Philip L Ballard

Publications (41)224.33 Total impact

• Article: Late surfactant replacement therapy increases surfactant protein-B content in a randomized pilot study.

  Roberta L Keller, Jeffrey D Merrill, Dennis M Black, Robin H Steinhorn, Eric C Eichenwald, David J Durand, Rita M Ryan, William E Truog, Sherry E Courtney, Philip L Ballard, Roberta A Ballard
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  ABSTRACT: Background:Surfactant dysfunction may contribute to the development of bronchopulmonary dysplasia in persistently ventilated preterm infants. We conducted a multicenter randomized, blinded pilot study to assess the safety and efficacy of late doses of a surfactant protein-B (SP-B) containing surfactant (calfactant) in combination with prolonged inhaled nitric oxide (iNO) in infants ≤ 1000g birth weight.Methods:We randomized 85 preterm infants ventilated at 7-14 d to late surfactant (up to 5 doses) and prolonged iNO versus iNO-alone. Large aggregate surfactant was isolated from daily tracheal aspirates for measurement of SP-B content, total protein and phospholipid.Results:Late surfactant was administered with minimal acute adverse effects. Clinical status and tracheal aspirate surfactant recovery and SP-B content were transiently improved compared to Controls; effects waned after 1 day. The change in SP-B content with surfactant dosing was positively correlated with SP-B levels during treatment (r=0.50, P=0.02).Conclusion:Low SP-B content increased with calfactant administration, but the relationship of response to SP-B levels suggests degradation as a contributing mechanism for SP-B deficiency and surfactant dysfunction. We conclude that late surfactant therapy in combination with iNO is safe and transiently increases surfactant SP-B content, which may lead to improved short- and long-term respiratory outcomes.Pediatric Research (2012); doi:10.1038/pr.2012.136.
  Pediatric Research 10/2012; · 2.70 Impact Factor
• Article: Distribution and surfactant association of carcinoembryonic cell adhesion molecule 6 in human lung.

  Cheryl Chapin, Nicole A Bailey, Linda W Gonzales, Jae-Woo Lee, Robert F Gonzalez, Philip L Ballard
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  ABSTRACT: Carcinoembryonic cell adhesion molecule 6 (CEACAM6) is a glycosylated, glycophosphatidylinositol-anchored protein expressed in epithelial cells of various primate tissues. It binds gram-negative bacteria and is overexpressed in human cancers. CEACAM6 is associated with lamellar bodies of cultured type II cells of human fetal lung and protects surfactant function in vitro. In this study, we characterized CEACAM6 expression in vivo in human lung. CEACAM6 was present in lung lavage of premature infants at birth and increased progressively in intubated infants with lung disease. Of surfactant-associated CEACAM6, ∼80% was the fully glycosylated, 90-kDa form that contains the glycophosphatidylinositol anchor, and the concentration (3.9% of phospholipid for adult lung) was comparable to that for surfactant proteins (SP)-A/B/C. We examined the affinity of CEACAM6 by purification of surfactant on density gradient centrifugation; concentrations of CEACAM6 and SP-B per phospholipid were unchanged, whereas levels of total protein and SP-A decreased by 60%. CEACAM6 mRNA content decreased progressively from upper trachea to peripheral fetal lung, whereas protein levels were similar in all regions of adult lung, suggesting proximal-to-distal developmental expression in lung epithelium. In adult lung, most type I cells and ∼50% of type II cells were immunopositive. We conclude that CEACAM6 is expressed by alveolar and airway epithelial cells of human lung and is secreted into lung-lining fluid, where fully glycosylated protein binds to surfactant. Production appears to be upregulated during neonatal lung disease, perhaps related to roles of CEACAM6 in surfactant function, cell proliferation, and innate immune defense.
  AJP Lung Cellular and Molecular Physiology 01/2012; 302(2):L216-25. · 3.66 Impact Factor
• Article: A small-molecule smoothened agonist prevents glucocorticoid-induced neonatal cerebellar injury.

  Vivi M Heine, Amelie Griveau, Cheryl Chapin, Philip L Ballard, James K Chen, David H Rowitch
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  ABSTRACT: Glucocorticoids are used for treating preterm neonatal infants suffering from life-threatening lung, airway, and cardiovascular conditions. However, several studies have raised concerns about detrimental effects of postnatal glucocorticoid administration on the developing brain leading to cognitive impairment, cerebral palsy, and hypoplasia of the cerebellum, a brain region critical for coordination of movement and higher-order neurological functions. Previously, we showed that glucocorticoids inhibit Sonic hedgehog-Smoothened (Shh-Smo) signaling, the major mitogenic pathway for cerebellar granule neuron precursors. Conversely, activation of Shh-Smo in transgenic mice protects against glucocorticoid-induced neurotoxic effects through induction of the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) pathway. Here, we show that systemic administration of a small-molecule agonist of the Shh-Smo pathway (SAG) prevented the neurotoxic effects of glucocorticoids. SAG did not interfere with the beneficial effects of glucocorticoids on lung maturation, and despite the known associations of the Shh pathway with neoplasia, we found that transient (1-week-long) SAG treatment of neonatal animals was well tolerated and did not promote tumor formation. These findings suggest that a small-molecule agonist of Smo has potential as a neuroprotective agent in neonates at risk for glucocorticoid-induced neonatal cerebellar injury.
  Science translational medicine 10/2011; 3(105):105ra104. · 7.80 Impact Factor
• Article: HTII-280, a biomarker specific to the apical plasma membrane of human lung alveolar type II cells.

  Robert F Gonzalez, Lennell Allen, Linda Gonzales, Philip L Ballard, Leland G Dobbs
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  ABSTRACT: The pulmonary alveolar epithelium is composed of two morphologically distinct cell types, type I (TI) and type II (TII) cells. Alveolar TII cells synthesize, secrete, and recycle surfactant components; contain ion transporters; and secrete immune effector molecules. In response to alveolar injury, TII cells have the capacity to act as progenitor cells, proliferating and transdifferentiating into TI cells. Although various proteins are associated with TII cells, a plasma membrane marker specific to human TII cells that would be useful for identification in tissue and for isolating this cell type has not been described previously. We devised a strategy to produce a monoclonal antibody (MAb) specific to the apical surface of human TII cells and developed an MAb that appears to be specific for human TII cells. The antibody recognizes a 280- to 300-kDa protein, HTII-280, which has the biochemical characteristics of an integral membrane protein. HTII-280 is detected by week 11 of gestation and is developmentally regulated. HTII-280 is useful for isolating human TII cells with purities and viabilities >95%. HTII-280 is likely to be a useful morphological and biochemical marker of human TII cells that may help to advance our understanding of various lung pathological conditions, including the origin and development of various lung tumors.
  Journal of Histochemistry and Cytochemistry 10/2010; 58(10):891-901. · 2.72 Impact Factor
• Article: Regulated gene expression in cultured type II cells of adult human lung.

  Philip L Ballard, Jae W Lee, Xiaohui Fang, Cheryl Chapin, Lennell Allen, Mark R Segal, Horst Fischer, Beate Illek, Linda W Gonzales, Venkatadri Kolla, Michael A Matthay
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  ABSTRACT: Alveolar type II cells have multiple functions, including surfactant production and fluid clearance, which are critical for lung function. Differentiation of type II cells occurs in cultured fetal lung epithelial cells treated with dexamethasone plus cAMP and isobutylmethylxanthine (DCI) and involves increased expression of 388 genes. In this study, type II cells of human adult lung were isolated at approximately 95% purity, and gene expression was determined (Affymetrix) before and after culturing 5 days on collagen-coated dishes with or without DCI for the final 3 days. In freshly isolated cells, highly expressed genes included SFTPA/B/C, SCGB1A, IL8, CXCL2, and SFN in addition to ubiquitously expressed genes. Transcript abundance was correlated between fetal and adult cells (r = 0.88), with a subset of 187 genes primarily related to inflammation and immunity that were expressed >10-fold higher in adult cells. During control culture, expression increased for 8.1% of expressed genes and decreased for approximately 4% including 118 immune response and 10 surfactant-related genes. DCI treatment promoted lamellar body production and increased expression of approximately 3% of probed genes by > or =1.5-fold; 40% of these were also induced in fetal cells. Highly induced genes (> or =10-fold) included PGC, ZBTB16, DUOX1, PLUNC, CIT, and CRTAC1. Twenty-five induced genes, including six genes related to surfactant (SFTPA/B/C, PGC, CEBPD, and ADFP), also had decreased expression during control culture and thus are candidates for hormonal regulation in vivo. Our results further define the adult human type II cell molecular phenotype and demonstrate that a subset of genes remains hormone responsive in cultured adult cells.
  AJP Lung Cellular and Molecular Physiology 04/2010; 299(1):L36-50. · 3.66 Impact Factor
• Article: Re: 'The use of iNO in the newborn period: results from the European iNO registry'.

  Sergio G Golombek, William E Truog, Roberta A Ballard, Philip L Ballard
  Acta Paediatrica 04/2010; 99(9):1283. · 2.07 Impact Factor
• Article: Two-year neurodevelopmental outcomes of ventilated preterm infants treated with inhaled nitric oxide.

  Michele C Walsh, Anna Maria Hibbs, Camilia R Martin, Avital Cnaan, Roberta L Keller, Eric Vittinghoff, Richard J Martin, William E Truog, Philip L Ballard, Arlene Zadell, Sandra R Wadlinger, Christine E Coburn, Roberta A Ballard
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  ABSTRACT: In a randomized multi-center trial, we demonstrated that inhaled nitric oxide begun between 7 and 21 days and given for 24 days significantly increased survival without bronchopulmonary dysplasia (BPD) in ventilated premature infants weighing <1250 g. Because some preventative BPD treatments are associated with neurodevelopmental impairment, we designed a follow-up study to assess the safety of nitric oxide. Our hypothesis was that inhaled nitric oxide would not increase neurodevelopmental impairment compared with placebo. We prospectively evaluated neurodevelopmental and growth outcomes at 24 months postmenstrual age in 477 of 535 surviving infants (89%) enrolled in the trial. In the treated group, 109 of 243 children (45%) had neurodevelopmental impairment (moderate or severe cerebral palsy, bilateral blindness, bilateral hearing loss, or score <70 on the Bayley Scales II), compared with 114 of 234 (49%) in the placebo group (relative risk, 0.92; 95% CI, 0.75-1.12; P = .39). No differences on any subcomponent of neurodevelopmental impairment or growth variables were found between inhaled nitric oxide or placebo. Inhaled nitric oxide improved survival free of BPD, with no adverse neurodevelopmental effects at 2 years of age.
  The Journal of pediatrics 02/2010; 156(4):556-61.e1. · 4.02 Impact Factor
• Article: Economic evaluation of inhaled nitric oxide in preterm infants undergoing mechanical ventilation.

  John A F Zupancic, Anna Maria Hibbs, Lisa Palermo, William E Truog, Avital Cnaan, Dennis M Black, Philip L Ballard, Sandra R Wadlinger, Roberta A Ballard
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  ABSTRACT: In the previously reported Nitric Oxide for Chronic Lung Disease (NO CLD) trial, ventilated preterm infants who received a course of inhaled nitric oxide (iNO) between 7 and 21 days of life had a significant improvement in survival without bronchopulmonary dysplasia (BPD), as well as a shorter duration of admission and ventilation. However, the price for the drug may be a barrier to widespread use. We sought to estimate the incremental cost-effectiveness of iNO therapy to prevent BPD in infants of <1250 g birth weight. We used patient-level data from the NO CLD randomized trial. The study took a third-party payer perspective and measured costs and effects through hospital discharge. We applied previously reported hospital per-diem costs stratified according to intensity of ventilatory support, nitric oxide costs from standard market prices, and professional (physician) fees from the Medicare fee schedule. We compared log transformed costs by using multivariable modeling and performed incremental cost-effectiveness analysis with estimation of uncertainty through nonparametric bootstrapping. The mean cost per infant was $193125 in the placebo group and $194702 in the iNO group (adjusted P = .17). The point estimate for the incremental cost per additional survivor without BPD was $21297. For infants in whom iNO was initiated between 7 and 14 days of life, the mean cost per infant was $187407 in the placebo group and $181525 in the iNO group (adjusted P = .46). In this group of early treated infants, there was a 71% probability that iNO actually decreased costs while improving outcomes. Despite its higher price relative to many other neonatal therapies, iNO in this trial was not associated with higher costs of care, an effect that is likely due to its impact on length of stay and ventilation. Indeed, for infants who receive nitric oxide between 7 and 14 days of life, the therapy seemed to lower costs while improving outcomes.
  PEDIATRICS 10/2009; 124(5):1325-32. · 4.47 Impact Factor
• Article: Carcinoembryonic cell adhesion molecule 6 in human lung: regulated expression of a multifunctional type II cell protein.

  Venkatadri Kolla, Linda W Gonzales, Nicole A Bailey, Ping Wang, Sreedevi Angampalli, Marye H Godinez, Muniswamy Madesh, Philip L Ballard
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  ABSTRACT: Carcinoembryonic cell adhesion molecule 6 (CEACAM6) is a glycosylated, glycosylphosphatidylinositol (GPI)-anchored protein expressed in epithelial cells of various human tissues. It binds gram-negative bacteria and is overexpressed in cancers, where it is antiapoptotic and promotes metastases. To characterize CEACAM6 expression in developing lung, we cultured human fetal lung epithelial cells and examined responses to differentiation-promoting hormones, adenovirus expressing thyroid transcription factor-1 (TTF-1), and silencing of TTF-1 with small inhibitory RNA. Glucocorticoid and cAMP had additive stimulatory effects on CEACAM6 content, and combined treatment maximally increased transcription rate, mRNA, and protein approximately 10-fold. Knockdown of TTF-1 reduced hormone induction of CEACAM6 by 80%, and expression of recombinant TTF-1 increased CEACAM6 in a dose-dependent fashion. CEACAM6 content of lung tissue increased during the third trimester and postnatally. By immunostaining, CEACAM6 was present in fetal type II cells, but not mesenchymal cells, and localized to both the plasma membrane and within surfactant-containing lamellar bodies. CEACAM6 was secreted from cultured type II cells and was present in both surfactant and supernatant fractions of infant tracheal aspirates. In functional studies, CEACAM6 reduced inhibition of surfactant surface properties by proteins in vitro and blocked apoptosis of electroporated cultured cells. We conclude that CEACAM6 in fetal lung epithelial cells is developmentally and hormonally regulated and a target protein for TTF-1. Because CEACAM6 acts as an antiapoptotic factor and stabilizes surfactant function, in addition to a putative role in innate defense against bacteria, we propose that it is a multifunctional alveolar protein.
  AJP Lung Cellular and Molecular Physiology 04/2009; 296(6):L1019-30. · 3.66 Impact Factor
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  Available from: Brigham C Willis

  Article: Postnatal estradiol up-regulates lung nitric oxide synthases and improves lung function in bronchopulmonary dysplasia.

  Donald C McCurnin, Richard A Pierce, Brigham C Willis, Ling Yi Chang, Bradley A Yoder, Ivan S Yuhanna, Philip L Ballard, Ronald I Clyman, Nahid Waleh, William Maniscalco, James D Crapo, Peter H Grubb, Philip W Shaul
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  ABSTRACT: Nitric oxide (NO) plays an important role in lung development and perinatal lung function, and pulmonary NO synthases (NOS) are decreased in bronchopulmonary dysplasia (BPD) following preterm birth. Fetal estradiol levels increase during late gestation and estradiol up-regulates NOS, suggesting that after preterm birth estradiol deprivation causes attenuated lung NOS resulting in impaired pulmonary function. To test the effects of postnatal estradiol administration in a primate model of BPD over 14 days after delivery at 125 days of gestation (term = 185 d). Cardiopulmonary function was assessed by echocardiography and whole body plethysmography. Lung morphometric and histopathologic analyses were performed, and NOS enzymatic activity and abundance were measured. Estradiol caused an increase in blood pressure and ductus arteriosus closure. Expiratory resistance and lung compliance were also improved, and this occurred before spontaneous ductal closure. Furthermore, both oxygenation and ventilation indices were improved with estradiol, and the changes in lung function and ventilatory support requirements persisted throughout the study period. Whereas estradiol had negligible effect on indicators of lung inflammation and on lung structure assessed after the initial 14 days of ventilatory support, it caused an increase in lung neuronal and endothelial NOS enzymatic activity. In a primate model of BPD, postnatal estradiol treatment had favorable cardiovascular impact, enhanced pulmonary function, and lowered requirements for ventilatory support in association with an up-regulation of lung NOS. Estradiol may be an efficacious postnatal therapy to improve lung function and outcome in preterm infants.
  American Journal of Respiratory and Critical Care Medicine 02/2009; 179(6):492-500. · 11.08 Impact Factor
• Article: Expression of nitric oxide synthases and endogenous NO metabolism in bronchopulmonary dysplasia.

  Christiana W Davis, Linda W Gonzales, Roberta A Ballard, Philip L Ballard, Changjiang Guo, Andrew J Gow
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  ABSTRACT: Bronchopulmonary dysplasia (BPD), a multifactorial disease of preterm neonates of complex etiology, is a significant problem within very low birth weight infants. Nitric oxide (NO) has been implicated in both the pathogenesis and as a potential therapeutic of this disease. At this time, there is little direct evidence of the changes in NO production and metabolism that occur within BPD in humans. Animal models have implied that reduced nitric oxide synthase (NOS) expression and NO production in the early stages of the disease may be critical factors. However, inflammation and hence iNOS expression, is also thought to play a role. In the present study we have utilized pathological samples to determine changes in the expression of NOS and NO metabolites within late stage BPD. It is our contention that within these samples iNOS expression is increased and associated with increased NO metabolite production. Mild immunostaining of all three nitric oxide synthase (NOS) enzymes (neuronal, inducible and endothelial) is observed in control lung with tight localization to the endothelium and epithelial airway. This tight localization was lost in samples from subjects with BPD. There was also a marked increase in iNOS expression throughout the lung tissue with strong coexistence with an epithelial cell marker cytokeratin. NO reaction products are altered with BPD as evidenced by increased S-nitrosothiol (SNO) and strong nitrotyrosine (NO(2)Y) imunoreactivity. This study demonstrates a strong correlation between products of NO reactivity and NOS localization in BPD.
  Pediatric Pulmonology 08/2008; 43(7):703-9. · 2.53 Impact Factor
• Article: One-year respiratory outcomes of preterm infants enrolled in the Nitric Oxide (to prevent) Chronic Lung Disease trial.

  Anna Maria Hibbs, Michele C Walsh, Richard J Martin, William E Truog, Scott A Lorch, Evaline Alessandrini, Avital Cnaan, Lisa Palermo, Sandra R Wadlinger, Christine E Coburn, Philip L Ballard, Roberta A Ballard
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  ABSTRACT: To identify whether inhaled nitric oxide treatment decreased indicators of long-term pulmonary morbidities after discharge from the neonatal intensive care unit. The Nitric Oxide (to Prevent) Chronic Lung Disease trial enrolled preterm infants (<1250 g) between 7 to 21 days of age who were ventilated and at high risk for bronchopulmonary dysplasia. Follow-up occurred at 12 +/- 3 months of age adjusted for prematurity; long-term pulmonary morbidity and other outcomes were reported by parents during structured blinded interviews. A total of 456 infants (85%) were seen at 1 year. Compared with control infants, infants randomized to inhaled nitric oxide received significantly less bronchodilators (odds ratio [OR] 0.53 [95% confidence interval 0.36-0.78]), inhaled steroids (OR 0.50 [0.32-0.77]), systemic steroids (OR 0.56 [0.32-0.97]), diuretics (OR 0.54 [0.34-0.85]), and supplemental oxygen (OR 0.65 [0.44-0.95]) after discharge from the neonatal intensive care unit. There were no significant differences between parental report of rehospitalizations (OR 0.83 [0.57-1.21]) or wheezing or whistling in the chest (OR 0.70 [0.48-1.03]). Infants treated with inhaled nitric oxide received fewer outpatient respiratory medications than the control group. However, any decision to institute routine use of this dosing regimen should also take into account the results of the 24-month neurodevelopmental assessment.
  The Journal of pediatrics 07/2008; 153(4):525-9. · 4.02 Impact Factor
• Article: Plasma biomarkers of oxidative stress: relationship to lung disease and inhaled nitric oxide therapy in premature infants.

  Philip L Ballard, William E Truog, Jeffrey D Merrill, Andrew Gow, Michael Posencheg, Sergio G Golombek, Lance A Parton, Xianqun Luan, Avital Cnaan, Roberta A Ballard
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  ABSTRACT: Inhaled nitric oxide treatment for ventilated premature infants improves survival without bronchopulmonary dysplasia. However, there has been no information regarding possible effects of this therapy on oxidative stress. We hypothesized that inhaled nitric oxide therapy would not influence concentrations of plasma biomarkers of oxidative stress. As part of the Nitric Oxide Chronic Lung Disease Trial, we collected blood samples at specified intervals from a subpopulation of 100 infants of <1250 g birth weight who received inhaled nitric oxide (20 ppm, weaned to 2 ppm) or placebo gas for 24 days. Plasma was assayed for total protein and for 3-nitrotyrosine and carbonylation by using immunoassays. The demographic characteristics and primary outcome for the infants were representative of the entire group of infants who were in the Nitric Oxide Chronic Lung Disease Trial. For all infants at baseline, before receiving study gas, the concentration of total protein was inversely correlated with the respiratory severity score, and plasma carbonyl was positively correlated with severity score, supporting an association between oxidative stress and severity of lung disease. Infants who survived without bronchopulmonary dysplasia had 30% lower protein carbonylation concentrations at study entry than those who had an adverse outcome. At each of 3 time points (1-10 days) during exposure to study gas, there were no significant differences between control and treated infants for concentrations of plasma protein, 3-nitrotyrosine, and carbonylation. Inhaled nitric oxide treatment for premature infants who are at risk for bronchopulmonary dysplasia does not alter plasma biomarkers of oxidative stress, which supports the safety of this therapy.
  PEDIATRICS 03/2008; 121(3):555-61. · 4.47 Impact Factor
• Article: Surfactant function and composition in premature infants treated with inhaled nitric oxide.

  Philip L Ballard, Jeffrey D Merrill, William E Truog, Rodolfo I Godinez, Marye H Godinez, Theresa M McDevitt, Yue Ning, Sergio G Golombek, Lance A Parton, Xianqun Luan, Avital Cnaan, Roberta A Ballard
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  ABSTRACT: We hypothesized that inhaled nitric oxide treatment of premature infants at risk for bronchopulmonary dysplasia would not adversely affect endogenous surfactant function or composition. 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. 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. 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.
  PEDIATRICS 08/2007; 120(2):346-53. · 4.47 Impact Factor
• Article: Developmental regulation of DUOX1 expression and function in human fetal lung epithelial cells.

  Horst Fischer, Linda K Gonzales, Venkatadri Kolla, Christian Schwarzer, Françoise Miot, Beate Illek, Philip L Ballard
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  ABSTRACT: The purpose of this study was to determine the expression and cellular functions of the epithelial NADPH oxidase DUOX1 during alveolar type II cell development. When human fetal lung cells (gestational age 11-22 wk) were cultured to confluency on permeable filters, exposure of cells to a hormone mixture (dexamethasone, 8-Br-cAMP, and IBMX, together referred to as DCI) resulted in differentiation of cells into a mature type II phenotype as assessed by expression of lamellar bodies, surfactant proteins, and transepithelial electrical parameters. After 6 days in culture in presence of DCI, transepithelial resistance (2,616 +/- 529 Omega.cm(2)) and potential (-8.5 +/- 0.6 mV) indicated epithelial polarization. At the same time, treatment with DCI significantly increased the mRNA expression of DUOX1 ( approximately 21-fold), its maturation factor DUOXA1 ( approximately 12-fold), as well as DUOX protein ( approximately 12-fold), which was localized near the apical cell pole in confluent cultures. For comparison, in fetal lung specimens, DUOX protein was not detectable at up to 27 wk of gestational age but was strongly upregulated after 32 wk. Function of DUOX1 was assessed by measuring H(2)O(2) and acid production. Rates of H(2)O(2) production were increased by DCI treatment and blocked by small interfering RNA directed against DUOX1 or by diphenylene iodonium. DCI-treated cultures also showed increased intracellular acid production and acid release into the mucosal medium, and acid production was largely blocked by knockdown of DUOX1 mRNA. These data establish the regulated expression of DUOX1 during alveolar maturation, and indicate DUOX1 in alveolar H(2)O(2) and acid secretion by differentiated type II cells.
  AJP Lung Cellular and Molecular Physiology 07/2007; 292(6):L1506-14. · 3.66 Impact Factor
• Article: Inflammatory markers and mediators in tracheal fluid of premature infants treated with inhaled nitric oxide.

  William E Truog, Philip L Ballard, Michael Norberg, Sergio Golombek, Rashmin C Savani, Jeffrey D Merrill, Lance A Parton, Avital Cnaan, Xianqun Luan, Roberta A Ballard
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  ABSTRACT: 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. 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-1beta, interleukin-8, transforming growth factor-beta, N-acetylglucosaminidase, 8-epi-prostaglandin F2alpha, and hyaluronan. Results were normalized to total protein and secretory component of immunoglobulin A. 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 F2alpha levels for infants who did (n = 21) and did not (n = 26) develop bronchopulmonary dysplasia at postmenstrual age of 36 weeks. Inhaled nitric oxide, as administered in this study, seemed to be safe. Its use was not associated with any increase in airway inflammatory substances.
  PEDIATRICS 04/2007; 119(4):670-8. · 4.47 Impact Factor
• Article: Thyroid transcription factor in differentiating type II cells: regulation, isoforms, and target genes.

  Venkatadri Kolla, Linda W Gonzales, John Gonzales, Ping Wang, Sreedevi Angampalli, Sheldon I Feinstein, Philip L Ballard
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  ABSTRACT: Thyroid transcription factor-1 (TTF-1, product of the Nkx2.1 gene) is essential for branching morphogenesis of the lung and enhances expression of surfactant proteins by alveolar type II cells. We investigated expression of two TTF-1 mRNA transcripts, generated by alternative start sites and coding for 42- and 46-kD protein isoforms in the mouse, during hormone-induced differentiation of human fetal lung type II cells in culture. Transcript for 42-kD TTF-1 was 20-fold more abundant than TTF-1(46) mRNA by RT-PCR. Only 42-kD protein was detected in lung cells, and its content increased during in vivo development and in response to in vitro glucocorticoid plus cAMP treatment. To examine TTF-1 target proteins, recombinant, phosphorylated TTF-1(42) was expressed in nuclei of cells by adenovirus transduction. By microarray analysis, 14 genes were comparably induced by recombinant TTF-1 (rTTF-1) and hormone treatment, and 9 additional hormone-responsive genes, including surfactant proteins-A/B/C, were partially induced by rTTF-1. The most highly (approximately 10-fold) TTF-1-induced genes were DC-LAMP (LAMP3) and CEACAM6 with induction confirmed by Western analysis and immunostaining. Treatment of cells with hormones plus small inhibitory RNA directed toward TTF-1 reduced TTF-1 content by approximately 50% and inhibited hormone induction of the 23 genes induced by rTTF-1. In addition, knockdown of TTF-1 inhibited 72 of 274 other genes induced by hormones. We conclude that 42-kD TTF-1 is required for induction of a subset of regulated genes during type II cell differentiation.
  American Journal of Respiratory Cell and Molecular Biology 03/2007; 36(2):213-25. · 5.13 Impact Factor
• Article: Role of endogenous TGF-beta in glucocorticoid-induced lung type II cell differentiation.

  Theresa M McDevitt, Linda W Gonzales, Rashmin C Savani, Philip L Ballard
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  ABSTRACT: In the fetal lung, endogenous transforming growth factor (TGF)-beta inhibits early morphogenesis and blocks hormone-induced type II cell differentiation. We hypothesized that endogenous TGF-beta inhibits type II cell differentiation and that the stimulatory effects of glucocorticoids result in part from suppression of TGF-beta. Epithelial cells were isolated from human fetal lung and cultured under defined conditions with and without dexamethasone plus cAMP to promote type II cell differentiation. Control cells produced TGF-beta, which was activated in part by alpha(V)beta(6)-integrin. Treatment with dexamethasone, but not cAMP, reduced TGF-beta1 and -beta2 transcripts and TGF-beta bioactivity in culture medium. To examine the effects of decreased TGF-beta in the absence of glucocorticoid, cells were treated with antibodies to TGF-beta and its receptors. By real-time RT-PCR, antibody blockade of TGF-beta reduced serpine1, a TGF-beta-inducible gene, and increased gene expression for sftpa, sftpb, sftpc, and titf1, mimicking the response to hormone treatment. By microarray analysis, 29 additional genes were induced by both TGF-beta antibody and hormone treatment, and 20 other genes were repressed by both treatments. For some genes, the fold response was comparable for antibody and hormone treatment. We conclude that endogenous TGF-beta suppresses expression of surfactant proteins and selected other type II cell genes in fetal lung, in part secondary to increased expression of titf1, and we propose that the mechanism of glucocorticoid-induced type II cell differentiation includes antagonism of TGF-beta gene suppression. Surfactant production during fetal development is likely influenced by relative levels of TGF-beta and glucocorticoids.
  AJP Lung Cellular and Molecular Physiology 02/2007; 292(1):L249-57. · 3.66 Impact Factor
• Article: Hop functions downstream of Nkx2.1 and GATA6 to mediate HDAC-dependent negative regulation of pulmonary gene expression.

  Zhan Yin, Linda Gonzales, Venkatadri Kolla, Nibedita Rath, Yuzhen Zhang, Min Min Lu, Shioko Kimura, Philip L Ballard, Michael F Beers, Jonathan A Epstein, Edward E Morrisey
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  ABSTRACT: Hop is an unusual homeodomain protein that was first identified in the developing heart where it functions downstream of Nkx2.5 to modulate cardiac gene expression. Hop functions through interactions with histone deacetylase (HDAC) 2 to mediate repression of cardiac-specific genes, and recent studies show that HDAC activity and HDAC2 expression are decreased in people with chronic obstructive pulmonary disease. Here, we show that Hop is expressed in airway epithelium coincident with HDAC2, and expression is induced by the combination of dexamethasone and cAMP in parallel with induction of surfactant protein gene expression. Hop functions in the developing pulmonary airway, acting downstream of Nkx2.1 and GATA6, to negatively regulate surfactant protein expression. Loss of Hop expression in vivo results in defective type 2 pneumocyte development with increased surfactant production and disrupted alveolar formation. Thus Hop represents a novel regulator of pulmonary maturation that is induced by glucocorticoids to mediate functionally important HDAC-dependent negative feedback regulation.
  AJP Lung Cellular and Molecular Physiology 09/2006; 291(2):L191-9. · 3.66 Impact Factor
• Article: Inhaled nitric oxide in preterm infants undergoing mechanical ventilation.

  Roberta A Ballard, William E Truog, Avital Cnaan, Richard J Martin, Philip L Ballard, Jeffrey D Merrill, Michele C Walsh, David J Durand, Dennis E Mayock, Eric C Eichenwald, [......], Sergio G Golombek, Sherry E Courtney, Dan L Stewart, Stephen E Welty, Roderic H Phibbs, Anna Maria Hibbs, Xianqun Luan, Sandra R Wadlinger, Jeanette M Asselin, Christine E Coburn
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  ABSTRACT: Bronchopulmonary dysplasia in premature infants is associated with prolonged hospitalization, as well as abnormal pulmonary and neurodevelopmental outcome. In animal models, inhaled nitric oxide improves both gas exchange and lung structural development, but the use of this therapy in infants at risk for bronchopulmonary dysplasia is controversial. We conducted a randomized, stratified, double-blind, placebo-controlled trial of inhaled nitric oxide at 21 centers involving infants with a birth weight of 1250 g or less who required ventilatory support between 7 and 21 days of age. Treated infants received decreasing concentrations of nitric oxide, beginning at 20 ppm, for a minimum of 24 days. The primary outcome was survival without bronchopulmonary dysplasia at 36 weeks of postmenstrual age. Among 294 infants receiving nitric oxide and 288 receiving placebo birth weight (766 g and 759 g, respectively), gestational age (26 weeks in both groups), and other characteristics were similar. The rate of survival without bronchopulmonary dysplasia at 36 weeks of postmenstrual age was 43.9 percent in the group receiving nitric oxide and 36.8 percent in the placebo group (P=0.042). The infants who received inhaled nitric oxide were discharged sooner (P=0.04) and received supplemental oxygen therapy for a shorter time (P=0.006). There were no short-term safety concerns. Inhaled nitric oxide therapy improves the pulmonary outcome for premature infants who are at risk for bronchopulmonary dysplasia when it is started between 7 and 21 days of age and has no apparent short-term adverse effects. (ClinicalTrials.gov number, NCT00000548 [ClinicalTrials.gov] .).
  New England Journal of Medicine 08/2006; 355(4):343-53. · 53.30 Impact Factor