PULMONARY ALVEOLAR PROTEINOSIS: A REVIEW

Pulmonary alveolar proteinosis (PAP) is a disorder that rapidly leads to respiratory failure, because the alveolar spaces fill with a lipid-rich, proteinaceous material that impedes gas exchange. The pathogenesis of this life-threatening process remained an enigma for decades. Recent analysis of the lung pathology and molecular genetics of affected families has provided a molecular basis for some cases of PAP - deficiency of surfactant protein SP-B. This lack results from mutations in the gene for SP-B. The common mutation, 121ins2, is present in about two-thirds of the patients with SP-B deficiency. Additional insights into the mechanism for this lipoproteinaceous accumulation within alveoli were contributed by serendipity in a granulocyte-macrophage - colony stimulating factor (GM-CSF) knock-out mouse model developed to study basal hematopoiesis. In this model, hematopoiesis was unaffected, but the animals developed pulmonary alveolar proteinosis. Subsequently, mutations in the genes for GM-CSF or its receptor were identified as the cause for pulmonary alveolar proteinosis in some patients. In our review, we discuss the known clinical, pathologic, and molecular genetic aspects of pediatric PAP and consider avenues for future research.     

Congenital alveolar proteinosis caused by a novel mutation of the surfactant protein B gene and misalignment of lung vessels in consanguineous kindred infants

Congenital alveolar proteinosis and misalignment of lung vessels are rare disorders. We report on five infants of consanguineous kindred. All infants were delivered at term after uneventful pregnancies. Shortly after birth they developed respiratory failure and severe persistent pulmonary hypertension. All died despite intensive care. Lung tissue of two infants was studied. Histological examination revealed combination of alveolar proteinosis and misalignment of lung vessels in one patient, alveolar proteinosis in the other. Immunostaining demonstrated surfactant protein B (SP-B) deficiency in both patients' lungs. In a further sibling, analysis of broncho-alveolar lavage fluid showed decreased surfactant protein. PCR and direct sequence analysis of the SP-B gene revealed three novel mutations. One of them, a single base deletion, shifts the reading frame at amino acid 122 and creates a premature termination of translation in exon 6. No mature SP-B protein is produced. Conclusion Surfactant protein B deficiency caused by mutations of the respective gene and misalignment of lung vessels can concur. Both diseases may have a pathogenetic factor in common. Received: 24 April 1998 / Accepted: 27 November 1998     

Pulmonary alveolar proteinosis in infants

We investigated the histological and molecular characteristics of pulmonary alveolar proteinosis (PAP) in two siblings (a brother and sister) who did not exhibit respiratory distress at birth but who each developed symptoms during infancy. Histological analysis of lung specimens showed positive staining for surfactant proteins in both patients. The polymerase chain reaction revealed expression of messenger RNA for surfactant protein B (SP-B) in the lung specimens. No defect in SP-B which is characteristic of the congenital form of PAP was observed. The concentration of surfactant protein A (SP-A) in bronchial alveolar lavage (BAL) fluid was elevated in patient 1 suggesting the BAL concentration of SP-A may be a clue to the diagnosis of this form of PAP. Conclusion The accumulation of surfactant protein A in two siblings with an infantile form of pulmonary alveolar proteinosis could be a clue to the diagnosis. Received: 6 July 1997 / Accepted in revised form: 15 September 1998     

Absence of Lamellar Bodies with Accumulation of Dense Bodies Characterizes a Novel Form of Congenital Surfactant Defect

Two female sibling full-term newborns developed respiratory distress shortly after birth, which progressed to respiratory failure. Tracheal lavage demonstrated presence of surfactant protein A (SP-A), but little surfactant protein B (SP-B), without aberrant surfactant protein C (SP-C). On a lung biopsy performed in both infants, prominent type II pneumocyte hyperplasia was evident. Through ultrastructural examination an absence of normally formed lamellar bodies was determined, with numerous irregular electron dense bodies within the type II pneumocytes. These electron dense bodies could also be identified in the alveolar spaces and alveolar macrophages. No alveolar tubular myelin was present. Abnormally high immunoreactivity for surfactant proteins SP-A, proSP-B, SP-B, and proSP-C was demonstrated by light microscopy. Presence of incompletely processed immunopositive proSP-B, but not proSP-C was observed in the alveolar lumina. No mutations in either the SP-B or SP-C gene were identified by sequence analysis of amplified cDNA. We conclude that these siblings exhibit an inherited surfactant deficiency characterized by abnormal accumulations of surfactant proteins within the pneumocytes. This abnormal accumulation may be due to a primary secretory defect, a defect in surfactant phospholipids, or an abnormal interaction between the phospholipids and surfactant proteins. Received November 17, 1998; accepted August 19, 1999.     

Heterozygous <Emphasis Type="Italic">ABCA3</Emphasis> mutation associated with non-fatal evolution of respiratory distress

A boy without symptoms up to 12 months of age started with persisting cough followed by respiratory failure at 18 months of age, resulting in mechanical ventilation because of alveolar proteinosis. Lung biopsy showed PAS-positive material. PCR was negative for CMV, Pneumocystis jiroveci and adenovirus. BALF showed mature SP-B. Analysis of the ATP-binding cassette transporter A3 (ABCA3; OMIM 601615) gene showed a compound heterozygous mutation from paternal W1148X and maternal T1114A. Alveolar lavage with 720 mg of bovine surfactant allowed weaning from ventilator support. Heterozygous mutation in the ABCA3 gene could be associated with a milder evolution as compared to the homozygous frequently lethal evolution. Takuya Yokota and Yoshihiro Matsumura contributed equally to this work.     

Exudative lung injury is associated with decreased levels of surfactant proteins in a rat model of meconium aspiration

OBJECTIVE: Meconium aspiration syndrome remains a common cause of respiratory failure in neonates. The acute effects of meconium aspiration are inactivation of lung surfactant in vivo and in vitro. This study investigated the delayed effects of meconium on alveolar surfactant phospholipids and protein levels in spontaneously breathing animals. METHODS: Twenty-two adult rats were given 4.3 mg of dry weight human meconium after endotracheal intubation. Rats were briefly mechanically ventilated in room air, extubated, then killed after 16 (n = 6), 24 (n = 6), 48 (n = 6), and 72 hours (n = 4). Control animals received the same volume of normal saline (n = 7) or no meconium (n = 7). Bronchoalveolar lavage and tissue specimens were evaluated for inflammatory cells, total proteins, surfactant phospholipids, and surfactant proteins. RESULTS: Meconium caused exudative lung injury that was reflected in increased cell counts and proteins in alveolar lavage fluid. The peak injury occurred at 16 hours after instillation, whereas recovery occurred by 72 hours. Although total lavage fluid phospholipids did not change over time, phospholipid and dipalmitoyl phosphatidylcholine in large aggregates tended to decrease at 24 hours. Western blot analysis demonstrated time-dependent qualitative decreases in surfactant proteins A and B (SP-A, SP-B) in meconium-instilled animals compared with the controls. ELISA for SP-B confirmed the Western blot findings with total SP-B in large aggregate decreasing from 25 +/- 4 microg in controls to 6.6 +/- 0.8 microg at 24 hours of injury. CONCLUSIONS: Our study suggests that the exudative lung injury with meconium instillation is associated with decreased levels of SP-A and SP-B in the large aggregate fraction of lung surfactant. We speculate that decreased secretion and/or increased degradation accounts for lower levels of SP-B in bronchoalveolar lavage fluid.     

Surfactant protein B deficiency: Clinical, histological and molecular evaluation

Congenital alveolar proteinosis due to surfactant protein B deficiency is an inherited disease which results in severe respiratory failure in term infants soon after birth. The pathophysiologic basis of this disease is now known to be an inability to synthesise adequate quantities of normally functioning surfactant protein B. We report a male infant with fatal respiratory failure of neonatal onset, and histopathological features typical of those seen in congenital alveolar proteinosis. Molecular analysis of genomic DNA revealed two mutations, the 'common' 121ins2 mutation in exon 4, and a novel 2bp frameshift mutation in exon 5. We believe this is the first Australian case of surfactant protein B deficiency confirmed by molecular analysis.     

Constitutional deficiency of pulmonary surfactant protein B: clinical presentation, histologic and molecular diagnosis]

We report a female full-term infant with fatal respiratory failure of early onset due to inherited SP-B deficiency. Lung biopsy was performed at 18 days after birth, with histopathological characterization indicating congenital alveolar proteinosis. Immunohistochemical studies of lung tissue revealed the absence of SP-B and the presence of intra-alveolar SP-A normal quantities. Analysis of genomic DNA showed homozygosity for the 121ins2 mutation of the SFTPB gene. The infant died 21 days after birth. Both parents were heterozygotes for the mutation. Chorionic villus sampling was performed at the first trimester of the following pregnancy. Restriction analysis of amplified fetal DNA, studies of microsatellite segregation and direct sequencing led to the diagnosis of homozygosity for the parental wild-type allele. The diagnosis of congenital SP-B deficiency should be suspected whenever an early and acute respiratory failure in a term or near-term infant does not resolve after five days of age: diagnostic confirmation can be easily and rapidly obtained with the analysis of genomic DNA and immunohistochemical characterization of lung tissue.     

盐酸氨溴索和地塞米松对胎鼠肺表面活性蛋白基因表达的影响

目的　探讨盐酸氨溴索和地塞米松对发育期胎鼠肺表面活性蛋白 (SPs)基因表达的影响。方法　将胎鼠 4 2只随机分为生理盐水对照组和盐酸氨溴索、地塞米松两个干预组 ,剖宫取孕19d的胎鼠肺组织作为早产鼠肺模型 ,用原位杂交研究胎鼠肺泡Ⅱ型上皮细胞内SP B基因的表达 ,逆转录PCR(RT PCR)观察分析各组肺表面活性蛋白SP A、SP B和SP CmRNA表达变化 ,用 β actinmRNA扩增产物为内参照密度扫描半定量分析。结果　 (1)发育 19d的胎鼠肺泡Ⅱ型上皮细胞内SP BmRNA表达阳性 ;(2 )鼠胚胎发育后期 ,支气管周围也分布着肺泡Ⅱ型上皮细胞 ;(3)生理盐水对照组SP A、SP B、SP CmRNA表达与 β actinmRNA比值分别为 0 81± 0 2 6、0 97± 0 2 0、0 88± 0 11。盐酸氨溴索干预SP A、SP B、SP CmRNA表达比值分别为 1 0 4± 0 16、1 2 8± 0 2 9、1 0 9± 0 2 5 ,与对照组比较显著性增加 (P <0 0 5 )。地塞米松干预后的胎鼠肺SP A、SP B、SP CmRNA表达比值分别为 1 0 8±0 2 5、1 2 3± 0 35、1 2 1± 0 2 5 ,与对照组比较显著性增加 (P <0 0 5 )。 (4 )对比盐酸氨溴索与地塞米松在促进SP A、SP B、SP C基因表达上 ,两者差异没有显著意义 (P >0 0 5 )。结论　盐酸氨溴索和地塞米松产前用药对发育期的胎鼠SPs基     

Experimental neonatal respiratory failure induced by a monoclonal antibody to the hydrophobic surfactant-associated protein SP-B

The present experiments were designed to test whether selective blocking of the surfactant-associated hydrophobic polypeptide SP-B (8.7 kD) would interfere with lung function during neonatal adaptation. A MAb to porcine SP-B was produced by hybridoma cell line (8B5E); this antibody cross-reacts with rabbit SP-B. Six mg of MAb to SP-B, dissolved in 0.2 mL of saline, was instilled into the airways of near-term newborn rabbits (gestational age 29 d), before the onset of ventilation. Control animals received the same amount of nonspecific rabbit IgG in saline, or were untreated. The animals were ventilated for 120 min with a standardized tidal volume (10 mL/kg). The specific antibody caused a prominent, immediate decrease in lung-thorax compliance, associated with acute inflammatory and exudative lung lesions including hyaline membranes. IgG alone had no such effects. Our data suggest that the MAb to SP-B inhibits surfactant function in the neonatal period by blocking one of the mechanisms responsible for fast adsorption of the surfactant phospholipids to the alveolar air-liquid interface. In addition, an acute inflammatory reaction is probably triggered in the lung parenchyma by the immune reaction.     

Leptin enhances lung maturity in the fetal rat

Pulmonary alveolar type II cells synthesize and secrete phospholipids and surfactant proteins. In most mammalian species, the synthesis of phospholipids and proteins of lung surfactant increases with fetal lung maturation, which occurs late in gestation. Factors that may promote lung maturation and surfactant production include the placental hormone, leptin, whose expression increases with advancing gestational age. We demonstrate that physiologic concentrations of leptin (1 and 10 ng/mL) increase the levels of surfactant proteins (SP) A, B, and C mRNA as well as SP-A and SP-B protein in d-17 fetal rat lung explants in vitro. To determine whether leptin exerts similar effects in vivo, we administered leptin antenatally to pregnant rats and compared its effects to that of dexamethasone, a known mediator of fetal lung development. Antenatal treatment with leptin for 2 d significantly increased the average weight of the fetal lungs in relation to their body weight. Histologic analysis revealed that the increase in fetal lung weight was accompanied by an increase in the number and maturation of type II alveolar cells and the expression of surfactant proteins B and C in these cells. Collectively, these results suggest that leptin is a cytokine regulator of rat fetal lung maturity.     

LIPID-PROTEIN INTERACTIONS OF HYDROPHOBIC PROTEINS SP-B AND SP-C IN LUNG SURFACTANT ASSEMBLY AND DYNAMICS

Phospholipids have the major role in pulmonary surfactant concerning its biophysical function of reducing surface tension at the alveolar air-liquid interface to facilitate respiratory mechanics. However, the presence of some specific, highly hydrophobic polypeptides is essential to modulate the physical behavior of phospholipids and to promote rapid formation of stable surface films that are able to produce surface tensions in the range of 0 dynes/cm during cyclic compression. The present review summarizes the available data on the parameters governing lipid-protein interactions of the hydrophobic surfactant proteins SP-B and SP-C with the main surfactant phospholipids. Lipid-protein interactions in surfactant have been studied in vitro using preparations reconstituted with very different methodological procedures. Conclusions concerning the role of hydrophobic surfactant proteins on the assembly of lipid-protein surfactant structures in vivo have been revised in this respect. This review presents the knowledge available on the disposition of SP-B and SP-C in surfactant structures, the mode, extent, selectivity, and stoichiometry of their lipid-protein interactions, and the effect of the proteins on structure and dynamics of surfactant bilayers and monolayers. Some considerations are given to possible concerted actions, under physiological conditions, of both proteins SP-B and SP-C.     

Surfactant protein deficiency in familial interstitial lung disease.

OBJECTIVE: To determine the contribution of surfactant protein abnormalities to the development of chronic lung injury in a familial form of interstitial lung disease. STUDY DESIGN: An 11-year-old girl, her sister, and their mother who were diagnosed with chronic interstitial lung disease underwent laboratory investigation of surfactant protein expression in bronchoalveolar lavage fluid and lung biopsy specimens. Nineteen patients with idiopathic pulmonary fibrosis and 9 patients who were investigated for pulmonary malignancy but who did not have interstitial lung disease served as control subjects. RESULTS: The 3 family members were found to have absent surfactant protein C (SP-C) and decreased levels of SP-A and SP-B in bronchoalveolar lavage fluid (BALF). Immunostaining for pulmonary surfactant proteins in lung biopsy specimens obtained from both children demonstrated a marked decrease of pro-SP-C in the alveolar epithelial cells but strong staining for pro-SP-B, SP-B, SP-A, and SP-D. No deviations from published surfactant protein B or C coding sequences were identified by DNA sequence analysis. All control subjects had a detectable level of SP-C in the BALF. CONCLUSION: The apparent absence of SP-C and a decrease in the levels of SP-A and SP-B are associated with familial interstitial lung disease.     

Exogenous porcine surfactant (Curosurf) is inactivated by monoclonal antibody to the surfactant-associated hydrophobic protein SP-B

A monoclonal antibody to the surfactant-associated hydrophobic protein SP-B was added at various concentrations to a standard preparation of porcine surfactant (Curosurf 10 mg/ml), and surface properties were evaluated with pulsating bubble. Retarded adsorption of surfactant was observed at antibody concentrations > or = 0.5 mg/ml and significantly increased values for minimum surface tension were observed at antibody concentrations > or = 1 mg/ml. In vivo effects of the antibody were tested in immature newborn rabbits ventilated with a standardized sequence of insufflation pressures. Animals receiving 0.1 ml surfactant (80 mg/ml) mixed with antibody at concentrations > or = 4 mg/ml had low tidal volumes, poor lung stability in pressure-volume recordings, poor alveolar expansion in histological sections and widespread epithelial necrosis in peripheral airways. Admixtures of IgG had no such effects. We conclude that this monoclonal antibody inactivates exogenous porcine surfactant, probably by preventing fast adsorption of surfactant lipids to the alveolar air-liquid interfaces.     

Inherited disorders of neonatal lung diseases

Although genetic factors are assumed to have a role in the etiology of respiratory distress syndrome (RDS), specific genes underlying this susceptibility are incompletely known. The most promising candidates are the genes coding for the lung-specific protein components of the surfactant. In congenital absence of surfactant protein A in mice, lung mechanics or surfactant homeostasis is normal. However, there is an increased susceptibility to infections. The major surfactant protein A alleles, 6A(2) and 1A(0), are the general high-risk RDS alleles, while the allele 6A(3) carries a decreased risk of RDS. The allele 6A(6) is also over-represented in infants with bronchopulmonary dysplasia. To date, no human infants who lack surfactant protein A have been identified, and the human respiratory phenotype associated with the 1A(0) allele has been demonstrated to be variable, therefore, surfactant protein A polymorphisms are not currently useful for estimation of individual risk of having an affected infant. Surfactant protein B (SP-B) plays an essential role in the structure of tubular myelin. Mutations resulting in an absence of surfactant protein B have been identified. They cause a recessively inherited, progressive respiratory disease. More than 27 loss of function mutations have been identified in the surfactant protein B gene that result in lethal neonatal respiratory failure. Of the several known common variants of the surfactant protein B gene, the most common mutation is 121ins22 that accounts for 60-70% of the mutant cases. Although the frequency of the 121ins2 mutation is rare, the consistent phenotype is exhibited by infants with a homozygous genotype. The clinical presentation in infants homozygous for the 121ins2 mutation is full-term infants who develop respiratory distress within the first 12-24 hours of life. Surfactant replacement therapy fails to reverse this outcome, and without lung transplantation, they expire within the first 1-6 months of life. Surfactant protein B gene mutations may also result in milder phenotypes. These mutations resulting in reduced synthesis of SP-B appear to be family-specific and result in respiratory distress, but sometimes with more gradually progressive or chronic respiratory failure. Surfactant protein C plays a role in the stabilization of surfactant and may also have a role in the intracellular processing of the surfactant complex. Surfactant protein B is important in the intracellular processing and production of surfactant protein C. Although surfactant protein C-deficient mice are viable and survive to adulthood without obvious pulmonary abnormalities, their lung have reduced viscoelasticty. Human respiratory disease in the neonatal period caused by loss-of-function mutations in the surfactant protein C gene has not been identified. However, an autosomal dominant inherited mutation at the surfactant protein C gene causes chronic interstitial lung disease. Surfactant protein D is a member of the collectin family like surfactant protein A, therefore it opsonizes pathogens and enhances their phagocytosis by alveolar macrophages and neutrophils. Unlike surfactant protein A, it does not contribute to lowering surface tension. Surfactant protein D-deficient mice have no respiratory abnormalities at birth, but it causes development of emphysema and predisposition to specific infections. No human infant or child with respiratory distress and mutation in the surfactant protein D gene has been identified.     

A major deletion in the surfactant protein-B gene causing lethal respiratory distress

BACKGROUND: Loss of function mutations in the surfactant protein-B gene (SFTPB) cause lethal neonatal respiratory distress due to reduced or absent expression of mature surfactant protein B (SP-B, encoded in exons 6 and 7). No large deletions in SFTPB have been previously identified. AIM: Genomic, proteomic and immunohistochemical characterization of a 3 kb deletion in SFTPB. METHODS: A full-term newborn presented with refractory respiratory failure. We amplified and sequenced SFTPB from the infant and both parents, determined SP-B protein expression in tracheal aspirate samples using Western-blot analysis, and performed immunohistochemical staining and electron microscopy of lung biopsy tissue. RESULTS: The infant was homozygous for a 2958 bp deletion in SFTPB that included exons 7 and 8. Both asymptomatic parents were heterozygous for the deletion. A truncated mature SP-B peptide was detected on Western blotting of tracheal aspirate. Amino acid sequence specific to that encoded in exon 5 was present, but that encoded by exon 7 was absent. ProSP-B expression was robust within alveolar type II cells and lamellar body structure was disrupted. CONCLUSIONS: This deletion in SFTPB resulted in SP-B deficiency due to absence of elements in mature SP-B that are critical for appropriate peptide folding, trafficking and processing.     

Surfactant composition and function in patients with ABCA3 mutations

Mutations in the gene encoding the ATP binding cassette transporter member A3 (ABCA3) are associated with fatal surfactant deficiency. ABCA3 lines the limiting membrane of lamellar bodies within alveolar type-II cells, suggesting a role in surfactant metabolism. The objective of this study was to determine the surfactant phospholipid composition and function in patients with mutations in the ABCA3 gene. Bronchoalveolar lavage (BAL) fluid was analyzed from three groups of infants: 1) Infants with ABCA3 mutations, 2) infants with inherited surfactant protein-B deficiency (SP-B), and 3) patients without parenchymal lung disease (CON). Surfactant phospholipid profile was determined using two-dimensional thin-layer chromatography, and surface tension was measured with a pulsating bubble surfactometer. Phosphatidylcholine comprised 41 +/- 19% of the total phospholipid in the BAL fluid of the ABCA3 group compared with 78 +/- 3% and 68 +/- 18%, p = 0.008 and 0.05, of the CON and SP-B groups, respectively. Surface tension was 31.5 +/- 9.3 mN/m and was significantly greater than CON but no different from SP-B. We conclude that mutations in ABCA3 are associated with surfactant that is deficient in phosphatidylcholine and has decreased function, suggesting that ABCA3 plays an important role in pulmonary surfactant phospholipid homeostasis.     

内蒙古西部地区汉族新生儿呼吸窘迫综合征与SP-B外显子7区域R236C位点的相关性研究

目的通过检测和分析肺表面活性物质蛋白B基因外显子7(exon7)区域上是否存在基因变异,探讨其与内蒙古西部地区汉族新生儿呼吸窘迫综合征(NRDS)发病的关系。方法采用病例对照研究方法,选择祖上三代都居住在内蒙古西部地区的汉族NRDS患儿47例作为病例组,选择同民族和同群体中未发生NRDS的新生儿47例为对照组。通过PCR基因分析技术检测SP-B基因外显子7区域上有无突变,以及SP-B外显子7(R236C)位点的基因型、等位基因分布。结果在内蒙古西部地区汉族新生儿中,SP-B基因外显子7区域无突变发生;SP-B外显子7(R236C)位点基因型均可检出两种基因型(CC、CT),两组中均未检出TT基因型。病例组CC和CT基因型频率分别为72%和28%,C等位基因频率为85%,T等位基因频率为15%。对照组此两种基因型分别为85%和15%,C等位基因频率为93%,T等位基因频率为7%。病例组与对照组此位点基因多态性相比等位基因及基因型频率在两组之间差异无统计学意义(P0.05)。结论内蒙古西部地区汉族NRDS患儿的SP-B基因第7外显子未发现基因突变。未发现SP-B基因外显子7(R236C)位点基因多态性与该地区汉族NRDS的发生有明显相关性。