RESUMEN
BACKGROUND: A core outcomes set (COS) is an agreed minimum set of outcomes that should be measured and reported in all clinical trials for a specific condition. Hidradenitis suppurativa (HS) has no agreed-upon COS. A central aspect in the COS development process is to identify a set of candidate outcome domains from a long list of items. Our long list had been developed from patient interviews, a systematic review of the literature and a healthcare professional survey, and initial votes had been cast in two e-Delphi surveys. In this manuscript, we describe two in-person consensus meetings of Delphi participants designed to ensure an inclusive approach to generation of domains from related items. OBJECTIVES: To consider which items from a long list of candidate items to exclude and which to cluster into outcome domains. METHODS: The study used an international and multistakeholder approach, involving patients, dermatologists, surgeons, the pharmaceutical industry and medical regulators. The study format was a combination of formal presentations, small group work based on nominal group theory and a subsequent online confirmation survey. RESULTS: Forty-one individuals from 13 countries and four continents participated. Nine items were excluded and there was consensus to propose seven domains: disease course, physical signs, HS-specific quality of life, satisfaction, symptoms, pain and global assessments. CONCLUSIONS: The HISTORIC consensus meetings I and II will be followed by further e-Delphi rounds to finalize the core domain set, building on the work of the in-person consensus meetings.
Asunto(s)
Hidradenitis Supurativa/terapia , Ensayos Clínicos como Asunto , Consenso , Conferencias de Consenso como Asunto , Técnica Delphi , Salud Global , Humanos , Resultado del TratamientoRESUMEN
BACKGROUND: We began to recover lungs from uncontrolled donation after circulatory determination of death to assess for transplant suitability by means of ex vivo lung perfusion (EVLP) and computerized tomographic (CT) scan. Our first case had a cold agglutinin with an interesting outcome. CASE REPORT: A 60-year-old man collapsed at home and was pronounced dead by Emergency Medical Services personnel. Next-of-kin consented to lung retrieval, and the decedent was ventilated and transported. Lungs were flushed with cold Perfadex, removed, and stored cold. The lungs did not flush well. Medical history revealed a recent hemolytic anemia and a known cold agglutinin. Warm nonventilated ischemia time was 51 minutes. O2-ventilated ischemia time was 141 minutes. Total cold ischemia time was 6.5 hours. At cannulation for EVLP, established clots were retrieved from both pulmonary arteries. At initiation of EVLP with Steen solution, tiny red aggregates were observed initially. With warming, the aggregates disappeared and the perfusate became red. After 1 hour, EVLP was stopped because of florid pulmonary edema. The lungs were cooled to 20°C; tiny red aggregates formed again in the perfusate. Ex vivo CT scan showed areas of pulmonary edema and a pyramidal right middle lobe opacity. Dissection showed multiple pulmonary emboli-the likely cause of death. However, histology showed agglutinated red blood cells in the microvasculature in pre- and post-EVLP biopsies, which may have contributed to inadequate parenchymal preservation. CONCLUSIONS: Organ donors with cold agglutinins may not be suitable owing to the impact of hypothermic preservation.
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Trasplante de Pulmón , Preservación de Órganos/efectos adversos , Perfusión/efectos adversos , Recolección de Tejidos y Órganos/efectos adversos , Isquemia Fría , Crioglobulinas/análisis , Circulación Extracorporea/métodos , Humanos , Pulmón/fisiopatología , Masculino , Persona de Mediana Edad , Preservación de Órganos/métodos , Perfusión/métodos , Arteria Pulmonar/cirugía , Donantes de Tejidos/provisión & distribución , Recolección de Tejidos y Órganos/métodosRESUMEN
The evolutionary origins of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) are unknown. Current evidence suggests that insectivorous bats are likely to be the original source, as several 2c CoVs have been described from various species in the family Vespertilionidae Here, we describe a MERS-like CoV identified from a Pipistrellus cf. hesperidus bat sampled in Uganda (strain PREDICT/PDF-2180), further supporting the hypothesis that bats are the evolutionary source of MERS-CoV. Phylogenetic analysis showed that PREDICT/PDF-2180 is closely related to MERS-CoV across much of its genome, consistent with a common ancestry; however, the spike protein was highly divergent (46% amino acid identity), suggesting that the two viruses may have different receptor binding properties. Indeed, several amino acid substitutions were identified in key binding residues that were predicted to block PREDICT/PDF-2180 from attaching to the MERS-CoV DPP4 receptor. To experimentally test this hypothesis, an infectious MERS-CoV clone expressing the PREDICT/PDF-2180 spike protein was generated. Recombinant viruses derived from the clone were replication competent but unable to spread and establish new infections in Vero cells or primary human airway epithelial cells. Our findings suggest that PREDICT/PDF-2180 is unlikely to pose a zoonotic threat. Recombination in the S1 subunit of the spike gene was identified as the primary mechanism driving variation in the spike phenotype and was likely one of the critical steps in the evolution and emergence of MERS-CoV in humans.IMPORTANCE Global surveillance efforts for undiscovered viruses are an important component of pandemic prevention initiatives. These surveys can be useful for finding novel viruses and for gaining insights into the ecological and evolutionary factors driving viral diversity; however, finding a viral sequence is not sufficient to determine whether it can infect people (i.e., poses a zoonotic threat). Here, we investigated the specific zoonotic risk of a MERS-like coronavirus (PREDICT/PDF-2180) identified in a bat from Uganda and showed that, despite being closely related to MERS-CoV, it is unlikely to pose a threat to humans. We suggest that this approach constitutes an appropriate strategy for beginning to determine the zoonotic potential of wildlife viruses. By showing that PREDICT/PDF-2180 does not infect cells that express the functional receptor for MERS-CoV, we further show that recombination was likely to be the critical step that allowed MERS to emerge in humans.
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Quirópteros/virología , Coronavirus del Síndrome Respiratorio de Oriente Medio/clasificación , Coronavirus del Síndrome Respiratorio de Oriente Medio/aislamiento & purificación , Filogenia , Acoplamiento Viral , Animales , Evolución Molecular , Genoma Viral , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/fisiología , Receptores Virales/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Sintenía , UgandaRESUMEN
It has been postulated that mucus stasis is central to the pathogenesis of obstructive lung diseases. In Scnn1b-transgenic (Scnn1b-Tg⺠mice, airway-targeted overexpression of the epithelial Na⺠channel ß subunit causes airway surface dehydration, which results in mucus stasis and inflammation. Bronchoalveolar lavage from neonatal Scnn1b-Tg⺠mice, but not wild-type littermates, contained increased mucus, bacteria, and neutrophils, which declined with age. Scnn1b-Tg⺠mice lung bacterial flora included environmental and oropharyngeal species, suggesting inhalation and/or aspiration as routes of entry. Genetic deletion of the Toll-interleukin-1 receptor adapter molecule MyD88 in Scnn1b-Tg⺠mice did not modify airway mucus obstruction, but caused defective neutrophil recruitment and increased bacterial infection, which persisted into adulthood. Scnn1b-Tg⺠mice derived into germ-free conditions exhibited mucus obstruction similar to conventional Scnn1b-Tg⺠mice and sterile inflammation. Collectively, these data suggest that dehydration-induced mucus stasis promotes infection, compounds defects in other immune mechanisms, and alone is sufficient to trigger airway inflammation.
Asunto(s)
Pulmón/inmunología , Pulmón/metabolismo , Moco/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Neumonía/inmunología , Neumonía/metabolismo , Animales , Infecciones Bacterianas/genética , Infecciones Bacterianas/inmunología , Modelos Animales de Enfermedad , Canales Epiteliales de Sodio/genética , Pulmón/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neutrófilos/inmunología , Neumonía/microbiología , Transducción de SeñalRESUMEN
Immortalization of human bronchial epithelial (hBE) cells often entails loss of differentiation. Bmi-1 is a protooncogene that maintains stem cells, and its expression creates cell lines that recapitulate normal cell structure and function. We introduced Bmi-1 and the catalytic subunit of telomerase (hTERT) into three non-cystic fibrosis (CF) and three DeltaF508 homozygous CF primary bronchial cell preparations. This treatment extended cell life span, although not as profoundly as viral oncogenes, and at passages 14 and 15, the new cell lines had a diploid karyotype. Ussing chamber analysis revealed variable transepithelial resistances, ranging from 200 to 1,200 Omega.cm(2). In the non-CF cell lines, short-circuit currents were stimulated by forskolin and inhibited by CFTR(inh)-172 at levels mostly comparable to early passage primary cells. CF cell lines exhibited no forskolin-stimulated current and minimal CFTR(inh)-172 response. Amiloride-inhibitable and UTP-stimulated currents were present, but at lower and higher amplitudes than in primary cells, respectively. The cells exhibited a pseudostratified morphology, with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells, and occasional ciliated cells. CF and non-CF cell lines produced similar levels of IL-8 at baseline and equally increased IL-8 secretion in response to IL-1beta, TNF-alpha, and the Toll-like receptor 2 agonist Pam3Cys. Although they have lower growth potential and more fastidious growth requirements than viral oncogene transformed cells, Bmi-1/hTERT airway epithelial cell lines will be useful for several avenues of investigation and will help fill gaps currently hindering CF research and therapeutic development.
Asunto(s)
Bronquios/citología , Técnicas de Cultivo de Célula/métodos , Fibrosis Quística/patología , Células Epiteliales/citología , Mucosa Respiratoria/citología , Adolescente , Adulto , Línea Celular Transformada , Senescencia Celular/fisiología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Cámaras de Difusión de Cultivos , Células Epiteliales/metabolismo , Femenino , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 1 , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Virus 40 de los Simios , Telomerasa/genéticaRESUMEN
Currently, there is great enthusiasm about potential stem cell therapies for intractable diseases. We previously reviewed the topic of stem cells in lung injury and repair, including the role of endogenous, tissue (somatic) stem cells and the contribution of circulating cells to the lung parenchyma. Our purpose here is to provide a concise update in this fast-moving field. New information and ongoing debate focus attention on basic issues in lung stem cell biology and highlight the need for additional studies to establish the feasibility of cell therapies to prevent or treat lung diseases.
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Enfermedades Pulmonares/terapia , Pulmón/citología , Trasplante de Células Madre , Células Madre , Animales , Diferenciación Celular , Células Cultivadas , Estudios de Factibilidad , Femenino , Citometría de Flujo , Predicción , Células Madre Hematopoyéticas/citología , Humanos , Inmunohistoquímica , Enfermedades Pulmonares/prevención & control , Trasplante de Pulmón , Masculino , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Seguridad , Células Madre/citología , Células Madre/fisiologíaRESUMEN
We describe procedures for isolating and culturing airway epithelial cells from chronically infected human lungs. Experience in our laboratory demonstrated the need to balance pathogen eradication against antibiotic toxicity to epithelial cells. To provide a logical basis for antibiotic selection and dose, we systematically analyzed the cytotoxicity of antibiotics useful against typical pathogens. Alone, colistin, ciprofloxacin, doxycycline, and tobramycin were moderately toxic at concentrations close to those used in cell culture, whereas amphotericin, ceftazidime, chloramphenicol, imipenem, meropenem, piperacillin, sulfamethoxazole/trimethoprim, and vancomycin were nontoxic even at concentrations many times the antimicrobial level. Epithelial cytotoxicity of combined antibiotics was additive, with no evidence of competition or synergism. Antibiotics had little effect on initial cell attachment and did not acutely lyse cells, but inhibited subsequent growth. Interestingly, cytotoxicity decreased markedly with increasing epithelial cell density. Cystic fibrosis (CF) and non-CF epithelial cells showed no differences in sensitivity to the antibiotics tested and initial exposure to antibiotics did not affect the electrophysiologic properties of resistance or short circuit current in well-differentiated cells. Tailored combinations of antibiotics at appropriate doses killed even multidrug-resistant bacteria. Thus, epithelial cells can usually be cultured from chronically infected CF airways.
Asunto(s)
Antibacterianos/administración & dosificación , Infecciones Bacterianas/patología , Separación Celular/métodos , Epitelio/patología , Enfermedades Pulmonares/patología , Antibacterianos/toxicidad , Infecciones Bacterianas/tratamiento farmacológico , Bronquios/patología , Adhesión Celular/efectos de los fármacos , Recuento de Células , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Enfermedad Crónica , Fibrosis Quística/patología , Resistencia a Múltiples Medicamentos , Quimioterapia Combinada , Humanos , Enfermedades Pulmonares/microbiologíaRESUMEN
In the experiment reported here, the lactulose/rhamnose urinary excretion test was used to compare intestinal permeability between four breeds of healthy adult dogs and a group of healthy adult cats. A significant difference in permeability was found between dogs and cats (P <0.001) and between different breeds of dogs (P <0.005). The range of urinary lactulose/rhamnose ratios in the dogs in this study (0.07-0.61) was wider than previously reported (0.03-0.12). The mean value for dogs was 0.19. The range in cats was 0.41-1.25 and the mean 0.52. The results of this study suggest that breed or some other factor such as environment, diet or sexual status as well as species should be taken into account when assessing intestinal permeability using the lactulose/rhamnose urinary excretion test.
Asunto(s)
Gatos/metabolismo , Perros/metabolismo , Intestino Delgado/metabolismo , Lactulosa/farmacocinética , Ramnosa/farmacocinética , Animales , Gatos/orina , Perros/orina , Femenino , Absorción Intestinal , Lactulosa/administración & dosificación , Lactulosa/orina , Modelos Lineales , Masculino , Permeabilidad , Valores de Referencia , Ramnosa/administración & dosificación , Ramnosa/orinaRESUMEN
It is generally important to elucidate airway epithelial cell lineages and to identify multipotent progenitors as targets for gene therapy. Stem (S) cells are typically present in specialized compartments spatially proximal to their differentiated progeny, but an equivalent paradigm has not been demonstrated in the airway. We discovered a distinct population of cells displaying high levels of keratin expression in murine tracheal submucosal gland ducts, and tested the hypothesis that bromodeoxyuridine (BrdU) label-retaining cells (LRCs), thought to represent the S-cells, were present in this compartment. Mice received weekly epithelial damage by intratracheal detergent or SO(2) inhalation for 4 wk and received intraperitoneal injections of BrdU every 48 h during the injury and repair period. At 3 and 6 d after injury, BrdU-positive epithelial cells were noted along the entire tracheal length in both basal and lumenal cell positions. At later time points (20 and 95 d) LRCs were localized to gland ducts in the upper trachea and to systematically arrayed foci in the lower trachea, typically near the cartilage-intercartilage junction. LRCs were not pulmonary neuroendocrine cells. Heterotopic tracheal grafts after surface epithelial removal demonstrated reconstitution of a surface-like epithelium from gland remnants. These results suggest that airway epithelial S cells are localized to specific niches.
Asunto(s)
Regeneración , Mucosa Respiratoria/fisiología , Células Madre/fisiología , Tráquea/fisiología , Animales , Queratinas/biosíntesis , Ratones , Ratones Transgénicos , Mucosa Respiratoria/citología , Mucosa Respiratoria/lesiones , Mucosa Respiratoria/trasplante , Trasplante de Células Madre , Células Madre/citología , Tráquea/citología , Tráquea/lesiones , Tráquea/trasplanteRESUMEN
The goal of this study was to develop a primary culture model of differentiated murine tracheal epithelium. When grown on semipermeable membranes at an air interface, dissociated murine tracheal epithelial cells formed confluent polarized epithelia with high transepithelial resistances ( approximately 12 kOmega. cm(2)) that remained viable for up to 80 days. Immunohistochemistry and light and electron microscopy demonstrated that the cells were epithelial in nature (cytokeratin positive, vimentin and alpha-smooth muscle actin negative) and differentiated to form ciliated and secretory cells from day 8 after seeding onward. With RT-PCR, expression of the cystic fibrosis transmembrane conductance regulator (Cftr) and murine beta-defensin (Defb) genes was detected (Defb-1 was constitutively expressed, whereas Defb-2 expression was induced by exposure to lipopolysaccharide). Finally, Ussing chamber experiments demonstrated an electrophysiological profile compatible with functional amiloride-sensitive sodium channels and cAMP-stimulated CFTR chloride channels. These data indicate that primary cultures of murine tracheal epithelium have many characteristics similar to those of murine tracheal epithelium in vivo. This method will facilitate the establishment of primary cultures of airway epithelium from transgenic mouse models of human diseases.
Asunto(s)
Diferenciación Celular , Mucosa Respiratoria/citología , Mucosa Respiratoria/fisiología , Amilorida/farmacología , Animales , Bumetanida/farmacología , Técnicas de Cultivo de Célula/métodos , Polaridad Celular , Separación Celular/métodos , Células Cultivadas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Defensinas/genética , Femenino , Humanos , Queratinas/análisis , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mucosa Respiratoria/ultraestructura , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tráquea , Vimentina/análisisRESUMEN
The induction of host antimicrobial molecules following binding of pathogen components to pattern recognition receptors such as CD14 and the Toll-like receptors (TLRs) is a key feature of innate immunity. The human airway epithelium is an important environmental interface, but LPS recognition pathways have not been determined. We hypothesized that LPS would trigger beta-defensin (hBD2) mRNA in human tracheobronchial epithelial (hTBE) cells through a CD14-dependent mechanism, ultimately activating NF-kappa B. An average 3-fold increase in hBD2 mRNA occurs 24 h after LPS challenge of hTBE cells. For the first time, we demonstrate the presence of CD14 mRNA and cell surface protein in hTBE cells and show that CD14 neutralization abolishes LPS induction of hBD2 mRNA. Furthermore, we demonstrate TLR mRNA in hTBE cells and NF-kappa B activation following LPS. Thus, LPS induction of hBD2 in hTBE cells requires CD14, which may complex with a TLR to ultimately activate NF-kappa B.
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Defensinas/fisiología , Células Epiteliales/fisiología , Receptores de Lipopolisacáridos/fisiología , Transducción de Señal , Bronquios/metabolismo , Células Cultivadas , Humanos , Lipopolisacáridos/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Smooth muscle constriction in asthma causes the airway to buckle into a rosette pattern, folding the epithelium into deep crevasses. The epithelial cells in these folds are pushed up against each other and thereby experience compressive stresses. To study the epithelial cell response to compressive stress, we subjected primary cultures of rat tracheal epithelial cells to constant elevated pressures on their apical surface (i.e., a transmembrane pressure) and examined changes in the expression of genes that are important for extracellular matrix production and maintenance of smooth muscle activation. Northern blot analysis of RNA extracted from cells subjected to transmembrane pressure showed induction of early growth response-1 (Egr-1), endothelin-1, and transforming growth factor-beta1 in a pressure-dependent and time-dependent manner. Increases in Egr-1 protein were detected by immunohistochemistry. Our results demonstrate that airway epithelial cells respond rapidly to compressive stresses. Potential transduction mechanisms of transmembrane pressure were also investigated.
Asunto(s)
Proteínas Inmediatas-Precoces , Tráquea/fisiología , Animales , Northern Blotting , Muerte Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Densitometría , Proteína 1 de la Respuesta de Crecimiento Precoz , Endotelina-1/genética , Endotelina-1/metabolismo , Células Epiteliales/fisiología , Inmunohistoquímica , Masculino , Membranas/fisiología , Estimulación Física , Presión , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Factores de Tiempo , Tráquea/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
OBJECTIVE: To determine whether plasma concentrations of benzodiazepines (BDZ) in dogs following intranasal (IN) administration of diazepam are comparable to concentrations following IV administration. ANIMALS: 6 (4 male, 2 female) healthy adult Greyhounds. PROCEDURE: Dogs were randomly assigned to 2 groups of 3 dogs in a crossover design. Diazepam (0.5 mg/kg of body weight) was administered intravenously to dogs in group 1 and intranasally to dogs in group 2. Blood was collected from the jugular vein of each dog into tubes containing lithium heparin before and 3, 6, 9, 12, 15, 20, 30, 60, 120, 240, and 480 minutes following diazepam administration. After a 4-day washout period, dogs in group 1 received diazepam intranasally, dogs in group 2 received diazepam intravenously, and blood was again collected. Plasma concentration of BDZ was determined by use of a fluorescence polarization immunoassay. RESULTS: Mean (+/- SD) peak plasma concentration of BDZ following IV administration (1,316 +/- 216 microg/L) was greater than that following IN administration (448 +/- 41 microg/L). Time to peak concentration was < or = 3 minutes following IV administration and 4.5 +/- 1.5 minutes following IN administration. Mean bioavailability of BDZ following IN administration was 80 +/- 9%. CONCLUSIONS AND CLINICAL RELEVANCE: Diazepam is rapidly and efficiently absorbed following IN administration of the parenteral formulation. Plasma concentrations match or exceed the suggested therapeutic concentration (300 microg/L). Intranasal administration of diazepam may be useful for treatment of seizures in dogs by owners or when intravenous access is not readily available.
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Anticonvulsivantes/farmacocinética , Diazepam/farmacocinética , Perros/metabolismo , Administración Intranasal , Animales , Anticonvulsivantes/administración & dosificación , Anticonvulsivantes/sangre , Anticonvulsivantes/uso terapéutico , Área Bajo la Curva , Estudios Cruzados , Diazepam/administración & dosificación , Diazepam/sangre , Diazepam/uso terapéutico , Femenino , Inmunoensayo de Polarización Fluorescente/veterinaria , Semivida , Inyecciones Intravenosas/veterinaria , Masculino , Distribución Aleatoria , Estado Epiléptico/tratamiento farmacológico , Estado Epiléptico/veterinariaRESUMEN
OBJECTIVE: This study examined the response of middle ear tissue to establish the lowest dose of lipopolysaccharide to induce mucin production in a rat otitis media model. METHODS: Twenty-six male Sprague-Dawley rats' eustachian tubes were obstructed before transtympanic inoculation of the bulla tympanica with 35 microL of Krebs Ringer or 1, 10, 100, or 1000 microgram/mL lipopolysaccharide. After 7 days the effusion and a lavage were collected for mucin ELISA measurement, and tissue was collected for histologic evaluation. RESULTS: Mucin secretion was significantly increased in the 100 microgram/mL 51.20 +/- 13.6 microgram/mL (SE) and 1000 microgram/mL 69.42 +/- 8.57 microgram/mL groups when compared with the Krebs Ringer control group 1.84 +/- 0.28 microgram/mL (P < 0.05). Histologic evaluation shows goblet cell metaplasia and hyperplasia in the middle ear epithelium in the 1000 and 100 microgram/mL groups. CONCLUSIONS: The histology and ELISA results suggest that a middle ear effusion is generated with a dose of lipopolysaccharide as low as 100 microgram/mL.
Asunto(s)
Oído Medio/metabolismo , Lipopolisacáridos/farmacología , Mucinas/metabolismo , Otitis Media con Derrame/metabolismo , Animales , Modelos Animales de Enfermedad , Oído Medio/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Lipopolisacáridos/administración & dosificación , Masculino , Membrana Mucosa/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Mucus hypersecretion is characteristic of chronic airway diseases. However, regulatory mechanisms are poorly understood. Human airway epithelial cells grown on permeable supports at the air-liquid interface (ALI) develop a mucociliary morphology resembling that found in vivo. Such cultures provide a model for studying secretory cell lineage, differentiation, and function, and may provide insight regarding events leading to mucus hypersecretion. The mucin gene expression profile of well-differentiated human airway epithelial cells in culture has not yet been established. We compared expression of all the currently described mucin genes in poorly differentiated (conventional cultures on plastic) and well-differentiated (ALI) human nasal and bronchial epithelial cells. Differentiation-dependent upregulation of MUC3, MUC5AC, MUC5B, and MUC6 messenger RNA (mRNA) was demonstrated using reverse transcriptase-polymerase chain reaction (RT-PCR). Northern blot analysis showed a similar increase for MUC4 and demonstrated that induction of MUC4 and MUC5B expression depended on retinoic acid. MUC1, MUC2, MUC7, and MUC8 mRNAs were also detected by RT-PCR, but these genes did not appear to be strongly regulated as a function of differentiation. Mucin gene expression was similar in bronchial and nasal cells. Thus, mucociliary differentiation of human airway epithelia in vitro entails upregulation of several mucin genes.
Asunto(s)
Células Epiteliales/citología , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Pulmón/citología , Mucinas/genética , Mucosa Nasal/citología , Mucosa Nasal/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Cromosomas Humanos Par 11 , Cartilla de ADN , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mucina 4 , Mucina 5B , Mucinas/biosíntesis , Familia de Multigenes , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tretinoina/farmacologíaAsunto(s)
Neoplasias Óseas/veterinaria , Enfermedades de los Perros/diagnóstico por imagen , Ilion/patología , Cojera Animal/diagnóstico por imagen , Osteosarcoma/veterinaria , Animales , Neoplasias Óseas/diagnóstico , Neoplasias Óseas/diagnóstico por imagen , Enfermedades de los Perros/diagnóstico , Perros , Femenino , Prótesis de Cadera/efectos adversos , Prótesis de Cadera/veterinaria , Ilion/diagnóstico por imagen , Osteosarcoma/diagnóstico , Osteosarcoma/diagnóstico por imagen , RadiografíaRESUMEN
Airway surface liquid is comprised of mucus and an underlying, watery periciliary liquid (PCL). In contrast to the well-described axial transport of mucus along airway surfaces via ciliary action, theoretical analyses predict that the PCL is nearly stationary. Conventional and confocal microscopy of fluorescent microspheres and photoactivated fluorescent dyes were used with well-differentiated human tracheobronchial epithelial cell cultures exhibiting spontaneous, radial mucociliary transport to study the movements of mucus and PCL. These studies showed that the entire PCL is transported at approximately the same rate as mucus, 39.2+/-4.7 and 39.8+/-4.2 micrometer/sec, respectively. Removing the mucus layer reduced PCL transport by > 80%, to 4.8+/-0.6 micrometer/sec, a value close to that predicted from theoretical analyses of the ciliary beat cycle. Hence, the rapid movement of PCL is dependent upon the transport of mucus. Mucus-dependent PCL transport was spatially uniform and exceeded the rate expected for pure frictional coupling with the overlying mucus layer; hence, ciliary mixing most likely accelerates the diffusion of momentum from mucus into the PCL. The cephalad movement of PCL along airway epithelial surfaces makes this mucus-driven transport an important component of salt and water physiology in the lung in health and disease.
Asunto(s)
Depuración Mucociliar/fisiología , Fenómenos Fisiológicos Respiratorios , Transporte Biológico , Líquidos Corporales/metabolismo , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/fisiología , Humanos , Modelos Biológicos , Moco/metabolismoRESUMEN
Normal lung morphogenesis and cytodifferentiation require interactions between epithelium and mesenchyme. We have previously shown that distal lung mesenchyme (LgM) is capable of reprogramming tracheal epithelium (TrE) from day 13-14 rat fetuses to branch in a lung-like pattern and express a distal lung epithelial phenotype. In the present study, we have assessed the effects of tracheal mesenchyme (TrM) on branching and cytodifferentiation of distal lung epithelium (LgE). Tracheae and distal lung tips from day 13 rat fetuses were separated into purified epithelial and mesenchymal components, then recombined as homotypic (LgM + LgE or TrM + TrE) or heterotypic (LgM + TrE or TrM + LgE) recombinants and cultured for 5 days; unseparated lung tips and tracheae served as controls. Control lung tips, LgM + LgE, and LgM + TrE recombinants all branched in an identical pattern. Epithelial cells, including those from the induced TrE, contained abundant glycogen deposits and lamellar bodies, and expressed surfactant protein C (SP-C) mRNA. Trachea controls, and both TrM + TrE, and TrM + LgE recombinants did not branch, but instead formed cysts. The epithelium contained ciliated and mucous secretory cells; importantly, no cells containing lamellar bodies were observed, nor was SP-C mRNA detected. Mucin immunostaining showed copious production of mucous in both LgE and TrE when recombined with TrM. These results demonstrate that epithelial differentiation in the recombinants appears to be wholly dependent on the type of mesenchyme used, and that the entire respiratory epithelium has significant plasticity in eventual phenotype at this stage in development.
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Células Epiteliales/metabolismo , Pulmón/embriología , Pulmón/crecimiento & desarrollo , Mesodermo/metabolismo , Fracciones Subcelulares/metabolismo , Tráquea/embriología , Tráquea/crecimiento & desarrollo , Animales , Diferenciación Celular , Células Cultivadas , Células Epiteliales/ultraestructura , Femenino , Pulmón/citología , Pulmón/ultraestructura , Mesodermo/citología , Mesodermo/ultraestructura , Microscopía Electrónica , Embarazo , Ratas , Ratas Sprague-Dawley , Tráquea/citología , Tráquea/ultraestructuraRESUMEN
Investigations of the efficiency and safety of human adenovirus vector (AdV)-mediated gene transfer in the airways of patients with cystic fibrosis (CF) in vivo have demonstrated little success in correcting the CF bioelectrical functional defect, reflecting the inefficiency of AdV-mediated gene transfer to the epithelial cells that line the airway luminal surface. In this study, we demonstrate that low AdV-mediated gene transfer efficiency to well-differentiated (WD) cultured airway epithelial cells is due to three distinct steps in the apical membrane of the airway epithelial cells: (i) the absence of specific adenovirus fiber-knob protein attachment receptors; (ii) the absence of alphavbeta3/5 integrins, reported to partially mediate the internalization of AdV into the cell cytoplasm; and (iii) the low rate of apical plasma membrane uptake pathways of WD airway epithelial cells. Attempts to increase gene transfer efficiency by increasing nonspecific attachment of AdV were unsuccessful, reflecting the inability of the attached vector to enter (penetrate) WD cells via nonspecific entry paths. Strategies to improve the efficiency of AdV for the treatment of CF lung disease will require methods to increase the attachment of AdV to and promote its internalization into the WD respiratory epithelium.
Asunto(s)
Adenoviridae/genética , Bronquios/virología , Técnicas de Transferencia de Gen , Vectores Genéticos , Receptores de Vitronectina , Tráquea/virología , Animales , Células CHO , Diferenciación Celular , Cricetinae , Dinamina III , Dinaminas , Endocitosis , GTP Fosfohidrolasas/fisiología , Células HeLa , Humanos , Integrinas/análisis , RatasRESUMEN
The pathogenesis of cystic fibrosis (CF) airways infection is unknown. Two hypotheses, "hypotonic [low salt]/defensin" and "isotonic volume transport/mucus clearance," attempt to link defects in cystic fibrosis transmembrane conductance regulator-mediated ion transport to CF airways disease. We tested these hypotheses with planar and cylindrical culture models and found no evidence that the liquids lining airway surfaces were hypotonic or that salt concentrations differed between CF and normal cultures. In contrast, CF airway epithelia exhibited abnormally high rates of airway surface liquid absorption, which depleted the periciliary liquid layer and abolished mucus transport. The failure to clear thickened mucus from airway surfaces likely initiates CF airways infection. These data indicate that therapy for CF lung disease should not be directed at modulation of ionic composition, but rather at restoring volume (salt and water) on airway surfaces.