RESUMO
OBJECTIVE: Bronchopulmonary dysplasia (BPD) is a pulmonary disease associated with poor neurodevelopmental and medical outcomes. Patients with BPD are medically fragile, at high risk for complications and require interdisciplinary care. We tested the hypothesis that a chronic care approach for BPD would improve neurodevelopmental outcomes relative to the National Institute of Child and Human Development Neonatal Research Network (NICHD NRN) and reduce medical complications. STUDY DESIGN: Infants were followed as inpatients and outpatients. Bayley developmental exams were carried out at 18-24 months of age and compared with the NICHD NRN report. Finally, rates of readmission (a proxy for medical complications) were compared before and after implementation of the Comprehensive Center for BPD (CCBPD). RESULT: Developmental scores obtained in 2007 and 2008 show that 12 and 10% of patients with moderate BPD (n=61) had Bayley Scores <70 for mental and motor indices respectively, whereas corresponding national rates were 35 and 26%. For patients with severe BPD (n=46), 15 and 11% of patients within the CCBPD vs 50 and 42% of national patients scored <70 for mental and motor indices, respectively. Finally, readmission rates dropped from 29% in the year before the implementation of the CCPD (n=269) to 5% thereafter (n=866, P<0.0001). CONCLUSION: The encouraging neurodevelopmental outcomes and readmission rates associated with a chronic care approach to BPD suggest these infants may be best served by a comprehensive interdisciplinary approach to care that focuses on neurodevelopment throughout the hospital stay.
Assuntos
Displasia Broncopulmonar/terapia , Deficiências do Desenvolvimento/prevenção & controle , Equipe de Assistência ao Paciente , Assistência ao Paciente/métodos , Readmissão do Paciente/estatística & dados numéricos , Displasia Broncopulmonar/complicações , Desenvolvimento Infantil , Pré-Escolar , Deficiências do Desenvolvimento/etiologia , Seguimentos , Humanos , Lactente , Recém-NascidoRESUMO
AIM: To test the hypothesis that implementing guidelines for the standardized care of the extremely premature infant (<27 weeks) in the first week of life would improve patient outcomes in an all referral NICU. METHODS: Data were collected on all infants <27 weeks gestational age and <7 days of age on admission cared for using these small baby guidelines (SBG), as well as on all age-matched infants admitted the year prior (comparison). RESULTS: Thirty-seven patients were cared for utilizing the SBG and 40 patients were in the comparison group. There were no differences between the groups in gestational age, birthweight or age on admission. There was no difference in survival to discharge (73% SBG, 70% comparison). The mean length of stay for survivors was 112 +/- 38 days SBG and 145 +/- 76 days (p < 0.05) comparison group. Survival without BPD was greater in the SBG group (24%) than in the comparison group (9%; p < 0.05), and survival without severe IVH was greater in the SBG group (65%) than in the comparison group (38%; p < 0.01). CONCLUSIONS: These data demonstrate that applying a unified approach to the care of the extremely premature infant in the first week of life resulted in a decrease in the length of hospitalization and improved patient outcomes.
Assuntos
Doenças do Prematuro/terapia , Unidades de Terapia Intensiva Neonatal/organização & administração , Terapia Intensiva Neonatal/normas , Idade Gestacional , Hospitalização , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Doenças do Prematuro/mortalidade , Tempo de Internação/estatística & dados numéricos , Guias de Prática Clínica como Assunto , Análise de Sobrevida , Resultado do TratamentoRESUMO
OBJECTIVE: Premature infants, especially those born less than 1500 g, often exhibit slow overall growth after birth and lack of early nutritional support may be an important element. We tested the hypothesis that early administration of amino acids (within the first few hours of life) to infants born at less than 1500 g would be associated with fewer infants that were less than the 10th percentile at 36 weeks post-conceptual age than infants that received amino acids after the first 24 h of life. STUDY DESIGN: A prospective intervention of early amino-acid (EAA) supplementation, began before 24 h of life, in preterm infants, <1500 g, was compared to a retrospective cohort of preterm infants receiving late amino-acid (LAA) supplementation, began after 24 h of life. The primary outcome variable was the proportion of infants at less than the 10th percentile at 36 weeks post-conceptual age. RESULT: Fewer infants fell below the 10th percentile (P<0.001) in the EAA group. Furthermore, infants in the EAA groups had significantly greater weight gains than did the LAA group (P<0.003) after adjusting for gestational age and time from birth to discharge. In addition, shorter duration of parenteral nutrition was associated with EAA supplementation (P<0.001). CONCLUSION: A prospective strategy of EAA in preterm infants <1500 g was associated with an improved weight gain, suggesting that nutrition that included amino acids may be critical during the first 24 h of life.
Assuntos
Aminoácidos/administração & dosagem , Recém-Nascido de Baixo Peso/crescimento & desenvolvimento , Recém-Nascido Prematuro/crescimento & desenvolvimento , Nutrição Parenteral/métodos , Aumento de Peso , Esquema de Medicação , Humanos , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Estudos ProspectivosRESUMO
The Clara cell secretory protein (CCSP) imparts a protective effect to the lung during oxidant injury. However, exposure to supplemental oxygen, a common therapeutic modality for lung disease, represses the expression of CCSP in the adult mouse lung. We investigated the mechanisms of hyperoxia-induced repression of the mouse CCSP promoter. Deletion experiments in vivo and in vitro indicated that the hyperoxia-responsive elements are localized to the proximal -166 bp of the CCSP promoter. Electrophoretic mobility shift and supershift analyses demonstrated increased binding of c-Jun at the activator protein-1 site, increased binding of CCAAT/enhancer binding protein (C/EBP) beta at the C/EBP sites, and decreased binding at the Nkx2.1 sites. Western analyses revealed that hyperoxia exposure induced an increase in the expression of the C/EBPbeta isoform liver-inhibiting protein (LIP) and an increase in cytoplasmic Nkx2.1. Cotransfection of LIP or c-Jun expression plasmids decreased the transcriptional activity of the proximal -166-bp CCSP promoter. These observations suggest that hyperoxia-induced repression of the CCSP gene is mediated, at least in part, at the level of transcription and that multiple mechanisms mediate this repression. Moreover, these novel observations may provide insights for generation of therapeutic interventions for the amelioration of oxidant-induced lung injury.
Assuntos
Inativação Gênica , Proteínas/genética , Uteroglobina , Região 5'-Flanqueadora , Animais , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular Transformada , Citoplasma/química , Proteínas de Homeodomínio/análise , Camundongos , Modelos Genéticos , Oxigênio/toxicidade , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Interferon gamma (IFN-gamma), a potent cytokine inducing a wide range of immunologic activities, is increased in the airway secondary to viral infection or during an inflammatory response. This increase in IFN-gamma concentration may alter the expression of specific airway epithelial cell genes that regulate adaptation of airway inflammatory responses. One protein induced by IFN-gamma is Clara cell secretory protein (CCSP), which may contribute to the attenuation of airway inflammation. This study was done to investigate the molecular mechanism by which IFN-gamma stimulates the expression of the CCSP gene in mouse transformed Clara cells and transgenic mice. Deletion mapping and linker-scanning mutations demonstrated that IFN-gamma-induced expression of CCSP was regulated, in part, at the level of transcription. In vitro and in vivo studies verified that the minimal IFN-gamma-responsive segment was localized to the proximal 166 bp of the 5'-flanking region. Additionally, IFN-gamma-induced expression of CCSP was mediated indirectly through an interferon regulatory factor-1-mediated increase in hepatocyte nuclear factor-3beta.
Assuntos
Regulação da Expressão Gênica/fisiologia , Interferon gama/fisiologia , Proteínas/genética , Fatores de Transcrição , Transcrição Gênica/fisiologia , Uteroglobina , Animais , Sítios de Ligação/fisiologia , Linhagem Celular Transformada , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Expressão Gênica/fisiologia , Fator 3-beta Nuclear de Hepatócito , Fator Regulador 1 de Interferon , Camundongos , Proteínas Nucleares/metabolismo , Fosfoproteínas/fisiologia , Regiões Promotoras Genéticas , Proteínas/química , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Estereoisomerismo , Transativadores/metabolismoRESUMO
Patients with poorly functioning lungs often require treatment with high concentrations of supplemental oxygen, which, although often necessary to sustain life, can cause lung injury. The mechanisms responsible for hyperoxic lung injury have been investigated intensely and most probably involve oxidant stress responses, but the details are not well understood. In the present studies, we exposed adult male C57/Bl6 mice to >95% O2 for up to 72 h and obtained lung and liver samples for assessment of lung injury, measurements of tissue concentrations of coenzyme A (CoASH) and the corresponding mixed disulfide with glutathione (CoASSG), as possible biomarkers of intramitochondrial thiol redox status. Subcellular fractions were prepared from both tissues for determination of glutathione reductase (GR) activities. Lung injury in the hyperoxic mice was demonstrated by increases in lung weight to body weight ratios at 48 h and by increases in bronchoalveolar lavage protein concentrations at 72 h. Lung CoASH concentrations declined in the hyperoxic mice, but CoASSG concentrations were not increased nor were CoASH/CoASSG ratios decreased, as would be expected for an oxidant shift in mitochondrial thiol-disulfide status. Interestingly, CoASSG concentrations increased (from 6.72+/-0.54 to 14.10+/-1.10 nmol/g of liver in air-breathing controls and 72 h of hyperoxia, respectively, P<0.05), and CoASH/CoASSG ratios decreased in the livers of mice exposed to hyperoxia. Some apparent effects of duration of hyperoxia on GR activities in lung or liver cytosolic, mitochondrial, or nuclear fractions were observed, but the changes were not consistent or progressive. Yields of isolated hepatic nuclear protein were decreased in the hyperoxic mice within 24 h of exposure, and by 72 h of hyperoxia, protein recoveries in purified nuclear fractions had declined from 41.8 to 14.8 mg of protein/g animal body weight. Concentrations of 10-formyltetrahydrofolate dehydrogenase were diminished in hepatic mitochondria of hyperoxic mice. A second protein in hepatic mitochondria of approximately 25 kDa showed apparent decreases in thiol content, as determined by fluorescence intensities of monobromobimane derivatives separated by SDS-PAGE. The mechanisms responsible for the observed effects and the possible implications for the adverse effects of hyperoxic therapies are not known and need to be investigated.
Assuntos
Hiperóxia/fisiopatologia , Fígado/metabolismo , Mitocôndrias Hepáticas/metabolismo , Compostos de Sulfidrila/metabolismo , Animais , Peso Corporal , Líquido da Lavagem Broncoalveolar/química , Coenzima A/metabolismo , Eletroforese em Gel de Poliacrilamida , Glutationa Redutase/metabolismo , Fígado/patologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Oxirredução , Fatores de TempoRESUMO
Bronchopulmonary dysplasia (BPD) is a chronic lung disease that affects prematurely born infants and appears to evolve in part from early inflammatory responses in the lung. The inflammatory responses have been associated with protein and lipid oxidation in tracheal aspirate fluids. The present study was designed to test the hypothesis that in the first week of life specific oxidations and/ or altered expressions of proteins would be observed in tracheal aspirate fluids in infants who would subsequently develop BPD. We obtained tracheal aspirate fluids on Days of life 1, 3, and 6 from infants born at < or = 29 wk gestation, incubated the fluids with 2,4-dinitrophenylhyrazine (DNPH), separated the proteins electrophoretically, and assessed DNPH reactivity by immunonblots. DNPH reactivity of a protein that was identified as Clara cell secretory protein (CCSP) was observed more consistently in tracheal aspirate fluids from infants who later developed BPD than from infants who did not develop BPD. Tracheal aspirate fluid levels of immunoreactive CCSP were also lower on Day of life 1 in infants who developed BPD than in those who did not develop BPD. Increased CCSP oxidation and decreased immunoreactive CCSP expression in infants who subsequently developed BPD suggest that Clara cell function and CCSP expression may be critical for normal bronchoalveolar fluid homeostasis and that maintaining CCSP expression and function may be useful goals for targeted therapies for inhibition of the development of BPD.
Assuntos
Displasia Broncopulmonar/metabolismo , Inibidores Enzimáticos/metabolismo , Recém-Nascido Prematuro , Proteínas/metabolismo , Traqueia/metabolismo , Uteroglobina , Análise de Variância , Western Blotting , Humanos , Recém-Nascido , Oxirredução , Valor Preditivo dos Testes , Estudos Prospectivos , Proteínas/isolamento & purificaçãoRESUMO
OBJECTIVE: Liquid lung ventilation has been demonstrated to improve cardiorespiratory function after cardiopulmonary bypass. We hypothesized that liquid lung ventilation (LLV) would decrease the pulmonary inflammatory response after cardiopulmonary bypass (CPB). DESIGN: Prospective, randomized, experimental, controlled, nonblinded study. SETTING: Animal research laboratory at a university setting. SUBJECTS: A total of 24 neonatal piglets. INTERVENTIONS: After intubation with a cuffed endotracheal tube, swine were conventionally ventilated. After surgical cannulation, each piglet was placed on conventional nonpulsatile CPB and cooled to 18 degrees C (64.4 degrees F). Subsequently, the animals were exposed to 90 mins of low-flow CPB (35 mL/kg/min). Animals were rewarmed to 37 degrees C (98.6 degrees F), removed from CPB, and ventilated for 90 min. Ten animals received conventional gas ventilation only (control), seven received initiation of LLV before CPB (prevention), and seven received initiation of LLV during the rewarming phase of CPB (treatment). After the animals were killed, the lungs were removed en bloc. The left lobe was dissected and formalin-fixed at 20 cm H2O overnight, followed by paraffin embedding. Sections were taken from the paraffin-embedded lungs. Neutrophil accumulation and lung injury were assessed by histochemical staining with leukocyte esterase and morphometrics, respectively. One hundred microscopic images were digitized from each tissue sample for lung morphometrics, and neutrophil counts were obtained from every fifth image. MEASUREMENTS AND MAIN RESULTS: Lung tissue sections showed a significantly lower number of neutrophils per alveolar area in the prevention and treatment groups than in the control group (control 681 +/- 65, prevention 380 +/- 49, treatment 412 +/- 101 neutrophils per alveolar area [cells/mm2]; p <.05 for both prevention and treatment compared with control). There were no differences in lung injury as assessed with morphometrics or hemodynamic measurements between any of the three groups. CONCLUSIONS: The data suggest that LLV reduces the CPB-induced neutrophil sequestration in the pulmonary parenchyma independent of its effects on the circulatory physiology or evidence of early lung injury.
Assuntos
Ponte Cardiopulmonar , Fluorocarbonos/uso terapêutico , Ventilação Líquida , Pulmão/metabolismo , Neutrófilos/metabolismo , Animais , Animais Recém-Nascidos , Hidrolases de Éster Carboxílico/metabolismo , Pulmão/enzimologia , Pulmão/patologia , SuínosRESUMO
Bronchopulmonary dysplasia (BPD) is a chronic lung disease common in premature infants that can cause severe complications. BPD's pathogenesis is multifactorial but oxidative processes during the first week of life are thought to play a key role in the development of the disease. Prevention of this oxidation through antioxidant therapy is therefore of interest. Unfortunately, this therapy has not proven effective, most likely owing to the nonspecific strategy used. This review focuses on the challenges facing researchers and clinicians in improving the antioxidant status of premature infants in order to prevent or lessen the severity of BPD. This review will focus on the particular oxidations that may lead to BPD and the specific therapies that can be used to counter these processes.
Assuntos
Antioxidantes/uso terapêutico , Displasia Broncopulmonar/tratamento farmacológico , Displasia Broncopulmonar/prevenção & controle , Humanos , Recém-Nascido , Recém-Nascido Prematuro , OxirreduçãoRESUMO
Bronchopulmonary dysplasia (BPD) is a chronic lung disease first described in 1967 as a complication of therapy for premature infants with hyaline membrane disease, and treatment with high concentrations of oxygen was thought to be a major contributor to its development. Thus, interventions to enhance lung antioxidants to prevent the development of BPD were considered appropriate therapeutic strategies. In the last decades, advances in the acute care of premature infants has reduced the reliance on therapy with high concentrations of supplemental oxygen. However, the incidence of BPD has not changed significantly. The changing clinical context in which BPD develops begs the question of whether oxidation is important in the development of BPD and, therefore, whether designing interventions enhancing lung antioxidants is still warranted. This review presents evidence that premature infants that will develop BPD have qualitative and quantitative differences in oxidation of lipids and proteins when compared to infants that do not develop BPD. Such differences in oxidation patterns are the most obvious in the first few days of life. The emerging evidence thus supports the concept that the lung injury process leading to the development of BPD occurs within hours to days of delivery and that oxidation is a major contributor to this pathological process. Unfortunately, early attempts at delivery of antioxidants to the lung have not been successful, perhaps because of an inability to deliver antioxidants in a timely manner to the areas in the lung in which deleterious oxidations are occurring. Further research is necessary to determine both the nature and the location of the oxidative events that lead to the development of early lung injury, so that more appropriate and specific antioxidant interventions can be designed.
Assuntos
Antioxidantes/uso terapêutico , Displasia Broncopulmonar/terapia , Estresse Oxidativo , Displasia Broncopulmonar/etiologia , Displasia Broncopulmonar/prevenção & controle , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Metabolismo dos Lipídeos , Oxirredução , Oxigenoterapia , Proteínas/metabolismoRESUMO
We used mice with a targeted disruption in g-glutamyl transpeptidase (GGT-deficient mice) to study the role of glutathione (GSH) in protection against oxygen-induced lung injury. These mice had reduced levels of lung GSH and restricted ability to synthesize GSH because of low levels of cysteine. When GGT-deficient mice were exposed to 80% oxygen, they developed diffuse pulmonary injury and died within eight days. Ten of 12 wild-type mice were alive after 18 days. Administration of N-acetylcysteine (NAC) to GGT-deficient mice corrected GSH values and prevented the development of severe pulmonary injury and death. Oxygen exposure induced an increase in lung GSH levels in both wild-type and GGT-deficient mice, but induced levels in the mutant mice were <50% of those in wild-type mice. Cysteine levels were approximately 50-fold lower than GSH levels the lungs of both wild-type and GGT-deficient mice. Levels of lung RNA coding for the heavy subunit of g-glutamyl cysteine synthetase rose three- to fourfold after oxygen exposure in both wild-type and GGT-deficient mice. In contrast, oxygen exposure failed to provoke increases in glutathione synthetase, glutathione peroxidase, glutaredoxin, or thioredoxin.
Assuntos
Acetilcisteína/farmacologia , Glutationa/fisiologia , Hiperóxia/metabolismo , Lesão Pulmonar , gama-Glutamiltransferase/deficiência , Animais , Cisteína/metabolismo , Glutamato-Cisteína Ligase/metabolismo , Hiperóxia/etiologia , Pulmão/metabolismo , Camundongos , RNA/genéticaRESUMO
Reactive oxygen species (ROS) are implicated as agents of cellular damage in pulmonary oxygen toxicity. Glutathione (GSH) and GSH-dependent antioxidant enzymes protect against damage by ROS, and recycling of glutathione disulfide (GSSG) to GSH by glutathione reductase (GR) is essential for the optimum functioning of this system. Exposure to hyperoxia inhibits lung development in newborn animals and humans, and attenuates cell growth in proliferating cell cultures. Considerable evidence supports a role for ROS as growth-altering molecules. Previously, we have observed that gene transfer of GR to mitochondria in H441 cells, using a vector containing a mitochondrial leader sequence (LGR), protected these cells against t-BuOOH-induced cytotoxicity. The present studies tested the hypothesis that gene transfer of LGR would attenuate the cytostatic effects of hyperoxia exposure in H441 cells. H441 cells (0.9 x 10(6) cells/plate) transfected with adenovirus containing LGR or the complementary DNA (cDNA) for manganese superoxide dismutase in reverse orientation (DOS) as a control construct, and untransfected cells (CON) were maintained in 21% oxygen (normoxia) or 95% oxygen (hyperoxia) for 48 h, and cell growth was assessed by cell counts and by reduction of the tetrazolium dye 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan. Cells maintained in normoxia achieved normal growth (CON, 1.98; DOS, 1.91; LGR, 2.0 x 10(6) cells/plate). Hyperoxia inhibited cell growth and the reduction of MTT; however, cells transfected with LGR had greater mitochondrial GR activities (CON, 16+/-2; DOS, 19+/-3; LGR, 322+/-18 mU/mg of protein), sustained more normal growth patterns (CON, 1.25+/-0.12; DOS, 1.24 +/-0.21, LGR, 1.8+/-0.25 x 10(6) cells/plate), and had less inhibition of MTT reduction (CON, 29; DOS, 27; LGR, 16% inhibition, P<0.01) after exposure to hyperoxia for 48 h than was observed in cells transfected with DOS or in control cells not infected with virus. In addition, resistant cells had higher mitochondrial GSH levels and maintained mitochondrial GSH/GSSG ratios in hyperoxia, suggesting that maintaining mitochondrial GSH homeostasis determined critical aspects of cell division in these studies. The mechanisms for sustaining cell growth during hyperoxia in H441 cells with enhanced mitochondrial GR activities are unknown, but similar effects in infants exposed to supplemental oxygen could be highly beneficial.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Glutationa Redutase/genética , Hiperóxia/metabolismo , Pulmão/citologia , Mitocôndrias/enzimologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular Transformada , Regulação Enzimológica da Expressão Gênica , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Glutationa Redutase/metabolismo , Inibidores do Crescimento/genética , Humanos , L-Lactato Desidrogenase/metabolismo , Oxigênio/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sequências Reguladoras de Ácido Nucleico , TransfecçãoRESUMO
Redox cycling metabolism of diquat catalyzes generation of reactive oxygen species, and diquat-induced acute hepatic necrosis in male Fischer 344 (F344) rats has been studied as a model of oxidant mechanisms of cell killing in vivo. At equal doses of diquat, female F344 rats sustained less hepatic damage than did male rats, as estimated by plasma alanine aminotransferase (ALT) activities after 6 h. Biliary efflux of glutathione disulfide (GSSG) was greater in male than in female rats at each dose of diquat, but even comparable rates of GSSG excretion were associated with less hepatic injury in female rats. Hepatic activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were similar in the two genders, and activities of glutathione reductase (GR) and glutathione S-transferase-alpha (GST-alpha) activities were higher in the male rats. Previous studies in male rats have implicated formation of 2,4-dinitrophenylhydrazine (DNPH)-reactive "protein carbonyls" and related iron chelate-catalyzed redox reactions as mechanisms critical to diquat-induced acute cell death in vivo. However, diquat-treated female rats showed higher levels of DNPH-reactive proteins in livers and in bile than did males, both at identical doses of diquat and at doses that produced similar elevations in plasma ALT activities. In female rats, fragmentation of hepatic deoxyribonucleic acids (DNA) was increased by doses of diquat that did not increase plasma ALT activities, and increased fragmentation was observed prior to elevation of plasma ALT activities. In the present studies, hepatic necrosis was most closely associated with DNA fragmentation, although additional studies are needed to determine the mechanisms responsible for and the pathophysiological consequences of the fragmentation.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Diquat/toxicidade , Herbicidas/toxicidade , Alanina Transaminase/metabolismo , Animais , Ductos Biliares , Western Blotting , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Fragmentação do DNA/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Feminino , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Glutationa Transferase/metabolismo , Masculino , Necrose , Proteínas/metabolismo , Ratos , Ratos Endogâmicos F344 , Caracteres Sexuais , Superóxido Dismutase/metabolismoRESUMO
OBJECTIVE AND DESIGN: Lung intercellular adhesion molecule-1 (ICAM-1) expression is increased by LPS or hyperoxia on type II cells in vivo. The goals of the present study were to determine the mechanisms of ICAM-1 expression in a lung alveolar epithelial cell line (A549) exposed to lipopolysaccharide (LPS). MATERIALS: A549 cells, a transformed human cell line with characteristics of alveolar epithelial cells, were used. TREATMENT: Cells were exposed to LPS, TNF-alpha, IL-1beta, or media alone for up to 12 h. METHODS: Northern blot analyses were done to determine mRNA expression of ICAM-1 after exposures. Protein binding to NF-kappaB sequences were determined by gel mobility shift assays and super-shift analysis. RESULTS: ICAM-1 mRNA expression was induced in A549 cells with exposure to LPS for 1 to 4 h, and was diminished to baseline at 8 h, and the inductions were independent of TNF-alpha and IL-1beta expression. Nuclear protein extracts from LPS-exposed cells bound to a NF-kappaB sequence and the timing of increased binding correlated closely with ICAM-1 mRNA induction. Super-shift studies indicated that p65 was involved in the binding to the NF-kappaB sequence and p50 was not. CONCLUSION: LPS inducibility of ICAM-1 mRNA in A549 cells is independent of TNF- and IL-1 in A549 cells, and the similar time course of mRNA induction and NF-kappaB activation suggest the induction of ICAM-1 is mediated, in part, by NF-kappaB.
Assuntos
Expressão Gênica/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/genética , Lipopolissacarídeos/farmacologia , Alvéolos Pulmonares/metabolismo , Sítios de Ligação , Northern Blotting , Linhagem Celular Transformada , Células Epiteliais/metabolismo , Humanos , Interleucina-1/farmacologia , NF-kappa B/metabolismo , RNA Mensageiro/biossíntese , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Bronchopulmonary dysplasia (BPD) is recognized as an important cause of morbidity and mortality in preterm infants. Because the role of congenital infections in BPD has been debated, the purpose of this study was to test the hypothesis that detection of infectious agents in tracheal aspirate samples was associated with the development of BPD. Tracheal aspirate samples were obtained within the 1st week of life and screened by polymerase chain reaction for adenovirus, cytomegalovirus, parvovirus, enteroviruses, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma pneumoniae, and Chlamydia species. BPD was defined as persistent oxygen dependence at 28 d of age and 36 wk postconceptional age (PCA). Infants that expired before these time points were excluded from statistical analysis. Out of 89 infants studied, at 28 d of life, 13 had expired, 45 had BPD, and 31 had no BPD (controls). At 36 wk PCA, 15 infants expired, 39 still had BPD, and 35 did not. A significant increase in the frequency of adenovirus genome was identified in BPD patients compared with controls, both at 28 d of life (12/45 = 27% versus 1/31 = 3%: p< or =0.01) and at 36 wk PCA (10/39 = 29% versus 2/35 = 6%: p = 0.01). Other microorganisms were rarely detected and not associated with the development of BPD. This is the first study reporting the frequency of detection of adenovirus DNA in tracheal aspirate samples obtained during the 1st week of life from infants with BPD and suggests that prenatal acquisition may be important in the development of BPD.
Assuntos
Infecções por Adenoviridae/diagnóstico , Displasia Broncopulmonar/microbiologia , Recém-Nascido Prematuro , Traqueia/microbiologia , Adenoviridae/isolamento & purificação , Infecções por Adenoviridae/microbiologia , Chlamydia/isolamento & purificação , Citomegalovirus/isolamento & purificação , DNA Bacteriano/análise , Enterovirus/isolamento & purificação , Células HeLa , Humanos , Recém-Nascido , Mycobacterium/isolamento & purificação , Parvovirus/isolamento & purificação , Reação em Cadeia da Polimerase/métodosRESUMO
In this investigation, we tested the hypothesis that the cytochrome P-450 (CYP) inhibitor 1-aminobenzotriazole (ABT) alters the susceptibility of rats to hyperoxic lung injury. Male Sprague-Dawley rats were treated i.p. with ABT (66 mg/kg), i.v. with N-benzyl-1-aminobenzotriazole (1 micromol/kg), or the respective vehicles, followed by exposure to >95% oxygen for 24, 48, or 60 h. Pleural effusion volumes were measured as estimates of hyperoxic lung injury, and lung microsomal ethoxyresorufin O-deethylation (EROD) (CYP1A1) activities and CYP1A1 apoprotein levels were determined by Western blotting. ABT-pretreated animals exposed to hyperoxia died between 48 and 60 h, whereas no deaths were observed with up to 60 h of hyperoxia in vehicle-treated animals. In addition, three of four ABT-treated rats exposed to hyperoxia for 48 h showed marked pleural effusions. Exposure of vehicle-treated rats to hyperoxia led to 6.3-fold greater lung EROD activities and greater CYP1A1 apoprotein levels than in air-breathing controls after 48 h, but both declined to control levels by 60 h. Liver CYP1A1/1A2 enzymes displayed responses to hyperoxia and ABT similar to the effects on lung CYP1A1. N-Benzyl-1-aminobenzotriazole markedly inhibited lung microsomal pentoxyresorufin O-depentylation (principally CYP2B1) activities in air-breathing and hyperoxic animals but did not affect lung EROD or liver CYP activities. In conclusion, the results suggest that induction of CYP1A enzymes may serve as an adaptive response to hyperoxia, and that CYP2B1, the major pulmonary CYP isoform, does not contribute significantly to hyperoxic lung injury.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Hiperóxia/patologia , Oxigênio/toxicidade , Síndrome do Desconforto Respiratório/induzido quimicamente , Triazóis/farmacologia , Animais , Western Blotting , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Hiperóxia/mortalidade , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Oxazinas/farmacocinética , Derrame Pleural , Isoformas de Proteínas/farmacologia , Ratos , Ratos Sprague-Dawley , Síndrome do Desconforto Respiratório/mortalidade , Fatores de TempoAssuntos
Linhagem Celular Transformada/metabolismo , Regulação da Expressão Gênica/fisiologia , Hiperóxia/genética , Pulmão/metabolismo , Proteínas/genética , Mucosa Respiratória/metabolismo , Uteroglobina , Animais , Linhagem Celular Transformada/patologia , Hiperóxia/metabolismo , Hiperóxia/fisiopatologia , Pulmão/patologia , Pulmão/fisiopatologia , Camundongos , Pneumonia/genética , Pneumonia/metabolismo , Pneumonia/fisiopatologia , RNA Mensageiro/metabolismo , Mucosa Respiratória/patologiaRESUMO
OBJECTIVE AND DESIGN: To test the hypothesis that glucocorticoid administration would diminish the lung expression of P-selectin mRNA in hyperoxia-exposed rats. ANIMALS: Adult male Sprague-Dawley rats were divided into 6 separate groups containing 10 to 13 animals per group. TREATMENT: Rats were dosed with 1 mg/kg of dexamethasone or vehicle only, ip. Immediately after dosing, animals were placed in > 95 % oxygen. Some animals were maintained in room air and are presented as 0 h of exposure to hyperoxia. Another group of animals was dosed with 10 mg/kg lipopolysaccharide (LPS) ip immediately after dosing with either dexamethasone or vehicle as above. METHODS: At 24 or 48 h, lung samples were obtained, and lung weight to body weight ratios calculated. In the LPS studies, samples were obtained 4 h after LPS dosing. In a subset of animals, lung sections were hybridized for P-selectin mRNA. All data except for hybridizations were analyzed with three-way ANOVA, with subsequent post-hoc testing. P-selectin hybridizations were quantified by counting the number of positive vessels per high-powered field, and subsequently analyzed by unpaired Student's t-test. Immunohistochemical analyses for P-selectin expression were also performed to determine whether changes in P-selectin mRNA were associated with differences in protein expression. All data are expressed as means +/- SEM. RESULTS: Rats dosed with dexamethasone had higher lung/body weight ratios after 24 and 48 h of exposure to hyperoxia than did similarly exposed rats dosed only with vehicle (at 48 h, 0.87 +/- 0.04 versus 0.65 +/- 0.06, respectively, P < 0.05). The higher ratios in hyperoxic animals dosed with dexamethasone were associated with much higher levels of lung expression for P-selectin mRNA than was observed in similarly exposed rats dosed with vehicle alone (at 48 h, 3.93 +/- 1.02, versus 0.20 +/- 0.06, respectively, P < 0.01). In contrast dexamethasone dosing lead to lower lung P-selectin mRNA expression in animals exposed to LPS (1.23 +/- 1.08 in dexamethasone dosed animals versus 6.80 +/- 0.92 in vehicle only dosed animals). Consistent with the mRNA data, P-selectin immunoreactivity increased as a function of hyperoxia-exposure time in animals dosed with dexamethasone, while immunoreactivity decreased as a function of hyperoxia-exposure time in animals dosed with vehicle only. CONCLUSIONS: Increased P-selectin mRNA combined with increased P-selectin protein expression in animals exposed to hyperoxia and dosed with dexamethasone suggests that enhanced expression of P-selectin may contribute to the greater lung injury and inflammation caused by hyperoxia in rats treated with dexamethasone.
Assuntos
Anti-Inflamatórios/farmacologia , Dexametasona/farmacologia , Hiperóxia/metabolismo , Pulmão/metabolismo , Selectina-P/biossíntese , RNA Mensageiro/biossíntese , Animais , Toxinas Bacterianas/toxicidade , Peso Corporal/efeitos dos fármacos , Endotoxinas/toxicidade , Hemoglobinas/metabolismo , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Hibridização In Situ , Lipopolissacarídeos/toxicidade , Pulmão/anatomia & histologia , Pulmão/efeitos dos fármacos , Masculino , Tamanho do Órgão/efeitos dos fármacos , Biossíntese de Proteínas , Ratos , Ratos Sprague-DawleyRESUMO
Oxidative modification of LDL may be important in the initiation and/or progression of atherosclerosis, but the precise mechanisms through which low density lipoprotein (LDL) is oxidized are unknown. Recently, evidence for the existence of HOCl-oxidized LDL in human atherosclerotic lesions has been reported, and myeloperoxidase (MPO), which is thought to act through production of HOCl, has been identified in human atherosclerotic lesions. In the present report we describe the formation of 2,4-dinitrophenylhydrazine (DNPH)-reactive modifications in the apolipoprotein (apo) by exposure of LDL to myeloperoxidase in vitro. In contrast with the complex mixture of peptides from oxidation of LDL with reagent HOCl, oxidation with MPO in vitro produced a major tryptic peptide showing absorbance at 365 nm. This peptide was isolated and characterized as VELEVPQL(*C)SFILK..., corresponding to amino acid residues 53-66...on apoB-100. Mass spectrometric analyses of two tryptic peptides from oxidation of LDL by HOCl indicated formation of the corresponding methionine sulfoxide (M=O), cysteinyl azo (*C), RS -N= N-DNP, derivatives of EEL(*C)T(M=O)FIR and LNDLNS VLV(M=O)PTFHVPFTDLQVPS(*C)K, which suggest oxidation to the corresponding sulfinic acids (RSO2H) by HOCl. The present results demonstrate that DNPH-reactive modifications other than aldehydes and ketones can be formed in the oxidation of proteins and illustrate how characterization of specific products of protein oxidation can be useful in assessing the relative contributions of different and unexpected mechanisms to the oxidation of LDL and other target substrates. The data also suggest a direct interaction of the LDL particle with the active site on myeloperoxidase and indicate that effects of the protein microenvironment can greatly influence product formation and stability.
Assuntos
Apolipoproteínas B/metabolismo , Lipoproteínas LDL/metabolismo , Peroxidase/metabolismo , Sequência de Aminoácidos , Apolipoproteína B-100 , Apolipoproteínas B/química , Cromatografia Líquida de Alta Pressão , Glutationa/química , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/química , Ácido Hipocloroso/química , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Molecular , Oxirredução , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fenil-Hidrazinas/química , Ácidos Sulfínicos/química , Tripsina/metabolismoRESUMO
Increased generation of reactive oxygen species (ROS) and low levels of antioxidants may cause morbidity in premature infants on supplemental oxygen. Glutathione (GSH)-dependent antioxidant systems protect against ROS, and regenerating GSH from GSH disulfide (GSSG) by the flavoenzyme GSH reductase (GR) is essential for the optimal function of this system. Previously, we have observed enhanced resistance to t-butyl hydroperoxide (t-BuOOH) in Chinese hamster ovary cells stably transfected with a vector (leader sequence GR [LGR]) for human GR cDNA that contained a functional synthetic mitochondrial targeting signal. The present studies were designed to investigate adenovirus-mediated gene transfer of LGR to H441 cells and resistance of such cells to t-BuOOH. Adenovirus-mediated transfection of H441 cells with LGR increased total GR activities more than 11-fold (mitochondria more than 10-fold and cytosolic more than 7-fold) and protected against t-BuOOH cytotoxicity, as indicated by lower fractional release of cellular lactate dehydrogenase (LDH) than was observed in wild-type untransfected cells (CON) or in cells transfected with a control gene (human manganese superoxide dismutase in the antisense orientation [DOS]) (*LGR 6.6 +/- 1.7; DOS 16 +/- 1.8; CON 16.6 +/- 0.7% LDH release). In addition, cells transfected with LGR retained higher GSH/GSSG ratios (*LGR 66 +/- 0.4; DOS 47 +/- 1; CON 52.6 +/- 2.3) and released less GSH + GSSG to the media in response to challenge with t-BuOOH (*LGR 0.05 +/- 0.01; DOS 0.08 +/- 0.01; CON 0.07 +/- 0.01 nmol/mg of protein) than did wild-type cells or cells transfected with a control vector, indicating an enhanced ability of the LGR cells to reduce GSSG formed in response to exposure to t-BuOOH. In conclusion, adenovirus-mediated gene transfer of LGR enhanced cellular GR activities and protected H441 cells from oxidant stresses.