Published online August 1, 2007
PEDIATRICS Vol. 120 No. 2 August 2007, pp. 346-353 (doi:10.1542/10.1542/peds.2007-0095)
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ARTICLE

Surfactant Function and Composition in Premature Infants Treated With Inhaled Nitric Oxide

Philip L. Ballard, MD, PhDa, Jeffrey D. Merrill, MDb, William E. Truog, MDc, Rodolfo I. Godinez, MD, PhDd, Marye H. Godinez, MDd, Theresa M. McDevitt, MSb, Yue Ning, MSb, Sergio G. Golombek, MD, MPHe, Lance A. Parton, MDe, Xianqun Luan, MSf, Avital Cnaan, PhDf and Roberta A. Ballard, MDa

a Department of Pediatrics, University of California, San Francisco, California
b Departments of Pediatrics
d Critical Care/Anesthesia
f Biostatistics, Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, Pennsylvania
c Department of Pediatrics, Children's Mercy Hospitals and Clinics/University of Missouri-Kansas City School of Medicine, Kansas City, Missouri
e Department of Pediatrics, New York Medical College/Marie Fareri Children's Hospital at Westchester Medical Center, Valhalla, New York


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
OBJECTIVES. We hypothesized that inhaled nitric oxide treatment of premature infants at risk for bronchopulmonary dysplasia would not adversely affect endogenous surfactant function or composition.

METHODS. As part of the Nitric Oxide Chronic Lung Disease Trial of inhaled nitric oxide, we examined surfactant in a subpopulation of enrolled infants. Tracheal aspirate fluid was collected at specified intervals from 99 infants with birth weights <1250 g who received inhaled nitric oxide (20 ppm, weaned to 2 ppm) or placebo gas for 24 days. Large-aggregate surfactant was analyzed for surface activity with a pulsating bubble surfactometer and for surfactant protein contents with an immunoassay.

RESULTS. At baseline, before administration of study gas, surfactant function and composition were comparable in the 2 groups, and there was a positive correlation between minimum surface tension and severity of lung disease for all infants. Over the first 4 days of treatment, minimum surface tension increased in placebo-treated infants and decreased in inhaled nitric oxide–treated infants. There were no significant differences between groups in recovery of large-aggregate surfactant or contents of surfactant protein A, surfactant protein B, surfactant protein C, or total protein, normalized to phospholipid.

CONCLUSIONS. We conclude that inhaled nitric oxide treatment for premature infants at risk of bronchopulmonary dysplasia does not alter surfactant recovery or protein composition and may improve surfactant function transiently.

Key Words: nitric oxide • premature infant • surface tension • surfactant dysfunction • respiratory severity score

Abbreviations: BPD—bronchopulmonary dysplasia • SP—surfactant protein • iNO—inhaled nitric oxide • NO—nitric oxide • TAF—tracheal aspirate fluid • CLD—chronic lung disease

Pulmonary surfactant is a complex mixture of lipids and proteins that reduces alveolar surface tension and prevents atelectasis. An inadequate amount of surfactant in prematurely born infants contributes to respiratory distress syndrome, and occurrence of this disorder is decreased by surfactant replacement at birth.1 Reduced function of surfactant occurs in infants undergoing chronic ventilation, resulting from delayed and/or inhibited production of surfactant protein (SP)-B and SP-C, and episodes of surfactant dysfunction in these infants are associated with respiratory decompensation.2 Similar observations of surfactant dysfunction and reduced content of SPs have been made in adults with acute respiratory distress syndrome.3

In addition to developmental regulation, surfactant production is influenced by exposure to a variety of agents. Synthesis and secretion are increased by glucocorticoids and by agents that increase cyclic adenosine monophosphate content, and production is decreased by several cytokines and growth factors.4 In this study, we investigated the effects of inhaled nitric oxide (iNO) treatment of premature infants on surfactant function and composition. Previous studies with lung cells in vitro demonstrated inhibitory effects of iNO on surfactant, including decreased levels of phosphatidylcholine and SPs.5–11 Brief exposure to relatively high concentrations of iNO in several animal studies caused surfactant dysfunction, decreased SP content, and increased inhibitory protein levels in lavage fluid.12–14 In contrast, chronic exposure of infant baboons to a clinically relevant dose of iNO had primarily beneficial effects on surfactant. Although surfactant recovery was reduced slightly, iNO-treated animals had an improved surfactant phospholipid/protein ratio and increased efficiency of SP-B/SP-C to promote low surface tension.15 Currently, there is no information regarding the effects of iNO on the surfactant of human infants.

Although the benefits and safety of iNO for the treatment of persistent pulmonary hypertension in term and near-term infants are well established, iNO treatment of premature infants with respiratory failure or chronic lung disease (CLD) is still under investigation.16–20 Controlled clinical trials of iNO for moderately ill, premature infants undergoing ventilation indicated benefits in preventing bronchopulmonary dysplasia (BPD) without adverse effects, particularly for infants with birth weights between 1000 and 1250 g.16,19,20 In addition, there was evidence of neurologic protection in treated infants.16,20 Because of the reported negative effects of iNO on surfactant in cell and animal studies, as well as the vulnerability of the surfactant system in preterm infants, we examined prospectively surfactant function and composition as part of the Nitric Oxide (NO) CLD Trial, which treated with iNO infants who were still intubated between 7 and 21 days of age.19 We collected tracheal aspirates from a subpopulation of study infants and determined the effects of iNO on surface tension properties and SP concentrations of large-aggregate surfactant preparations. In addition, we examined the relationship between surfactant function and severity of respiratory disease, in a follow-up study of previous observations regarding surfactant dysfunction.2,21


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Study Population
The NO CLD Trial was a randomized, blinded, controlled trial of iNO treatment performed at 21 centers. Infants with birth weights of 500 to 1250 g who still required ventilatory support between 7 and 21 days of age were enrolled, with consent, to receive either iNO (INO Therapeutics, Clinton, NJ) or placebo for 24 days. The study gas was administered at 20 ppm for 48 to 96 hours and then decreased to 10 ppm for 7 days, to 5 ppm for 7 days, and to 2 ppm until discontinuation. Clinical data, including the respiratory severity score (fraction of inspired oxygen x mean airway pressure) as an index of severity of lung disease, were collected. The respiratory severity score was used rather than oxygenation index, which requires an arterial line for measurement of PaO2. Most infants received replacement surfactant at birth, but none received later surfactant treatment. After entry into the study, no infants had pulmonary hemorrhage, and the rates of sepsis, diagnosed on the basis of positive blood or cerebrospinal fluid cultures, were similar in the control (52.4%) and iNO-treated (43.9%; not significant) groups. Details of the NO CLD Trial protocol, infant characteristics, and outcomes have been published.19

Tracheal Aspirate Samples
Specimens of tracheal aspirate fluid (TAF) were collected from a subpopulation of infants who were enrolled in the NO CLD Trial and provided consent for TAF collection, at 4 centers, namely, Children's Hospital of Philadelphia, Hospital of the University of Pennsylvania, Children's Mercy Hospitals and Clinics in Kansas City, and Marie Fareri Children's Hospital at Westchester Medical Center. There were a total of 99 infants, with 519 TAF samples that were collected at study entry (just before study gas treatment) and at 24 to 48 hours, 4 days, 11 days, 18 days, and 25 days after initiation of study gas treatment. Because some samples were not obtained on the predetermined day, analyses of time course data combined samples for 1 to 2 days, 3 to 5 days, 9 to 12 days, 16 to 19 days, and 23 to 26 days; only 1 sample per infant was collected within the time intervals. All analyses were limited to the 83 infants with a preentry sample and ≥1 postentry TAF sample of sufficient amount for surfactant isolation and assays. The TAF sampling procedure involved instillation of 0.5 mL of saline solution into the trachea of infants in clinically stable condition, brief ventilation, and suction at the end of the endotracheal tube to recover saline solution containing lung epithelial lining fluid. This procedure was repeated twice, and the combined aliquots constituted 1 TAF sample. The samples were centrifuged at 500 x g for 5 minutes for removal of cells, and the supernatant was frozen for shipment to Philadelphia. After thawing, the supernatant was centrifuged at 27000 x g for 60 minutes to yield a large-aggregate surfactant pellet, which was used for all assays without refreezing. Supernatants were stored for assays of various inflammatory markers, NO metabolites, and protein adducts, and these results are reported separately.

Surface Tension Measurements
Surface tension properties were measured in a pulsating bubble surfactometer as described previously.22,23 In brief, large-aggregate surfactant was resuspended in buffer at a concentration of 1.5 mg of phospholipid per mL, and an aliquot was cycled at 0.33 Hz in a surfactometer (Electronics Corp, Buffalo, NY). Data for minimum surface tension at 3 minutes of pulsation, time to reach minimum surface tension, and maximum surface tension were obtained. For samples with sufficient amounts of phospholipid, measurements were repeated and results were averaged; duplicate determinations agreed within 10%. Some samples contained an insufficient amount of phospholipid for analysis of surface tension or were lost during pulsation. There were insufficient amounts of high-speed supernatant for assay of phospholipid as a measure of small-aggregate surfactant.

SP Assay
SP-A, SP-B, and SP-C were measured in large-aggregate surfactant by using an immunodot assay, as described.23,24 Serial dilutions of surfactant samples, with Infasurf (Calfactant; Forest Pharmaceuticals, St Louis, MO) as a standard for SP-B and SP-C or purified SP-A (generously provided by J. R. Wright), were applied to nitrocellulose and exposed to primary and secondary antibodies; chemiluminescence in the presence of enhanced chemiluminescence reagent (Pierce Biotechnology, Rockford, IL) was detected through exposure to radiographic film and quantitated through scanning. Specific antibodies against SP-A and SP-B have been described.25,26 Antibody to mature SP-C was provided by ALTANA Pharma (Kontanz, Germany) and was generated against recombinant human SP-C containing phenylalanine substituted for cysteine at residues 3 and 4 and isoleucine substituted for methionine at residue 32. These assays are specific for each SP, are sensitive, and have interassay coefficients of variation of <8%. SP results are expressed as percentages of phospholipid, by weight.

Other Assays
Lipids were extracted from large-aggregate surfactant by using the method described by Bligh and Dyer,27 and the phospholipid content of the extract was determined with the phosphorus assay described by Dittmer and Wells.28 Total protein content was determined by using the Bradford assay (BioRad Laboratories, Hercules, CA).

Statistical Analyses
Tracheal aspirate samples were collected from 99 infants, and data on surfactant composition and/or function were available for 83 patients with both a baseline sample and ≥1 postentry sample. Reasons for unavailable or censored data included inadequate volume of tracheal aspirate sample or content of phospholipid or total protein, loss of sample during bubble surfactometry, collection of only 1 (pretreatment) sample from an infant, and collection of a sample outside the period for each time point during treatment. Data for both composition and surface properties were skewed and were logarithmically transformed for statistical analyses, with presentation of the geometric mean. To test for significant differences between iNO and placebo groups, we used Fisher's exact test for categorical data and Student's t test for continuous variables. A mixed-effect model was used to examine the effect of treatment on changes in surfactant parameters from baseline values with time after entry into the study. To test for equivalence of iNO and placebo data during treatment, we used logarithmically transformed data and the two one-sided test approach, with 90% confidence intervals.29 This analysis examined the equivalence of data within limits assigned by likely physiologic significance. We used marginal endurable difference values for each variable, as follows: minimum surface tension, 2.5 mN/m; maximum surface tension, 2.5 mN/m; time to minimum surface tension, 30 seconds; SP-A, 2.5% of phospholipid; SP-B, 0.5% of phospholipid; SP-C, 5% of phospholipid; total protein, 35% of phospholipid. On the basis of the confidence intervals in the two one-sided test analysis, results for the 2 treatment groups were judged to be equivalent or not. A logistic model was used to explore the relationship between the primary outcome (survival without BPD) and the change in minimum surface tension with study gas. The statistical software package SAS 9.13 (SAS Institute, Cary, NC) was used for analyses, with .05 as the significance level in all tests.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Demographic Characteristics
In the subpopulation of NO CLD Trial infants with TAF collections (n = 99), surfactant function and/or composition data were obtained at study entry for 83 infants (41 iNO-treated infants and 42 placebo-treated infants) (Table 1). The mean gestational ages (25.5 weeks), birth weights (725–755 g), and racial distributions were similar for the groups. The mean age at enrollment was 15 days for both groups, with more infants enrolled in the third week of life, compared with week 2. The respiratory severity scores (measuring the severity of lung disease) at enrollment were similar for the groups. The infants in this study were representative of the entire population of infants in the NO CLD Trial with respect to the listed demographic features.19


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  		TABLE 1 Demographic Characteristics of Study Population

 
Baseline Characteristics
We previously described episodes of dysfunctional surfactant, as assessed with a pulsating bubble surfactometer, in premature infants undergoing ventilation; the occurrence of inactive surfactant was associated temporally with worsening respiratory status and infection.2 To examine this relationship further, we determined the association between surfactant function (minimum surface tension) and respiratory severity score for all infants before initiation of study gas treatment. As shown in Fig 1, there was a positive correlation (r = 0.43; P < .001) between surface tension and respiratory severity scores, which supported the previous observation of surfactant dysfunction being associated with worsening respiratory status. This correlation was also observed for TAF samples collected during administration of study gas (r = 0.34; P < .01; n = 347), with similar slopes for the iNO-treated and placebo-treated groups (data not shown). Surfactant for 31 infants (43%) at study entry had minimum surface tension of >5 mN/m (Fig 1), which was defined as the upper limit of the reference range.


Figure 1
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  		FIGURE 1 Relationship of respiratory severity scores to surfactant function at study entry. Data are for 63 infants at 7 to 21 days of age with data for both surface tension and severity score on the day of study entry. Increasing minimum surface tension of surfactant was associated with higher severity scores (r = 0.43; P < .001).

 
The protein compositions of surfactant for the 2 groups of infants at study entry are shown in Table 2. The total protein and SP contents, normalized to surfactant phospholipid, were not different between the groups, and concentrations were comparable to those reported previously for intubated premature infants. The geometric mean values for minimum surface tension were <5 mN/m for both iNO-treated and placebo-treated infants.


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  		TABLE 2 Surfactant Function and Composition at Study Entry

 
Surfactant Recovery, Function, and Composition During Study Gas Administration
The recovery of surfactant from TAF samples was determined through phospholipid assay of the large-aggregate fraction and was normalized to total protein levels in the TAF. The surfactant contents of TAF were similar for placebo- and iNO-treated infants (mean ± SE: 180.1 ± 8.5 µg of phospholipid per mg of protein vs 178.3 ± 8.4 µg of phospholipid per mg of protein; not significant), which suggests that NO therapy did not influence surfactant production or secretion.

Values for minimum and maximum surface tension, as well as the time required to achieve minimum surface tension, were obtained through pulsating bubble surfactometric testing of surfactant isolated from TAF samples during study gas treatment (Table 3). Minimum surface tension values were of particular importance because they reflect the ability of surfactant to form a stable surface film. After initiation of treatment, minimum surface tension increased for placebo-treated infants and decreased for iNO-treated infants, with the largest difference occurring on day 4 (P = .02, Student's t test). Mean values for the time required to achieve minimum surface tension and for maximum surface tension were also less for iNO-treated infants on day 4 (P = .02 and P = .04, respectively). At later time points, there were no significant differences between groups in surface tension parameters. The data in Table 3 were also analyzed for equivalence by using the two one-sided equivalence test approach and were assigned marginal endurable differences (see "Methods"). On the basis of the assigned difference values and confidence intervals (data not shown), none of the results for the iNO and placebo groups was statistically equivalent.


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  		TABLE 3 Surfactant Function Before and During Study Gas Administration

 
We examined further the effect of iNO on minimum surface tension by calculating the change over time from the baseline value for each infant (Fig 2A). Surface tension values over time were significantly different between groups during study gas exposure, in mixed-effect analysis (P = .039).


Figure 2
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  		FIGURE 2 Time course of changes in minimum surface tension (A) and SP-B concentrations (B) during administration of study gas. Data (mean ± SE) for the changes from baseline for each infant are shown for iNO- and placebo-treated infants. The patterns for minimum surface tension (STmin) were significantly different between iNO and placebo groups, according to mixed-effect analysis (P = .039; for SP-B concentrations, P = .13). PL indicates phospholipid.

 
Improved clinical outcomes in the NO CLD Trial were restricted to infants who were entered into the study between day 7 and day 14; the rate of survival without BPD was 49.1% for iNO-treated infants, compared with 27.8% for placebo-treated infants (P = .006 for interaction with entry age).19 Therefore, we examined surfactant function for infants entered at 7 to 14 days (n = 24) and 15 to 21 days (n = 39) of age. After 4 days of therapy with study gas, lower minimum surface tension was observed for iNO-treated infants with both times of entry (7–14 days: iNO: 4 ± 1.3 mN/m; placebo: 11 ± 2.6 mN/m; P = .045; 15–21 days: iNO: 8 ± 2.4 mN/m; placebo: 13 ± 1.8 mN/m; P = .06). There were no significant differences in minimum surface tension at other times of treatment.

Total protein, SP-A, SP-B, and SP-C contents, normalized to phospholipid of large-aggregate surfactant, were determined with immunodot assays and are expressed as changes from baseline values in Table 4. There were no significant differences (or equivalence) between groups in changes in SP or total protein contents at individual time points or over the 25-day time course.


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  		TABLE 4 Protein Contents of Surfactant Before and During Study Gas Administration

 
Levels of SP-B, which is critical for surfactant function, were stable for 10 days in iNO-treated infants and tended to decrease in placebo-treated infants during this time (Fig 2B); the time courses for SP-B contents in the 2 groups of infants were not significantly different (P = .13). We also analyzed the relationship between SP-B content and minimum surface tension for all samples (placebo- and iNO-treated combined; n = 400). As observed previously, higher SP-B content of surfactant was significantly (P = .007) associated with lower minimum surface tension (data not shown).

Surfactant Function and Clinical Outcomes
Because the data of Table 3 and Fig 2A indicated transiently improved surfactant function in iNO-treated infants, we evaluated the relationship between surface tension and the primary clinical outcome in the trial. The rate of survival without BPD in the iNO-treated group was 60% for infants with a decrease in minimum surface tension between day 3 and day 7 of study entry, compared with 25% for infants without improved surfactant function. In the placebo-treated group, 25% of infants with decreased minimum surface tension had favorable outcomes, compared with 53% of the infants without improved surfactant function. None of these differences was statistically significant, although the study was not sufficiently powered to address this issue.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
This study of surfactant was designed primarily to provide information regarding the safety of iNO treatment for 3 weeks for premature infants in the NO CLD Trial.19 We found no evidence that iNO therapy affected adversely SPs or in vitro function of surfactant; therefore, the results complement the clinical findings of no short-term adverse effects. In addition, we assessed the possible impact of iNO on the inflammatory profile (proinflammatory cytokine levels, cell counts, and hyaluronan levels) and generation of protein adducts in the same TAF samples; these associated studies also indicated a lack of NO effects, and results are reported separately.30 Combined with the overall improvement in rates of survival without BPD found in the NO CLD Trial, the surfactant data increase confidence in the favorable benefit/risk ratio for iNO therapy.

At the initiation of this study, most published data related to NO and surfactant in vitro and in animal models indicated concern regarding adverse responses.5–14 The reported inhibitory effects of NO exposure on SPs and surfactant lipids in these studies might reflect modification of tyrosine and thiol residues of proteins, which can alter protein activity, decrease or increase degradation, or inhibit synthesis by modifying regulatory factors. The apparent absence of such effects in infants might result from lower iNO concentrations, different NO metabolism pathways, or species differences. In the baboon model, 2 weeks of iNO treatment had no effect on the minimum surface tension of surfactant but did decrease the amount of total protein associated with large-aggregate surfactant, presumably reflecting decreased pulmonary edema.15 We did not observe this effect in human infants.

The transient improvement in surfactant function in iNO-treated infants was an unexpected finding. Improved function, relative to both baseline values and the control group, was restricted to samples collected during the first 4 days after initiation of therapy. At that time, infants had received iNO at 20 ppm for at least 2 of the 4 days, with subsequent weaning of the dose to 10 ppm. We cannot establish from this study whether improved function occurred only at the higher NO dose or was a transient response unrelated to dose. The mechanism of improved surface tension properties is uncertain. One possibility is decreased amounts of inhibitory proteins associated with surfactant; however, iNO had no effect on the total protein/phospholipid ratio in the surfactant. Infection and inflammation can affect surfactant production and function; however, we found no differences between control and treated groups in the incidence of sepsis or levels of TAF inflammatory markers during administration of study gas.30 Another possibility is improved composition of surfactant phospholipid, particularly increased concentrations of disaturated phosphatidylcholine, which we were unable to examine with the amounts of available samples. It is also possible that iNO reduced oxidative inactivation of surfactant lipids or proteins, thereby improving function. Surfactant function in vivo and in vitro is directly related to SP-B concentrations, and low levels of SP-B are frequently observed in surfactant of premature infants undergoing ventilation.2 Therefore, improved surfactant function in iNO-treated infants may involve increased function of SP-B, although contents were not different between the groups.

It seems unlikely that the transient improvement in surfactant function with iNO therapy was the primary factor in the observed reduction in the incidence of BPD in the treated group of the NO CLD Trial.19 An attempt to examine this possibility was limited by the small numbers in each outcome group (Table 5).


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  		TABLE 5 Infant Outcomes Related to Changes in Surfactant Function After Initiation of Therapy

 
There are some limitations of this study. Tracheal aspiration provides samples of upper airway fluid, rather than fluid from respiratory airways and alveoli. Although earlier studies found comparable surfactant compositions and function for upper and lower airway fluids, likely resulting from mucociliary and capillary spreading actions of alveolar surfactant,31–33 our results may not reflect properties of alveolar surfactant. In the longitudinal studies, the number of available samples decreased with time as infants were extubated; accordingly, comparisons between treated and control infants at later time points are less reliable. We compensated for this in part by examining changes from baseline for each infant. The studies of surfactant function were performed in vitro, and it is possible that these results may not reflect in vivo activity. In an attempt to assess surfactant in its native state, we did not purify the large-aggregate pellet after isolation; this approach retains proteins loosely associated with surfactant, which potentially can affect function. In addition to traditional statistical analyses examining differences between treatment groups, we tested for equivalence with the hypothesis that surfactant parameters differed between groups by assigned amounts of likely physiologic importance. This analysis mostly did not demonstrate statistical equivalence (or nonequivalence) between groups, which reflects the variance in the data and inadequate power. Despite this limitation, the equivalence testing results are consistent with statistical significance in difference testing for surfactant function at 4 days of study gas treatment.

Results of this study confirm previous observations that respiratory decompensations in infants are related, at least in part, to surfactant dysfunction and SP-B deficiency.2 We found a positive correlation between minimum surface tension of surfactant and severity of respiratory disease. This association was observed for data obtained before entry and during administration of study gas for both groups. Moreover, minimum surface tension was related inversely to SP-B concentrations in surfactant, as described previously. These findings have prompted pilot studies of booster surfactant treatment beyond the first week for infants at risk for BPD, and transient improvements in respiratory status have been observed.21,34 It is possible that repeated booster doses would reduce overall oxygen- and pressure-related damage to the lungs, improving rates of survival without BPD. Moreover, because the pathogenesis of BPD is multifactorial, additional surfactant therapy might be additive with the effects of NO, which involve structural changes, including enhanced alveogenesis and reduced muscularization of vessels and airways.35–37


   CONCLUSIONS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
This study provides the first information on the interaction of exogenous NO with endogenous surfactant of premature infants undergoing ventilation. Under the conditions of iNO use in this trial, we found no evidence of deleterious effects of NO therapy on recovery or protein composition of surfactant, and we observed transient improvement in surfactant function. These findings support the safety of iNO therapy for prevention of BPD.


   ACKNOWLEDGMENTS
 
This research was supported by grants from the National Institutes of Health (grants U01 HL62514, P50 HL56401, P30 HD26979, and Mental Retardation and Developmental Disabilities Research Center P30 HD26979) and from the General Clinical Research Centers Program (grants M01 RR00240, M01 RR00084, M01 RR00425, M01 RR001271, M01 RR00064, and M01 RR00080).

We thank the nurses, respiratory therapists, and physicians at participating hospitals; the study coordinators, S. Wadlinger and C. Coburn; the families and infants who participated in this study; and INO Therapeutics for providing study equipment and gas.


   FOOTNOTES
 
Accepted Mar 16, 2007.

Address correspondence to Philip L. Ballard, MD, PhD, University of California, San Francisco, 3333 California St, Suite 150, San Francisco, CA 94118. E-mail: ballardp{at}peds.ucsf.edu

Financial Disclosure: Drs P. L. Ballard and R. A. Ballard acknowledge research grant support from INO Therapeutics. Drs Golombek and Parton have received speaker fees from INO Therapeutics.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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PEDIATRICS (ISSN 1098-4275). ©2007 by the American Academy of Pediatrics

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