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BACKGROUND: In Canada, severe asthma affects an estimated 5-10% of people with asthma and is associated with frequent exacerbations, poor symptom control and significant morbidity from the disease itself, as well as the high dose inhaled, and systemic steroids used to treat it. Significant heterogeneity exists in service structure and patient access to severe asthma care, including access to biologic treatments. There appears to be over-reliance on short-acting beta agonists and frequent oral corticosteroid use, two indicators of uncontrolled asthma which can indicate undiagnosed or suboptimally treated severe asthma. The objective of this modified Delphi consensus project was to define standards of care for severe asthma in Canada, in areas where the evidence is lacking through patient and healthcare professional consensus, to complement forthcoming guidelines. METHODS: The steering group of asthma experts identified 43 statements formed from eight key themes. An online 4-point Likert scale questionnaire was sent to healthcare professionals working in asthma across Canada to assess agreement (consensus) with these statements. Consensus was defined as high if ≥ 75% and very high if ≥ 90% of respondents agreed with a statement. RESULTS: A total of 150 responses were received from HCPs including certified respiratory educators, respirologists, allergists, general practitioners/family physicians, nurses, pharmacists, and respiratory therapists. Consensus amongst respondents was very high in 37 (86%) statements, high in 4 (9%) statements and was not achieved in 2 (5%) statements. Based on the consensus scores, ten key recommendations were proposed. These focus on referrals from primary and secondary care, accessing specialist asthma services, homecare provision for severe asthma patients and outcome measures. CONCLUSIONS: Implementation of these recommendations across the severe asthma care pathway in Canada has the potential to improve outcomes for patients through earlier detection of undiagnosed severe asthma, reduction in time to severe asthma diagnosis, and initiation of advanced phenotype specific therapies.
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INTRODUCTION: Lipid nanoparticles (LNP) have been successfully used as a platform technology for delivering nucleic acids to the liver. To broaden the application of LNPs in targeting non-hepatic tissues, we developed LNP-based RNA therapies (siRNA or mRNA) for the respiratory tract. Such optimized LNP systems could offer an early treatment strategy for viral respiratory tract infections such as COVID-19. METHODS: We generated a small library of six LNP formulations with varying helper lipid compositions and characterized their hydrodynamic diameter, size distribution and cargo entrapment properties. Next, we screened these LNP formulations for particle uptake and evaluated their potential for transfecting mRNA encoding green fluorescence protein (GFP) or SARS-CoV2 nucleocapsid-GFP fusion reporter gene in a human airway epithelial cell line in vitro. Following LNP-siGFP delivery, GFP protein knockdown efficiency was assessed by flow cytometry to determine %GFP+ cells and median fluorescence intensity (MFI) for GFP. Finally, lead LNP candidates were validated in Friend leukemia virus B (FVB) male mice via intranasal delivery of an mRNA encoding luciferase, using in vivo bioluminescence imaging. RESULTS: Dynamic light scattering revealed that all LNP formulations contained particles with an average diameter of <100 nm and a polydispersity index of <0.2. Human airway epithelial cell lines in culture internalized LNPs with differential GFP transfection efficiencies (73-97%). The lead formulation LNP6 entrapping GFP or Nuc-GFP mRNA demonstrated the highest transfection efficiency (97%). Administration of LNP-GFP siRNA resulted in a significant reduction of GFP protein expression. For in vivo studies, intranasal delivery of LNPs containing helper lipids (DSPC, DOPC, ESM or DOPS) with luciferase mRNA showed significant increase in luminescence expression in nasal cavity and lungs by at least 10 times above baseline control. CONCLUSION: LNP formulations enable the delivery of RNA payloads into human airway epithelial cells, and in the murine respiratory system; they can be delivered to nasal mucosa and lower respiratory tract via intranasal delivery. The composition of helper lipids in LNPs crucially modulates transfection efficiencies in airway epithelia, highlighting their importance in effective delivery of therapeutic products for airways diseases.
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COVID-19 , Nanopartículas , Animales , Proteínas Fluorescentes Verdes/genética , Humanos , Lípidos , Liposomas , Masculino , Ratones , ARN Mensajero/genética , ARN Interferente Pequeño , ARN Viral , Sistema Respiratorio/metabolismo , SARS-CoV-2RESUMEN
Genome-wide association studies have linked gene variants of the receptor patched homolog 1 (PTCH1) with chronic obstructive pulmonary disease (COPD). However, its biological role in the disease is unclear. Our objective was to determine the expression pattern and biological role of PTCH1 in the lungs of patients with COPD. Airway epithelial-specific PTCH1 protein expression and epithelial morphology were assessed in lung tissues of control and COPD patients. PTCH1 mRNA expression was measured in bronchial epithelial cells obtained from individuals with and without COPD. The effects of PTCH1 siRNA knockdown on epithelial repair and mucous expression were evaluated using human epithelial cell lines. Ptch1+/- mice were used to assess the effect of decreased PTCH1 on mucous expression and airway epithelial phenotypes. Airway epithelial-specific PTCH1 protein expression was significantly increased in subjects with COPD compared to controls, and its expression was associated with total airway epithelial cell count and thickness. PTCH1 knockdown attenuated wound closure and mucous expression in airway epithelial cell lines. Ptch1+/- mice had reduced mucous expression compared to wildtype mice following mucous induction. PTCH1 protein is up-regulated in COPD airway epithelium and may upregulate mucous expression. PTCH1 provides a novel target to reduce chronic bronchitis in COPD patients.
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Bronquios/metabolismo , Receptor Patched-1/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Transducción de Señal , Adulto , Anciano , Animales , Epitelio/metabolismo , Femenino , Silenciador del Gen , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Receptor Patched-1/genéticaRESUMEN
BACKGROUND: Conjugated linoleic acids (CLA) are naturally occurring fatty acids that have multiple biological properties including the regulation of metabolic, proliferative and immune processes. OBJECTIVE: The aim of this study was to investigate the efficacy and safety of CLA as a dietary supplement in mild asthma. METHODS: This was a prospective, randomized, double-blind, placebo-controlled study. Twenty-eight adult subjects (aged 19-40 years) with mild asthma (FEV(1)>70% predicted) were randomized to CLA 4.5 g/day or placebo for 12 weeks in addition to usual treatment. On average, subjects were overweight with a mean body mass index (BMI) of 27.9 kg/m(2). RESULTS: Subjects in the CLA group had a significant improvement in airway hyperresponsiveness (AHR) at week 12 compared with week 0 [PC(20) 6.6 (2.1) mg/mL vs. 2.2 (0.7) mg/mL; P<0.05]. The CLA group had a significant reduction in weight and BMI compared with placebo and this was associated with a reduction in leptin/adiponectin ratio. There were no differences in systemic cytokine levels, induced sputum cell counts, quality-of-life scores or adverse events. CONCLUSIONS: CLA treatment as an adjunct to usual care in overweight mild asthmatics was well tolerated and was associated with improvements in AHR and BMI.
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Antiasmáticos/uso terapéutico , Asma/complicaciones , Asma/tratamiento farmacológico , Ácidos Linoleicos Conjugados/uso terapéutico , Sobrepeso/complicaciones , Adulto , Asma/inmunología , Suplementos Dietéticos , Método Doble Ciego , Femenino , Humanos , MasculinoRESUMEN
BACKGROUND: Epidermal growth factor receptor (EGFR) is a major regulator of airway epithelial cell (AEC) functions such as migration, proliferation and differentiation, which play an essential role in epithelial repair. EGFR is a glycoprotein with 12 potential N-glycosylation sites in its extracellular domain. Glycosylation of EGFR has been shown to modulate its function. Previously, our laboratory demonstrated an important role of the carbohydrate structure sialyl Lewis x (sLe(x)) in airway epithelial repair. OBJECTIVE: To examine whether an sLe(x) decoration of EGFR can modulate receptor function during AEC repair. METHODS: Primary normal human bronchial epithelial (NHBE) cells were cultured in vitro. Co-localization of sLe(x) and EGFR was examined using confocal microscopy. Expressions of RNA and protein were analysed using RT-PCR and Western blotting. The final step in the synthesis of sLe(x) was catalysed by a specific alpha-1,3-fucosyltransferase (FucT-IV). To evaluate the role of sLe(x) in EGFR activation, a knockdown of the FucT-IV gene with small interfering RNA (siRNA) and an inhibitory anti-sLe(x) antibody (KM-93) was used. RESULTS: We demonstrated a co-localization of sLe(x) with EGFR on NHBE cells using confocal microscopy. Using a blocking antibody for sLe(x) after a mechanical injury, we observed a reduction in EGFR phosphorylation and epithelial repair following injury. FucT-IV demonstrates a temporal expression coordinate with epithelial repair. Down-regulation of FucT-IV expression in NHBE by specific siRNA suppressed sLe(x) expression. The use of FucT-IV siRNA significantly reduced phosphorylation of EGFR and prevented epithelial repair. An immunohistochemical analysis of human normal and asthmatic airways showed a significant reduction in sLe(x) and tyrosine-phosphorylated EGFR (pY(845)-EGFR) in the epithelium of asthmatic subjects compared with that of normal subjects. CONCLUSION: The present data demonstrate that sLe(x), in association with EGFR, in NHBE is coordinate with repair. This glycosylation is important in modulating EGFR activity to affect the repair of normal primary AEC.
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Bronquios , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Regulación de la Expresión Génica , Oligosacáridos/metabolismo , Cicatrización de Heridas/fisiología , Bronquios/lesiones , Bronquios/metabolismo , Células Cultivadas , Receptores ErbB/genética , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Glicosilación , Humanos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Antígeno Sialil Lewis XRESUMEN
Fas-mediated apoptosis of the alveolar epithelium is important in the pathogenesis of acute respiratory distress syndrome. Erythropoietin (EPO) has cytoprotective properties in other organ systems, and is relatively deficient in critical illness. This study investigates a potential role for EPO in reducing apoptosis in a model of acute lung injury. Apoptosis was induced in human alveolar epithelial (A549) cells or normal human bronchial epithelial (NHBE) cells by Fas activation with CH-11 Fas-crosslinking antibody or by co-culture with polymorphonuclear neutrophils in a transwell system. The effect of recombinant human (rh)EPO on apoptosis was measured by poly(ADP-ribose) polymerase cleavage and cell death detection assay. The specific EPO-EPO receptor (EPOR)-mediated effect was determined using an EPO-blocking antibody or EPOR small interfering RNA. Expression of EPOR was demonstrated in A549, NHBE and normal human alveolar epithelium. Fas- and neutrophil-mediated apoptosis of A549 and NHBE cells was inhibited by rhEPO by a specific EPO-EPOR-mediated mechanism. This anti-apoptotic effect was associated with induction of a pro-apoptotic Bcl-xL/Bax ratio. EPO has cytoprotective properties in respiratory epithelium in an in vitro model, which may indicate a potential therapeutic role in acute lung injury.
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Lesión Pulmonar Aguda/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Células Epiteliales/metabolismo , Eritropoyetina/farmacología , Análisis de Varianza , Western Blotting , Células Cultivadas , Técnicas de Cocultivo , Citometría de Flujo , Humanos , Inmunohistoquímica , Lipopolisacáridos , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Regulación hacia Arriba , Receptor fas/metabolismoRESUMEN
BACKGROUND: The airway epithelium acts as a protective barrier, separating the external environment from the underlying tissue. Daily challenges result in damage to the epithelium that, in normal individuals, is quickly and effectively repaired. In respiratory diseases, such as asthma, this repair is compromised. Following injury to the epithelium, plasma leaks into the airway lumen acting as a protective protein cap. Carbohydrates are mediators of epithelial repair, however, the associated regulatory proteins remain unknown. OBJECTIVE: To identify mediators of epithelial repair based on their carbohydrate moieties using an in vitro wound repair culture model of human airway epithelial cells (1HAEo(-)). METHODS: Using the lectin Allomyrina dichotoma agglutinin (AlloA) as a tool, ligands essential in the repair of damaged epithelium were characterized. AlloA was subsequently used to purify and identify a glycoprotein associated with epithelial repair. RESULTS: The addition of AlloA to the media of mechanically wounded monolayers inhibited repair. Fetuin, a highly glycosylated serum protein, was identified as a glycoprotein bound by AlloA. The addition of fetuin to serum starved monolayers stimulated wound closure. CONCLUSION: These results indicate that following mechanical injury to the epithelium, serum glycoproteins, not only provide a protective barrier, but also are involved in the initiation of wound closure.
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Asma/fisiopatología , Células Epiteliales/fisiología , Lectinas/farmacología , Sistema Respiratorio/patología , Animales , Asma/patología , Proteínas Sanguíneas/metabolismo , Células Cultivadas , Precipitación Química , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Glicoproteínas/aislamiento & purificación , Glicoproteínas/metabolismo , Humanos , Lectinas/metabolismo , Modelos Biológicos , Lectinas de Plantas/metabolismo , Lectinas de Plantas/farmacología , Sistema Respiratorio/efectos de los fármacos , Cicatrización de Heridas/fisiología , alfa-Fetoproteínas/farmacología , Proteínas Séricas GlicadasRESUMEN
Epithelia are the layers of cells that form barriers between external milieu and underlying tissues and thus, are important components of most organs of the body. Epithelial layers of organs, such as the lung, are exposed to various challenges resulting in frequent injury. Epithelial wound healing represents an important process by which repair restores the physical barrier lost as a result of cell damage and apoptosis. The repair of epithelial layers consists of a series of ordered events including epithelial cell spreading, migration proliferation and, differentiation. Carbohydrates attached to cell surface proteins and lipids can modulate the function of structures that they are conjugated to and therefore, can affect cell behavior. Although the basic mechanisms of epithelial repair are not entirely understood, many studies suggest glycoconjugates attached to proteins on the cell surface of epithelial cells play important roles in many of these cellular processes. In the present review, the role of carbohydrates in epithelial repair of different organs, including the sources of epithelial injury and current models of epithelial repair will be discussed with a focus on our understanding of the airway epithelium. With a better understanding of carbohydrates and their role in epithelial repair, new therapeutic targets for diseases involving damage to the epithelium can be identified.
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Carbohidratos/fisiología , Epitelio/fisiología , Animales , Anexinas/fisiología , Comunicación Celular , Movimiento Celular , Transformación Celular Neoplásica , Galectinas/fisiología , Glicoconjugados/fisiología , Glicosilación , Humanos , Polisacáridos/fisiología , Procesamiento Proteico-Postraduccional , Regeneración , Cicatrización de HeridasRESUMEN
Damage to the airway epithelium is one prominent feature of chronic asthma. Corticosteroids induce apoptosis in inflammatory cells, which in part explains their ability to suppress airway inflammation. However, corticosteroid therapy does not necessarily reverse epithelial damage. We hypothesized that corticosteroids may induce airway epithelial cell apoptosis as one potential explanation for persistent damage. We tested this hypothesis in cultured primary central airway epithelial cells and in the cell line 1HAEo(-). Treatment with dexamethasone, beclomethasone, budesonide, or triamcinolone each elicited a time-dependent and concentration-dependent cell death. This cell death was associated with cleavage of nuclear chromatin, mitochondrial depolarization, cytochrome c extrusion, activation of caspase-9, and expression of phosphatidylserine on the outer cell membrane. Inhibitors of caspase activity blocked apoptotic cell death, as did overexpression of the apoptosis regulators Bcl-2 or Bcl-x(L). We demonstrated that CD95 ligation is not essential for the corticosteroid-induced apoptosis in airway epithelial cells. These data demonstrate that corticosteroids induce apoptotic cell death of airway epithelium. This raises the possibility that at least one of the major components of chronic airway damage in asthma, epithelial shedding and denudation, may in part result from a major therapy for the disease.
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Antiasmáticos/efectos adversos , Antiinflamatorios/efectos adversos , Apoptosis/efectos de los fármacos , Asma/tratamiento farmacológico , Asma/patología , Beclometasona/efectos adversos , Broncodilatadores/efectos adversos , Budesonida/efectos adversos , Dexametasona/efectos adversos , Receptores de Interleucina-2/efectos de los fármacos , Mucosa Respiratoria/citología , Mucosa Respiratoria/efectos de los fármacos , Triamcinolona/efectos adversos , Apoptosis/fisiología , Asma/inmunología , Caspasa 9 , Caspasas/efectos de los fármacos , Caspasas/fisiología , Células Cultivadas/efectos de los fármacos , Enfermedad Crónica , Grupo Citocromo c/efectos de los fármacos , Grupo Citocromo c/fisiología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Genes bcl-2/efectos de los fármacos , Genes bcl-2/fisiología , Humanos , Inflamación , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Receptores de Interleucina-2/fisiología , Mucosa Respiratoria/inmunología , Factores de Tiempo , Proteína bcl-X , Receptor fasRESUMEN
Our laboratory recently demonstrated the pattern of cell surface glycosylation of nonsecretory central airway epithelium (Dorscheid DR, Conforti AE, Hamann KJ, Rabe KF, and White SR. Histochem J 31: 145-151, 1999), but the role of glycosylation in airway epithelial cell migration and repair is unknown. We examined the functional role of cell surface carbohydrates in wound repair after mechanical injury of 1HAEo(-) human airway epithelial and primary bronchial epithelial monolayers. Wound repair stimulated by epidermal growth factor was substantially attenuated by 10(-7) M tunicamycin (TM), an N-glycosylation inhibitor, but not by the inhibitors deoxymannojirimycin or castanospermine. Wound repair of 1HAEo(-) and primary airway epithelial cells was blocked completely by removal of cell surface terminal fucose residues by alpha-fucosidase. Cell adhesion to collagen matrix was prevented by TM but was only reduced ~20% from control values with prior alpha-fucosidase treatment. Cell migration in Blind Well chambers stimulated by epidermal growth factor was blocked by pretreatment with TM but alpha-fucosidase pretreatment produced no difference from control values. These data suggest that cell surface N-glycosylation has a functional role in airway epithelial cell adhesion and migration and that N-glycosylation with terminal fucosylation plays a role in the complex process of repair by coordination of certain cell-cell functions.
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Fucosa/metabolismo , Proteínas de la Membrana/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Antibacterianos/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Línea Celular Transformada , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Glicosilación/efectos de los fármacos , Humanos , Lectinas , Tunicamicina/farmacología , Cicatrización de Heridas/fisiología , alfa-L-Fucosidasa/farmacologíaRESUMEN
Changes in epithelial cell shape can lead to cell death and detachment. Actin filaments are cleaved during apoptosis, but whether disruption in the actin cytoskeletal network, as one manifestation of cell shape change, can itself induce apoptosis is not known. We tested this hypothesis in the airway epithelial cell line 1HAEo(-) and in primary airway epithelial cells by preventing actin filament elongation with cytochalasin D or by aggregating actin filaments with jasplakinolide. Disruption of actin filament integrity promptly induced apoptosis in adherent epithelial cells within 5 h. Jasplakinolide-induced apoptosis did not disrupt focal adhesions, whereas cytochalasin D-induced apoptosis decreased focal adhesion protein expression and occurred despite ligation of the fibronectin receptor. Death induction was abrogated by the caspase inhibitors z-VAD-fmk and Ac-DEVD-cho but not by blocking the Fas (CD95) receptor. Whereas cytochalasin D--induced apoptosis was associated with cleavage of pro-caspase-8, jasplakinolide-induced apoptosis was not. Both agents induced formation of a death-inducing signaling complex. These data demonstrate that disruption of actin filament integrity with either cytochalasin D or jasplakinolide induces apoptosis in airway epithelial cells but by different mechanisms, and suggest that actin may be an early modulator of apoptotic commitment.
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Actinas/fisiología , Apoptosis/fisiología , Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , Depsipéptidos , Mucosa Respiratoria/citología , Mucosa Respiratoria/fisiología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 8 , Caspasa 9 , Caspasas/metabolismo , Adhesión Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Citocalasina D/farmacología , Proteínas del Citoesqueleto/análisis , Perros , Adhesiones Focales , Humanos , Riñón , Cinética , Paxillin , Péptidos Cíclicos/farmacología , Fosfoproteínas/análisis , Receptores de Fibronectina/fisiología , Vinculina/análisisRESUMEN
Dystroglycans (DGs) bind laminin matrix proteins in skeletal and cardiac muscle and are expressed in other nonmuscle tissues. However, their expression in airway epithelial cells has not been demonstrated. We examined expression of DGs in the human airway epithelial cell line 1HAEo(-), and in human primary airway epithelial cells. Expression of the common gene for alpha- and beta-DG was demonstrated by reverse transcriptase/ polymerase chain reaction in 1HAEo(-) cells. Protein expression of beta-DG was demonstrated by both Western blot and flow cytometry in cultured cells. Localization of alpha-DG, using both a monoclonal antibody and the alpha-DG binding lectin wheat-germ agglutinin (WGA), was to the cell membrane and nucleus. We then examined the function of DGs in modulating wound repair over laminin matrix. Blocking alpha-DG binding to laminin in 1HAEo(-) monolayers using either glycosyaminoglycans or WGA attenuated cell migration and spreading after mechanical injury. alpha-DG was not expressed in epithelial cells at the wound edge immediately after wound creation, but localized to the cell membrane in these cells within 12 h of injury. These data demonstrate the presence of DGs in airway epithelium. alpha-DG is dynamically expressed and serves as a lectin to bind laminin during airway epithelial cell repair.
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Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/fisiología , Glicoproteínas de Membrana/metabolismo , Cicatrización de Heridas/fisiología , Unión Competitiva , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/patología , Movimiento Celular , Células Cultivadas/citología , Proteínas del Citoesqueleto/genética , Cartilla de ADN/química , Distroglicanos , Células Epiteliales/citología , Células Epiteliales/patología , Técnica del Anticuerpo Fluorescente , Humanos , Laminina/metabolismo , Pulmón/fisiología , Neoplasias Pulmonares , Glicoproteínas de Membrana/genética , Músculo Esquelético/patología , Reacción en Cadena de la Polimerasa , Receptores de Laminina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Tráquea/fisiología , Heridas y LesionesRESUMEN
Glycosylated structures on the cell surface have a role in cell adhesion, migration, and proliferation. Repair of the airway epithelium after injury requires each of these processes, but the expression of cell surface glycosylation of airway epithelial cells after injury is not known. We examined cell surface glycosylation using lectin-binding profiles of normal and repairing epithelia in Hartley guinea pigs from 0 to 14 days after mechanical injury. The epithelium regenerated completely-over 7 days. In normal trachea, galactose- or galactosamine-specific lectins (14 of 20 tested) labelled epithelial cells, but fucose, mannose, and other sugar-specific lectins (15 tested) did not. GSA-2, a glucosamine-specific lectin, labelled epithelial cells weakly in uninjured tracheas, but intense labelling was noted in basal and non-ciliated columnar cells adjacent to the injury site over 3 h to 14 days after injury. Labelling of these cells peaked at 12 h and 5 days after injury respectively. Similar patterns were seen with lectins AlloA and HAA but not with CPA during repair. The binding of the lectin DSA to proteins collected from primary cultures of airway epithelial cells decreased substantially after treatment for 24 h with either transforming growth factor-beta or interleukin-1beta, but that of the CPA lectin did not. We demonstrate changes in glycosylation profiles of airway epithelial cells coordinate with repair after mechanical injury. These changes may be useful to study mechanisms by which repair is regulated.
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Lectinas/metabolismo , Mucosa Respiratoria/metabolismo , Tráquea/metabolismo , Animales , Western Blotting/métodos , Glicosilación , Cobayas , Masculino , Mucosa Respiratoria/lesiones , Mucosa Respiratoria/patología , Tráquea/lesiones , Tráquea/patologíaRESUMEN
Hypertrophy and hyperplasia lead to excess accumulation of smooth muscle in the airways of human asthmatic subjects. However, little is known about mechanisms that might counterbalance these processes, thereby limiting the quantity of smooth muscle in airways. Ligation of Fas on the surface of vascular smooth muscle cells and nonmuscle airway cells can lead to apoptotic cell death. We therefore tested the hypotheses that 1) human airway smooth muscle (HASM) expresses Fas, 2) Fas cross-linking induces apoptosis in these cells, and 3) tumor necrosis factor (TNF)-alpha potentiates Fas-mediated airway myocyte killing. Immunohistochemistry using CH-11 anti-Fas monoclonal IgM antibody revealed Fas expression in normal human bronchial smooth muscle in vivo. Flow cytometry using DX2 anti-Fas monoclonal IgG antibody revealed that passage 4 cultured HASM cells express surface Fas. Surface Fas decreased partially during prolonged serum deprivation of cultured HASM cells and was upregulated by TNF-alpha stimulation. Fas cross-linking with CH-11 antibody induced apoptosis in cultured HASM cells, and this effect was reduced by long-term serum deprivation and synergistically potentiated by concomitant TNF-alpha exposure. TNF-alpha did not induce substantial apoptosis in the absence of Fas cross-linking. These data represent the first demonstration that Fas is expressed on HASM and suggest a mechanism by which Fas-mediated apoptosis could act to oppose excess smooth muscle accumulation during airway remodeling in asthma.
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Anticuerpos Monoclonales/farmacología , Apoptosis/fisiología , Bronquios/fisiología , Músculo Liso Vascular/fisiología , Receptor fas/inmunología , Receptor fas/fisiología , Apoptosis/efectos de los fármacos , Bronquios/citología , Bronquios/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Sinergismo Farmacológico , Humanos , Inmunohistoquímica , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Receptor fas/metabolismoRESUMEN
Glycosylated structures on the cell surface have a role in cell adhesion, migration, and proliferation. Repair of the airway epithelium after injury requires each of these processes, but the normal cell surface glycosylation of non-mucin producing airway epithelial cells is unknown. We examined cell surface glycosylation in human airway epithelial cells in tissue sections and in human airway epithelial cell lines in culture. Thirty-eight lectin probes were used to determine specific carbohydrate residues by lectin-histochemistry. Galactose or galactosamine-specific lectins labeled basal epithelial cells, lectins specific for several different carbohydrate structures bound columnar epithelial cells, and fucose-specific lectins labeled all airway epithelial cells. The epithelial cell lines 1HAEo- and 16HBE14o- bound lectins that were specific to basal epithelial cells. Flow cytometry of these cell lines with selected lectins demonstrated that lectin binding was to cell surface carbohydrates, and revealed possible hidden tissue antigens on dispersed cultured cells. We demonstrate specific lectin-binding patterns on the surface of normal human airway epithelial cells. The expression of specific carbohydrate residues may be useful to type epithelial cells and as a tool to examine cell events involved in epithelial repair.
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Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Lectinas/metabolismo , Pulmón/citología , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/metabolismo , Línea Celular Transformada/química , Línea Celular Transformada/ultraestructura , Citometría de Flujo , Glicoconjugados/biosíntesis , Histocitoquímica , Humanos , Pulmón/metabolismoRESUMEN
Repair of the airway epithelium after injury requires that processes such as adhesion and cell migration occur in a defined order. Both of these processes depend on interactions between extracellular matrix (ECM) proteins and appropriate integrins. To study these interactions, we examined monolayer wound repair in a cultured human airway epithelial cell line, 16HBE14o-. Wounds created in confluent monolayers grown on either collagen-IV, laminin-1, or laminin-2 matrix closed quickly in response to 15 ng/ml epidermal growth factor (EGF). Concurrent treatment of cells grown on each matrix protein with EGF and a monoclonal antibody (mAb) to beta1-integrin inhibited wound closure. Treatment with a mAb to alpha2-, alpha3-, and alpha6-integrin blocked wound repair in monolayers grown on collagen-IV but did not do so in monolayers grown either on laminin-1 or laminin-2. Inhibition was not due to cell detachment or apoptosis. These data demonstrate that integrins expressed by airway epithelial cells mediate wound closure on different constitutive ECM proteins. These data suggest that beta1-integrin subunit function is required to permit migration and spreading of epithelial cells, and that alpha-integrin subunits alone do not mediate migration of epithelial cells grown on either laminin-1 or laminin-2. These differences may become important if the matrix protein composition of airway basement membrane changes in disease states such as asthma.
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Apoptosis , Células Epiteliales/fisiología , Integrina beta1/fisiología , Receptores de Antígeno muy Tardío/fisiología , Cicatrización de Heridas , Anticuerpos Monoclonales/farmacología , Bronquios , División Celular , Línea Celular Transformada , Colágeno/fisiología , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/citología , Células Epiteliales/patología , Proteínas de la Matriz Extracelular/fisiología , Citometría de Flujo , Humanos , Integrina beta1/inmunología , Laminina/fisiología , Receptores de Antígeno muy Tardío/biosíntesis , Virus 40 de los SimiosRESUMEN
The cell surface molecule Fas (CD95) is a member of the tumor necrosis factor receptor family. Ligation of the Fas receptor can lead to induction of apoptosis in inflammatory cells. It has been suggested that expression of the Fas receptor and its ligand (FasL) in airway epithelium may modulate the inflammatory response commonly found in asthmatic lungs. We examined Fas and FasL expression on primary human tissues, on bronchial epithelial cells in primary culture, and on the immortalized human airway epithelial cell line, 1HAEo-. Receptor and ligand expression were demonstrated using multiple antibodies and multiple techniques, including immunohistochemistry, flow cytometry, Western blots, and reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemical staining demonstrated that both columnar and basal cells of intact human lung tissues expressed cell surface Fas and FasL. In addition, both primary cultured and immortalized 1HAEo- cells expressed cell surface Fas and FasL, as demonstrated by flow cytometry; expression of Fas and FasL was confirmed at the transcription level using RT-PCR and, for additional confirmation of FasL, using Western blots. We demonstrate that both Fas and FasL are expressed by human airway epithelial cell subtypes. Expression of these molecules may play an important role in regulation of the inflammatory response.
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Bronquios/citología , Células Epiteliales/fisiología , Glicoproteínas de Membrana/genética , Receptor fas/genética , Apoptosis/inmunología , Western Blotting , Bronquios/inmunología , Células Cultivadas , Células Epiteliales/química , Células Epiteliales/citología , Proteína Ligando Fas , Citometría de Flujo , Expresión Génica/inmunología , Humanos , Glicoproteínas de Membrana/análisis , Neumonía/inmunología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/inmunología , Receptor fas/análisisRESUMEN
The current management of malignant gliomas is unsatisfactory compared to that of other solid tumors; the expected median survival period is less than 1 year with the patient undergoing conventional surgery, radiotherapy, and chemotherapy treatment. Immunological reagents could be a useful adjunct. Human monoclonal antibodies derived from patients with astrocytic tumors might recognize subtle antigenic specificities that would differ from those recognized by xenogeneic (murine) systems. Five hybridomas, designated as BT27/1A2, BT27/2A3, BT32/A6, BT34/A5, and BT54/B8, were produced from the fusion of peripheral blood lymphocytes of four patients with astrocytic tumors to the human myeloma-like cell line TM-H2-SP2. This cell line has a 46, XX karyotype and is negative for hypoxanthine guanine phosphoribosyltransferase. All five human monoclonal antibodies produced 2.4 to 44 micrograms/ml of immunoglobulin M, had a similar but not identical pattern of reactivity against a panel of human tumor cell lines, and failed to react with normal human astrocytes. Labeling of four neuroectodermal tumor explant cultures by BT27/2A3 was demonstrated by flow cytometry. Karyotyping of three of the five hybridomas demonstrated that two were pseudodiploid (2-3n) and one hypodiploid (less than 2n). The monoclonality of the hybridomas was evaluated by Southern blot analysis of JH gene rearrangements, revealing two types of rearrangements for each hybridoma, both consistent with monoclonality. Preliminary antigen characterization indicated that at least four of the five human monoclonal antibodies were directed to cell-surface glycolipids.
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Anticuerpos Monoclonales/biosíntesis , Glioma/inmunología , Hibridomas/inmunología , Inmunoglobulina M/biosíntesis , Adolescente , Adulto , Southern Blotting , Neoplasias Encefálicas/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Reordenamiento Génico , Genes de Inmunoglobulinas , Glioma/genética , Humanos , Cariotipificación , Masculino , Persona de Mediana EdadRESUMEN
In 38 yeast genera tested, utilization of D-glucarate was uncommon, occurring with only 10 strains out of 373. The ability was prominent among Cryptococcus strains, with 8 out of 8 tested being positive, including the pathogen Cryptococcus neoformans. The ability was present also in Trichosporon where 2 out of the 4 strains tested were positive. There was a correlation between ability to utilize D-glucarate, galactarate, L-tartarate, and D-glucuronate. Use of L-ascorbate occurred in more genera than use of D-glucarate, but all strains that grew on D-glucarate grew on L-ascorbate. The utilization of certain hydroxylated carboxylates by strains, mainly found in two genera, is of interest in identifying the catabolic pathways involved, in taxonomic studies, and in developing rapid methods of yeast identification.