Heber C. Nielsen

Association of pulmonary surfactant protein A (SP-A) gene and respiratory distress syndrome : Interaction with SP-B

Deficiency of the lipoprotein complex, surfactant, can lead to respiratory distress syndrome (RDS) in the prematurely born infant. The surfactant proteins (SP) play important roles in the function of surfactant. Previously, we have characterized four allelic variants of the SP-A1 gene (6A, 6A2, 6A3, and 6A4) and five allelic variants of the SP-A2 gene (1A, 1A0, 1A1, 1A2, and 1A3). We hypothesized that specific SP-A alleles/genotypes are associated with increased risk of RDS. Because race, gestational age (GA), and sex are risk factors for RDS, we first studied the distribution and frequencies of SP-A alleles/genotypes while adjusting for these factors as confounders or effect modifiers in control(n = 86 white and 12 black subjects) and RDS (n = 106 white and 37 black subjects) populations with GAs ranging from 24 wk to term. Although the odds ratios of several alleles and genotypes were in the opposite directions for black and white subjects, the homogeneity of odds ratio reached statistical significance only in the case of 6A3/6A3. Although differences were observed in subgroups with different GAs (≤28 and >28 wk) of the RDS white population, definitive conclusions cannot be made regarding the effect of modification by GA. No differences were observed as a function of sex. Second, we compared the frequencies of SP-A genotypes and alleles between control (n = 83) and RDS (n = 82) patients in the >28-wk white population. Differences between the two groups were observed for the 1A0 allele and 1A0 genotypes. Moreover, a significant synergistic positive association was observed between 1A0 allele + SP-B polymorphic variant and RDS. We conclude that 1) the genetic analyses of RDS and SP-A locus should be performed separately for black and white populations and 2) SP-A alleles/genotypes and SP-B variant may contribute to the etiology of RDS and/or may serve as markers for disease subgroups.

Dihydrotestosterone Inhibits Fetal Rabbit Pulmonary Surfactant Production

Males have a higher morbidity and mortality for neonatal respiratory distress syndrome (RDS) than females, and respond less well to hormone therapy designed to prevent RDS by stimulating fetal pulmonary surfactant production. We have shown that male fetuses exhibit delayed production of pulmonary surfactant. We tested the hypothesis that the sex difference in fetal pulmonary surfactant production is under hormonal control. Pulmonary surfactant was measured as the saturated phosphatidylcholine/sphingomyelin ratio (SPC/S) in the lung lavage of fetal rabbits at 26 d gestation. There was an association between the sex of neighboring fetuses and the SPC/S ratio of the female fetuses, such that with one or two male neighbors, respectively, females had decreasing SPC/S ratios (P < 0.05). We injected dihydrotestosterone (DHT) into pregnant does from day 12 through day 26 of gestation in doses of 0.1, 1.0, 10, and 25 mg/d, and measured the SPC/S ratio in fetal lung lavage on day 26. In groups with the normal sex difference in fetal serum androgen levels (controls, 0.1 mg DHT/d) the normal sex difference in the SPC/S ratio was also present (females > males, P = 0.03). In the 1-mg/d group there was no sex difference in androgen levels and the sex difference in the SPC/S ratio was also eliminated as the female values were lowered to the male level. Higher doses of DHT (10, 25 mg/d) further reduced the SPC/S ratios. We injected the anti-androgen Flutamide (25 mg/d) from day 12 through day 26 of gestation. This treatment eliminated the normal sex difference in the lung lavage SPC/S ratio by increasing the male ratios to that of the females. We conclude that androgens inhibit fetal pulmonary surfactant production. An understanding of the mechanism of the sex difference in surfactant production may allow development of therapy that is as effective in males as in females for preventing RDS.

Early Minimal Feedings Promote Growth in Critically III Premature Infants

Critically ill premature infants requiring mechanical ventilation and an umbilical artery catheter usually do not receive enteral feedings during the acute phase of their illness. We studied the safety and benefit of early minimal enteral feedings during this time in a prospective, controlled, and randomized study. Twenty-nine infants were randomly assigned to receive only standard intravenous fluid and nutrition (nothing per OS, NPO group; n = 13), or in addition to receive small-volume hypocaloric continuous feedings (1 ml/kg/h), beginning at 24 h of age (early-feeding group; n = 16). Standard enteral feedings were begun in both groups at the resolution of the acute phase of the illness and advanced by protocol. The two groups were of comparable birth weight, gestational age, and Apgar scores. There were no significant differences in the episodes of feeding intolerance. Two infants in the NPO group developed clinical signs of necrotizing enterocolitis. Serum diamine oxidase and somatomedin C were measured weekly until 30-60 days of age and were not different between the two groups. The early-feeding group required fewer days to reach 120 ml/kg/day enteral intake (early-feeding group 10 +/- 3 days, NPO group 13 +/- 4 days; p < 0.05). On day 30 of life the early-feeding group was 223 +/- 125 g above birth weight, while the NPO group was 95 +/- 161 g above birth weight (p < 0.05). The average intake (kcal/kg/day) from day 6 to day 30 was not different between the two groups. We conclude that early minimal feedings in critically ill very-low-birth-weight infants requiring mechanical ventilation are well tolerated and result in reduced time to reach 120 ml/kg/day of enteral feeding and in a greater weight gain by day 30 of life.

Sex differences in fetal rabbit pulmonary surfactant production.

Summary: Male infants have a higher risk of respiratory distress syndrome than females at concurrent gestations. Recent evidence in humans has linked fetal sex with differences in amniotic fluid indices of lung maturation. We tested the hypothesis that the late gestation surge in pulmonary surfactant production occurs later in the male fetus than in the female fetus in the rabbit model. We measured saturated phosphatidylcholine and total phosphatidylcholine in lung lavage at 26, 28, and 30 days gestation and in amniotic fluid at 24, 26, 28, and 30 days gestation (term = 31 days). The saturated phosphatidylcholine/sphingomyelin ratios were 158 and 55% higher in female fetal lung lavage at 26 and 28 days, respectively, and 75% higher in amniotic fluid at 28 days (P < 0.05). The total phosphatidylcholine/sphingomyelin ratios were 39% higher in female fetal lung lavage and 35% higher in female amniotic fluid at 28 days (P < 0.05). Significant differences were not detected in the very immature (24 day) or the mature (30 day) fetuses. This provides clear evidence of a biochemical difference according to fetal sex in the maturation of pulmonary surfactant production.Speculation: This is the first documentation of a biochemical difference according to fetal sex arising in the course of normal fetal development which is directly related to an increase in morbidity and mortality in the newborn. Further investigations using this model may help to define the mechanism by which the sex difference in fetal lung maturation occurs. This may lead to development of better methods for preventing respiratory distress syndrome in the premature infant. Understanding the causes of the male disadvantage for respiratory distress syndrome may also help to define the causes of the male disadvantage for a variety of other disorders.

Neonatal outcome of very premature infants from multiple and singleton gestations

OBJECTIVES Our purpose was to determine whether, in the era of surfactant treatment, very premature neonates from multiple gestations have outcomes similar to those of singletons. STUDY DESIGN We collected data on 572 infants (369 singletons, 203 multiple gestation) born and cared for at a single institution from July 1, 1992, through Dec, 31, 1994, of gestational ages 24 to 32 weeks. We compared singleton infants with infants from multiple gestations within gestational age categories 24 to 26 weeks, 27 to 29 weeks, and 30 to 32 weeks. RESULTS Infants of multiple gestations were more likely to have been born by cesarean section. The incidences of respiratory distress syndrome and bronchopulmonary dysplasia were similar, except that respiratory distress syndrome was more frequent in infants of multiple gestations at 30 to 32 weeks. Infants of multiple gestations from 27 to 29 weeks were more likely to have at least one of the following complications: patent ductus arteriosus, intraventricular hemorrhage, necrotizing enterocolitis, or retinopathy of prematurity. Further analysis suggested that this increase is unlikely to cause a difference in long-term outcome. The survival to discharge increased from 79% (multiples) and 81% (singletons) at 24 to 26 weeks to 98% (multiples) and 96% (singletons) at 30 to 32 weeks. CONCLUSIONS Incidences of significant neonatal problems in very premature infants from multiple gestations who are born alive are little different from those of singletons. These data should have an impact on decision making in the perinatal and neonatal care of infants of multiple gestations.

Insulin-like Growth Factor-I signaling mechanisms, type I collagen and alpha smooth muscle actin in human fetal lung fibroblasts.

Bronchial wall remodeling is a major morbidity component in oxidant injury in bronchopulmonary dysplasia (BPD) and asthma. Hypothesis: IGF-1 enhances alpha smooth muscle expression and collagen synthesis in developing lung fibroblasts leading to fibrosis through nuclear NF-kB -dependent transcription. We studied NF-kB dependent transcription by transfecting HFLF with a NF-kB responsive promoter driving the luciferase gene and treating with IGF-1 (100 ng/mL) and measuring luciferase activity. We exposed cells to the PI-3 kinase inhibitor or the Erk1/2 inhibitor one hr before stimulating with IGF-1. We also used IGF-1 receptor antibody to inhibit the action of IGF-1 and studied its effect on alpha-sma and type I collagen. IGF-1 treatment significantly increased luciferase activity. This was attenuated by PI-3 kinase and MAP-Kinase inhibitors. Western blot analysis showed PI-3 kinase mediates IGF-1 activation of NF-kB independent of IKB phosphorylation. We found an up-regulation of phospho NF-kB in the nuclear extract compared with total NFKB showing that IGF-1 regulates NF-kB transcriptional activity downstream of NF-kB nuclear translocation. IGF-1-induced increase in alpha-sma expression and type-I collagen was significantly inhibited by pretreatment with LY294002 and IGF-1 receptor antibody. IGF-1 cell signaling leading to collagen synthesis in fetal lung fibroblasts is mediated by PI3 Kinase acting through NF-kB in HFLF.

Pulse oximetry: What's normal in the newborn nursery?

The objective of this study was to establish normal values for pulse oximetry saturation (POS) in healthy newborn infants in the nursery. POS values were obtained from the right (R) hand and R foot at admission, 24 hr, and at discharge. The following information was recorded: postnatal age, activity state, gender, gestational age (GA), birth weight (BW), mode of delivery (MOD), and Apgar scores. Charts were reviewed and follow‐up information was obtained for newborns with measurements ≤92%. The study group consisted of a convenience sample of newborn infants, excluding those on supplemental oxygen.

Role of matrix metalloprotease-9 in hyperoxic injury in developing lung.

Matrix metalloprotease-9 (MMP-9) is increased in lung injury following hyperoxia exposure in neonatal mice, in association with impaired alveolar development. We studied the role of MMP-9 in the mechanism of hyperoxia-induced functional and histological changes in neonatal mouse lung. Reduced alveolarization with remodeling of ECM is a major morbidity component of oxidant injury in developing lung. MMP-9 mediates oxidant injury in developing lung causing altered lung remodeling. Five-day-old neonatal wild-type (WT) and MMP-9 (-/-) mice were exposed to hyperoxia for 8 days. The lungs were inflation fixed, and sections were examined for morphometry. The mean linear intercept and alveolar counts were evaluated. Immunohistochemistry for MMP-9 and elastin was performed. MMP-2, MMP-9, type I collagen, and tropoelastin were measured by Western blot analysis. Lung quasistatic compliance was studied in anaesthetized mice. MMP-2 and MMP-9 were significantly increased in lungs of WT mice exposed to hyperoxia compared with controls. Immunohistochemistry showed an increase in MMP-9 in mesenchyme and alveolar epithelium of hyperoxic lungs. The lungs of hyperoxia-exposed WT mice had less gas exchange surface area and were less compliant compared with room air-exposed WT and hyperoxia-exposed MMP-9 (-/-) mice. Type I collagen and tropoelastin were increased in hyperoxia-exposed WT with aberrant elastin staining. These changes were ameliorated in hyperoxia-exposed MMP-9 (-/-) mice. MMP-9 plays an important role in the structural changes consequent to oxygen-induced lung injury. Blocking MMP-9 activity may lead to novel therapeutic approaches in preventing bronchopulmonary dysplasia.

Dihydrotestosterone Stimulates Branching Morphogenesis, Cell Proliferation, and Programmed Cell Death in Mouse Embryonic Lung Explants

Early gestation lung development is characterized by branching morphogenesis of the airways and basic lung structure formation. Androgens delay late-gestation lung development if the androgen exposure begins in early gestation. We hypothesized that there would be effects of early gestation androgens on lung development. Embryonic mouse lungs (d 11.5) were cultured with dihydrotestosterone (DHT), DHT plus flutamide, or with nothing as controls. Branching morphogenesis was significantly increased after 24, 48, and 72 h of culture. This effect was blocked by simultaneous flutamide treatment. Fetal sex did not influence the DHT response. DHT increased cell proliferation as measured by [3H]thymidine incorporation into DNA. Autoradiography showed prominent [3H]thymidine labeling of epithelia and mesenchyme in regions of new bud formation. DHT treatment significantly increased the thymidine-labeling index of fibroblasts and airway epithelial cells. Programmed cell death, which is found in developing organs in association with cell proliferation during structure formation and tissue remodeling, was studied using terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling assay. In control lungs, programmed cell death occurred in the peripheral mesenchyme surrounding newly forming buds and underlying airway branch points. DHT treatment increased programmed cell death in association with increased branching morphogenesis. Evaluation of near-adjacent sections (control and DHT-treated lungs) showed that apoptotic mesenchymal cells were flanked by [3H]thymidine-labeled fibroblasts and epithelial cells, suggesting a coordination of these processes in the progression of branching morphogenesis. We conclude that androgen enhances the process of early lung morphogenesis by increasing cell proliferation and programmed cell death and by promoting the structural progression of branching morphogenesis.

Epidermal growth factor influences the developmental clock regulating maturation of the fetal lung fibroblast

Growth factors may play a significant role in regulating the orderly progression of organ growth and differentiation during fetal development. We hypothesized that epidermal growth factor (EGF) would help regulate the development of surfactant synthesis in the fetal lung by influencing fibroblast-epithelial cell interactions. The effect of EGF (10 ng per ml) on the ability of the fetal lung fibroblast to produce fibroblast pneumonocyte factor (FPF) was studied in sex-specific fibroblasts cultured from day 16, day 17 or day 18 fetal mouse lungs. FPF which is normally not produced by day 16 fibroblasts, is found only in female fibroblasts on day 17, and then in both males and females on day 18. EGF advanced this pattern such that female fibroblasts produced activity on day 16 and fibroblasts from both sexes produced FPF activity on day 17 and day 18. Fibroblasts from an androgen receptor-deficient mouse model confirmed that the effect of EGF was sex-specific and related to the state of development of the fetal lung. We conclude that EGF advances the fetal lung fibroblast through specific stages of development. It appears, therefore, to help control the timing of the clock regulating fetal lung maturation.