PEDIATRICS Vol. 119 No. 4 April 2007, pp. 670-678 (doi:10.1542/peds.2006-2683)
ARTICLE |
Inflammatory Markers and Mediators in Tracheal Fluid of Premature Infants Treated With Inhaled Nitric Oxide
a Section of Neonatology, Children's Mercy Hospitals and Clinics, University of Missouri-Kansas City School of Medicine, Kansas City, Missouri
b Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
c Maria Fareri Children's Hospital at Westchester Medical Center, New York Medical College, Valhalla, New York
d Department of Pediatrics, University of Texas Southwestern, Dallas, Texas
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
OBJECTIVE. We compared serial measurements of inflammatory mediators and markers in infants treated with inhaled nitric oxide or placebo to assess the effects of inhaled nitric oxide therapy on lung inflammation during bronchopulmonary dysplasia. We investigated relationships between respiratory severity scores and airway concentrations of inflammatory markers/mediators.
METHODS. As part of the Nitric Oxide (to Prevent) Chronic Lung Disease trial, a subset of 99 infants (52 placebo-treated infants and 47 inhaled nitric oxide-treated infants; well matched at baseline) had tracheal aspirate fluid collected at baseline, at 2 to 4 days, and then weekly while still intubated during study gas treatment (minimum of 24 days). Fluid was assessed for interleukin-1ß, interleukin-8, transforming growth factor-ß, N-acetylglucosaminidase, 8-epi-prostaglandin F2
, and hyaluronan. Results were normalized to total protein and secretory component of immunoglobulin A.
RESULTS. At baseline, there was substantial variability of each measured substance and no correlation between tracheal aspirate fluid levels of any substance and respiratory severity scores. Inhaled nitric oxide administration did not result in any time-matched significant change for any of the analytes, compared with the placebo-treated group. There was no correlation between any of the measured markers/mediators and respiratory severity scores throughout the 24 days of study gas administration. In the posthoc analysis of data for inhaled nitric oxide-treated infants, there was a difference at baseline in 8-epi-prostaglandin F2 levels for infants who did (n = 21) and did not (n = 26) develop bronchopulmonary dysplasia at postmenstrual age of 36 weeks.
CONCLUSIONS. Inhaled nitric oxide, as administered in this study, seemed to be safe. Its use was not associated with any increase in airway inflammatory substances.
Key Words: bronchopulmonary dysplasia • nitric oxide • inflammation
Abbreviations: BPD—bronchopulmonary dysplasia • iNO—inhaled nitric oxide • MLEC—mink lung epithelial cell • NAG—N-acetylglucosaminidase • IgA—immunoglobulin A • NO—nitric oxide • TAF—tracheal aspirate fluid • TGF—transforming growth factor • NO-CLD—nitric oxide (to prevent) chronic lung disease • 8-epi-PGF2—8-epi-prostaglandin F2 • FIO2—fraction of inspired oxygen • IL-1ß—interleukin-1ß • IL-8—interleukin-8 • ELISA—enzyme-linked immunosorbent assay • bHABP—biotinylated hyaluronic acid-binding peptide
Of the >4 million births in the United States each year, premature births account for 12.3%; very low birth weight infants account for 1.4%.1 Although the survival rate for these premature newborns has improved, morbidity and long-term complications persist. Approximately 10000 to 15000 of these infants develop bronchopulmonary dysplasia (BPD) of prematurity each year in the United States,2 with mortality rates varying from 5% to 30%.3 Therapy to prevent or to ameliorate BPD should reduce the duration of positive pressure ventilation and elevated fraction of inspired oxygen (FIO2) and reduce overall pulmonary morbidity.
One recently completed, randomized, blinded, multicenter, placebo-controlled trial of inhaled nitric oxide (iNO), the Nitric Oxide (to Prevent) Chronic Lung Disease (NO-CLD) trial, tested whether iNO administered between 7 and 21 days of age to infants with birth weights of 500 to 1250 g would be efficacious and safe for prevention or amelioration of BPD.4 The results demonstrated significant reductions in total BPD and in the more severe variants of BPD, as assessed at postmenstrual ages of 36, 40, and 44 weeks.4 There was no increase in the incidence of the common morbidities of prematurity in the infants treated with iNO, compared with those treated with placebo.4
A second goal of the NO-CLD clinical trial was to determine the safety of iNO, as used in the trial. The impact of iNO administered for a clinically meaningful time into already inflamed, immaturely developed lungs is uncertain. Persistence of elevated tracheal aspirate fluid (TAF) concentrations of the innate proinflammatory substances interleukin-1ß (IL-1ß) and interleukin-8 (IL-8) at 1 week of age has been associated with increased risk of BPD.5–7 The concentration of the extracellular matrix component and cell migration-promoting substance hyaluronan has been shown to be altered by respiratory distress syndrome of prematurity8 and by inflammation-associated lung injury.9 How iNO administration affects the presence of these substances has not been evaluated.
NO can have both proinflammatory and antiinflammatory effects. In the presence of elevated FIO2, NO is converted to NO2, peroxynitrite, and other oxides of nitrogen, which may initiate or exacerbate pulmonary inflammation. iNO may also modulate the pulmonary inflammatory response by downregulating the production of inflammatory cytokines10,11 and by decreasing lung neutrophil accumulation.12 Hyperoxia and elevated NO levels have been shown to induce cell death in in vitro lung fibroblast cultures.13 Because iNO is coadministered with high FIO2 into an environment with preexisting inflammation when given to infants at risk for BPD, it is important to evaluate the specific effects of the dose and duration of treatment used in the NO-CLD study on lung inflammation.14
Another form of lung injury results from cell membrane oxidation by reactive oxygen species. A marker of membrane oxidative injury, 8-epi-prostaglandin F2 (8-epi-PGF2), mediates neonatal pulmonary hypertension in animal models15 and is associated with pulmonary hypertension in human newborns.16 The effects of iNO on 8-epi-PGF2 production or presence in the airway are unknown. Assessing changes in 8-epi-PGF2, if any, with iNO administration could provide insight into the mechanism of action of iNO in this clinical setting.
The potent cytokine transforming growth factor-ß (TGF-ß), which is derived in part from alveolar macrophages, regulates fibroblast activity, reduces surfactant production, and has been found to be increased in TAF of infants developing BPD.17 Active TGF-ß also has been implicated in the development of pulmonary fibrosis in adults.18 Fibrosis and airway simplification are pathologic hallmarks of fully established BPD. Therefore, the effects of iNO administration on active TGF-ß could offer insights into its mechanisms of action. Our objectives in this study were (1) to determine the relationship between inflammatory markers/mediators and disease severity in premature infants at 7 to 21 days; (2) to investigate the effect of iNO on the inflammatory profile, to assess both safety and the potential mechanisms of NO in lung disease; and, (3) for the subset of infants who received iNO and from whom serial TAF samples were obtained, to identify whether the response to iNO was predictable from baseline TAF data.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
Summary of Parent Clinical Trial
The results reported in the present study were obtained from a subset of infants enrolled in the placebo-controlled NO-CLD trial.4 The study enrolled infants with birth weights of 500 to 1250 g who were between 7 and 21 days of age and required ventilatory support. Treated infants received decreasing concentrations of iNO, beginning at 7 to 21 days of age and 20 ppm, for a minimal duration of 24 days. The respiratory severity score was used as an index of respiratory support. The respiratory severity score is calculated as mean airway pressure x FIO2.
Infants who received iNO (n = 294) demonstrated a 43.9% rate of survival without BPD at postmenstrual age of 36 weeks, compared with 36.8% for the 288 infants in the placebo group (P = .042).4 There was no difference in mortality rates. Benefit was sustained at 40 and 44 weeks. There was no increase in the incidence of any of the common complications of prematurity, including intracranial injury, for the iNO-treated infants.
The TAF samples from which the current results were derived were collected from 99 of the 582 infants enrolled. Serial samples were collected routinely from infants enrolled in the study at the Children's Hospital of Philadelphia, at the Hospital of the University of Pennsylvania, at Westchester Medical Center (Valhalla, NY), and at Children's Mercy Hospitals and Clinics (Kansas City, MO). Of the 99 infants for whom serial TAF samples were obtained, 52 infants had been assigned randomly to placebo gas treatment and 47 to iNO treatment. Informed parental permission was obtained specifically for the collection of samples, in addition to permission for enrollment in the study. No parent who gave permission for the primary study declined permission for the TAF sampling.
TAF Collection and Processing
TAF was collected as described previously by our group,19,20 by using saline lavage, at baseline, at 24 to 48 hours, at 4 days, and then weekly during study gas treatment (minimum of 24 days). Aspirated samples were placed in sterile tubes and centrifuged at 500 x g for 10 minutes, and the supernatant was frozen immediately at –70°C. Aliquots of the supernatant and the cell pellet were shipped frozen, with overnight express delivery, to Philadelphia. The aliquots were thawed and centrifuged at 27000 x g for isolation of the surfactant fraction. Results of the surfactant function studies are being reported separately.21 Assays for TGF-ß, hyaluronan, N-acetylglucosaminidase (NAG), and total protein were performed in Philadelphia; assays for IL-1ß, IL-8, 8-epi-PGF2, and the secretory component of Immunoglobulin A (IgA) were performed in Kansas City. To account for dilution of epithelial lining fluid during collection, results were normalized to total protein and secretory component of IgA levels, both measured in the same sample (Fig 1).
|
Assays for IL-1ß and IL-8
IL-1ß and IL-8 (acute proinflammatory cytokines) were assayed by using commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN). These assays use a quantitative sandwich ELISA technique, with assay sensitivities of <1 pg/mL and <3.5 pg/mL, respectively and intraassay coefficients of variation of 2.4% and 4.6%, respectively.
Assay for TGF-ß
Active TGF-ß content was assayed by using the mink lung epithelial cell (MLEC) line, according to the protocol described by Abe et al.22 The MLEC line is a TGF-ß-responsive cell line that is stably transfected with the human plasminogen activator inhibitor promoter (a TGF-ß-responsive gene) fused to a luciferase reporter gene. MLECs were plated in a 96-well plate at 1.8 x 104 cells per well, allowed to attach for 3 hours, and then cultured overnight with 30 µL of TAF supernatant or 40 to 1200 pg/mL TGF-ß standard. MLEC extracts were lysed the next day and assayed for luciferase activity by using the luciferase assay system (Promega, Madison, WI). Recombinant human TGF-ß was purchased from Sigma (St Louis, MO).
Assay for NAG
TAF supernatant and cell pellets were assayed for NAG activity as an index of macrophage content. The NAG assay measures the spectrophotometric release of p-nitrophenol from a conjugated NAG substrate and is based on the method described by Nitta et al.23 In a 96-well plate, 20 µL of TAF supernatant or cell pellet (diluted 1:10) or 25 to 400 µU of ß-nitro-acetylglucosaminidase standard (Sigma) was incubated with 10 µL of Triton X-100 and 20 µL of substrate (15 mmol/L p-nitrophenyl-N-acetyl-ß-glucosaminidase; Sigma) for 30 minutes at 37°C, to allow for product formation. The reaction was stopped with the addition of 200 µL of 0.2 mol/L Na2CO3 to each well. The change in absorbance at 450 nm of each well was measured, and NAG activity in the samples was calculated from the standard curve.
Assay for 8-Epi-PGF2
The non-cyclooxygenase-derived prostaglandin 8-epi-PGF2, a biomarker of lipid oxidative injury, was assayed by using a commercial ELISA kit (Assay Designs, Ann Arbor, MI). The direct competitive ELISA technique measures both free and esterified isoprostane after alkaline hydrolysis of phospholipid-coupled 8-epi-PGF2. The assay sensitivity is <40 pg/mL, and the intraassay coefficient of variation is 11.3%.
Assay for Hyaluronan
Hyaluronan was measured by using a competitive ELISA-like method, as published previously by Lokeshwar et al24 and by Maeda et al.25 Hyaluronic acid (ICN Biochemicals, Aurora, OH) at 0.2 mg/mL was coated on microtiter plates for binding to a biotinylated hyaluronic acid-binding peptide (bHABP) (Seikagaku, Tokyo, Japan). Samples and standards were digested with protease overnight at 37°C, to remove any potential interfering proteins. The next day, samples were diluted either 1:2 or 1:4 in phosphate-buffered saline, and 60 µL of sample or standard was incubated with 60 µL of bHABP for 1 hour at 37°C. Serial dilutions of hyaluronic acid standards (Healon; Pharmacia and Upjohn, Kalamazoo, MI), ranging from 0 to 3000 ng/mL, were included; 100 µL of samples or standards were transferred to hyaluronic acid-coated plates and allowed to react for 1 hour at 37°C. Binding to bHABP was detected with the addition of streptavidin-biotin complex (Vectastain; Vector Laboratories, Burlingame, CA), followed by the addition of o-phenylenediamine (Sigma) for colorimetric analysis. The absorbance of each well was scanned at 405 nm, and hyaluronic acid content was normalized to TAF protein content and expressed as nanograms of hyaluronic acid per milligram of protein.
Total Cell Number Determination
For determination of total cell number in TAF cell pellets, the CyQuant cell proliferation assay kit (Invitrogen, Carlsbad, CA) was used to assay DNA content, according to manufacturer's instructions. In a 96-well plate, cells were lysed with a 1x CyQuant-GR cell lysis buffer and incubated for 5 minutes with a proprietary fluorescent dye that binds to nucleic acids. DNA standards ranged from 0 to 1000 ng/mL. The fluorescence of each well was measured by using a microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Conversion of DNA to cell number used a value of 6.5 pg of DNA per human diploid cell. The total cell count was obtained for each sample, to investigate the possibility of iNO-associated increase or decrease in airway inflammatory cell content.
Normalization of Analytes
Assays were normalized with 2 methods, to account for the inevitable variations in dilution during the collection process. Concentrations of the soluble secretory component of IgA and of total protein measured in the same sample served to normalize each TAF sample. The soluble secretory component of IgA was measured by using an established 96-well ELISA technique.26 Polyclonal rabbit antihuman secretory component served as a primary antibody, with horseradish peroxidase-conjugated rabbit antihuman immunoglobulin (Dako, Glostrup, Denmark) as a secondary antibody. Quantification was performed against a standard curve from 2.34 ng/mL to 300 ng/mL, with purified human colostrum. Total protein levels in the TAF supernatant were determined by using the bicinchoninic acid reagent (BioRad, Hercules, CA).
Statistical Analyses
On the basis of TAF measurements in a phase 2 study,20 we anticipated a wide range of values at baseline. A prospective power analysis indicated that a change in the mean equal to the SD (standardized event ratio of 1.0), tested at an of .05 (2-tailed) and a ß of 90%, would require 21 patients per group. A standardized effect size of 0.5 with the same assumptions would require a sample size of 63 patients per group. Given the anticipated rate of patient accrual at the 3 study sites committed to collecting TAF samples, we sought to enroll a minimum of 50 to 60 patients for each arm of the main trial for the TAF analysis.
For nonparametric data comparison (eg, the normalized TAF concentrations at each time point for each substance of interest), the Mann-Whitney test was used. Data are expressed as box plots demonstrating the median, 25th and 75th percentiles as the limits of the box, and 5th and 95th percentiles as the error bars. For continuous and normally distributed data (eg, severity score, birth weight, and gestational age), the unpaired t test was used, with correction for multiple comparisons. Categorical data (eg, gender and outcome) were evaluated with the 
2 test or Fisher's exact test. For the posthoc hypothesis-generating analysis, we did not adjust for multiple comparisons.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
The 99 infants had a total of 519 TAF samples collected and analyzed. Each analyte was assayed in duplicate in each sample, and the results were averaged.
The 47 iNO-treated infants and the 52 placebo-treated infants were well matched according to birth weight, gestational age, and postnatal age at the time of study entry (Table 1). In parallel with the overall study, there was a trend toward greater rates of survival without BPD for the iNO-treated infants (Table 1). The 99 patients were also representative of the infants in the 2 treatment arms of the parent study (Table 2). Of the 99 infants in the subset, 31 were treated in Philadelphia, 13 at Westchester Medical Center, and 55 at Children's Mercy Hospital. There were 4 deaths among the 99 infants, 2 in each randomized group. Because infants were extubated typically to continuous positive airway pressure as the trial progressed, the number of samples obtained diminished at the later data collection times. There were 64 infants (33 placebo-treated infants and 31 iNO-treated infants) who were still intubated and had sample collections at days 9 to 12; the total number at the day 23 to 26 collection time was 28 (17 placebo-treated infants and 11 iNO-treated infants).
|
|
At study entry, before treatment, there was substantial variability in each of the substances assessed (Figs 2–8). In no case was there a significant difference between the infants subsequently treated with iNO and those treated with placebo. There was no correlation between any of the substances measured and the respiratory severity score at baseline (data not shown).
|
|
iNO administration resulted in no significant changes, compared with the placebo-treated group, over the period of time assessed for any of the markers (Figs 2–8). Data are shown as normalized to total protein. The same statistical results were obtained if the results were normalized to soluble secretory component of IgA (data not shown). In addition, there were no differences in the concentrations of markers, or in the total cell counts, when they were assessed separately for infants entered early (7–14 days) or later (15–21 days) (data not shown). Posthoc analysis showed that clinical benefit was limited to early-entry infants.4 There were no correlations between any of the substances measured and respiratory severity scores measured throughout the first 3 weeks after initiation of the study gas.
One posthoc analysis limited to the 47 infants treated with iNO was performed. Of those 47 infants, 21 (44.7%) had a favorable outcome (survival without BPD at 36 weeks) and 26 demonstrated either BPD or death (n = 2) at 
36 weeks. Infants treated with iNO, when divided according to this primary outcome at postmenstrual age of 36 weeks, did not differ in any important demographic characteristic (Table 3). At baseline, the iNO-treated infants who had improved outcomes had higher normalized 8-epi-PGF2 levels in TAF than did the infants with unfavorable outcomes (P = .017) (Table 4). At day 4 of treatment, this difference no longer existed (data not shown).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
Overall Results
The current results were obtained from a representative subset of patients enrolled in a multicenter, double-blind, randomized trial of iNO (the NO-CLD study) designed to reduce the incidence or severity of BPD.4 iNO administration was associated with fewer infants developing BPD, and less severe BPD in those who did develop it, in the iNO-treated arm. The results of the current report both add to the safety profile of iNO administration and suggest that the mechanism of action through which iNO operates is not through any acute change in lung inflammatory status.
Our goal was to investigate the potential effect of iNO on a range of substances measurable in TAF. For this purpose, we assayed total cell count, the proinflammatory cytokines IL-1ß and IL-8, the multifunctional and profibrogenic cytokine TGF-ß, and a marker of alveolar macrophage activity, NAG. We also measured hyaluronan, an extracellular matrix component that is altered during lung injury, and one marker of lung oxidative injury, 8-epi-PGF2.
To our knowledge, these results are derived from the largest number of preterm patients at high risk for developing BPD reported to date. Most previously published studies reported serial results, without separate control and intervention groups. An important characteristic of our data set is that collection started at a minimum of 7 days of age and a median of 15 to 16 days and continued in many cases through the next 3 weeks. The results included no samples obtained at birth but only those collected after a minimum of 7 days, presumably after the mechanisms for developing BPD were already activated. In addition, given the inherent limitations of assaying substances in epithelial lining fluid, we normalized the results both according to total protein and according to the soluble secretory component of IgA. It is unclear which denominator is preferable for this population, because infants developing BPD demonstrate high alveolar capillary permeability.27 The results are presented by using the more familiar total protein, but the interpretation of the data was the same when soluble secretory component were used as the denominator.
Proinflammatory Cytokines
In the lung, IL-8 is generated both from alveolar macrophages and from epithelial cells, fibroblasts, microvascular epithelium, and apparently smooth muscle cells. Both IL-1ß and IL-8 participate in the recruitment of inflammatory leukocytes to the pulmonary interstitium and air space. One previous report demonstrated the abundant expression of IL-8 mRNA in infants who progressed to develop BPD.28 The lack of change in IL-1ß, IL-8, and TGF-ß levels in both groups during study gas administration is consistent with no significant change in levels of NAG, a marker for macrophages in airways. There was no correlation between NAG levels and total cell counts in the placebo-treated and iNO-treated groups.
Hyaluronan
Hyaluronan, a component of the extracellular matrix, helps stimulate inflammation and fibrosis after lung injury29 and has been associated with increased lung water content in adult animals. The hydrodynamic properties of hyaluronan support pulmonary cell migration; increased hyaluronan likely promotes pulmonary macrophage aggregation. We found no change in TAF hyaluronan levels with iNO treatment. The lack of change in hyaluronan levels also is consistent with the finding of no increase in NAG levels and no change in proinflammatory cytokine levels.
TGF-ß
TGF-ß is a fibrogenic cytokine thought to play a crucial role in lung parenchymal remodeling, particularly that which results in enlarged air spaces. It has been associated with prolonged oxygen therapy after BPD.30 In the present study, active TGF-ß was identified in the TAF but there was no significant change with iNO administration.
8-Epi-PGF2
The 8-isoprostanes (such as 8-epi-PGF2), a family of non-cyclooxygenase-derived prostaglandins, are generated through direct oxidation of membrane phospholipids. These substances serve as markers for pulmonary oxidative injury31 and may themselves contribute to pulmonary hypertension.16 No difference in TAF levels for control and treated patients was found at baseline or at any treatment time.
Reproducibility of Assays
Each of the assays was performed in duplicate or triplicate, and the results were averaged. In addition, we sought to make sure that the shipment of samples and the necessary second thaw/refreeze cycles did not alter measurements. We limited the number of thaw/refreeze cycles to 1 for 8-epi-PGF2, which is known to be sensitive to this process. All aliquots for measurement of the analytes were treated identically, so that any effects would be shared equally between samples from iNO-treated and placebo-treated infants. In a separate evaluation of 3 thaw/refreeze cycles for IL-1ß, we found no effect on the results of the assay (data not shown).
Limitations of the Study
TAF data may not represent in a predictable manner concentrations of inflammatory cells or their products in the pulmonary interstitial or vascular spaces. However, they do correlate with epithelial lining fluid measurements,32,33 and the use of either total protein or soluble secretory component of IgA provides a reasonable way of normalizing the data.34 No evidence of exacerbation of inflammation or oxidative injury was found with iNO administration.
Receptors for IL-1ß and IL-8 were not assayed, and the antiinflammatory cytokine IL-10 was not measured. Others have used IL-1/IL-10 and IL-8/IL-10 ratios as a way of expressing lung inflammatory status.35 Other vasoactive mediators that might have been affected by iNO (for example, endothelin-1) were not included in the panel of substances measured.36 Given the limited amount of TAF obtained at each time point, we were unable to perform all biologically plausible assays.
Infants needed to remain intubated for serial TAF collections to occur. As infants improved, many were treated with nasal continuous positive pressure ventilation systems and eventually with supplemental oxygen delivery through nasal cannulae. Our results were limited in that only the more severely affected infants, who needed prolonged support with endotracheal tube-delivered positive pressure ventilation, were available for serial sampling. However, the numbers of dropouts were similar for the 2 groups during the sampling times reported here. There was no evident trend, even in the full data set for infants who had baseline, day 4, and then serial weekly collections.
Posthoc Analysis
In the parent study, the number needed to treat to produce 1 less infant with BPD was 14 for the overall study and 5 for the early enrollment group.4 Because of the cost and complexity of iNO administration for many days, it is crucial to identify clinical or laboratory markers predicting benefit with iNO treatment.
In the posthoc analysis, we sought to identify from within the panel of markers used for this study whether 1 of the substances measured at baseline (mean of 16 days of age) would predict a favorable response to iNO. Baseline TAF 8-epi-PGF2 levels were higher for the iNO-treated infants who responded favorably to iNO. One speculation from this hypothesis-generating activity is that NO may produce a more favorable response if introduced into a pulmonary environment that includes factors contributing to elevated pulmonary vascular resistance. However, no decrease in severity score was noted for the 21 infants between baseline and 4 days of treatment. A decrease in severity score might have been expected if a decrease in pulmonary vascular resistance, with concomitant improvement in ventilation perfusion matching and improved arterial oxygen tension and saturation, had occurred. Alternatively, the elevated 8-epi-PGF2 levels at baseline might indicate an environment of increased oxidative injury, a specific situation in which iNO would be beneficial.
|
CONCLUSIONS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
Our findings of no identifiable significant change in the panel of proinflammatory or lung injury markers and mediators used for this study suggest that the administration of iNO in the dose and duration used here was safe. No iNO-associated evidence of an increase in lung inflammation or injury was identified. This suggests that, if the iNO-associated clinical improvements result from changes in the proinflammatory milieu of the lung, then the mechanism must be an indirect one. In the subpopulation of infants who received iNO, only 8-epi-PGF2
was identified as possibly associated with infants who progressed to BPD, compared with those who did not. Additional work differentiating the iNO-treated favorable- and unfavorable-outcome groups is underway.
|
|
|
|
|
|
ACKNOWLEDGMENTS |
|---|
This work was supported by grants from the National Institutes of Health (grants U01 HL62514, P50 HL56401, P30 HD26979, MRDDRC P30 HD26979, R01 HL070560, HL62472, HL62868, HL75930, and HL 73896 and General Clinical Research Centers Program grants M01 RR00240, M01 RR00084, M01 RR00425, M01 RR001271, M01 RR00064, and M01 RR00080). All investigators received support from the National Heart, Lung, and Blood Institute.
We thank the NO-CLD investigators, nurses, residents, fellows, staff physicians, and respiratory therapists who made this work possible. We thank Cheri Castor in Kansas City and Theresa McDevitt in Philadelphia for overseeing the processing of the samples. We thank Mary S. Bailey for help with manuscript preparation and Christopher Norberg, MS, and Steve Simon, PhD, for help with data management. We thank INO Therapeutics for supplying study gas and equipment for the parent trial.
|
FOOTNOTES |
|---|
Accepted Dec 13, 2006.
Address correspondence to William E. Truog, MD, Department of Pediatrics, Section of Neonatology, Children's Mercy Hospitals and Clinics, 2401 Gillham Rd, Kansas City, MO 64108. E-mail: wtruog{at}cmh.edu
The authors have indicated they have no financial relationships relevant to this article to disclose.
Dr P. L. Ballard's and Dr R. A. Ballard's current affiliation is Department of Pediatrics, University of California, San Francisco, School of Medicine, San Francisco, CA.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
- March of Dimes. Prematurity facts. Available at: www.marchofdimes.com/prematurity/21191.asp. Accessed February 6, 2007
- Jobe AH, Bancalari E. Bronchopulmonary dysplasia.
Am J Respir Crit Care Med. 2001;163
:1723
–1729
[Free Full Text] - Van Marter LJ, Allred EN, Pagano M, et al. Do clinical markers of barotrauma and oxygen toxicity explain interhospital variation in rates of chronic lung disease? The Neonatology Committee for the Developmental Network.
Pediatrics. 2000;105
:1194
–1201
[Abstract/Free Full Text] - Ballard RA, Truog WE, Cnaan A, et al. Improved outcome with inhaled nitric oxide in preterm infants mechanically ventilated at 7–21 days of age.
N Engl J Med. 2006;355
:1
–11
[Free Full Text] - Kotecha S, Wilson L, Wangoo A, Silverman M, Shaw RJ. Increase in interleukin (IL)-1ß and IL-6 in bronchoalveolar lavage fluid obtained from infants with chronic lung disease of prematurity. Pediatr Res. 1996;40 :250 –256[Web of Science][Medline]
- Rindfleisch MS, Hasday JD, Taciak V, Broderick K, Viscardi RM. Potential role of interleukin-1 in the development of bronchopulmonary dysplasia. J Interferon Cytokine Res. 1996;16 :365 –373[Web of Science][Medline]
- Groneck P, Schmale J, Soditt V, Stutzer H, Gotze-Speer B, Speer CP. Bronchoalveolar inflammation following airway infection in preterm infants with chronic lung disease. Pediatr Pulmonol. 2001;31 :331 –338[CrossRef][Web of Science][Medline]
- Juul SE, Kinsella MG, Jackson JC, Truog WE, Standaert TA, Hodson WA. Changes in hyaluronan deposition during early respiratory distress syndrome in premature monkeys. Pediatr Res. 1994;35 :238 –243[Web of Science][Medline]
- Juul SE, Kinsella MA, Truog WE, Gibson RG, Redding GJ. Lung hyaluronan decreases during GBS pneumonia in piglets. Am J Respir Crit Care Med. 1996;153 :1567 –1571[Abstract]
- Kuo HP, Hwang KH, Lin HC, Wang CH, Lu LC. Effect of endogenous nitric oxide on tumour necrosis factor--induced leukosequestration and IL-8 release in guinea-pigs airways in vivo.
Br J Pharmacol. 1997;122
:103
–111[CrossRef][Web of Science][Medline]
- Thomassen MJ, Buhrow LT, Connors MJ, Kaneko FT, Erzurum SC, Kavuru MS. Nitric oxide inhibits inflammatory cytokine production by human alveolar macrophages.
Am J Respir Cell Mol Biol. 1997;17
:279
–283
[Abstract/Free Full Text] - Chollet-Martin S, Gatecel C, Kermarrec N, Gougerot-Pocidalo MA, Payen DM. Alveolar neutrophil functions and cytokine levels in patients with the adult respiratory distress syndrome during nitric oxide inhalation. Am J Respir Crit Care Med. 1996;153 :985 –990[Abstract]
- Raghuram N, Fortenberry JD, Owens ML, Brown LA. Effects of exogenous nitric oxide and hyperoxia on lung fibroblast viability and DNA fragmentation. Biochem Biophys Res Commun. 1999;262 :685 –691[CrossRef][Web of Science][Medline]
- Auten RL, Ekekezie II. Blocking leukocyte influx and function to prevent chronic lung disease of prematurity. Pediatr Pulmonol. 2003;35 :335 –341[CrossRef][Web of Science][Medline]
- Truog WE, Norberg M, Thibeault DW. Effects of 8-epi-prostaglandin F2 and U46,619 on pulmonary hemodynamics in piglets.
Biol Neonate. 1997;71
:306
–316[CrossRef][Web of Science][Medline]
- Goil S, Truog WE, Barnes C, et al. Eight-epi-PGF2: a possible marker of lipid peroxidation in term infants with severe pulmonary disease.
J Pediatr. 1998;132
:349
–351[CrossRef][Web of Science][Medline]
- Kotecha S, Wangoo A, Silverman M, Shaw RJ. Increase in the concentration of transforming growth factor ß1 in bronchoalveolar lavage fluid before development of chronic lung disease of prematurity. J Pediatr. 1996;128 :464 –469[CrossRef][Web of Science][Medline]
- Dhainaut JF, Charpentier J, Chiche JD. Transforming growth factor-ß: a mediator of cell regulation in acute respiratory distress syndrome. Crit Care Med. 2003;31(suppl) :S258 –S264[CrossRef][Web of Science][Medline]
- Ekekezie II, Thibeault DW, Simon SD, et al. Low levels of tissue inhibitors of metalloproteinases with a high matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio are present in tracheal aspirate fluids of infants who develop chronic lung disease.
Pediatrics. 2004;113
:1709
–1714
[Abstract/Free Full Text] - Clark PL, Ekekezie II, Kaftan HA, Castor CA, Truog WE. Safety and efficacy of nitric oxide in chronic lung disease.
Arch Dis Child Fetal Neonatal Ed. 2002;86
:F41
–F45
[Abstract/Free Full Text] - Ballard PL, Merrill JD, Truog WE, et al. The NO CLD Trial: effect of inhaled nitric oxide in premature infants on surfactant function and composition [abstract]. E-PAS. 2006;59 :4840.139
- Abe M, Harpel JG, Metz CN, Nunes I, Loskutof DJ, Rifkin DB. An assay for transforming growth factor-ß using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. Anal Biochem. 1994;216 :276 –284[CrossRef][Web of Science][Medline]
- Nitta T, Okumura K, Sato K. Lysosomal enzymic activity of astroglial cells. Pathobiology. 1992;60 :42 –44[Web of Science][Medline]
- Lokeshwar VB, Obek C, Soloway MS, Block NL. Tumor-associated hyaluronic acid: a new sensitive and specific urine marker for bladder cancer.
Cancer Res. 1997;57
:773
–777
[Abstract/Free Full Text] - Maeda H, Fujita H, Sakura Y, Miyazaki K, Goto M. A competitive enzyme-linked immunosorbent assay-like method for the measurement of urinary hyaluronan. Biosci Biotechnol Biochem. 1999;63 :892 –895[CrossRef][Medline]
- Goil S, Barnes C, Thibeault D, Truog WE. Simplified method for purification of colostrums to obtain secretory component of immunoglobulin A, using secretory component as a reference protein in tracheal aspirate fluid. J Chromatogr B Biomed Sci Appl. 1998;705 :203 –211[CrossRef][Medline]
- Seeger W, Grube C, Gunther A, Schmidt R. Surfactant inhibition by plasma proteins: differential sensitivity of various surfactant preparations. Eur Respir J. 1993;6 :971 –977[Abstract]
- Jones CA, Cayabyab RG, Kwong KY, et al. Undetectable interleukin (IL)-10 and persistent IL-8 expression early in hyaline membrane disease: a possible developmental basis for the predisposition to chronic lung inflammation in preterm newborns. Pediatr Res. 1996;39 :966 –975[Web of Science][Medline]
- Savani RC, Hou G, Liu P, et al. A role for hyaluronan in bleomycin-induced pulmonary inflammation.
Am J Respir Cell Mol Biol. 2000;23
:475
–484
[Abstract/Free Full Text] - Lecart C, Cayabyab R, Buckley S, et al. Bioactive transforming growth factor-ß in the lungs of extremely low birth weight neonates predicts the need for home oxygen supplementation. Biol Neonate. 2000;77 :217 –223[CrossRef][Web of Science][Medline]
- Morrow JD, Hill KE, Burk RF, Namour TM, Badr KF, Roberts LJ II. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase free radical-catalyzed mechanism.
Proc Natl Acad Sci USA. 1990;87
:9383
–9387
[Abstract/Free Full Text] - D'Angio CT, Basavegowda K, Avissar NE, Finkelstein JN, Sinkin RA. Comparison of tracheal aspirate and bronchoalveolar lavage specimens from premature infants. Biol Neonate. 2002;82 :145 –149[CrossRef][Web of Science][Medline]
- Dargaville PA, South M, McDougall PN. Comparison of two methods of diagnostic lung lavage in ventilated infants with lung disease.
Am J Respir Crit Care Med. 1999;160
:771
–777
[Abstract/Free Full Text] - Dargaville PA, South M, Vervaart P, McDougall PN. Validity of markers of dilution in small volume lung lavage.
Am J Respir Crit Care Med. 1999;160
:778
–784
[Abstract/Free Full Text] - Huang HC, Tai FY, Feng-Sheng W, et al. Correlation of augmented IL-8 production to premature chronic lung disease: implication of posttranscriptional regulation. Pediatr Res. 2005;58 :216 –221[CrossRef][Web of Science][Medline]
- Truog WE, Pallotto E, Clark P, et al. Interaction of endogenous endothelin-1 and inhaled nitric oxide in term and preterm infants. Clin Sci. 2002;103(suppl) :294S –297S[Web of Science][Medline]
PEDIATRICS (ISSN 1098-4275). ©2007 by the American Academy of Pediatrics
CiteULike 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  C L Bose, C E L Dammann, and M M Laughon Bronchopulmonary dysplasia and inflammatory biomarkers in the premature neonate Arch. Dis. Child. Fetal Neonatal Ed., November 1, 2008; 93(6): F455 - F461. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. E. Truog, R. J. Martin, R. A. Ballard, D. Black, A. Cnaan, and For the NO CLD Study Group Inhaled Nitric Oxide for Preterm Infants Pediatrics, June 1, 2008; 121(6): 1287 - 1288. [Full Text] [PDF]
|
|
|
|
|
|
P. L. Ballard, W. E. Truog, J. D. Merrill, A. Gow, M. Posencheg, S. G. Golombek, L. A. Parton, X. Luan, A. Cnaan, and R. A. Ballard Plasma Biomarkers of Oxidative Stress: Relationship to Lung Disease and Inhaled Nitric Oxide Therapy in Premature Infants Pediatrics, March 1, 2008; 121(3): 555 - 561. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. L. Ballard, J. D. Merrill, W. E. Truog, R. I. Godinez, M. H. Godinez, T. M. McDevitt, Y. Ning, S. G. Golombek, L. A. Parton, X. Luan, et al. Surfactant Function and Composition in Premature Infants Treated With Inhaled Nitric Oxide Pediatrics, August 1, 2007; 120(2): 346 - 353. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
