RESUMO
OBJECTIVE: Many premature infants at risk for bronchopulmonary dysplasia experience episodes of surfactant dysfunction with reduced surfactant protein B (SP-B). In this study, we investigated the safety and responses to booster doses of surfactant. STUDY DESIGN: A total of 87 infants, 500 to 1250 g birth weight, who were ventilated at 7 to 10 days received 2 or 3 doses of Infasurf (Calfactant, Forest Pharmaceuticals, St Louis, MO, USA) within a 1-week period. RESULT: For 184 doses, occurrence rates of transient bradycardia (13) and plugged endotracheal tube (5) were low, and no other adverse effects were noted. Treatment transiently improved the respiratory severity score (FiO(2) × mean airway pressure), SP-B content (+75%) and surface properties of isolated surfactant. Levels of eight proinflammatory cytokines in tracheal aspirate were interrelated and unchanged from baseline after surfactant treatment. CONCLUSION: Booster doses of surfactant for premature infants with lung disease are safe and transiently improve respiratory status as well as composition and function of endogenous surfactant.
Assuntos
Displasia Broncopulmonar/terapia , Surfactantes Pulmonares/administração & dosagem , Respiração Artificial , Displasia Broncopulmonar/tratamento farmacológico , Feminino , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Masculino , Projetos Piloto , Resultado do TratamentoRESUMO
OBJECTIVE: Inhaled nitric oxide (iNO) is a potential new therapy for prevention of bronchopulmonary dysplasia and brain injury in premature infants. This study examined dose-related effects of iNO on NO metabolites as evidence of NO delivery. STUDY DESIGN: A subset of 102 premature infants in the NO CLD trial, receiving 24 days of iNO (20 p.p.m. decreasing to 2 p.p.m.) or placebo, were analyzed. Tracheal aspirate (TA) and plasma samples collected at enrollment and at intervals during study gas were analyzed for NO metabolites. RESULT: iNO treatment increased NO metabolites in TA at 20 and 10 p.p.m. (1.7- to 2.3-fold vs control) and in plasma at 20, 10, and 5 p.p.m. (1.6- to 2.3-fold). In post hoc analysis, treated infants with lower metabolite levels at entry had an improved clinical outcome. CONCLUSION: iNO causes dose-related increases in NO metabolites in the circulation as well as lung fluid, as evidenced by TA analysis, showing NO delivery to these compartments.
Assuntos
Recém-Nascido Prematuro/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico/uso terapêutico , Nitritos/sangue , Terapia Respiratória/métodos , Displasia Broncopulmonar/prevenção & controle , Idade Gestacional , Humanos , Recém-Nascido , Recém-Nascido Prematuro/sangue , Nitratos/sangue , TraqueiaRESUMO
We report a simplified culture system for human fetal lung type II cells that maintains surfactant expression. Type II cells isolated from explant cultures of hormone-treated lungs (18-22 wk gestation) by collagenase + trypsin digestion were cultured on plastic for 4 days in serum-free medium containing dexamethasone (Dex, 10 nM) + 8-bromo-cAMP (0.1 mM + isobutylmethylxanthine (0.1 mM) or were untreated (control). Surfactant protein (SP) mRNAs decreased markedly in control cells between days 1 and 4 of culture, but mRNA levels were high in treated cells on day) 4 (SP-A, SP-B, SP-C, SP-D; 600%, 100%, 85%, 130% of day 0 content, respectively). Dex or cAMP alone increased SP-B, SP-C, and SP-D mRNAs and together had additive effects. The greatest increase in SP-A mRNA occurred with cAMP alone. Treated cells processed pro-SP-B and pro-SP-C proteins to mature forms and had a higher rate of phosphatidylcholine (PC) synthesis (2-fold) and higher saturation of PC (approximately 34% versus 27%) than controls. Only treated cells maintained secretagogue-responsive phospholipid synthesis. By electron microscopy, the treated cells retained lamellar bodies and extensive microvilli. We conclude that Dex and cAMP additively stimulate expression of surfactant components in isolated fetal type II cells, providing a simplified culture system for investigation of surfactant-related, and perhaps other, type II cell functions.
Assuntos
Técnicas de Cultura de Células/métodos , Células Epiteliais/citologia , Pulmão/embriologia , Tensoativos/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Colagenases/metabolismo , Corantes/farmacologia , Meios de Cultura Livres de Soro/farmacologia , AMP Cíclico/metabolismo , DNA Complementar/metabolismo , Dexametasona/farmacologia , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Glicoproteínas/biossíntese , Humanos , Immunoblotting , Imuno-Histoquímica , Pulmão/citologia , Microscopia Eletrônica , Oxazinas/farmacologia , Fosfatidilcolinas/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Fosfolipídeos/metabolismo , Plásticos , Testes de Precipitina , Proteolipídeos/biossíntese , Proteína A Associada a Surfactante Pulmonar , Proteína D Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/biossíntese , RNA Mensageiro/metabolismo , Fatores de Tempo , Transfecção , Tripsina/metabolismoRESUMO
Intratracheal bleomycin in rats is associated with respiratory distress of uncertain etiology. We investigated the expression of surfactant components in this model of lung injury. Maximum respiratory distress, determined by respiratory rate, occurred at 7 days, and surfactant dysfunction was confirmed by increased surface tension of the large-aggregate fraction of bronchoalveolar lavage (BAL). In injured animals, phospholipid content and composition were similar to those of controls, mature surfactant protein (SP) B was decreased 90%, and SP-A and SP-D contents were increased. In lung tissue, SP-B and SP-C mRNAs were decreased by 2 days and maximally at 4--7 days and recovered between 14 and 21 days after injury. Immunostaining of SP-B and proSP-C was decreased in type II epithelial cells but strong in macrophages. By electron microscopy, injured lungs had type II cells lacking lamellar bodies and macrophages with phagocytosed lamellar bodies. Surface activity of BAL phospholipids of injured animals was restored by addition of exogenous SP-B. We conclude that respiratory distress after bleomycin in rats results from surfactant dysfunction in part secondary to selective downregulation of SP-B and SP-C.
Assuntos
Bleomicina/administração & dosagem , Surfactantes Pulmonares/deficiência , Insuficiência Respiratória/induzido quimicamente , Animais , Líquido da Lavagem Broncoalveolar/química , Técnica Indireta de Fluorescência para Anticorpo , Injeções , Pulmão/patologia , Masculino , Microscopia Eletrônica , Fosfolipídeos/análise , Proteolipídeos/farmacologia , Proteolipídeos/fisiologia , Surfactantes Pulmonares/farmacologia , Surfactantes Pulmonares/fisiologia , Ratos , Ratos Sprague-Dawley , Insuficiência Respiratória/patologia , Insuficiência Respiratória/fisiopatologia , Distribuição Tecidual , TraqueiaRESUMO
BACKGROUND: It has been suggested that preterm infants may have developmental immaturity of the hypothalamic-pituitary-adrenal axis, and that decreased cortisol response to stress increases risk of chronic lung disease (CLD) secondary to inflammatory lung injury. METHODS: To investigate the relationship between endogenous corticosteroid and CLD, we measured plasma cortisol during the first 28 days of life in a subset of neonates in the North American Thyrotropin-Releasing Hormone (TRH) Collaborative Trial. Analyses were performed on 314 infants, 24 to 32 weeks' gestation, whose mothers received 1 or 2 courses of antenatal corticosteroids plus TRH or placebo. RESULTS: Mean cortisol was 3.1 microg/dL (range: 0.1-17.9) at birth, reached maximal levels at 24 hours (19.4 microg/dL, range: 0.8-124.6), and decreased to 5.9 microg/dL (range: 0.2-24.7) at 14 to 28 days of age; levels during the first week were not associated with gestational age. The Clinical Risk Index for Babies (CRIB), a neonatal assessment tool that is correlated with risk of mortality, was positively associated with cortisol level on days 1 and 3 through 7. TRH versus placebo treatment did not influence cortisol levels at any time point. To examine the relationship between cortisol and adverse outcome of death or CLD at 36 weeks' postmenstrual age (CLD36), logistic regression models adjusting for known contributing clinical factors (gestational age and CRIB score) were fit. There was a statistically borderline negative association between median cortisol level at 3 to 7 days and CLD36. After adjusting for gestational age and CRIB score, the predicted probability of CLD36 was only minimally influenced by the cortisol concentration. CONCLUSION: In preterm infants, basal plasma cortisol concentration during the first week is a weak predictor for CLD36. Possible benefits as well as risks of supplemental, low-dose cortisol treatment of high-risk preterm infants remain to be determined.
Assuntos
Displasia Broncopulmonar/sangue , Hidrocortisona/sangue , Doenças do Prematuro/sangue , Pneumopatias/sangue , Displasia Broncopulmonar/prevenção & controle , Doença Crônica , Feminino , Idade Gestacional , Glucocorticoides/uso terapêutico , Humanos , Sistema Hipotálamo-Hipofisário/fisiologia , Recém-Nascido , Recém-Nascido Prematuro , Doenças do Prematuro/prevenção & controle , Pneumopatias/prevenção & controle , Sistema Hipófise-Suprarrenal/fisiologia , Gravidez , Análise de Regressão , Risco , Índice de Gravidade de Doença , Hormônio Liberador de Tireotropina/uso terapêuticoRESUMO
The transforming growth factor-beta (TGF beta) polypeptides control a variety of cellular processes including organogenesis and cellular proliferation and differentiation. In the developing lung, TGF beta(1) treatment inhibits airway branching and expression of the genes for surfactant proteins (SP). Many effects of TGF beta are mediated at the level of gene transcription but there is limited information regarding signaling pathways and target transcription factors. In this study with human pulmonary adenocarcinoma H441 cells, we investigated TGF beta(1) effects on SP-B, a protein which is essential for normal function of pulmonary surfactant. TGF beta(1) (10 ng/ml) reduced SP-B mRNA content in a time-dependent fashion, and transient transfection studies localized responsiveness to the region of the SP-B promoter (-112/-72 bp) containing binding sites for thyroid transcription factor-1 (TTF-1) and hepatocyte nuclear factor 3 (HNF3), transcription factors that are important enhancers of SP gene expression. Using electrophoretic mobility shift assay and immunofluorescence, we demonstrated rapid accumulation of these transcription factors in the cytoplasm and subsequent loss from the nucleus on TGF beta(1) treatment of both adenocarcinoma cells and cultured human fetal lung. TGF beta(1) treatment caused intracellular translocation of protein kinase C and effects of TGF beta(1) were mostly abrogated in the presence of the protein kinase inhibitor calphostin C. We conclude that TGF beta(1), acting via protein phosphorylation, blocks nuclear translocation of TTF-1 and HNF3 which results in down-regulation of the SP-B gene and presumably other pulmonary genes which are transactivated by these factors.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica , Proteínas Nucleares/fisiologia , Precursores de Proteínas/genética , Proteolipídeos/genética , Fatores de Transcrição/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Regiões 5' não Traduzidas , Transporte Biológico , Citoplasma/metabolismo , Feto/metabolismo , Fator 3-alfa Nuclear de Hepatócito , Humanos , Pulmão/metabolismo , Proteína Quinase C/fisiologia , Proteínas Quinases/fisiologia , RNA Mensageiro/metabolismo , Fator Nuclear 1 de Tireoide , Células Tumorais CultivadasAssuntos
Desenvolvimento Embrionário e Fetal/efeitos dos fármacos , Glucocorticoides/farmacologia , Glucocorticoides/fisiologia , Pulmão/efeitos dos fármacos , Animais , Esquema de Medicação , Feminino , Glucocorticoides/farmacocinética , Glucocorticoides/uso terapêutico , Humanos , Pulmão/embriologia , Gravidez , Surfactantes Pulmonares/efeitos dos fármacos , OvinosRESUMO
Infants with inherited deficiency of pulmonary surfactant protein (SP) B develop respiratory failure at birth and die without lung transplantation. We examined aspects of surfactant metabolism in lung tissue and lavage fluid acquired at transplantation or postmortem from ten infants born at term with inherited deficiency of SP-B; comparison groups were infants with other forms of chronic lung disease (CLD) and normal infants. In pulse/chase labeling studies with cultured deficient tissue, no immunoprecipitable SP-B was observed and an approximately 6-kD form of SP-C accumulated that was only transiently present in CLD tissue. SP-B messenger RNA (mRNA) was approximately 8% of normal in deficient specimens, and some intact message was observed after, but not before, explant culture. Transcription rates for SP-B, assessed by nuclear run-on assay using probes for sequences both 5' and 3' of the common nonsense mutation (121ins2), were comparable in all lungs examined. The minimal surface tension achieved with lavage surfactant was similarly elevated in both deficient and CLD infants (26-31 mN/m) compared with normal infants (6 mN/m). Both SP-B-deficient and CLD infants had markedly decreased phosphatidylglycerol content of lavage and tissue compared with normal lung, whereas synthetic rates for phospholipids, including phosphatidylglycerol, were normal. We conclude that the mutated SP-B gene is transcribed normally but produces an unstable mRNA and that absence of SP-B protein blocks processing of SP-C. Chronic infant lung disease, of various etiologies, reduces surfactant function and apparently alters phosphatidylglycerol degradation.
Assuntos
Proteolipídeos/genética , Proteolipídeos/metabolismo , Surfactantes Pulmonares/genética , Surfactantes Pulmonares/metabolismo , Síndrome do Desconforto Respiratório do Recém-Nascido/metabolismo , Acetatos/metabolismo , Acetatos/farmacologia , Western Blotting , Cisteína/farmacocinética , Feto/metabolismo , Expressão Gênica/fisiologia , Genótipo , Humanos , Lactente , Recém-Nascido , Metionina/farmacocinética , Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/metabolismo , Proteolipídeos/análise , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/análise , RNA Mensageiro/análise , Radioisótopos de Enxofre , Transcrição Gênica/fisiologia , TrítioRESUMO
Glucocorticoid treatment increases content of surfactant protein (SP) A and SP-B in lung tissue and lavage fluid of preterm lambs. To investigate this process, we determined the ontogeny and glucocorticoid induction of SP mRNAs. In separate treatment protocols, each with its own controls, sheep were injected with betamethasone 15 h, 48 h, or weekly for 1-4 doses before preterm delivery. Using ovine SP cDNAs, we found an increase equal to or more than threefold in basal levels of all three SP mRNAs between 125 days and term. After betamethasone treatment, SP-B and SP-C mRNA levels increased by 15 h and all SP mRNAs were elevated after 24 h (>/=2-fold); mRNA levels in fetuses delivered 1-3 wk after betamethasone were not different from control. We conclude that in vivo betamethasone rapidly induces a coordinated increase in SP mRNAs, which is fully reversible within 7 days despite repetitive doses of betamethasone. Similar increases in mRNA and protein contents for SP-A and SP-B suggest that glucocorticoid regulation of these SPs in vivo is primarily pretranslational.
Assuntos
Betametasona/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glucocorticoides/farmacologia , Proteolipídeos/genética , Alvéolos Pulmonares/embriologia , Surfactantes Pulmonares/genética , Animais , Northern Blotting , Feminino , Idade Gestacional , Gravidez , Alvéolos Pulmonares/crescimento & desenvolvimento , Alvéolos Pulmonares/fisiologia , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , RNA Mensageiro/metabolismo , OvinosRESUMO
OBJECTIVES: We sought to examine outcome for premature neonates after multiple courses of antenatal corticosteroids compared with a single course. STUDY DESIGN: We performed a post hoc nonrandomized analysis on 710 neonates of 25-32 weeks' gestation who were born to mothers enrolled in the North American Thyrotropin-Releasing Hormone Trial and who received 1, 2, or >/=3 courses of antenatal corticosteroids. RESULTS: There was no detectable clinical difference in incidence of respiratory distress syndrome, chronic lung disease, and intraventricular hemorrhage related to courses of antenatal corticosteroids, and outcome was similar for infants delivered at 7-13 days compared with those delivered at 1-6 days after receiving antenatal corticosteroids. Compared with those who received a single course, neonates who received >/=2 courses had lower birth weights (-39 g, P =.02), and those receiving >/=3 courses had increased risk of death (adjusted odds ratio, 2.8; 95% confidence interval, 1.3-5.9; P =.01) and lower levels of plasma cortisol at age 2 hours. CONCLUSION: In this retrospective analysis multiple courses of antenatal corticosteroids did not improve outcome and were associated with increased mortality, decreased fetal growth, and prolonged adrenal suppression.
Assuntos
Corticosteroides/administração & dosagem , Recém-Nascido Prematuro , Pneumopatias/prevenção & controle , Trabalho de Parto Prematuro , Resultado do Tratamento , Corticosteroides/efeitos adversos , Corticosteroides/uso terapêutico , Insuficiência Adrenal/induzido quimicamente , Feminino , Idade Gestacional , Humanos , Hidrocortisona/sangue , Mortalidade Infantil , Recém-Nascido de Baixo Peso , Recém-Nascido , Razão de Chances , Gravidez , Estudos RetrospectivosRESUMO
Fatty acid synthase (FAS; EC 2.3.1.85) supplies de novo fatty acids for pulmonary surfactant synthesis, and FAS gene expression is both developmentally and hormonally regulated in the fetal lung. To further examine hormonal regulation of FAS mRNA and to determine the cellular localization of FAS gene expression, we cultured human fetal lungs (18-22 wk gestation) as explants for 1-4 days in the absence (control) or presence of glucocorticoid [dexamethasone (Dex), 10 nM] and/or cAMP agents (8-bromo-cAMP, 0.1 mM and IBMX, 0.1 mM). FAS protein content and activity increased similarly in the presence of Dex (109 and 83%, respectively) or cAMP (87 and 111%, respectively), and responses were additive in the presence of both hormones (230 and 203%, respectively). With a rabbit anti-rat FAS antibody, FAS immunoreactivity was not detected in preculture lung specimens but appeared in epithelial cells lining the tubules with time in culture. Dex and/or cAMP markedly increased staining of epithelial cells, identified as type II cells, whereas staining of mesenchymal fibroblasts was very low under all conditions. With in situ hybridization, FAS mRNA was found to be enriched in epithelial cells lining the alveolar spaces, and the reaction product increased in these cells when the explants were cultured with the hormones. The increased FAS mRNA content in the presence of Dex and/or cAMP is primarily due to increased stabilization of mRNA, although Dex alone increased the transcription rate by approximately 30%. We conclude that hormonal treatment of cultured human fetal lungs increases FAS gene expression primarily by increasing stability of the message. The induction of FAS during explant culture and by hormones occurs selectively in type II epithelial cells, consistent with the regulatory role of this enzyme in de novo synthesis of fatty acid substrate for surfactant synthesis in perinatal lungs.
Assuntos
Ácido Graxo Sintases/metabolismo , Hormônios/farmacologia , Pulmão/embriologia , Estabilidade de Medicamentos , Feto/citologia , Feto/efeitos dos fármacos , Feto/metabolismo , Humanos , Hibridização In Situ , Técnicas de Cultura de Órgãos , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Distribuição Tecidual , Transcrição Gênica/efeitos dos fármacos , Receptor fas/genética , Receptor fas/metabolismoRESUMO
Thyroid hormones are important for both perinatal adaptation and long-term psychomotor development; however, there is limited information on the effects of extreme prematurity and antenatal TSH-releasing hormone (TRH) treatment on pituitary-thyroid function. In this study we assayed plasma triiodothyronine (T3) and TSH in infants who were part of a collaborative trial of antenatal maternal TRH therapy. Within the control population (n = 166), infants of 24-28-wk and 28-32-wk gestational age had comparable levels of T3 (0.94 and 1.06 nmol/L, respectively) and TSH (5.7 and 7.2 mU/L) at birth, but the increases at 2 h and subsequent T3 levels were less in the 24-28 wk versus 28-32-wk gestation infants. In the TRH-treated group (n = 131), T3 was lower in the first day for infants delivered 7-72 h after antenatal TRH compared with control infants. TSH at birth was approximately 3.5-fold greater for infants delivered at 0-6 h after the last TRH dose compared with the control group and was suppressed in infants delivering at 7-36 h. T3 and TSH levels were not different between control and TRH-treated groups at 3-28 d of age. In TRH stimulation tests on d 28, control and TRH-treated groups had similar peak levels of TSH and incidence of exaggerated response (TSH > or = 35 mU/L). We conclude that extremely premature infants have a reduced postnatal surge in TSH and T3 and maintain lower T3 concentrations, probably reflecting tertiary hypothyroidism. The stimulatory and suppressive effects of antenatal TRH treatment observed at birth are transient and do not affect pituitary-thyroid responsiveness at 28 d of age.
Assuntos
Hipotireoidismo Congênito , Idade Gestacional , Doenças do Prematuro/tratamento farmacológico , Recém-Nascido Prematuro/sangue , Hormônios Tireóideos/sangue , Hormônio Liberador de Tireotropina/uso terapêutico , Humanos , Hipotireoidismo/tratamento farmacológico , Recém-Nascido , Diagnóstico Pré-Natal , Tireotropina/sangue , Tri-Iodotironina/sangueRESUMO
Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine shown to play a critical role in organ morphogenesis, development, growth regulation, cellular differentiation, gene expression, and tissue remodeling after injury. We examined the effect of exogenously administered TGF-beta1 on the expression of surfactant proteins (SPs) and lipids, fatty acid synthetase, and ultrastructural morphology in human fetal lung cultured for 5 days with and without dexamethasone (10 nM). Expression of the type II cell-specific marker surfactant proprotein C (proSP-C), studied by [35S]Met incorporation and immunoprecipitation, increased sevenfold with dexamethasone treatment. TGF-beta1 (0.1-100 ng/ml) in the presence of dexamethasone inhibited 21-kDa proSP-C expression in a dose-dependent manner (maximal inhibition 31% of control level at 100 ng/ml). There was no change in [35S]Met incorporation into total protein in any of the treatment groups vs. the control group. In immunoblotting experiments, TGF-beta1 blocked culture-induced accumulation of SP-A and SP-B. Under the same conditions, TGF-beta1 reduced mRNA content for SP-A, SP-B, and SP-C to 20, 38, and 41%, respectively, of matched control groups but did not affect levels of beta-actin mRNA. SP transcription rates after 24 h of exposure to TGF-beta1 were reduced to a similar extent (20-50% of control level). In both control and dexamethasone-treated explants, TGF-beta1 (10 ng/ml) also decreased fatty acid synthetase mRNA, protein, and enzyme activity and the rate of [3H]choline incorporation into phosphatidylcholine. By electron microscopy, well-differentiated type II cells lining potential air spaces were present in explants cultured with dexamethasone, whereas exposure to TGF-beta1 with or without dexamethasone resulted in epithelial cells lacking lamellar bodies. We conclude that exogenous TGF-beta1 disrupts culture-induced maturation of fetal lung epithelial cells and inhibits expression of surfactant components through effects on gene transcription.
Assuntos
Apoproteínas/genética , Células Epiteliais/fisiologia , Pulmão/fisiologia , Proteolipídeos/genética , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/genética , Transcrição Gênica , Fator de Crescimento Transformador beta/farmacologia , Apoproteínas/biossíntese , Células Cultivadas , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feto , Humanos , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/ultraestrutura , Proteolipídeos/biossíntese , Surfactantes Pulmonares/biossíntese , RNA Mensageiro/análise , Proteínas Recombinantes/farmacologia , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Surfactant protein B (SP-B8), an 8-kDa hydrophobic protein essential for surfactant and normal lung function, is produced from the intracellular processing of preproSP-B. To characterize SP-B processing in human type 2 cells, we used human fetal lung in explant culture and polyclonal antibodies to human SP-B8 (Phe201-Met279) and to specific epitopes within the NH2- and COOH-terminal propeptide domains (Ser145-Leu160, Gln186-Gln200, and Gly284-Ser304). Western blot analysis revealed a novel intermediate at approximately 9 kDa, representing mature SP-B8, with a residual NH2-terminal peptide of approximately 10 amino acids. Pulse-chase studies showed a precursor-product relationship between the 9- and 8-kDa forms. During differentiation of type 2 cells in explant culture, the rate of proSP-B conversion to 25-kDa intermediate remained constant, whereas the rate of 25-kDa intermediate conversion to SP-B8 increased, resulting in a net increase in tissue SP-B8. Dexamethasone did not affect the rate of proSP-B processing but markedly enhanced the rate of SP-B8 accumulation. We conclude that NH2-terminal propeptide cleavage of proSP-B is a multistep process and that more distal processing events are rate limiting and both developmentally and hormonally regulated.
Assuntos
Pulmão/embriologia , Proteolipídeos/biossíntese , Surfactantes Pulmonares/biossíntese , Sequência de Aminoácidos , Anticorpos , Epitopos/análise , Feminino , Feto , Idade Gestacional , Humanos , Pulmão/metabolismo , Gravidez , Segundo Trimestre da Gravidez , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/metabolismo , Proteolipídeos/química , Proteolipídeos/genética , Surfactantes Pulmonares/química , Surfactantes Pulmonares/genéticaRESUMO
Human pulmonary surfactant protein A (SP-A) is genetically complex and its regulation may also be complex, reflecting genotypic variability. Fetal lung explants were used to study the regulation of the SP-A genes, SP-A1 and SP-A2, by dexamethasone, interferon, gamma (IFN gamma), cyclic 3',-5' adenosine monophosphate (cAMP), and tumor necrosis factor alpha (TNF alpha). For comparison, the mRNA levels of surfactant protein B (SP-B) and its response to test substances were also examined. Results showed: (a) In control culture total SP-A mRNA varied widely among explants (C.V. = 0.70) compared with SP-B (C.V. = 0.26) (b) IFN gamma significantly increased total SP-A mRNA but there were marked differences among fetal lungs in response to all treatments. (c) SP-A1 mRNA concentration is higher than SP-A2 in both control and treated explants. (d) SP-A1 alleles are inhibited to a greater degree by dexamethasone than SP-A2 alleles. The relative effect of cAMP and IFN gamma on SP-A1 and SP-A2 mRNA varied widely among explants. We conclude that SP-A genotype may account in part for the marked differences in SP-A mRNA concentration among fetal lungs and that the SP-A genes and/or alleles may be differentially regulated.
Assuntos
Genes/genética , Proteolipídeos/genética , Surfactantes Pulmonares/genética , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Alelos , Processamento Alternativo/genética , Antivirais/farmacologia , Técnicas de Cultura , AMP Cíclico/farmacologia , Dexametasona/farmacologia , Feto , Expressão Gênica/genética , Regulação da Expressão Gênica , Variação Genética/genética , Glucocorticoides/farmacologia , Hormônios/farmacologia , Humanos , Interferon gama/farmacologia , Pulmão , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Projetos de Pesquisa , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVE: Premature infants are susceptible to bronchopulmonary dysplasia (BPD), a chronic lung disease of infancy that appears to be caused in part by oxidative stress from hyperoxia. To investigate the possible role of nitric oxide-derived oxidants such as peroxynitrite in the etiology of BPD, we measured levels of plasma 3-nitrotyrosine, which is produced by the reaction of peroxynitrite with proteins. PATIENTS AND METHODS: Ten premature infants who developed BPD, defined as requiring supplemental oxygen beyond 36 weeks' postmenstrual age, were identified retrospectively from a group of subjects enrolled in a clinical trial of antenatal therapy. Serial plasma samples had been collected on these infants during the first month of life as part of the trial. Sixteen comparison premature infants were identified from the same population: 5 had no lung disease, 6 had respiratory distress syndrome that resolved, and 5 had residual lung disease at 28 days of life that resolved by 36 weeks' postmenstrual age. Plasma 3-nitrotyrosine levels were measured using a solid phase immunoradiochemical method. RESULTS: All 3-nitrotyrosine values in infants without BPD were <0.25 ng/mg protein, and levels did not change with postnatal age. Plasma 3-nitrotyrosine concentrations were significantly higher in infants with BPD, increasing approximately fourfold during the first month of life. For the 20 infants who had blood samples available at 28 days of life, plasma 3-nitrotyrosine levels correlated with the fraction of inspired oxygen that the infant was receiving (r = 0.7). CONCLUSION: Plasma 3-nitrotyrosine content is increased during the first month of life in infants who develop BPD. This suggests that peroxynitrite-mediated oxidant stress may contribute to the development of this disease in premature infants and that 3-nitrotyrosine may be useful as an early plasma indicator of infants at risk for developing BPD.
Assuntos
Displasia Broncopulmonar/sangue , Recém-Nascido Prematuro/sangue , Tirosina/análogos & derivados , Displasia Broncopulmonar/etiologia , Feminino , Humanos , Recém-Nascido , Masculino , Nitratos/metabolismo , Oxidantes/metabolismo , Estresse Oxidativo , Projetos Piloto , Estudos Retrospectivos , Tirosina/sangueRESUMO
BACKGROUND: Pulmonary disease is common in preterm infants, despite antenatal glucocorticoid therapy. The addition of antenatal thyrotropin-releasing hormone therapy has been reported to decrease pulmonary morbidity in these infants. METHODS: We enrolled 996 women at 13 North American centers who were in preterm labor at <30 weeks' gestation in a double-blind, placebo-controlled, randomized trial of antenatal thyrotropin-releasing hormone, given intravenously in four doses of 400 microg each at eight-hour intervals. The primary outcome was chronic lung disease or death of the infant on or before the 28th day after delivery, and secondary outcomes were respiratory distress syndrome and chronic lung disease or death at 36 weeks' postmenstrual age. Complete data were available for 981 women and their 1134 live-born infants. The 769 infants born at < or = 32 weeks' gestation were defined as the group at risk. RESULTS: There were no significant differences between the at-risk treatment and placebo groups in mean (+/-SD) birth weight (1109+/-354 vs. 1097+/-355 g), gestational age (27.9+/-2.1 vs. 27.9+/-2.1 weeks), sex, or race. The frequencies of respiratory distress syndrome (66 percent vs. 65 percent), death at 28 days (11 percent vs. 11 percent), chronic lung disease or death at 28 days (45 percent vs. 42 percent) and at 36 weeks (32 percent vs. 34 percent), and other neonatal complications as well as the severity of lung disease were not significantly different in the at-risk treatment and placebo groups. Similarly, there were no differences in outcome between the treatment and placebo groups for the infants born at >32 weeks' gestation. CONCLUSIONS: In preterm infants at risk for lung disease, antenatal administration of thyrotropin-releasing hormone and glucocorticoid is no more beneficial than glucocorticoid alone.
Assuntos
Doenças do Prematuro/prevenção & controle , Pneumopatias/prevenção & controle , Cuidado Pré-Natal , Hormônio Liberador de Tireotropina/uso terapêutico , Adulto , Doença Crônica , Dexametasona/uso terapêutico , Método Duplo-Cego , Quimioterapia Combinada , Feminino , Glucocorticoides/uso terapêutico , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Masculino , Trabalho de Parto Prematuro , Gravidez , Síndrome do Desconforto Respiratório do Recém-Nascido/prevenção & controle , Hormônio Liberador de Tireotropina/administração & dosagem , Hormônio Liberador de Tireotropina/efeitos adversos , Resultado do TratamentoRESUMO
We examined the ability of the human surfactant protein B (SP-B) promoter to confer cell specificity of transgene expression in an adenoviral vector. Using similar replication-deficient adenoviruses (rAd), we compared lacZ reporter gene expression driven by the human SP-B promoter (rAd.SPBlacZ) with the ubiquitously expressed Rous sarcoma virus promoter (rAd.RSVlacZ). rAd.SPBlacZ expressed lacZ in H-441 and A549 lung epithelial cell lines and not in HeLa cells whereas rAd.RSVlacZ expressed in all three cell lines. In primary human fetal lung fibroblasts, beta-galactosidase activity from rAd.RSVlacZ transduction increased in a dose-dependent manner whereas activity from rAd.SPBlacZ remained low. In mixed cell cultures prepared from human fetal lung explants that contained fibroblasts and type II cells, X-Gal staining localized rAd.SPBlacZ expression to only type II cells whereas rAd.RSVlacZ expressed in both cell types. In 24-wk gestation human fetal tissue explants infected ex vivo, the RSV promoter directed lacZ expression in lung, trachea, heart, liver, and esophagus, whereas with the SP-B promoter lacZ was expressed only in lung, specifically in air space-lining cells. This specificity was maintained in vivo. lacZ expression was undetectable in lung and other tissues after intravenous administration of rAd.SPBlacZ whereas rAd.RSV-lacZ expressed primarily in liver. After intratracheal instillation of rAd.SPBlacZ into mice, X-Gal staining localized expression to type II and Clara cells. In contrast, rAd.RSVlacZ expressed in all pulmonary epithelial cell types. Our results indicate that the SP-B promoter may be useful in targeting type II and Clara cells for gene therapy of conditions such as inherited deficiency of SP-B.
Assuntos
Adenoviridae/genética , Expressão Gênica , Técnicas de Transferência de Genes , Pulmão/citologia , Regiões Promotoras Genéticas , Proteolipídeos/genética , Surfactantes Pulmonares/genética , Adenocarcinoma , Animais , Northern Blotting , Western Blotting , Feminino , Marcação de Genes , Células HeLa , Humanos , Imuno-Histoquímica , Pulmão/metabolismo , Neoplasias Pulmonares , Camundongos , Plasmídeos/genética , Proteolipídeos/análise , Surfactantes Pulmonares/análise , Proteínas Recombinantes , Células Tumorais Cultivadas , beta-Galactosidase/análiseRESUMO
To assess effects of dose and duration of glucocorticoid exposure on maturation of the fetal lung, we administered single or multiple doses of betamethasone (0.5 mg/kg im) to pregnant sheep for 2 or 21 days before preterm delivery at 125 days of gestation. Lung function (compliance, lung volume at 40 cmH2O pressure, and ventilatory efficiency index) was increased after two to four weekly doses of glucocorticoid (2.5- to 4-fold increase) and after 48 h of exposure (1.4- to 2.3-fold). Total protein of lavage fluid decreased similarly with three doses, four doses, and 48 h of treatment. In lambs with long-term exposure to betamethasone, there was a similar, dose-dependent increase in concentrations of saturated phosphatidylcholine and surfactant proteins A (SP-A) and B (SP-B) (maximal 2- to 3-fold in tissue and 10- to 15-fold in lavage fluid). Levels of SP-A and SP-B were closely correlated in lavage fluid. In animals treated for 48 h, only tissue SP-B was increased (2.7-fold). We conclude that 48 h of glucocorticoid treatment improves lung function in the premature lamb without a detectable increase in lavage surfactant components and that longer exposure to antenatal glucocorticoid increases surfactant lipid and proteins in a coordinated fashion. The enhanced response with repetitive dosing indicates that the process of glucocorticoid-induced lung maturation is either reversible and/or gestational age dependent.
Assuntos
Betametasona/farmacologia , Glucocorticoides/farmacologia , Pulmão/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Proteolipídeos/metabolismo , Surfactantes Pulmonares/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Líquido da Lavagem Broncoalveolar/química , Feminino , Pulmão/embriologia , Pulmão/fisiologia , Tamanho do Órgão/efeitos dos fármacos , Fosfatidilcolinas/metabolismo , Gravidez , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Análise de Regressão , Respiração/efeitos dos fármacos , Testes de Função Respiratória , OvinosRESUMO
The hydrophobic surfactant protein C (SP-C) is known to modulate the biophysical properties of surfactant phospholipid. Although SP-C mRNA has been demonstrated in human fetal lung, there is limited information regarding developmental expression and processing of proSP-C protein. Two epitope-specific human proSP-C antisera, anti-hCPROSP-C (His59-Ser72) and anti-hCTERMSP-C (Gly162-Gly175), were generated to complement previously produced anti-NPROSP-C (Met10-Gln23) for the study of proSP-C expression in human fetal lung. Western blotting and immunocytochemistry detected expression of proSP-C protein by 12-16 wk of gestation. ProSP-C immunoreactivity of preculture lung, limited to expression of proSP-C21 in airway epithelial cells, was markedly enhanced by culture of lung explants in dexamethasone. To examine synthesis of proSP-C, homogenates from explants were labeled with 35S-Met/Cys for 0.5-4 h. Immunoprecipitation with anti-NPROSP-C detected 35S-proSP-C21 by 30 min and, after 2 h of labeling, there was a 15-fold increase in 35S-proSP-C21 in dexamethasone-treated lungs versus controls. Synthesis of proSP-C21 was followed by the appearance of a 24-kD form and smaller processing intermediates including 6-10-kD forms. Posttranslational processing of proSP-C21 was not observed in control explants. SP-C(6-10) were not recognized by either anti-CPROSP-C or anti-hCTERMSP-C. These results indicate that low level expression of proSP-C protein first occurs in epithelial cells early in the second trimester and that expression can be enhanced by dexamethasone. Initial posttranslational processing of human proSP-C involves modification of proSP-C21 to SP-C24 and subsequent proteolysis of C-terminal propeptide domains. We speculate that absence of low Mr intermediates in unstimulated second trimester fetal lung tissue reflects developmental and glucocorticoid dependent regulation of proSP-C21 synthesis and posttranslational processing.