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Objective: To evaluate lung transplantation for treatment of surfactant protein B (SP-B) deficiency., Study Design: We compared surfactant composition and function from pretransplantation and posttransplantation samples of bronchoalveolar lavage fluid, somatic and lung growth, neurodevelopmental progress, pulmonary function, and pulmonary immunohistology in 3 infants with SP-B deficiency who underwent bilateral lung transplantation at 2 months of age and 3 infants who underwent lung transplantation for other reasons., Results: Two years after transplantation, the 2 surviving infants with SP-B deficiency exhibited comparable somatic growth and cognitive development to the comparison infants. All infants had delays in gross motor development that improved with time. Both groups have exhibited normal gas exchange, lung growth, and pulmonary function. The SP-B-deficient infants have also exhibited normal SP-B expression and pulmonary surfactant function after lung transplantation. In two SP-B-deficient infants antibody to SP-B developed. No pathologic consequences of this antibody were identified., Conclusions: Apart from the development of anti-SP-B antibody, the outcomes for SP-B-deficient infants after lung transplantation are similar to those of infants who undergo lung transplantation for other reasons. Lung transplantation offers a successful interim therapy until gene replacement for this disease is available.

The 5' flanking region of the Clara cell secretory protein (CCSP) gene contains two cis-acting elements which bind hepatocyte nuclear factor (HNF)-3 alpha and HNF-3 beta in vitro. To determine the role of these proteins in mediating CCSP gene expression in the bronchiolar epithelium, chimeric CCSP-reporter gene constructs containing various regions of the CCSP 5' flanking region were co-transfected into H-441 cells with HNF-3 alpha or HNF-3 beta expression plasmids. These studies indicate that each of these transcription factors positively regulates CCSP gene expression and revealed that CCSP region I (-132 to -76) is sufficient to mediate this effect. Gel-mobility-shift assays with oligonucleotides corresponding to CCSP region I, nuclear extract from bronchiolar epithelial cells and HNF-3-specific antibodies indicate that HNF-3 alpha and HNF-3 beta are the only proteins in bronchiolar epithelial cells which directly interact with this region. Consistent with these observations, HNF-3 alpha and HNF-3 beta transcripts were found to be enriched in this cell population and in situ hybridization of adult lung revealed HNF-3 gene expression in non-ciliated bronchiolar epithelial cells expressing the CCSP gene. Finally, experiments with CCSP region I and a heterologous promoter indicate that this region acts in a promoter-specific context, suggesting that additional factors interacting via the minimal CCSP promoter region are essential in determining the effects of HNF-3 on cell-specific CCSP gene expression in the bronchiolar epithelium.

An infant with a family history of congenital alveolar proteinosis associated with surfactant protein B (SP-B) deficiency was identified when SP-B was not detected in amniotic fluid obtained at 37, 38, and 40 weeks of gestation. Surfactant replacement with commercially available preparations that contained SP-B was begun soon after delivery. Progressive respiratory failure developed despite continued surfactant replacement, corticosteroid therapy, and extracorporeal membrane oxygenation. The infant died at 54 days of age while awaiting lung transplantation. Surfactant extracted from amniotic fluid, bronchoalveolar lavage fluid, and lung tissue had no phosphatidylglycerol; surface tension was 24 dynes/cm (normal, < 10 dynes/cm) and did not decrease with in vitro addition of exogenous SP-B. Pulmonary vascular permeability measured with positron emission tomography was twice normal. At autopsy the alveolar proteinosis pattern was less prominent than that seen in affected siblings. Immunoreactivity of SP-B was absent in type II cells, but numerous foreign body granulomas with central immunoreactivity for SP-B and surfactant protein C were present. We conclude that exogenous surfactant replacement did not normalize surfactant composition, activity, or pulmonary vascular permeability. These findings suggest that endogenous SP-B synthesis is necessary for mature surfactant metabolism and function.

Surfactant protein D (SP-D) is a collagenous, surfactant-associated, carbohydrate-binding protein that is synthesized by alveolar type II epithelial cells. To further characterize SP-D, we isolated RNA from adult rat lungs and rat type II cells and translated mRNAs in vitro. [35S]methionine-labeled translation products were precipitated with antibodies to rat SP-D, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by fluorography. Immune precipitates of translation reactions for rat lung or rat type II cells demonstrated a single collagenous polypeptide (39.3 kDa) that was smaller than surfactant-associated SP-D (43 kDa, reduced) but larger than the mature secreted form of rat SP-A. This component was not identified in translation reactions of rat liver, gut, brain, mammary gland, or rat L2 cell RNA. There was a fivefold enrichment of SP-D mRNA in freshly isolated type II cells relative to lung; however, the levels of translatable SP-D mRNA decreased rapidly during the first 24 h of cell culture. The SP-D translation product migrated faster than the major cellular form of SP-D but approximately 1 kDa slower than cellular SP-D synthesized in the presence of 2,2'-dipyridyl plus tunicamycin. Translation in the presence of canine pancreatic microsomes gave a single glycosylated, endoglycosidase F-sensitive form (40.6 kDa) and demonstrated cleavage of a small signal peptide. These results indicate that SP-D is a secretory product of differentiated type II epithelial cells and that SP-D is secreted in a mature form that does not undergo further proteolytic processing in vivo.

Human lymphoblastoid cell lines evolve in vitro by the emergence of successive waves of clones which are often chromosomally marked. This offers the opportunity to compare tissue samples of the same genetic origin but differing in certain defined parts of the karyotype. Using selected sets of lines in which the members of genetically matched pairs differed in the number of copies of 8p or of 12p, levels of GSR and LDH B respectively have been shown to correlate with the specific chromosome aberrations, supporting existing data on the regional assignment of these two structural loci. This approach represents a useful addition to established methods for human gene mapping.

Conventional cleavage of linked polypeptide chains by heating in SDS can so alter molecular structure as to interfere with antibody binding, on which both immunoprecipitation and 'western blotting' depend. As an alternative, gentle treatment with acid at room temperature or at 0 degrees C was effective in separating the alpha and beta chains of human MHC Class II glycoprotein dimers and proved superior in terms of preservation of at least one labile epitope on the beta chain.

CP4 is a collagenous glycoprotein (43 kDa, reduced) synthesized by rat type II pulmonary epithelial cells in primary culture (Persson et al., 1988). In order to better characterize this protein, CP4 was isolated from rat bronchoalveolar lavage and EDTA extracts of lung surfactant by adsorption to barium sulfate and elution with sodium citrate followed by reverse-phase HPLC. Amino acid analysis of purified CP4 demonstrated 4-hydroxyproline (Hyp), hydroxylysine (Hyl), and acid-labile components coeluting with Hyl glycosides. In addition, gas-phase amino-terminal microsequencing of two CP4 CNBr peptides demonstrated nonoverlapping collagenous sequences comprised of nine and six Gly-X-Y triplets, containing a total of four residues of Hyp and two of Hyl. There was less than 50% sequence homology of these peptides with the cDNA-derived sequence of the collagenous domain of rat SP-A. Two-dimensional IEF/SDS-PAGE resolved the protein into a charge train of basic isoforms (pI approximately 6-8), similar to those of newly synthesized CP4 and the class D surfactant proteins (Phelps & Taeusch, 1985). Gel filtration of nondenatured CP4 on 4% agarose showed a high apparent molecular mass complex comprised of disulfide-bonded trimers of the 43-kDa subunits. Antibodies to purified lavage CP4 showed specific binding to newly synthesized and surfactant-associated CP4. We propose that CP4 be designated "surfactant protein D" (SP-D) in accordance with an accepted nomenclature for surfactant-associated proteins.

Ia antigens were shown to be present in the cell almost exclusively as mature alpha beta dimers which split into separate alpha and beta chains after boiling in SDS. In contrast metabolically labelling the cells with [35S]methionine resulted in only free alpha and beta chains being labelled. It is concluded that this widely used type of labelling, although useful for studying intermediate synthesis, should not be used for labelling mature cell surface molecules.

Monoclonal antibodies directed against human Ia alpha- and beta-subunit chains have been used as probes to detect polypeptides carrying recognized antigenic determinants or epitopes following two-dimensional PAGE separation. Approximately 4 sets of components differing in isoelectric point but not in molecular weight are recognized by each antibody. The anti-alpha-chain, McAb, reacts weakly with spots designated as epsilon but neither antibody recognizes Im, Ii or delta determinants under the conditions tested.

Type II pneumocytes secrete pulmonary surfactant and are known to synthesize SP-35, a collagenous surfactant-associated protein. Freshly isolated type II cells also synthesize other bacterial collagenase-sensitive and hydroxyproline-containing proteins, including a glycoprotein designated CP4. CP4 was isolated from rat pneumocyte culture medium by immune precipitation with polyclonal antibodies to rat surfactant proteins or by DEAE chromatography and reverse-phase or gel permeation HPLC. CP4 did not cross-react with polyclonal antibodies to SP-35 and was completely resolved from SP-35 by SDS-PAGE (Mr 43K reduced) or isoelectric focusing. Unlike SP-35, which consists of acidic isoforms assembled as disulfide-bonded dimers and multimers, CP4 was secreted as basic isoforms assembled as disulfide-bonded trimers. Differences in primary structure were demonstrated by CNBr and V8 protease peptide mapping. The secretion of both proteins was inhibited by 2,2'-dipyridyl, an inhibitor of posttranslational prolyl and lysyl hydroxylation and collagen triple helix formation. CP4 was isolated from EDTA extracts of rat surfactant. These studies provide evidence for the heterogeneity of pneumocyte-derived collagenous surfactant-associated proteins.

Four mouse monoclonal antibodies to human B cell surface determinants previously described as being directed against Ia like (MHC class II) antigens, have been shown to precipitate Ia alpha and beta chains. Electrophoretic transfer experiments showed one antibody to be directed against Ia alpha chains and two others to be against Ia beta chains. The antibodies were then used to analyse a range of cell types and a large number of lymphoblastoid and lymphoma cell lines. Ia antigens could not be detected on peripheral blood T cells, cord endothelium or T cell lines but their presence was confirmed on activated T cells and peripheral blood non-T cells. There was both qualitative and quantitative variation of Ia like antigen expression on B cell lines, including an apparent genetic polymorphism in alpha chain structure unrelated to DR allotypes and a single instance of a beta chain of abnormally high molecular weight.