Tracheal occlusion did not improve survival or morbidity rates in this cohort of fetuses with congenital diaphragmatic hernia.

ABSTRACTare collectins, members of the C-type lectin family (4). The collectins are composed of four domains: a short aminoterminal region with interchain disulfide bonds, a long collagen-like domain, a coiled-coil neck region and a calciumdependent carbohydrate recognition domain. The basic structural unit of each collectin is a trimer based on the collagenlike triple helix, but the arrangement of multiple trimers into higher order oligomers varies (4). The close linkage of the mouse collectin genes on chromosome 14 suggests the collectins arose by ancestral gene duplication (5).It is not known whether the genetic and structural relationships among the collectins lead to related functions. There are variable degrees of evidence for each of the collectins having a role in innate immunity (for review, see ref. 6). Humans with low levels of MBP secondary to gene mutations are predisposed to infections (7,8), and mice deficient in SP-A secondary to gene targeting have delayed clearing of certain intratracheal bacterial challenges (9). Consistent with a possible role in innate immunity, SP-D binds to both microbes and phagocytic cells in vitro (6, 10), yet there is no direct evidence that SP-D has a role in host defense in vivo.Although SP-D was initially called a SP because it was expressed in the alveolar type II cell and had striking biochemical similarities to SP-A (1), a role for SP-D in surfactant homeostasis has not been established. Some surfactant phospholipid is associated with SP-D purified from alveolar lavage (11), and SP-D will interact with surfactant phospholipids in vitro under certain circumstances (12-14). SP-D also binds to both type II cell apical membranes and alveolar macrophages (6), cells that participate in alveolar surfactant metabolism (15). To develop a model to test the role of SP-D, we have produced mice lacking in SP-D secondary to the disruption of the single copy mouse SP-D gene by homologous recombination. Initial characterization of the phenotype demonstrates a progressive alveolar surfactant accumulation and a striking increase in foamy alveolar macrophages and alteration in type II cell morphology. These findings differ markedly from the results of SP-A gene targeting (16,17) and show that deletion of SP-D alters surfactant homeostasis. MATERIALS AND METHODS Generation of SP-D-Deficient Mice.A murine 129 strain genomic library (Stratagene) was screened by using a 1.2-kb full-length SP-D cDNA to obtain a 15-kb genomic fragment containing all but the extreme 3Ј end of the structural gene. A replacement-type targeting vector containing 1.2-kb and 4.3-kb homology regions was constructed by standard methods (Fig. 1a). Pgk-neo (1.8 kb) for positive selection replaced all of exon 2, including the translation start site for murine SP-D, and short segments of flanking intronic sequence (2.8 kb). Pgk-tk was inserted 5Ј to the regions of homology for negative selection. The targeting vector was linearized by using a unique NotI site and electroporated into CB1-4 embryonic stem cells as des...

Pulmonary surfactant is a lipid-rich material that promotes alveolar stability by lowering the surface tension at the air-fluid interface in the peripheral air spaces. The turnover of surfactant phospholipids in the alveolar space is fast, and several lines of evidence suggest there is rapid formation and replenishment of the phospholipid surface film during normal respiration. Specific proteins may regulate these dynamic surface properties. The predominant surfactant protein is a well-characterized, lipid-associated glycoprotein, SP [28][29][30][31][32][33][34][35][36] Pulmonary surfactant is a lipid-rich material secreted as tightly packed lamellae into the extracellular alveolar fluid layer (1). Within the alveolar space, surfactant lipids are found in a number of different structural forms, including lamellar bodies, tubular myelin, and various vesicular structures (2). Although the lipid compositions of the surfactant structures are similar, the physical properties, particularly the ability ofthe lipoprotein complexes to form a surface film, are quite different (3). Specific proteins appear to influence the structure and surface activity of surfactant-lipid complexes (4-10).The predominant surfactant-associated protein is the glycoprotein SP 28-36 (28-36 kDa) characterized by a collagenlike NH2-terminal domain and variable N-linked glycosylation of the COOH-terminal region (11-13). SP 28-36 is water soluble but readily associates with phospholipids (PLs) (14). In the presence of calcium, SP 28-36 causes PL aggregation and increases the rate of adsorption of surfactant lipids to an air-fluid interface (6,8,14). A second group of very hydrophobic proteins has been identified in lamellar bodies isolated from lung homogenate and in surfactant isolated from the bronchoalveolar wash (4, 9, 10, 15-17). Very little is known about the homogeneity, structure, or function ofthis group of hydrophobic proteins, but it has been reported that at least one of these proteins enhances PL surface film formation (9,10,18 (20). The surfactant in water (-16 mg of PL per ml, 2 mg of protein per ml) was extracted in 1-butanol (1:50, vol/vol) at room temperature (21). The surfactant/butanol mixture was spun twice at 10,000 X gav for 20 min to sediment the butanolinsoluble protein (94% of the total). The butanol supernatant was dried by rotary evaporation and the residue was resuspended in chloroform/methanol/0.1 M HCl, 1:1:0.5, vol/vol). A small amount of insoluble material was removed by centrifugation and the supernatant, containing 30 mg ofPL in 1 ml, was applied to a 1 cm x 45 cm column of Sephadex and eluted at 4 ml/hr with the same solvent at 40C. The eluted fractions, 0.5 ml, were assayed for protein (22) in the presence of 1% NaDodSO4, phosphorus (23) for the calculation of the PL content, and cholesterol (24). An aliquot of each fraction containing protein was analyzed by NaDodSO4/PAGE (25) and silver staining (26). 66The publication costs of this article were defrayed in part by page charge payment. This article...

Pulmonary surfactant is a phospholipid-protein complex which serves to lower the surface tension at the air-liquid interface in the alveoli of the mammalian lung and is essential for normal respiration. Inadequate levels of surfactant at birth, a frequent situation in premature infants, results in respiratory failure. In all species examined, surfactant is composed primarily of dipalmitoylphosphatidylcholine and two major protein species of relative molecular mass (Mr) 32,000 (32K) and 10K (refs 2-5). Reconstitution in vitro of purified 32K pulmonary surfactant apoprotein (PSAP) with synthetic lipids forms a lipoprotein complex that lowers surface tension by spreading to create a thin interfacial film. Here we describe the cloning of the human PSAP gene and complementary DNA, and discuss features of the unusual encoded protein.

Previous studies have demonstrated that lung-specific proteins are associated with surfactant lipids, particularly the highly surface active subfraction known as tubular myelin. We have isolated a surfactant-associated protein complex with molecular weight components of 36 000, 32 000, and 28 000 and reassembled it with protein-free lung surfactant lipids prepared as small unilamellar liposomes. The effects of divalent cations on the structure and surface activity of this protein-lipid mixture were investigated by following (1) the state of lipid dispersion by changes in turbidity and by electron microscopy and (2) the ability of the surfactant lipids to form a surface film from an aqueous subphase at 37 degrees C. The protein complex markedly increased the rate of Ca2+-induced surfactant-lipid aggregation. Electron microscopy demonstrated transformation of the small unilamellar liposomes (median diameter 440 A) into large aggregates. The threshold Ca2+ concentration required for rapid lipid aggregation was reduced from 13 to 0.5 mM by the protein complex. This protein-facilitated lipid aggregation did not occur if Mg2+ was the only divalent cation present. Similarly, 5 mM Ca2+ but not 5 mM Mg2+ improved the ability of the protein-lipid mixture to form a surface film at 37 degrees C. Extensive aggregation of the surfactant lipids without protein by 20 mM Ca2+ or 20 mM Mg2+ did not promote rapid surface film formation. These results add to the growing evidence that specific Ca2+-protein-lipid interactions are important in determining both the structure and function of extracellular lung surfactant fractions.