Variability in the efficacy of a standardized antenatal steroid treatment was independent of maternal or fetal plasma drug levels: evidence from a sheep model of pregnancy.

BACKGROUND: Administration of antenatal steroids is standard of care for women assessed to be at imminent risk of preterm delivery. There is a marked variation in antenatal steroid dosing strategy, selection for treatment criteria, and agent choice worldwide. This, combined with very limited optimization of antenatal steroid use per se, means that treatment efficacy is highly variable, and the rate of respiratory distress syndrome is decreased to perhaps as low as 40%. In some cases, antenatal steroid use is associated with limited benefit and potential harm. OBJECTIVE: We hypothesized that individual differences in maternofetal steroid exposure would contribute to observed variability in antenatal steroid treatment efficacy. Using a chronically catheterized sheep model of pregnancy, we aimed to explore the relationship between maternofetal steroid exposure and antenatal steroid treatment efficacy as determined by functional lung maturation in preterm lambs undergoing ventilation. STUDY DESIGN: Ewes carrying a single fetus underwent surgery to catheterize a fetal and maternal jugular vein at 119 days' gestation. Animals recovered for 24 hours before being randomized to either (1) a single maternal intramuscular injection of 2 mL saline (negative control group, n=10) or (2) a single maternal intramuscular injection of 0.25 mg/kg betamethasone phosphate plus acetate (antenatal steroid group, n=20). Serial maternal and fetal plasma samples were collected from each animal after 48 hours before fetuses were delivered and ventilated for 30 minutes. Total and free plasma betamethasone concentration was measured by mass spectrometry. Fetal lung tissue was collected for analysis using quantitative polymerase chain reaction. RESULTS: One animal from the control group and one animal from the antenatal steroid group did not complete their treatment protocol and were removed from analyses. Animals in the antenatal steroid group were divided into a responder subgroup (n=12/19) and a nonresponder subgroup (n=7/19) using a cutoff of partial pressure of arterial CO2 at 30-minute ventilation within 2 standard deviations of the mean value from saline-treated negative control group animals. Although antenatal steroid improved fetal lung maturation in the undivided antenatal steroid group and in the responder subgroup both physiologically (blood gas- and ventilation-related data) and biochemically (messenger ribonucleic acid expression related to fetal lung maturation), these values did not improve relative to saline-treated control group animals in the antenatal steroid nonresponder subgroup. No differences in betamethasone distribution, clearance, or protein binding were identified between the antenatal steroid responder and nonresponder subgroups. CONCLUSION: This study correlated individual maternofetal steroid exposures with preterm lung maturation as determined by pulmonary ventilation. Herein, approximately 40% of preterm lambs exposed to antenatal steroids had lung maturation that was not significantly different to saline-treated control group animals. These nonresponsive animals received maternal and fetal betamethasone exposures identical to animals that had a significant improvement in functional lung maturation. These data suggest that the efficacy of antenatal steroid therapy is not solely determined by maternofetal drug levels and that individual fetal or maternal factors may play a role in determining treatment outcomes in response to glucocorticoid signaling.

Effect of Tocolytics on Surfactant Secretion When Administered with Betamethasone: An in Vitro Study.

OBJECTIVE: To analyze the amount of surfactant protein (SP)-B and lecithin/sphingomyelin (L/S) ratio in response to betamethasone (BMS) alone as compared with magnesium sulfate (Mg(2+)), indomethacin (Indo), and nifedipine (Nif) with or without BMS. STUDY DESIGN: NCI-H441 human lung cells were grown and distributed into eight plates. BMS and tocolytics were added and the final plates were: control, BMS only, and each tocolytic ± BMS. Cells were stained with SP-B antibodies and relative fluorescence was measured. Lipids were also extracted, identified, and examined for relative densities. The L/S ratio was calculated. RESULTS: Nine independent measurements were obtained for each plate. The protein analysis revealed that among all eight plates, SP-B levels were highest among BMS only. There was a nonsignificant decrease in SP-B in each of the combinations of tocolytics + BMS as compared with BMS only. Compared with BMS only, L/S ratio was decreased in Mg(2+) + BMS (p = 0.041), Indo + BMS (p = 0.042), and Nif + BMS (p = 0.025). CONCLUSION: In our in vitro human lung cell model, SP-B and L/S ratio increased in response to BMS administration alone. The addition of tocolytics to BMS resulted in no increase in L/S ratio and no changes seen in SP-B production compared with BMS alone.

Exogenous surfactant: intubated present, nebulized future?

BACKGROUND: exogenous surfactant is currently administered via intra-tracheal instillation, a method which can increase the possibility of clinical instability in the peri-surfactant administration period. Since its introduction, there has been an increase in understanding of the pathology of respiratory distress syndrome and surfactant biology. This includes development of a potential nebulized surfactant which has the potential to increase the number, safety and timely administration of the medication in preterm infants. DATA SOURCES: based on recent original publications in the field of surfactant biology, we reviewed our experience with surfactant administration and discussed the available evidence on nebulized surfactant and outlined potential barriers toward widespread introduction of this therapy. RESULTS: surfactant has revolutionized modern neonatal management and nebulized surfactant is attractive and a vector for administration. However, issues regarding costeffectiveness, development of nebulizer devices capable of administration, deposition of medication in the airway and dosing strategies remain unresolved. CONCLUSIONS: nebulized surfactant has the potential to be a therapeutic breakthrough by eliminating the potent volu-and-baro-traumatic effects of mechanical ventilation in the peri-surfactant period. Nebulization would likely lead to increased administration immediately after birth and more emphasis on noninvasive ventilator strategies. These features will aid clinical implementation of nebulized surfactant as a standard of treatment after introduction.

Inactivation of pulmonary surfactant by silicone oil in vitro and in ventilated immature rabbits.

OBJECTIVE: Surface activity of pulmonary surfactant is impaired by exposure to syringes lubricated with silicone oil (SO). These syringes are used daily in clinical practice. DESIGN: In vitro experiments were used for detection of SO, determination of surface activity, and semiquantitative measurement of surfactant protein (SP)-B and -C in SO/surfactant mixtures. Randomized, controlled animal studies were applied for determination of in vivo activity. SETTING: University research laboratory. INTERVENTIONS: Mass spectrometry of SO originating from syringes with and without surfactant was performed. The surface activity of SO plus surfactant phospholipids (PLs) or modified natural surfactant (Curosurf) was measured. SO/Curosurf preparations were further analyzed for changes in the content of SP-B and SP-C using immunoblotting. Neonatal rabbits received mixtures of SO/Curosurf (ratio 0-1.3 mg/mg PL) intratracheally and were then ventilated with a standardized sequence of peak insufflation pressures. Tidal volume curves were recorded, gas volumes of excised lungs were measured, and histologic analysis was performed. MEASUREMENTS AND MAIN RESULTS: Dissolved SO was found after rinsing syringes with organic solvents or Curosurf. Surface activity of Curosurf was significantly reduced after addition of 0.13-1.3 mg SO/mg PL. Immunoblotting revealed interference of SO with SP-B, but not with SP-C. With increasing SO/Curosurf ratios, patchy alveolar air expansion was observed, lung gas volumes were reduced, and time to inflate the lungs was increased, whereas compliance and tidal volumes remained unimpaired. CONCLUSIONS: In vitro SO impairs surface activity of Curosurf and leads to interference with SP-B. SO contamination of exogenous surfactant impairs lung function in animal studies and should be avoided.

Effects of keratinocyte growth factor on intra-alveolar surfactant fixed in situ: Quantitative ultrastructural and immunoelectron microscopic analysis.

Quantitative (immuno) transmission electron microscopy using design-based stereology was performed on specimens collected by means of systematic uniform random sampling of rat lungs, which were fixed by vascular perfusion to stabilize intra-alveolar surfactant in situ. This procedure ensures that the data recorded are representative of the whole organ. Ultrathin sections of specimens embedded at low temperature in Lowicryl HM20 were labeled by indirect immuno-gold staining for surfactant protein A. We observed that, 3 days after treatment of lungs in vivo with truncated keratinocyte growth factor (DeltaN23-KGF), a potent mitogen of alveolar epithelial type II cells, surfactant protein A associated with the tubular myelin fraction of intra-alveolar surfactant was increased by 47% in comparison with buffer-treated control lungs. Despite the marked type II cell hyperplasia, the relative amount of ultrastructural surfactant subtypes was not significantly affected. Because surfactant protein A reduces the sensitivity to inhibition of the biophysical activity of surfactant by exudating plasma proteins, we propose that pretreatment of lungs with DeltaN23-KGF ameliorates adverse effects observed in acute lung injury following, for example, ischemia and reperfusion.

Characterization of corticosteroid redosing in an in vitro cell line model.

OBJECTIVE: The purpose of this study was to investigate dexamethasone redosing as function of time and dose. STUDY DESIGN: We studied the effect of 48 hours' exposure to various concentrations of dexamethasone in a human pulmonary adenocarcinoma cell line (H-441). We measured the level of surfactant protein B (SP-B) mRNA by quantitative reverse transcription-PCR after initial dexamethasone exposure, and after redosing, 1 or 2 weeks later. Values are mean +/- SE for 5 experiments. Comparisons were made by Mann-Whitney and Kruskal-Wallis test with significance set at P < .05. RESULTS: Induction of SP-B mRNA was maximal within 48 hours of the initial dexamethasone exposure. Redosing with the same dexamethasone concentration resulted in levels more than double those initially observed. Redosing with dexamethasone concentration 10 times lower had an effect comparable to that of the initial, higher concentration. CONCLUSION: Our results suggest a residual effect of the initial exposure that potentiates redosing, allowing significant dose reductions.

The effects of dexamethasone and betamethasone on surfactant protein-B messenger RNA expression in human type II pneumocytes and human lung adenocarcinoma cells.

OBJECTIVE: The purpose of this study was to compare the effect of a single 48-hour exposure to betamethasone or dexamethasone in the NCI-H441 cell line and in human type II pneumocytes. STUDY DESIGN: NCI-H441 cells were exposed 48 hours to varying concentrations of betamethasone or dexamethasone (10(-10) to 10(-7) mol/L) alone or in combination with 1 mmol/L dibutyryl cyclic adenosine monophosphate. Likewise, human type II pneumocytes were exposed 48 hours to varying concentrations of betamethasone or dexamethasone (10(-9) to 10(-7) mol/L) alone or in combination with 1 mmol/L dibutyryl cyclic adenosine monophosphate. The measured outcome was the stimulatory effect on surfactant protein B gene transcription as expressed by surfactant protein B messenger RNA accumulation. The experiment was conducted 5 times in NCI-H441 cells and 6 times in type II cells, in parallel with control. Surfactant protein B messenger RNA was determined at control level and 48 hours after exposure by quantitative reverse transcription-polymerase chain reaction. RESULTS: A similar dose-dependent response in surfactant protein B messenger RNA expression was seen with both betamethasone and dexamethasone. In human type II pneumocytes, the inductive profile of surfactant protein B messenger RNA after 48-hour exposure to betamethasone or dexamethasone was similar to that seen in the NCI-H441 cells. CONCLUSION: Dexamethasone and betamethasone achieved similar dose-response patterns of surfactant protein-B expression in vitro.

Vitamin C rescues in part the effects of nitrofen on cultured human pneumocytes.

BACKGROUND: One of the mechanisms of action of nitrofen is intracellular oxidative stress. It is therefore likely that anti-oxidant agents can counteract its action. The aim of this study was to examine whether vitamin C mitigates the effects of nitrofen on the proliferation/apoptosis balance and functional maturation of cultured human pneumocytes. METHODS: Lung aCa type II pneumocytes (NIH-H441) in culture were exposed to 1.5 microM of nitrofen alone or followed 48 h later by 60 microM of vitamin C. The culture dishes were recovered at 72 h for immunohistochemical (PCNA for proliferation, bis-benzimide for apoptosis, anti-SpB and anti-TTF-1 antibodies for SpB and TTF-1 assessment) and molecular studies. Real-time PCR was used to quantitate TTF-1 RNAm, with S26 as internal control. ANOVA or Kruskall-Wallis with appropriate post hoc tests were used for comparison with p<0.05 as threshold of significance. RESULTS: Nitrofen decreased proliferation and TTF/1 and SpB expression with no apparent affect on apoptosis. Additional exposure to vitamin C reverted these effects. Furthermore, nitrofen decreased TTF/1 mRNA and vitamin C tended to rescue normal values. CONCLUSIONS: Vitamin C partially rescued proliferation and maturity in nitrofen-treated human pneumocytes. The likely beneficial influence of this prenatal anti-oxidant medication should be further investigated.