RESUMEN
BACKGROUND: The risks for infants and young children receiving inhaled corticosteroid (ICS) therapy are largely unknown. Recent clinical studies indicate that ICS therapy in pre-school children with symptoms of asthma result in decreased symptoms without influencing the clinical disease course, but potentially affect postnatal growth and development. The current study employs a primate experimental model to identify the risks posed by ICS therapy. OBJECTIVE: To (1) establish whether ICS therapy in developing primate lungs reverses pulmonary pathobiology associated with allergic airway disease (AAD) and (2) define the impact of ICS on postnatal lung growth and development in primates. METHODS: Infant rhesus monkeys were exposed, from 1 through 6 months, to filtered air (FA) with house dust mite allergen and ozone using a protocol that produces AAD (AAD monkeys), or to FA alone (Control monkeys). From three through 6 months, the monkeys were treated daily with ICS (budesonide) or saline. RESULTS: Several AAD manifestations (airflow restrictions, lavage eosinophilia, basement membrane zone thickening, epithelial mucin composition) were reduced with ICS treatment, without adverse effects on body growth or adrenal function; however, airway branching abnormalities and intraepithelial innervation were not reduced. In addition, several indicators of postnatal lung growth and differentiation: vital capacity, inspiratory capacity, compliance, non-parenchymal lung volume and alveolarization, were increased in both AAD and Control monkeys that received ICS treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Incomplete prevention of pathobiological changes in the airways and disruption of postnatal growth and differentiation of airways and lung parenchyma in response to ICS pose risks for developing primate lungs. These responses also represent two mechanisms that could compromise ICS therapy's ability to alter clinical disease course in young children.
Asunto(s)
Corticoesteroides/farmacología , Alérgenos/toxicidad , Antígenos Dermatofagoides/toxicidad , Asma , Pulmón , Animales , Asma/inducido químicamente , Asma/tratamiento farmacológico , Asma/patología , Asma/fisiopatología , Niño , Preescolar , Modelos Animales de Enfermedad , Femenino , Humanos , Lactante , Recién Nacido , Pulmón/crecimiento & desarrollo , Pulmón/patología , Pulmón/fisiopatología , Macaca mulatta , MasculinoRESUMEN
Increases in Clara cell abundance or cellular expression of Clara cell secretory protein (CCSP) may cause increased tolerance of the lung to acute oxidant injury by repeated exposure to ozone (O3). This study defines how disruption of the gene for CCSP synthesis affects the susceptibility of tracheobronchial epithelium to acute oxidant injury. Mice homozygous for a null allele of the CCSP gene (CCSP-/-) and wild type (CCSP+/+) littermates were exposed to ozone (0.2 ppm, 8 h; 1 ppm, 8 h) or filtered air. Injury was evaluated by light and scanning electron microscopy, and the abundance of necrotic, ciliated, and nonciliated cells was estimated by morphometry. Proximal and midlevel intrapulmonary airways and terminal bronchioles were evaluated. There was no difference in airway epithelial composition between CCSP+/+ and CCSP-/- mice exposed to filtered air, and exposure to 0.2 ppm ozone caused little injury to the epithelium of both CCSP+/+ and CCSP-/- mice. After exposure to 1.0 ppm ozone, CCSP-/- mice suffered from a greater degree of epithelial injury throughout the airways compared to CCSP+/+ mice. CCSP-/- mice had both ciliated and nonciliated cell injury. Furthermore, lack of CCSP was associated with a shift in airway injury to include proximal airway generations. Therefore, we conclude that CCSP modulates the susceptibility of the epithelium to oxidant-induced injury. Whether this is due to the presence of CCSP on the acellular lining layer surface and/or its intracellular distribution in the secretory cell population needs to be defined.
Asunto(s)
Bronquios/efectos de los fármacos , Ozono/toxicidad , Tráquea/efectos de los fármacos , Uteroglobina/genética , Animales , Bronquios/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/patología , Tráquea/patologíaRESUMEN
To establish whether allergic asthma could be induced experimentally in a nonhuman primate using a common human allergen, three female rhesus monkeys (Macaca mulatta) were sensitized with house dust mite (Dermatophagoides farinae) allergen (HDMA) by subcutaneous injection, followed by four intranasal sensitizations, and exposure to allergen aerosol 3 hours per day, 3 days per week for up to 13 weeks. Before aerosol challenge, all three monkeys skin-tested positive for HDMA. During aerosol challenge with HDMA, sensitized monkeys exhibited cough and rapid shallow breathing and increased airway resistance, which was reversed by albuterol aerosol treatment. Compared to nonsensitized monkeys, there was a fourfold reduction in the dose of histamine aerosol necessary to produce a 150% increase in airway resistance in sensitized monkeys. After aerosol challenge, serum levels of histamine were elevated in sensitized monkeys. Sensitized monkeys exhibited increased levels of HDMA-specific IgE in serum, numbers of eosinophils and exfoliated cells within lavage, and elevated CD25 expression on circulating CD4(+) lymphocytes. Intrapulmonary bronchi of sensitized monkeys had focal mucus cell hyperplasia, interstitial infiltrates of eosinophils, and thickening of the basement membrane zone. We conclude that a model of allergic asthma can be induced in rhesus monkeys using a protocol consisting of subcutaneous injection, intranasal instillation, and aerosol challenge with HDMA.
Asunto(s)
Asma/inmunología , Glicoproteínas/inmunología , Animales , Antígenos Dermatofagoides , Asma/patología , Bronquios/efectos de los fármacos , Bronquios/inmunología , Bronquios/metabolismo , Pruebas de Provocación Bronquial , Líquido del Lavado Bronquioalveolar/química , Exudados y Transudados/metabolismo , Femenino , Glicoproteínas/administración & dosificación , Histamina/administración & dosificación , Histamina/sangre , Histamina/inmunología , Inmunoglobulina E/sangre , Inmunofenotipificación , Inyecciones Subcutáneas , Pruebas Intradérmicas , Linfocitos/citología , Linfocitos/inmunología , Macaca mulatta , ÁcarosRESUMEN
Acute pulmonary epithelial injury produced by short-term exposure to ozone varies by site within the tracheobronchial tree. To test whether this variability is related to the local dose of ozone at the tissue site or to local concentrations of glutathione, we exposed adult male rhesus monkeys for 2 h to filtered air or to 0.4 or 1.0 ppm ozone generated from 18O2. Following exposure, lungs were split into lobes and specimens were selected by microdissection so that measurements could be made on airway tissue of similar branching history, including trachea, proximal (generation one or two) and distal (generation six or seven) intrapulmonary bronchi, and proximal respiratory bronchioles. One half of the lung was lavaged for analysis of extracellular components. In monkeys exposed to filtered air, the concentration of reduced glutathione (GSH) varied throughout the airway tree, with the proximal intrapulmonary bronchus having the lowest concentration and the parenchyma having the highest concentration. Exposure to 1.0 ppm ozone significantly reduced GSH only in the respiratory bronchiole, whereas exposure to 0.4 ppm increased GSH only in the proximal intrapulmonary bronchus. Local ozone dose (measured as excess 18O) varied by as much as a factor of three in different airways of monkeys exposed to 1.0 ppm, with respiratory bronchioles having the highest concentration and the parenchyma the lowest concentration. In monkeys exposed to 0.4 ppm, the ozone dose was 60% to 70% less than in the same site in monkeys exposed to 1.0 ppm. Epithelial disruption was present to some degree in all airway sites, but not in the parenchyma, in animals exposed to 1.0 ppm ozone. The mass of mucous and ciliated cells decreased in all airways, and necrotic and inflammatory cells increased. At 0.4 ppm, epithelial injury was minimal, except in the respiratory bronchiole, where cell loss and necrosis occurred, and was 50% that found in monkeys exposed to 1.0 ppm ozone. We conclude that there is a close association between site-specific O3 dose, the degree of epithelial injury, and glutathione depletion at local sites in the tracheobronchial tree.
Asunto(s)
Células Epiteliales/patología , Glutatión/metabolismo , Pulmón/patología , Ozono/toxicidad , Animales , Líquido del Lavado Bronquioalveolar/química , Histocitoquímica , Leucocitos/metabolismo , Pulmón/efectos de los fármacos , Macaca mulatta , Macrófagos/metabolismo , Masculino , Isótopos de Oxígeno , Proteínas/análisisRESUMEN
After acute exposure to oxidant gases in vivo, migration and accumulation of inflammatory cells in pulmonary epithelium coincides with epithelial cell necrosis. The present study was designed to test quantitatively the hypothesis that quiescent neutrophils enhance the removal of oxidant-injured pulmonary epithelial cells after exposure to ozone in vitro. Primary isolated rat alveolar type II cells were cultured as monolayers, using serum-free medium. After exposure to 0.1-0.5 ppm ozone for 0.5 h, apical sides of monolayers were administered either fresh nutrient medium only or medium containing quiescent human neutrophils. Monolayer bioelectric properties and cellular uptake of vital dye were recorded from 5 to 48 h after ozone exposure. Ozone dose-dependent increases in monolayer permeability were associated with proportionally higher numbers of injured epithelial cells. However, the direction and magnitude of neutrophil effects on monolayer permeability after ozone exposure were dependent on ozone concentration. Furthermore, neutrophil-treated monolayers exposed to 0.1 ppm ozone had significantly fewer attached cells positive for uptake of vital dye relative to monolayers exposed to the low level of ozone only; this effect was ablated with increasing ozone concentration. These data suggest that at high levels of ozone neutrophils may exacerbate injury to oxidant-impaired epithelial cells, whereas the presence of neutrophils after exposure to ambient concentrations of ozone may expedite the restoration of epithelial barrier function. We conclude that, by enhancing the removal of injured cells, neutrophils may facilitate the repair of centriacinar epithelium after ozone exposure in vivo.
Asunto(s)
Neutrófilos/fisiología , Alveolos Pulmonares/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Impedancia Eléctrica , Epitelio/efectos de los fármacos , Epitelio/patología , Humanos , Masculino , Ozono/farmacología , Alveolos Pulmonares/patología , Ratas , Ratas Sprague-DawleyRESUMEN
The objective of this study was to characterize an in vitro model of oxidant gas toxicity, using primary cultures of alveolar type II cells maintained in serum-free medium, by evaluating (1) epithelial barrier function, (2) the stability of cellular antioxidant defenses, and (3) the response of alveolar epithelial barrier properties to ozone exposure. Antioxidant enzyme activities and glutathione levels were measured in rat type II cells that were freshly isolated, cultured for 1 day in serum-supplemented medium, and subsequently grown in serum-free nutrient medium. After measurement of peak bioelectric properties on Day 4 in primary culture, alveolar epithelial monolayers were exposed to ozone at various concentrations and lengths of exposure. Ozone-induced alterations in monolayer bioelectric properties and impairment of cellular organization were used to evaluate oxidant injury. The primary effect of ozone exposure was a dose-dependent increase in monolayer permeability, which resulted from damage to intercellular junctions and/or loss of epithelial integrity. Extensive and persistent permeability increases correlated with focal areas of epithelial degradation. The focal nature of ozone injury to alveolar epithelium in vitro suggests that individual cell susceptibility to oxidant stress may account for the overall decrement in barrier function. However, this sensitivity does not result from overall loss of antioxidant defenses associated with cell culture, as these monolayers (when cultured in serum-free medium) maintained their antioxidant enzyme activities and glutathione content at levels found in freshly isolated cells. We conclude that the sensitivity of these monolayers to ozone injury in vitro reflects a disproportionate degree of oxidant stress on cell membranes relative to intracellular antioxidant defenses, i.e., cellular susceptibility to oxidant injury may depend on the ratio of the surface area of the cell to its cytoplasmic volume.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Ozono/toxicidad , Alveolos Pulmonares/efectos de los fármacos , Animales , Catalasa/metabolismo , Células Cultivadas , ADN/análisis , Relación Dosis-Respuesta a Droga , Electrofisiología , Epitelio/efectos de los fármacos , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Glutatión Transferasa/metabolismo , Masculino , Oxidación-Reducción , Proteínas/análisis , Alveolos Pulmonares/citología , Alveolos Pulmonares/metabolismo , Ratas , Ratas Sprague-Dawley , Superóxido Dismutasa/metabolismoRESUMEN
An in vitro system for exposing respiratory epithelial cells or explant tissues to ozone has been developed and characterized. This system is designed to generate and monitor consistent, reproducible levels of ozone, over a range of concentrations, in a humidified atmosphere, and to allow an exposure time of 24 h or longer. Based on chemical analysis, highly reproducible concentrations of ozone are delivered throughout the chamber, with a coefficient of variation of < 5% between five replicate vials exposed to 0.5 ppm of ozone for 50 min. The viability of cultured human tracheobronchial epithelial cells, as measured by the ability to oxidize a vital dye, and of rat tracheal epithelium, as measured by total numbers of necrotic cells in tracheal explants, after ozone exposure was examined in this system. Responses of cultured cells to ozone exposure as measured by bioassay were consistent with the observed low level of variability of ozone concentration between replicate incubation dishes or vials. Responses of cultured cells to ozone were proportional to duration of exposure and inversely proportional to the volume of medium covering the cells. We conclude that this newly developed in vitro exposure system will allow relatively simple and convenient exposure of cultured cells or organs to ozone or other gaseous agents under highly controlled and reproducible conditions.
Asunto(s)
Cámaras de Exposición Atmosférica , Ozono/toxicidad , Sistema Respiratorio/efectos de los fármacos , Tráquea/efectos de los fármacos , Contaminación del Aire , Animales , Epitelio/efectos de los fármacos , Humanos , Técnicas In VitroAsunto(s)
Supervivencia Celular/efectos de los fármacos , Técnicas de Cultivo/instrumentación , Ozono/toxicidad , Animales , Células Cultivadas/efectos de los fármacos , Técnicas de Cultivo/métodos , Relación Dosis-Respuesta a Droga , Exposición a Riesgos Ambientales , Humanos , Modelos Biológicos , Ozono/análisisRESUMEN
Aerosolized fluorescent microspheres were used to study particle deposition in site-specific regions of the lung with confocal laser scanning microscopy. A nebulizer was used to aerosolize microspheres followed by passage through a heated discharging column to reduce static charge and to remove water surrounding each microsphere. Precoating of microspheres with albumin helped to minimize displacement during vascular fixation of the lungs. Confocal laser microscopy facilitated visualization of microspheres throughout the bronchial tree, ducts, and alveoli of the lungs. The use of fluorescent microspheres and confocal laser imaging provided distinct advantages compared with other methods to study lung particle deposition due to (1) the generation of single microspheres of uniform size by nebulization, (2) easy detection of microspheres in large slabs of microdissected lung tissues, (3) excellent resolution of tissue surfaces and microspheres for an infinite number of orientations and planes of section, and (4) the ability to visualize microspheres below fluid lining layers and on surfaces that could not easily be done by other methods of microscopy.
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Aerosoles , Colorantes Fluorescentes , Pulmón/anatomía & histología , Microesferas , Animales , Masculino , Microscopía/métodos , Fagocitosis , Ratas , Ratas Sprague-DawleyRESUMEN
Nitrogen dioxide (NO2.) is often present in inhaled air and may be generated in vivo from nitric oxide. Exposure of human blood plasma to NO2. caused rapid losses of ascorbic acid, uric acid and protein thiol groups, as well as lipid peroxidation and depletions of alpha-tocopherol, bilirubin and ubiquinol-10. No increase in protein carbonyls was detected. Supplementation of plasma with ascorbate decreased the rates of lipid peroxidation, alpha-tocopherol depletion and loss of uric acid. Uric acid supplementation decreased rates of lipid peroxidation but not the loss of alpha-tocopherol. We conclude that ascorbic acid, protein -SH groups, uric acid and alpha-tocopherol may be important agents protecting against NO2. in vivo. If these antioxidants are depleted, peroxidation of lipids occurs and might contribute to the toxicity of NO2..
Asunto(s)
Antioxidantes/metabolismo , Dióxido de Nitrógeno/sangre , Adulto , Proteínas Sanguíneas/metabolismo , Cromatografía Líquida de Alta Presión , Radicales Libres , Humanos , Técnicas In Vitro , Peroxidación de Lípido , Mediciones Luminiscentes , Masculino , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Ozone has been shown to increase airway resistance and/or airway reactivity in vivo in animals and humans. Because of the complexities inherent in studying this phenomenon in whole animals, we developed a model of ozone-induced effects on airway physiology using the isolated perfused rat lung. Rat lungs were suspended in an airtight chamber and perfused via the pulmonary circulation with a modified Krebs-Henseleit buffer containing 4.5% bovine albumin. Ventilation of the lungs was achieved by generating a fluctuating negative pressure within the chamber (-2 to -7 cm H2O) at a rate of 60 breaths/min. The lungs were ventilated with humidified 95% air and 5% CO2 alone (control condition) or mixed with ozone at 1.0 or 2.0 ppm. Transpulmonary pressure, flow rate, and tidal volume were recorded at 0, 1, 2, and 3 hours, and pulmonary resistance (RL) and dynamic compliance (Cdyn) were calculated. There was no significant difference in lung weight/total body weight ratios between the three groups at the end of the 3-h period. RL increased and Cdyn decreased in a time- and dose-dependent manner with ozone exposure. The percent increase above baseline in RL +/- SEM at 3 h was 9.4 +/- 4.1% for control lungs, 21.0 +/- 3.2% for 1.0 ppm ozone-exposed lungs, and 63.6 +/- 13.5% for 2.0 ppm ozone-exposed lungs. The percent decrease below baseline in Cdyn +/- SEM at 3 h was 27.4 +/- 2.1% for control lungs, 37.1 +/- 2.7% for 1.0 ppm ozone-exposed lungs, and 55.2 +/- 7.3% for 2.0 ppm ozone-exposed lungs.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Pulmón/efectos de los fármacos , Ozono/efectos adversos , Resistencia de las Vías Respiratorias/efectos de los fármacos , Animales , Pulmón/fisiología , Rendimiento Pulmonar/efectos de los fármacos , Masculino , Microscopía Electrónica , Perfusión , Ratas , Ratas EndogámicasRESUMEN
Pulmonary carbon monoxide (CO) excretion rates (VeCO) were 50% greater, on average, in Bolivian squirrel monkeys (BoSMs) which exhibit a unique fasting hyperbilirubinemia (FH), than in fasted control Brazilian squirrel monkeys (BrSMs). Since the catabolism of heme produces equimolar amounts of CO and bilirubin, the increased VeCOs are consistent with concurrent increases in endogenous bilirubin production rates. Tin-protoporphyrin, a competitive inhibitor of heme oxygenase, significantly decreased both the VeCO and serum bilirubin level in fasted BoSMs. Overproduction of bilirubin may be responsible in part for the marked FH in BoSMs.
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Monóxido de Carbono/metabolismo , Hiperbilirrubinemia/veterinaria , Pulmón/metabolismo , Enfermedades de los Monos/metabolismo , Saimiri/metabolismo , Animales , Bilirrubina/sangre , Bolivia , Brasil , Modelos Animales de Enfermedad , Ayuno , Enfermedad de Gilbert/metabolismo , Hiperbilirrubinemia/metabolismo , Masculino , Metaloporfirinas/farmacología , Protoporfirinas/farmacologíaAsunto(s)
Sulfato de Amonio/toxicidad , Aerosoles/toxicidad , Animales , Peso Corporal/efectos de los fármacos , ADN/análisis , Peróxido de Hidrógeno/toxicidad , Pulmón/efectos de los fármacos , Masculino , Tamaño de los Órganos/efectos de los fármacos , Oxidación-Reducción , Ratas , Ratas Endogámicas , Tráquea/efectos de los fármacosRESUMEN
A method has been developed for generation of submicrometer aerosols of sodium sulfite suitable for use in inhalation toxicologic research. Concentrations ranging up to about 30 mg/m3 Na2SO3 were achieved in a 0.44 m3 exposure chamber with an air flow rate of 0.20 m3/min for periods up to 16 days. The coefficient of variation of the sulfite aerosol mass concentration was about 4% during a typical exposure period. The measured mass median aerodynamic diameters (MMADar) of the generated aerosols were 1.2 (+/- 0.2SD) microns with a geometric standard deviation (sigma g) of 1.9 (+/- 0.3SD). The chamber was sampled for gas phase SO2 concentration, and aerosol samples were analyzed for particulate sulfite and sulfate. The fraction of sulfur qas sulfite in the aerosol was usually 95% and was always greater than 90%. Gas phase SO2 amounted to less than 2% of the total S(IV) present in the chamber.
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Aerosoles , Sulfitos/síntesis química , Toxicología/métodos , Aerosoles/análisis , Tamaño de la Partícula , Sulfitos/análisis , Dióxido de Azufre/análisisAsunto(s)
Cromatos/farmacología , Tráquea/metabolismo , Aerosoles , Animales , Cromatos/administración & dosificación , Cromo/metabolismo , Glucosamina/metabolismo , Glicoproteínas/metabolismo , Semivida , Cinética , Masculino , Ratas , Tráquea/anatomía & histología , Tráquea/efectos de los fármacosRESUMEN
Experience with a high output sulfuric acid aerosol generator suitable for large chamber exposure studies is described. Sulfuric acid aerosol is produced by combining sulfur trioxide (SO3)(g) vapor with water vapor in the exposure chamber air supply. The mass output of the generator is stable over long periods, and the mass median aerodynamic diameter of the aerosol produced is in the submicron size range (0.3-0.6 micrometer).
Asunto(s)
Cámaras de Exposición Atmosférica , Ácidos Sulfúricos , Aerosoles , Animales , Cámaras de Exposición Atmosférica/instrumentación , Fenómenos Químicos , Química , Medicina del Trabajo , Óxidos de AzufreRESUMEN
Groups of Sprague-Dawley rats were exposed to ozone for either 8 or 24 hours a day for 7 consecutive days to evaluate morphologic changes of the respiratory system. Three levels of exposure (0.2, 0.5, and 0.8 p.p.m. of O3) were selected to simulate moderate to severe episodes of oxidant pollution in urban environments. Morphologic evaluation included light, scanning electron, and transmission electron microscopy. Biochemical parameters which were examined included succinate oxidase, glucose-6-phosphate dehydrogenase, and nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activities. The results indicated that (1) exposure to concentrations as low as 0.2 p.p.m. for 7 days induced pulmonary damage; (2) there was a dose-dependent pulmonary response to the three levels of ozone which was quantitated by alterations in biochemical marker enzyme activities and observed morphologically; (3) proportionate differences were not observed in morphologic characteristics of the lesions or detected in biochemical parameters between rats exposed continuously for 7 days and those exposed intermittently for 8 hours a day for 7 consecutive days; (4) alterations in surface height and granularity of the cytoplasmic luminal projection of Clara cells were subtle changes which were dose-dependent, occurring even at the lowest ozone concentration, and best detected by scanning electron microscopy; (5) alveolar macrophage accumulation within proximal alveoli of alveolar ducts was the most readily detectable morphologic indicator of pulmonary damage; and (6) although the brunt of ozone damage was borne by the centriacinar region, there was damage to cilia and increased ciliogenesis occurring in the trachea and larger conducting airways following exposure of 0.5 and 0.8 p.p.m. of ozone.
Asunto(s)
Pulmón/efectos de los fármacos , Ozono/efectos adversos , Esmog , Sacos Aéreos/citología , Sacos Aéreos/ultraestructura , Animales , Peso Corporal/efectos de los fármacos , Bronquios/patología , Exposición a Riesgos Ambientales , Células Epiteliales , Epitelio/ultraestructura , Pulmón/metabolismo , Pulmón/patología , Macrófagos/ultraestructura , Masculino , Microscopía Electrónica de Rastreo , Consumo de Oxígeno , Ratas , Tráquea/patologíaRESUMEN
Ozone exposure (0.5 p.p.m., 8 hours daily for 7 days) resulted in a 20-26% (P less than 0.05) increase in the level of reduced glutathione (GSH), and the activities of the GSH peroxidase system in rat lungs. The increases were of smaller magnitude (10-15%) in the lungs of ozone-exposed monkeys. No significant changes were observed in these parameters in the erythrocytes of ozone-exposed and control animals of the two species. The results suggest that rats may be more sensitive to ozone than monkeys in terms of biochemical lesions in the lung, and that ozone effects are manifested primarily in the lung.