células Cultivadas , Colágeno/metabolismo , Humanos , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/genética , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Proteínas rac de Ligação ao GTP/metabolismo , Proteína RAC2 de Ligação ao GTPRESUMO
Thy-1-negative lung fibroblasts are resistant to apoptosis. The mechanisms governing this process and its relevance to fibrotic remodeling remain poorly understood. By using either sorted or transfected lung fibroblasts, we found that Thy-1 expression is associated with downregulation of anti-apoptotic molecules Bcl-2 and Bcl-xL, as well as increased levels of cleaved caspase-9. Addition of rhFasL and staurosporine, well-known apoptosis inducers, caused significantly increased cleaved caspase-3, -8, and PARP in Thy-1-transfected cells. Furthermore, rhFasL induced Fas translocation into lipid rafts and its colocalization with Thy-1. These in vitro results indicate that Thy-1, in a manner dependent upon its glycophosphatidylinositol anchor and lipid raft localization, regulates apoptosis in lung fibroblasts via Fas-, Bcl-, and caspase-dependent pathways. In vivo, Thy-1 deficient (Thy1-/-) mice displayed persistence of myofibroblasts in the resolution phase of bleomycin-induced fibrosis, associated with accumulation of collagen and failure of lung fibrosis resolution. Apoptosis of myofibroblasts is decreased in Thy1-/- mice in the resolution phase. Collectively, these findings provide new evidence regarding the role and mechanisms of Thy-1 in initiating myofibroblast apoptosis that heralds the termination of the reparative response to bleomycin-induced lung injury. Understanding the mechanisms regulating fibroblast survival/apoptosis should lead to novel therapeutic interventions for lung fibrosis.
Assuntos
Apoptose/fisiologia , Fibroblastos/metabolismo , Lesão Pulmonar/metabolismo , Microdomínios da Membrana/metabolismo , Antígenos Thy-1/metabolismo , Receptor fas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Bleomicina , Caspase 9/metabolismo , Linhagem Celular , Embrião de Mamíferos/citologia , Proteína Ligante Fas/farmacologia , Fibroblastos/efeitos dos fármacos , Immunoblotting , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/prevenção & controle , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/prevenção & controle , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Estaurosporina/farmacologia , Antígenos Thy-1/genética , Proteína bcl-X/metabolismoRESUMO
OBJECTIVE: The purposes of this study were to evaluate the chest radiographic features of adult patients hospitalized for respiratory syncytial virus respiratory tract infections and to assess whether initial chest radiographic findings help predict clinical outcome. MATERIALS AND METHODS: All adult patients hospitalized from January 2009 to December 2011 with laboratory-confirmed respiratory syncytial virus infection were included in the study. Patient clinical data and admission chest radiographs were retrospectively reviewed. Adverse outcomes included need for supplemental oxygen, need for assisted ventilation, and death. RESULTS: Of 285 patients (mean age, 74 ± 16 years) included, 199 (69.8%) had abnormal chest radiographic findings: 49.5% (141/285) had acute changes, and 47.7% (136/285) had chronic changes. Consolidation (68/141 [48.2%]) and ground-glass opacity (57/141 [40.4%]) were the predominant types of acute changes and were most common in unilateral single-lower-zone involvement. Consolidation, ground-glass opacity, and chronic changes occurred with significantly higher frequency in patients with adverse outcomes. The presence of acute (odds ratio, 3.6) and chronic (odds ratio, 2.2) changes were independent risk factors for mortality. CONCLUSIONS: A large proportion of adult patients hospitalized with respiratory syncytial virus respiratory tract infection had changes on initial chest radiographs. Consolidation or ground-glass opacity in a unilateral single-lower-zone distribution were the most common findings. The presence of acute and chronic radiographic lung changes was associated with adverse outcomes.
Assuntos
Radiografia Torácica , Infecções por Vírus Respiratório Sincicial/diagnóstico por imagem , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Pacientes Internados , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos RetrospectivosRESUMO
In this study, we examined the role of neprilysin (NEP), a key membrane-bound endopeptidase, in the inflammatory response induced by diesel exhaust emissions (DEE) in the airways through a number of approaches: in vitro, animal, and controlled human exposure. Our specific aims were (1) to examine the role of NEP in inflammatory injury induced by diesel exhaust particles (DEP) using Nep-intact (wild-type) and Nep-null mice; (2) to examine which components of DEP are associated with NEP downregulation in vitro; (3) to determine the molecular impact of DEP exposure and decreased NEP expression on airway epithelial cells' gene expression in vitro, using a combination of RNA interference (RNAi) and microarray approaches; and (4) to evaluate the effects on NEP activity of human exposure to DEE. We report four main results: First, we found that exposure of normal mice to DEP consisting of standard reference material (SRM) 2975 via intratracheal installation can downregulate NEP expression in a concentration-dependent manner. The changes were accompanied by increases in the number of macrophages and epithelial cells, as well as proinflammatory cytokines, examined in bronchoalveolar lavage (BAL) fluid and cells. Nep-null mice displayed increased and/or additional inflammatory responses when compared with wild-type mice, especially in response to exposure to the higher dose of DEP that we used. These in vivo findings suggest that loss of NEP in mice could cause increased susceptibility to injury or exacerbate inflammatory responses after DEP exposure via release of specific cytokines from the lungs. Second, we found evidence, using in vitro studies, that downregulation of NEP by DEP in cultured human epithelial BEAS-2B cells was mostly attributable to DEP-adsorbed organic compounds, whereas the carbonaceous core and transition metal components of DEP had little or no effect on NEP messenger RNA (mRNA) expression. This NEP downregulation was not a specific response to DEP or its contents because the change also occurred after exposure to urban dust (SRM 1649a), which differs in physical and chemical composition from DEP. Third, we also collected the transcriptome profiles of the concentration-effects of SRM 2975 in cultured BEAS-2B cells through a 2 X 3 factorial design. DEP exposure upregulated 151 genes and downregulated 59 genes. Cells with decreased NEP expression (accomplished by transfecting an NEP-specific small interfering RNA [siRNA]) substantially altered the expression of genes (upregulating 17 and downregulating 14) associated with DNA/protein binding, calcium channel activities, and the cascade of intracellular signaling by cytokines. Data generated from the combined RNAi and microarray approaches revealed that there is a complex molecular cascade mediated by NEP in different subcellular compartments, possibly influencing the inflammatory response. Fourth, in a controlled human exposure study, we observed significant increases in soluble NEP in sputum after acute exposure to DEE, with an average net increase of 31%. We speculate that the change in NEP activity in sputum, if confirmed in larger epidemiologic investigations at ambient exposure levels to DEE, may provide a useful endpoint and promote insight into the mechanism of DEE-induced airway alterations.
Assuntos
Bronquite/induzido quimicamente , Bronquite/enzimologia , Neprilisina/metabolismo , Emissões de Veículos/intoxicação , Adulto , Animais , Regulação para Baixo , Células Epiteliais/enzimologia , Feminino , Expressão Gênica , Humanos , Inflamação , Masculino , Camundongos , Camundongos Knockout , Neprilisina/genética , Tamanho da Partícula , Escarro/enzimologia , Adulto JovemRESUMO
This study was designed to characterize and compare the effects of jet propellant-8 (JP-8) fuel and synthetic-8 (S-8) on cell viability and nitric oxide synthesis in cultured alveolar type II epithelial cells of rats. Exposure times varied from 0.25, 0.5, 1, and 6 hours at the following concentrations of jet fuel: 0.0, 0.1, 0.4, and 2.0 microg/mL. Data indicate that JP-8 presents a gradual decline in cell viability and steady elevation in nitric oxide release as exposure concentrations increase. At a 2.0 microg/mL concentration of JP-8, nearly all of the cells are not viable. Moreover, S-8 exposure to rat type II lung cells demonstrated an abrupt fall in percentage cell viability and increases in nitric oxide measurement, particularly after the 2.0 microg/mL was reached at 1 and 6 hours. At 0.0, 0.2, and 0.4 microg/mL concentrations of S-8, percentage viability was sustained at steady concentrations. The results suggest different epithelial toxicity and mechanistic effects of S-8 and JP-8, providing further insight concerning the impairment imposed at specific levels of lung function and pathology induced by the different fuels.
Assuntos
Hidrocarbonetos/toxicidade , Alvéolos Pulmonares/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Técnicas In Vitro , Óxido Nítrico/análise , Óxido Nítrico/biossíntese , Alvéolos Pulmonares/química , Ratos , Mucosa Respiratória/química , Fatores de TempoRESUMO
Neprilysin (NEP) is a key cell surface peptidase in the maintenance of airway homeostasis and the development of pulmonary disorders. However, little information is available about the effect of particulate matter (PM) on airway NEP. In this controlled human exposure study, changes in induced sputum were measured in 11 subjects at baseline, overshot (OS) mucking, and diesel exhaust (DE) exposure days. Neither OS condition nor DE exposure was found to induce significant changes in total protein, but DE induced significant increases in cell numbers of macrophages and epithelium. Moreover, significant increases in soluble NEP were observed following OS mining dust particulates (0.43 +/- 0.06 nmol/microg protein/min; p = .023) and DE exposure (0.40 +/- 0.03 nmol/microg protein/min; p = .035) when compared with the baseline control (0.30 +/- 0.04 nmol/microg protein/min), with 42% and 31% average net increase, respectively. Pearson's correlation analyses indicated that sputum NEP activity was significantly associated with personal exposure product (elemental carbon concentration [mg/m(3)] x time [min]; C x T). The data suggest that changes in NEP activity may be an early, accurate endpoint for airway epithelial injury and provide a new insight into the mechanism of airway effects following particulate exposure.
Assuntos
Poluentes Ocupacionais do Ar/toxicidade , Exposição por Inalação/análise , Mineração , Neprilisina/metabolismo , Material Particulado/toxicidade , Escarro/enzimologia , Adulto , Biomarcadores/análise , Contagem de Células , Feminino , Humanos , Exposição por Inalação/efeitos adversos , Masculino , Neprilisina/análise , Escarro/citologia , Testes de Toxicidade , Adulto JovemRESUMO
No current studies have systematically examined pulmonary health effects associated with Syntroleum S-8 synthetic jet fuel (S-8). In order to gain an understanding about the threshold concentration in which lung injury is observed, C57BL/6 male mice were nose-only exposed to S-8 for 1 h/day for 7 days at average concentrations of 0 (control), 93, 352, and 616 mg/m(3). Evaluation of pulmonary function, airway epithelial barrier integrity, and pathohistology was performed 24 h after the final exposures. Significant decreases were detected in expiratory lung resistance and total lung compliance of the 352 mg/m(3) group, for which no clear concentration-dependent alterations could be determined. No significant changes in respiratory permeability were exhibited, indicating that there was no loss of epithelial barrier integrity following S-8 exposure. However, morphological examination and morphometric analysis of distal lung tissue, by using transmission electron microscopy, revealed cellular damage in alveolar type II epithelial cells, with significant increases in volume density of lamellar bodies/vacuoles at 352 and 616 S-8 mg/m(3). Moreover, terminal bronchiolar Clara injury, as evidenced by apical membrane blebs, was observed at relatively low concentrations, suggesting if this synthetic jet fuel is utilized, the current permissible exposure limit of 350 mg/m(3) for hydrocarbon fuels should cautiously be applied.
Assuntos
Bronquíolos/efeitos dos fármacos , Hidrocarbonetos/toxicidade , Pulmão/efeitos dos fármacos , Alvéolos Pulmonares/efeitos dos fármacos , Resistência das Vias Respiratórias/efeitos dos fármacos , Análise de Variância , Animais , Bronquíolos/citologia , Desenho de Equipamento , Hidrocarbonetos/administração & dosagem , Exposição por Inalação , Pulmão/citologia , Pulmão/fisiologia , Complacência Pulmonar/efeitos dos fármacos , Masculino , Concentração Máxima Permitida , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/patologia , Testes de ToxicidadeRESUMO
This study was designed to characterize and compare the pulmonary effects in distal lung from a low-level exposure to jet propellant-8 fuel (JP-8) and a new synthetic-8 fuel (S-8). It is hypothesized that both fuels have different airway epithelial deposition and responses. Consequently, male C57BL/6 mice were nose-only exposed to S-8 and JP-8 at average concentrations of 53mg/m(3) for 1h/day for 7 days. A pulmonary function test performed 24h after the final exposure indicated that there was a significant increase in expiratory lung resistance in the S-8 mice, whereas JP-8 mice had significant increases in both inspiratory and expiratory lung resistance compared to control values. Neither significant S-8 nor JP-8 respiratory permeability changes were observed compared to controls, suggesting no loss of epithelial barrier integrity. Morphological examination and morphometric analysis of airway tissue demonstrated that both fuels showed different patterns of targeted epithelial cells: bronchioles in S-8 and alveoli/terminal bronchioles in JP-8. Collectively, our data suggest that both fuels may have partially different deposition patterns, which may possibly contribute to specific different adverse effects in lung ventilatory function.
Assuntos
Epitélio/efeitos dos fármacos , Hidrocarbonetos/farmacologia , Pulmão/efeitos dos fármacos , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Testes de Função RespiratóriaRESUMO
Four groups of Fischer Brown Norway hybrid rats were exposed for 5, 10, 15, or 20 d to aerosolized-vapor jet propulsion fuel 8 (JP-8) compared to freely moving (5 and 10-d exposures) or sham-confined controls (15 and 20-d exposures). Behavioral testing utilized the U.S. Environmental Protection Agency Functional Observational Battery. Exploratory ethological factor analysis identified three salient factors (central nervous system [CNS] excitability, autonomic 1, and autonomic 2) for use in profiling JP-8 exposure in future studies. The factors were used as dependent variables in general linear modeling. Exposed animals were found to engage in more rearing and hyperaroused behavior compared to controls, replicating prior JP-8 exposure findings. Exposed animals also showed increasing but rapidly decelerating stool output (autonomic 1), and a significant increasing linear trend for urine output (autonomic 2). No significant trends were noted for either of the control groups for the autonomic factors. Rats from each of the groups for each of the time frames were randomly selected for tissue assay from seven brain regions for neurotransmitter levels. Hippocampal DOPAC was significantly elevated after 4-wk JP-8 exposure compared to both control groups, suggesting increased dopamine release and metabolism. Findings indicate that behavioral changes do not appear to manifest until wk 3 and 4 of exposure, suggesting the need for longitudinal studies to determine if these behaviors occur due to cumulative exposure, or due to behavioral sensitization related to repeated exposure to aerosolized-vapor JP-8.
Assuntos
Aerossóis/toxicidade , Poluentes Ocupacionais do Ar/toxicidade , Comportamento Animal/efeitos dos fármacos , Hidrocarbonetos/toxicidade , Neurotransmissores/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Administração por Inalação , Análise de Variância , Animais , Nível de Alerta/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Modelos Animais de Doenças , Modelos Lineares , Atividade Motora/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344RESUMO
The effects of JP-8 on pro-inflammatory cytokine interleukin (IL)-1alpha,beta and nitric oxide (NO) secretion as well as the role of substance P (SP) in these processes were examined in cultured alveolar macrophages (AM), type II epithelial cells (AIIE), and AM/AIIE co-cultures. Exposure of AM to JP-8 for 24 hr exhibited release of IL-1alpha,beta, whereas exposure to AIIE showed a concentration-dependent NO overproduction. Data indicate that there are cell-dependent inflammatory mechanisms responsible for the actual level of JP-8 exposure in alveoli. However, treatment with substance P significantly attenuated JP-8 induced the IL-1alpha,beta secretion. This finding was confirmed by using [Sar(9) Met (O(2))(11)] SP (10(- 10) M), an agonist of substance P, suggesting that substance P may have signal pathway(s) to AM in the inflammatory response mediated by IL-1. Moreover, AM/AIIE co-culture obviously reduced NO overproduction observed in AIIE alone, suggesting that there may be cell interactions or communications between AM and AIIE in response to the JP-8 exposure.
RESUMO
Using an in-line, real-time, in vivo exposure system, we investigated whether acute adverse effects of diesel exhaust (DE*) exposure involve neurogenic inflammation in the lungs via sensory nerve C fibers. A total of 168 female F344 rats (175 g, 8 weeks old) were randomly assigned to pretreatment with capsaicin or saline to deplete C-fiber neurotransmitters. In a 2 x 3 factorial design, groups of animals were then exposed nose-only to a low level of DE (LDE, 35.3 microg/m3), a high level of DE (HDE, 632.9 microg/m3), or side-stream cigarette smoke (CS, 0.4 mg/m3). Two control groups were exposed whole body to filtered air in the animal room (fRA) or unfiltered air in the diesel engine room (eRA), respectively. DE was taken directly from a heavy-duty Cummins N14 research engine operated at 75% throttle (California Air Resources Board [CARB] 8, mode 6). Exposure to DE or air was 4 hours/day, 5 days/week, for 3 weeks. Exposure to CS was for 4 hours/day for 7 days. Involvement of neurogenic inflammation in the response to DE or CS was assessed via comparison of plasma extravasation, a sensitive endpoint of neurogenic inflammation, between rats with and without capsaicin pretreatment. Lung injury was assessed via analysis of proinflammatory cytokines, respiratory permeability, and histopathology. Moreover, whether DE exposure affected the molecular mechanisms of neurogenic inflammation was analyzed through quantification of substance P (SP) and its primary neurokinin-1 (NK1) receptor at the gene and protein levels and through neutral endopeptidase (NEP) activity. DE and CS exposure induced dose-dependent plasma extravasation, which may play an important role in initiating the associated lung inflammation and injury. Exposure of rats to DE affected the SP signaling pathway as indicated by overexpression of the NK1 receptor or reduction of SP in the lung tissue. DE exposure consistently inactivated tissue NEP, a key factor that switches neurogenic inflammation from its physiological and protective functions to a role that increases and perpetuates lung injury. The roles of these overlapping neurokininergic mechanisms in the initiation of DE-associated lung injury are plausible, and these changes may contribute to DE-associated respiratory disorders. Capsaicin rats followed the same trends as those of saline animals when exposed to DE or CS: capsaicin rats did not have significantly different plasma extravasation in the airways or lung parenchyma compared to their corresponding controls. Histopathology evaluation likewise demonstrated the same degree of tissue changes, such as edema and alveolar macrophage collection, in capsaicin and saline rats after the same level of DE exposure. In summary, our data suggest that neurokininergic mechanisms may have been involved in DE-induced inflammatory conditions in rat lung but that C fibers did not appear to be involved under these exposure conditions. We believe that time-course or protein knockdown/knockout animal studies are required to characterize further the role of neurokininergic mechanisms in DE-induced lung injury.
Assuntos
Pulmão/efeitos dos fármacos , Inflamação Neurogênica/induzido quimicamente , Emissões de Veículos/toxicidade , Administração Intranasal , Animais , Feminino , Pulmão/inervação , Ratos , Ratos Endogâmicos F344RESUMO
To evaluate the role of substance P (SP)-containing C-fiber nerves in the development of the inflammatory responses to sidestream cigarette smoke (SSCS), female Fischer 344 rats were randomly assigned into vehicle and capsaicin groups, respectively. Then, half the number in each group (N = 24) was nose-only exposed to air or 0.4 mg/m3 total particulate matter of SSCS for 4 h/day for 7 days. Exposure of the vehicle rats to SSCS induced obvious pulmonary neurogenic inflammation as indicated by elevations in plasma extravasation and proinflammatory cytokine secretions [interieukin (IL)-1beta and IL-12]. In addition, except for SP release, SSCS exposure significantly induced the tachykininergic toxicities at the gene level: upregulation of beta-preprotachykinin-I (beta-PPT-I) mRNA. However, neither SSCS exposure nor capsaicin pretreatment affects the immunolabeling density of neurokinin-1 receptor (NK-1R) in airway epithelium. SSCS also significantly inactivated pulmonary neutral endopeptidase (NEP) in lung tissue. Moreover, pretreatment with capsaicin significantly exacerbated the SSCS-induced inflammatory responses mentioned above as well as the release of plasma protein. Considering that capsaicin did not affect the normal control baselines of these parameters except for a decrease in NK-1R mRNA, we conclude that the degree of SSCS-induced inflammatory response was exacerbated because of the depletion of stored SP and/or inactivation of capsaicin-sensitive C-fiber nerves. Our data suggest the loss of afferent tachykinin SP signaling may lead to dysfunction of the sensory C-fiber nerve reflexes during exposure to SSCS, suggesting that SP serves a protective role.
Assuntos
Capsaicina/farmacologia , Pulmão/efeitos dos fármacos , Fumaça/efeitos adversos , Substância P/fisiologia , Taquicininas/efeitos dos fármacos , Animais , Citocinas/metabolismo , Feminino , Pulmão/enzimologia , Pulmão/metabolismo , Neprilisina/metabolismo , Ratos , Ratos Endogâmicos F344 , Receptores da Neurocinina-1/efeitos dos fármacos , Receptores da Neurocinina-1/genética , Substância P/deficiência , Taquicininas/metabolismoRESUMO
To characterize the tachykininergic effects in fire smoke (FS)-induced acute respiratory distress syndrome (ARDS), we designed a series of studies in rats. Initially, 20 min of FS inhalation induced a significant increase of substance P (SP) in bronchoalveolar lavage fluid (BALF) at 1 h and persisted for 24 h after insult. Conversely, FS disrupted 51.4, 55.6, 46.3, and 43.0% enzymatic activity of neutral endopeptidase (NEP, a primary hydrolyzing enzyme for SP) 1, 6, 12, and 24 h after insult, respectively. Immunolabeling density of NEP in the airway epithelium largely disappeared 1 h after insult due to acute cell damage and shedding. These changes were also accompanied by extensive influx of albumin and granulocytes/lymphocytes in BALF. Furthermore, levels of BALF SP and tissue NEP activity dose dependently increased and decreased, respectively, following 0, low (10 min), and high (20 min) levels of FS inhalation. However, neither the time-course nor the dose-response study observed a significant change in the highest affinity neurokinin-1 receptor (NK-1R) for SP. Finally, treatment (10 mg/kg im) with SR-140333B, an NK-1R antagonist, significantly prevented 20-min FS-induced hypoxemia and pulmonary edema 24 h after insult. Further examination indicated that SR-140333B (1.0 or 10.0 mg/kg im) fully abolished early (1 h) plasma extravasation following FS. Collectively, these findings suggest that a combination of sustained SP and NEP inactivity induces an exaggerated neurogenic inflammation mediated by NK-1R, which may lead to an uncontrolled influx of protein-rich edema fluid and cells into the alveoli as a consequence of increased vascular permeability.