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1.  ABCA3 Mutations Associated with Pediatric Interstitial Lung Disease 
Rationale: ABCA3 is a member of the ATP-binding cassette family of proteins that mediate the translocation of a wide variety of substrates, including lipids, across cellular membranes. Mutations in the gene encoding ABCA3 were recently identified in full-term neonates with fatal surfactant deficiency.
Objective: To test the hypothesis that ABCA3 mutations are not always associated with fatal neonatal lung disease but are a cause of pediatric interstitial lung disease.
Methods: DNA samples were obtained from 195 children with chronic lung disease of unknown etiology. The 30 coding exons of the ABCA3 gene were sequenced in four unrelated children with a referring diagnosis of desquamative interstitial pneumonitis and who were older than 10 years at the time of enrollment.
Results: Three of four patients (ages 16, 23, and 11 years) with desquamative interstitial pneumonitis had ABCA3 mutations identified on both alleles. All three had the same missense mutation (E292V) and a second unique mutation. The E292V mutation was not found on 200 control alleles from adults without lung disease, but seven additional patients of the remaining study patients had the E292V mutation on one allele. Immunohistochemical analysis of surfactant protein expression in three patients revealed a specific staining pattern for surfactant protein-B, which was the same pattern observed in several infants with fatal lung disease due to ABCA3 mutations.
Conclusion: ABCA3 mutations cause some types of interstitial lung disease in pediatric patients.
doi:10.1164/rccm.200503-504OC
PMCID: PMC1403838  PMID: 15976379
desquamative interstitial pneumonitis; pulmonary alveolar proteinosis; surfactant
2.  ABCA3 Mutations Associated with Pediatric Interstitial Lung Disease 
Rationale: ABCA3 is a member of the ATP-binding cassette family of proteins that mediate the translocation of a wide variety of substrates, including lipids, across cellular membranes. Mutations in the gene encoding ABCA3 were recently identified in full-term neonates with fatal surfactant deficiency.
Objective: To test the hypothesis that ABCA3 mutations are not always associated with fatal neonatal lung disease but are a cause of pediatric interstitial lung disease.
Methods: DNA samples were obtained from 195 children with chronic lung disease of unknown etiology. The 30 coding exons of the ABCA3 gene were sequenced in four unrelated children with a referring diagnosis of desquamative interstitial pneumonitis and who were older than 10 years at the time of enrollment.
Results: Three of four patients (ages 16, 23, and 11 years) with desquamative interstitial pneumonitis had ABCA3 mutations identified on both alleles. All three had the same missense mutation (E292V) and a second unique mutation. The E292V mutation was not found on 200 control alleles from adults without lung disease, but seven additional patients of the remaining study patients had the E292V mutation on one allele. Immunohistochemical analysis of surfactant protein expression in three patients revealed a specific staining pattern for surfactant protein-B, which was the same pattern observed in several infants with fatal lung disease due to ABCA3 mutations.
Conclusion: ABCA3 mutations cause some types of interstitial lung disease in pediatric patients.
doi:10.1164/rccm.200503-504OC
PMCID: PMC1403838  PMID: 15976379
desquamative interstitial pneumonitis; pulmonary alveolar proteinosis; surfactant
3.  Airway Epithelial Transcription Factor NK2 Homeobox 1 Inhibits Mucous Cell Metaplasia and Th2 Inflammation 
Rationale: Airway mucous cell metaplasia and chronic inflammation are pathophysiological features that influence morbidity and mortality associated with asthma and other chronic pulmonary disorders. Elucidation of the molecular mechanisms regulating mucous metaplasia and hypersecretion provides the scientific basis for diagnostic and therapeutic opportunities to improve the care of chronic pulmonary diseases.
Objectives: To determine the role of the airway epithelial–specific transcription factor NK2 homeobox 1 (NKX2-1, also known as thyroid transcription factor-1 [TTF-1]) in mucous cell metaplasia and lung inflammation.
Methods: Expression of NKX2-1 in airway epithelial cells from patients with asthma was analyzed. NKX2-1+/− gene targeted or transgenic mice expressing NKX2-1 in conducting airway epithelial cells were sensitized to the aeroallergen ovalbumin. In vitro studies were used to identify mechanisms by which NKX2-1 regulates mucous cell metaplasia and inflammation.
Measurements and Main Results: NKX2-1 was suppressed in airway epithelial cells from patients with asthma. Reduced expression of NKX2-1 in heterozygous NKX2-1+/− gene targeted mice increased mucous metaplasia in the small airways after pulmonary sensitization to ovalbumin. Conversely, mucous cell metaplasia induced by aeroallergen was inhibited by expression of NKX2-1 in the respiratory epithelium in vivo. Genome-wide mRNA analysis of lung tissue from ovalbumin-treated mice demonstrated that NKX2-1 inhibited mRNAs associated with mucous metaplasia and Th2-regulated inflammation, including Spdef, Ccl17, and Il13. In vitro, NKX2-1 inhibited SPDEF, a critical regulator of airway mucous cell metaplasia, and the Th2 chemokine CCL26.
Conclusions: The present data demonstrate a novel function for NKX2-1 in a gene network regulating mucous cell metaplasia and allergic inflammation in the respiratory epithelium.
doi:10.1164/rccm.201101-0106OC
PMCID: PMC3175541  PMID: 21562130
asthma; goblet cell; respiratory epithelium; NK2 homeobox 1
4.  Intersections between Pulmonary Development and Disease 
Recent advances in cellular, molecular, and developmental biology have revolutionized our concepts regarding the process of organogenesis that have important implications for our understanding of both lung formation and pulmonary disease pathogenesis. Pulmonary investigators have long debated whether developmental processes are recapitulated during normal repair of the lung or in the setting of chronic pulmonary diseases. Although the cellular events involved in lung morphogenesis and those causing pulmonary disease are likely to include processes that are distinct, there is increasing evidence that the pathogenesis of many lung disorders involves the same genetic machinery that regulates cell growth, specification, and differentiation during normal lung development.
doi:10.1164/rccm.201103-0495PP
PMCID: PMC3175542  PMID: 21642246
lung; morphogenesis; transcription; respiratory
5.  Conditional Depletion of Airway Progenitor Cells Induces Peribronchiolar Fibrosis 
Rationale: The respiratory epithelium has a remarkable capacity to respond to acute injury. In contrast, repeated epithelial injury is often associated with abnormal repair, inflammation, and fibrosis. There is increasing evidence that nonciliated epithelial cells play important roles in the repair of the bronchiolar epithelium after acute injury. Cellular processes underlying the repair and remodeling of the lung after chronic epithelial injury are poorly understood.
Objectives: To identify cell processes mediating epithelial regeneration and remodeling after acute and chronic Clara cell depletion.
Methods: A transgenic mouse model was generated to conditionally express diphtheria toxin A to ablate Clara cells in the adult lung. Epithelial regeneration and peribronchiolar fibrosis were assessed after acute and chronic Clara cell depletion.
Measurements and Main Results: Acute Clara cell ablation caused squamous metaplasia of ciliated cells and induced proliferation of residual progenitor cells. Ciliated cells in the bronchioles and pro–surfactant protein C–expressing cells in the bronchiolar alveolar duct junctions did not proliferate. Epithelial cell proliferation occurred at multiple sites along the airways and was not selectively associated with regions around neuroepithelial bodies. Chronic Clara cell depletion resulted in ineffective repair and caused peribronchiolar fibrosis.
Conclusions: Colocalization of proliferation and cell type–specific markers demonstrate that Clara cells are critical airway progenitor cells. Continuous depletion of Clara cells resulted in persistent squamous metaplasia, lack of normal reepithelialization, and peribronchiolar fibrosis. Induction of proliferation in subepithelial fibroblasts supports the concept that chronic epithelial depletion caused peribronchiolar fibrosis.
doi:10.1164/rccm.201005-0744OC
PMCID: PMC3056226  PMID: 20870756
chronic obstructive pulmonary disease; bronchiolitis obliterans syndrome; squamous metaplasia; diphtheria toxin; progenitor cells
6.  Hereditary Pulmonary Alveolar Proteinosis 
Rationale: We identified a 6-year-old girl with pulmonary alveolar proteinosis (PAP), impaired granulocyte-macrophage colony–stimulating factor (GM-CSF) receptor function, and increased GM-CSF.
Objectives: Increased serum GM-CSF may be useful to identify individuals with PAP caused by GM-CSF receptor dysfunction.
Methods: We screened 187 patients referred to us for measurement of GM-CSF autoantibodies to diagnose autoimmune PAP. Five were children with PAP and increased serum GM-CSF but without GM-CSF autoantibodies or any disease causing secondary PAP; all were studied with family members, subsequently identified patients, and controls.
Measurement and Main Results: Eight children (seven female, one male) were identified with PAP caused by recessive CSF2RA mutations. Six presented with progressive dyspnea of insidious onset at 4.8 ± 1.6 years and two were asymptomatic at ages 5 and 8 years. Radiologic and histopathologic manifestations were similar to those of autoimmune PAP. Molecular analysis demonstrated that GM-CSF signaling was absent in six and severely reduced in two patients. The GM-CSF receptor β chain was detected in all patients, whereas the α chain was absent in six and abnormal in two, paralleling the GM-CSF signaling defects. Genetic analysis revealed multiple distinct CSF2RA abnormalities, including missense, duplication, frameshift, and nonsense mutations; exon and gene deletion; and cryptic alternative splicing. All symptomatic patients responded well to whole-lung lavage therapy.
Conclusions: CSF2RA mutations cause a genetic form of PAP presenting as insidious, progressive dyspnea in children that can be diagnosed by a combination of characteristic radiologic findings and blood tests and treated successfully by whole-lung lavage.
doi:10.1164/rccm.201002-0271OC
PMCID: PMC3001266  PMID: 20622029
GM-CSF receptor; genetic disease; surfactant; alveolar macrophage; whole lung lavage
7.  Pulmonary Pathology in Thyroid Transcription Factor-1 Deficiency Syndrome 
Thyroid transcription factor-1 (TTF-1) deficiency syndrome is characterized by neurologic, thyroidal, and pulmonary dysfunction. Children usually have mild-to-severe respiratory symptoms and occasionally die of respiratory failure. Herein, we describe an infant with a constitutional 14q12–21.3 haploid deletion encompassing the TTF-1 gene locus who had cerebral dysgenesis, thyroidal dysfunction, and respiratory insufficiency. The clinical course was notable for mild hyaline membrane disease, continuous ventilatory support, and symmetrically distributed pulmonary cysts by imaging. He developed pneumonia and respiratory failure and died at 8 months. Pathologically, the lungs had grossly visible emphysematous changes with “cysts” up to 2 mm in diameter. The airway generations and radial alveolar count were diminished. In addition to acute bacterial pneumonia, there was focally alveolar septal fibrosis, pneumocyte hypertrophy, and clusters of airspace macrophages. Ultrastructurally, type II pneumocytes had numerous lamellar bodies, and alveolar spaces contained fragments of type II pneumocytes and extruded lamellar bodies. Although immunoreactivity for surfactant protein SP-A and ABCA3 was diminished, that for SP-B and proSP-C was robust, although irregularly distributed, corresponding to the distribution of type II pneumocytes. Immunoreactivity for TTF-1 protein was readily detected. In summation, we document abnormal airway and alveolar morphogenesis and altered expression of surfactant-associated proteins, which may explain the respiratory difficulties encountered in TTF-1 haploinsufficiency. These findings are consistent with experimental evidence documenting the important role of TTF-1 in pulmonary morphogenesis and surfactant metabolism.
doi:10.1164/rccm.201002-0167CR
PMCID: PMC2937244  PMID: 20203240
alveolar growth abnormality; cerebral dysgenesis; thyroid transcription factor-1; bronchopulmonary dysplasia; surfactant protein
8.  Patient-derived Granulocyte/Macrophage Colony–Stimulating Factor Autoantibodies Reproduce Pulmonary Alveolar Proteinosis in Nonhuman Primates 
Rationale: Granulocyte/macrophage colony–stimulating factor (GM-CSF) autoantibodies (GMAb) are strongly associated with idiopathic pulmonary alveolar proteinosis (PAP) and are believed to be important in its pathogenesis. However, levels of GMAb do not correlate with disease severity and GMAb are also present at low levels in healthy individuals.
Objectives: Our primary objective was to determine whether human GMAb would reproduce PAP in healthy primates. A secondary objective was to determine the concentration of GMAb resulting in loss of GM-CSF signaling in vivo (i.e., critical threshold).
Methods: Nonhuman primates (Macaca fascicularis) were injected with highly purified, PAP patient-derived GMAb in dose-ranging (2.2–50 mg) single and multiple administration studies, and after blocking antihuman immunoglobulin immune responses, in chronic administration studies maintaining serum levels greater than 40 μg/ml for up to 11 months.
Measurements and Main Results: GMAb blocked GM-CSF signaling causing (1) a milky-appearing bronchoalveolar lavage fluid containing increased surfactant lipids and proteins; (2) enlarged, foamy, surfactant-filled alveolar macrophages with reduced PU.1 and PPARγ mRNA, and reduced tumor necrosis factor-α secretion; (3) pulmonary leukocytosis; (4) increased serum surfactant protein-D; and (5) impaired neutrophil functions. GM-CSF signaling varied inversely with GMAb concentration below a critical threshold of 5 μg/ml, which was similar in lungs and blood and to the value observed in patients with PAP.
Conclusions: GMAb reproduced the molecular, cellular, and histopathologic features of PAP in healthy primates, demonstrating that GMAb directly cause PAP. These results have implications for therapy of PAP and help define the therapeutic window for potential use of GMAb to treat other disorders.
doi:10.1164/rccm.201001-0008OC
PMCID: PMC2902758  PMID: 20224064
alveolar macrophages; surfactant homeostasis; autoimmunity; neutrophils; anti–granulocyte/macrophage colony–stimulating factor therapy
9.  Surfactant Protein-D Inhibits Lung Inflammation Caused by Ventilation in Premature Newborn Lambs 
Rationale: Premature newborns frequently require manual ventilation for resuscitation during which lung injury occurs. Although surfactant protein (SP)-D regulates pulmonary inflammation, SP-D levels are low in the preterm lung. Commercial surfactants for treatment of respiratory distress syndrome do not contain SP-D.
Objectives: To determine whether addition of recombinant human SP-D (rhSP-D) to commercial surfactant influences lung inflammation in ventilated premature newborn lambs.
Methods: Prematurely delivered lambs (130 d gestation age) were resuscitated with 100% O2 and peak inspiratory pressure 40 cm H2O for 20 minutes and then treated with Survanta or Survanta containing rhSP-D. Ventilation was then changed to regulate tidal volume at 8 to 9 ml/kg. At 5 hours of age lambs were killed for sample collection.
Measurements and Main Results: Sequential blood gas and tidal volume were similar in lambs treated with or without rhSP-D, indicating that lung immaturity and ventilatory stress used to support premature lambs were comparable between the two groups. Ventilation caused pulmonary inflammation in lambs treated with surfactant alone. In contrast, surfactant containing rhSP-D decreased neutrophil numbers in bronchoalveolar lavage fluid and decreased neutrophil elastase activity in lung tissue. IL-8 mRNA and IL-8 protein were significantly decreased in the +rhSP-D group lamb lungs, to 20% of those in controls. The addition of rhSP-D also rendered Survanta more resistant to plasma protein inhibition of surfactant function.
Conclusions: Treatment with rhSP-D–containing surfactant inhibited lung inflammation and enhanced the resistance of surfactant to inhibition, supporting its potential usefulness for prevention of lung injury in the preterm newborn.
doi:10.1164/rccm.200912-1818OC
PMCID: PMC2874451  PMID: 20133924
respiratory distress syndrome; surfactant treatment; IL-8; newborn resuscitation; lung injury
10.  Intratracheal Recombinant Surfactant Protein D Prevents Endotoxin Shock in the Newborn Preterm Lamb 
Rationale: The susceptibility of neonates to pulmonary and systemic infection has been associated with the immaturity of both lung structure and the immune system. Surfactant protein (SP) D is a member of the collectin family of innate immune molecules that plays an important role in innate host defense of the lung.
Objectives: We tested whether treatment with recombinant human SP-D influenced the response of the lung and systemic circulation to intratracheally administered Escherichia coli lipopolysaccharides.
Methods: After intratracheal lipopolysaccharide instillation, preterm newborn lambs were treated with surfactant and ventilated for 5 h.
Measurement: Survival rate, physiologic lung function, lung and systemic inflammation, and endotoxin level in plasma were evaluated.
Main Results: In control lambs, intratracheal lipopolysaccharides caused septic shock and death associated with increased endotoxin in plasma. In contrast, all lambs treated with recombinant human SP-D were physiologically stable and survived. Leakage of lipopolysaccharides from the lungs to the systemic circulation was prevented by intratracheal recombinant human SP-D. Recombinant human SP-D prevented systemic inflammation and decreased the expression of IL-1β, IL-8, and IL-6 in the spleen and liver. Likewise, recombinant human SP-D decreased IL-1β and IL-6 in the lung and IL-8 in the plasma. Recombinant human SP-D did not alter pulmonary mechanics following endotoxin exposure. Recombinant human SP-D was readily detected in the lung 5 h after intratracheal instillation.
Conclusions: Intratracheal recombinant human SP-D prevented shock caused by endotoxin released from the lung during ventilation in the premature newborn.
doi:10.1164/rccm.200509-1485OC
PMCID: PMC2662974  PMID: 16556693
cytokines; lung compliance; pulmonary surfactant; respiratory distress syndrome; sepsis

Results 1-10 (10)