Association of peri-operative prescription of non-steroidal anti-inflammatory drugs with continued prescription of opioids after total knee arthroplasty: a retrospective claims-based cohort study.

Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the mainstays of multimodal pain management. While effective for acute pain control, recent pre-clinical evidence has raised concerns regarding an association between NSAIDs and chronic pain and potential opioid use. Our objective was to explore the association between peri-operative use of prescription NSAIDs and the need for continued opioid prescriptions lasting 90-180 days in previously opioid-naïve patients undergoing total knee arthroplasty. A database of health claims in the USA was used to identify all opioid-naïve adult patients who underwent primary knee arthroplasty between January 2010 and October 2021. We evaluated the magnitude of association between peri-operative prescription NSAID claims and claims for opioids at 90 days postoperatively using multivariable logistic regression models. Secondary outcomes included: the magnitude of association between peri-operative NSAID prescription and claims for opioids at 180 days postoperatively; and identifying other potential factors associated with opioid claims at 90 days postoperatively. After risk adjustment using multivariable logistic regression models in the 789,736-patient cohort, the adjusted odds ratio (95%CI) for a continuous claim of opioids at 90 and 180 days postoperatively among patients with a peri-operative NSAID prescription within 30 days was 1.32 (1.30-1.35), p < 0.001; and 1.12 (1.10-1.15), p < 0.001, respectively. This estimate of effect remained robust at 90 days after accounting for known potential confounders, including pre-existing knee pain and acute postoperative pain severity. Similar analysis of other pain medications (e.g. paracetamol) did not detect such an association. This population-based cohort study suggests that peri-operative prescription NSAID use may be associated with continued opioid prescription claims at 90 and 180 days after knee arthroplasty, even after adjusting for other observed covariates for continuous opioid claims. These novel findings can inform clinical decision-making for post-surgical pain management, risk-benefit discussions with patients and future research.

Bacterial killing is enhanced by exogenous administration of lysozyme in the lungs.

OBJECTIVE: Lysozyme, a 14-kDa protein, is one of the most abundant antimicrobials in the lungs. Its concentration in airway surface sufficient to kill several bacterial pathogens in vitro. The purpose of this study was to determine if administration of exogenous lysozyme would further enhance bacterial killing in vivo. METHODS: To assess the effect of acute lung infection on endogenous lysozyme protein levels, mice were infected by intratracheal instillation of Pseudomonas aeruginosa and bronchoalveolar (BAL) fluid assessed for lysozyme concentration and for muramidase activity. In order to inform in vivo testing, species-specific bacterial killing efficacy was determined by incubating mucoid P. aeruginosa with 2×105 units of chicken lysozyme, human lysozyme or with vehicle at 37°C for 2hours. Subsequently, mice challenged with intratracheally-administered mucoid P. aeruginosa, were reintubated and injected with 2×105 Units of native human lysozyme, recombinant human lysozyme or with vehicle. Lung bacterial burden was enumerated subsequently. RESULTS: The concentration of lysozyme protein in BAL fluid from mice challenged with mucoid clinical isolate of P. aeruginosa was increased 4-fold at 6hours post-infection. Quantitative culture showed that the number of recoverable bacteria was significantly decreased by both chicken and human lysozyme compared to vehicle but human lysozyme was significantly more effective than chicken egg lysozyme. In vivo, 24hours post-infection quantitative culture of lung homogenates showed that the number of viable bacteria recovered from mice treated with either native or recombinant lysozyme was decreased with 0.76±0.25×104 and 0.84±0.16×104, respectively, vs. 7.0±2.52×104 CFU/g protein in mice treated with HBSS, both P<0.05. CONCLUSIONS: These results indicate that endogenous lysozyme is increased during acute lung infection and that early administration of exogenous lysozyme further enhances bacterial killing in vivo.

Synthesis, processing and secretion of surfactant proteins B and C.

Two small, hydrophobic peptides, surfactant protein (SP)-B and SP-C, play important roles in the generation and maintenance of a surface active film in the alveolus. Isolation and characterization of the cDNAs encoding SP-B and SP-C indicate that both peptides are synthesized as larger proproteins which are proteolytically processed to peptides with Mr approx. 8000 and 4000, respectively. The biosynthetic pathway leading to generation and secretion of the biophysically active mature SP-B and SP-C peptides is reviewed.

In vitro sulfation of pulmonary surfactant-associated protein-35.

Surfactant-associated protein-35 consists of a group of phospholipid-associated proteins of 26-36 kDa isolated from pulmonary alveolar surfactant. In the rat, surfactant-associated protein-35 is synthesized from 26-kDa primary translation products which are cotranslationally acetylated and glycosylated to heterogeneous 30 and 34 kDa forms. High-mannose oligosaccharide-containing precursors of surfactant-associated protein-35 are processed in the rough endoplasmic reticulum and Golgi to complex-type oligosaccharides, resulting in a mature glycoprotein which exhibits extensive charge heterogeneity in two-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis. Much of this charge heterogeneity is related to terminal sialylation of the two asparagine-linked oligosaccharides. In the present study, we report that surfactant-associated protein-35 is also sulfated. Sulfation of the 30 and 34 kDa forms of surfactant-associated protein-35 was clearly detected in primary cultures of rat Type II epithelial cells. These sulfated isoforms were sensitive to endoglycosidase F digestion, but resistant to neuraminidase, suggesting that sulfation occurred at oligosaccharide residues other than sialic acid. The lack of sulfation of the 26 kDa forms of surfactant-associated protein-35 and the resistance of the sulfated isoforms to endoglycosidase H digestion are consistent with Golgi-associated sulfation of the complex type oligosaccharides of surfactant-associated protein-35. Thus, sulfation is another component of the complex post-translational processing of surfactant-associated protein-35, which includes acetylation, hydroxylation, glycosylation, sialylation, sulfhydryl-dependent oligomerization and sulfation.

Surfactant protein C precursor is palmitoylated and associates with subcellular membranes.

Surfactant protein C (SP-C) is a 3.7 kDa, hydrophobic protein that enhances the adsorption of phospholipids in pulmonary surfactant. SP-C is generated by proteolytic processing of a 21 kDa precursor. Murine fetal lung explant cultures and a Chinese hamster ovary cell line expressing recombinant human SP-C gene (CHO/SPC) were used to determine the subcellular location and post-translational modification(s) of proSP-C. After in vitro translation, proSP-C of Mr = 21,000 was generated. ProSP-C was associated with canine pancreatic microsomes during co-translation and was partially protected from digestion with proteinase K, supporting the concept that proSP-C enters but does not completely traverse the membrane of the endoplasmic reticulum (ER). Association of proSP-C isoforms of 21 and 26 kDa with intracellular membranes was demonstrated by subcellular fractionation of CHO/SPC cells. Pulse/chase experiments demonstrated that the 21 kDa SP-C proprotein was synthesized first and after 15 min was modified to produce a 26 kDa isoform in CHO/SPC cells or a 24 kDa isoform in murine fetal lung. Both the 21 and 26 kDa proSP-C isoforms were detected after labelling CHO/SPC cells with [3H]palmitic acid. The formation of the 26 kDa proSP-C isoform in CHO/SPC cells and the 24 kDa proSP-C isoform in murine fetal lung was blocked by cerulenin, an inhibitor of fatty acid synthesis. In conclusion, proSP-C is associated with subcellular membranes. ProSP-C is palmitoylated and undergoes additional post-translational modification that is blocked by an inhibitor of fatty acid synthesis.

Structural requirements for intracellular transport of pulmonary surfactant protein B (SP-B).

Human SP-B is synthesized by the alveolar Type II epithelial cell as a 381 amino acid preproprotein. The 79 residue mature SP-B peptide is extremely hydrophobic and flanked by propeptides of 200 and 102 amino acids at its NH2- and COOH-termini, respectively. The purpose of this study was to identify peptide domains of the SP-B proprotein necessary for trafficking of the mature peptide in the secretory pathway. To this end several constructs were generated, by subcloning the full length human SP-B (SP-B), COOH-terminally truncated SP-B (SP-B delta C, in which residues 201-381 were deleted), NH2-terminally deleted SP-B (SP-B delta N, in which residues 28-200 were deleted), NH2-terminal propeptide (SP-BN), mature SP-B (SP-BM) and COOH-terminal propeptide (SP-BC), into the mammalian expression vector pcDNA3. The resulting expression constructs were characterized by DNA sequencing and in vitro transcription/translation and subsequently transfected into Chinese hamster ovary cells. 48 h after transfection, cells were labeled with [35S]-met/cys and analyzed by immunoprecipitation, SDS-PAGE and autoradiography. Proteins encoded by SP-B, SP-B delta C, SP-BN and SP-BC constructs were secreted into media; in contrast, SP-B constructs lacking the NH2-terminal propeptide (SP-B delta N) remained in the endoplasmic reticulum (as assessed by endoglycosidase H sensitivity) and were rapidly degraded. We conclude that (1) 27 amino acids at the NH2-terminus of SP-B contain a functional signal peptide and (2) the NH2-terminal propeptide of the SP-B precursor is necessary and sufficient for intracellular trafficking of the mature peptide.

Intracellular and oligomeric forms of surfactant-associated apolipoproteins(s) A in the rat.

Sulfhydryl-dependent oligomeric forms of the surfactant-associated apolipoprotein(s) A, obtained from particulate preparations of adult rat lung lavage, were characterized by immunoblot analysis and by silver staining of proteins separated by one- and two-dimensional SDS-polyacrylamide gel electrophoresis. Under non-reducing conditions, these proteins migrated as oligomers, Mr approx. 50-70, 115, 160 kDa and greater. The large oligomers were reduced to the apolipoprotein(s) A subunits by treatment with beta-mercaptoethanol; Mr 38 (A3), 32 (A2) and 26 kDa (A1), pI 4.2-4.8. Mr 50 kDa protein was composed of sulfhydryl-dependent homo-dimers of protein(s) A1 (Mr 26 kDa). 55 kDa protein was a hetero-dimer composed primarily of A1 and A2 (Mr 26 and 32 kDa). 62 kDa protein was composed of hetero-dimers of A3 and apolipoprotein A2 (Mr 38 and 32 kDa). 70 kDa protein was a homodimer composed of apolipoprotein A3 A3 (38 kDa). Larger molecular forms were composed primarily of 38 and 32 kDa and lesser amounts of 26 kDa. Treatment with endoglycosidase F reduced A2 and A3 to 26 kDa. Apolipoprotein A1 co-migrated with a protein of Mr 26 kDa immunoprecipitated from [35S]methionine-labelled Type II epithelial cells. Chymotryptic-tryptic peptide maps of apolipoproteins A1, A2 and A3 were identical, suggesting that apolipoproteins A3 and A2 arise through extensive glycosylation of apolipoprotein A1.

In vitro acetylation of rat pulmonary surfactant-associated glycoprotein(s) A primary translation products.

The primary translation products of pulmonary surfactant-associated glycoprotein(s) A, the major apolipoprotein in mammalian surfactants, exhibit extensive charge heterogeneity. After in vitro translation of poly(A)+ mRNA from rat lung, the primary translation products of glycoprotein(s) A were identified as a charge train of five proteins of 26 kDa (pI 4.6-5.0), the predominant forms being the more acidic members (pI less than 4.8). Inhibition of acetylation during in vitro translation of rat lung poly(A)+ mRNA resulted in a predominance of the more basic isoforms (pI greater than or equal to 4.8). Intracellular forms of glycoprotein(s) A were immunoprecipitated from rat Type II epithelial cells after treatment with tunicamycin or after deglycosylation with endoglycosidase H. Five intracellular precursors consisting primarily of acidic members of the charge train were identified, this being consistent with the intracellular acetylation of the protein. In contrast, canine glycoprotein(s) A translation products consisted of only three proteins of 26 kDa (pI 4.8-5.0), in which most of the radiolabel was concentrated in the more basic components. Acetylation may account for some, but not all, of the charge heterogeneity in the primary translation products and processed forms of surfactant-associated glycoprotein(s) A in the rat.

Characterization of a rat cDNA clone encoding calcium/calmodulin-dependent protein kinase I.

Two cDNA clones encoding calcium/calmodulin-dependent (CaM) protein kinase I were isolated. In contrast to the previously reported CaM kinase I cDNA, which encodes a protein with a mass of 37 kDa, the clones identified in this study encode a protein (10-1/CaM kinase I) with a predicted mass of 42 kDa; the size of 10-1/CaM kinase I was verified by hybrid-selected translation.

In vitro translation, post-translational processing and secretion of pulmonary surfactant protein B precursors.

Surfactant proteolipid SP-B is a hydrophobic protein of Mr = 8000 identified in organic solvent extracts of pulmonary surfactant. Analysis of the human SP-B RNA predicts that the active surfactant peptide is derived by proteolysis of an Mr = 40,000 precursor. In the present work, characteristics of synthesis, secretion and processing of SP-B were demonstrated in a pulmonary adenocarcinoma cell line by immunoprecipitation of radiolabelled precursors. Treatment of cells with tunicamycin resulted in synthesis and secretion of unglycosylated proSP-B of Mr = 39,000. Immunoprecipitation of protein produced by in vitro translation of human lung poly(A)+ RNA detected an Mr = 40,000 protein; the size discrepancy is likely related to cleavage of a leader signal sequence. Endoglycosidase-H-sensitive precursors of Mr = 41,000-43,000, pI = 5.1-5.4 were the first isoforms detected within the cells and were processed to endoglycosidase-H-resistant isoforms and secreted. Neuraminidase and endoglycosidase-F-sensitive forms of proSP-B were first detected in the media at 60 min as Mr = 42-46,000 isoforms with pI = 4.6-5.1. Proteolytically processed isoforms of proSP-B were detected primarily in the media and were generated by cleavage of an amino-terminal Mr = 16,000 peptide resulting in Mr = 27,000-33,000 isoforms (pH = 5.6-6.8). The Mr = 27,000-33,000 isoforms were sensitive to neuraminidase, resulting in isoforms with pH = 6.0-6.8. Digestion of the Mr = 27,000-33,000 peptide with endoglycosidase-F resulted in isoforms of Mr = 23,000, pH = 6.0-6.8. The endoglycosidase-F-resistant peptide of Mr = 16,000, pI = 4.2-4.4 was identified with an antiserum generated against synthetic peptides derived from the amino-terminal domain, as deduced from the SP-B DNA sequence. Further proteolytic processing of the Mr = 27,000-33,000 isoforms to the Mr = 8000 peptide detected in surfactant was not observed in this cell line. Thus, in the H441-4 cells (a cell line with morphologic features of Clara cells), SP-B is synthesized as a preproprotein which undergoes cleavage of a signal sequence and addition of asparagine-linked carbohydrate; proSP-B is secreted by processes which are independent of glycosylation. SP-B peptides of Mr = 27,000-33,000 and Mr = 16,000, representing carboxy and amino-terminal domains, accumulate in the media.

The carboxy-terminal domain of human surfactant protein B is not required for secretion in milk of transgenic mice.

Previous studies in which human pulmonary surfactant protein B (SP-B) was targeted to the mammary gland of transgenic mice using the rat whey acidic protein (WAP) regulatory sequences resulted in secretion of only the unprocessed proprotein (42 kDa) in milk. To test the feasibility of producing a partially processed SP-B protein in milk, a new construct was designed in which the coding region for the carboxy-terminal domain was deleted. Expression of rWAP/SP-BDELTA C mRNA was detected in all three transgenic lines generated, and the expected carboxy-terminal deleted SP-B molecule (28 kDa), identified by using domain-specific antibodies, was secreted in the milk. Histochemical examination of lactating mammary tissue from the transgenic line expressing the highest levels of WAP/SP-BDelta C mRNA revealed an inhibition of lobulo-alveolar development, and led to growth retardation in pups, apparently due to the decreased milk production. Mothers from this line tended to cannibalize litters in mid-lactation. This phenotype has been observed previously with several other WAP-based transgenes. This phenotype suggests that there may be an upper limit to the level of SP-BDeltAC which can be produced in milk

Neurobehavioral consequences of arousals.

The neurobehavioral deficits of obstructive sleep apnea syndrome (OSAS) are often attributed to the rate of respiratory disturbance or rate of arousals during sleep. However, sleep disordered breathing is also associated with other changes in sleep infrastructure that may account for cumulative waking deficits. This was illustrated in polysomnographic data from 1,521 patients with OSAS where increasing arousal indices were associated with increased duration of stage 1 sleep and concomitant reduction in total sleep time. Similar results have been found in paradigms in which sleep was experimentally fragmented in healthy individuals. It appears that chronic fragmentation of sleep, whether by apneas or acoustic stimuli, leads to cumulative homeostatic pressure for sleep, which may explain a number of phenomenon characteristic of both untreated OSAS patients and experimentally fragmented sleepers: (1) increased arousal threshold, (2) rapid return to sleep after arousal, (3) fewer awakenings over time, (4) increased sleep inertia on awakenings, (5) increased amnesia for arousals, and (6) daytime sleepiness. Elevated homeostatic drive for sleep appears to be a function of both the frequency of arousals within a night and the chronicity of sleep fragmentation across nights, neither of which have been adequately modeled in experimental studies of healthy subjects.

An instrument to measure functional status outcomes for disorders of excessive sleepiness.

This article reports the development of the functional outcomes of sleep questionnaire (FOSQ). This is the first self-report measure designed to assess the impact of disorders of excessive sleepiness (DOES) on multiple activities of everyday living. Three samples were used in the development and psychometric analyses of the FOSQ: Sample 1 (n = 153) consisted of individuals seeking medical attention for a sleep problem and persons of similar age and gender having no sleep disorder; samples 2 (n = 24) and 3 (n = 51) were composed of patients from two medical centers diagnosed with obstructive sleep apnea (OSA). Factor analysis of the FOSQ yielded five factors: activity level, vigilance, intimacy and sexual relationships, general productivity, and social outcome. Internal reliability was excellent for both the subscales (alpha = 0.86 to alpha = 0.91) and the total scale (alpha = 0.95). Test-retest reliability of the FOSQ yielded coefficients ranging from r = 0.81 to r = 0.90 for the five subscales and r = 0.90 for the total measure. The FOSQ successfully discriminated between normal subjects and those seeking medical attention for a sleep problem (T157 = -5.88, p = 0.0001). This psychometric evaluation of the FOSQ demonstrated parameters acceptable for its application in research and in clinical practice to measure functional status outcomes for persons with DOES. Thus, the FOSQ can be used to determine how disorders of excessive sleepiness affect patients' abilities to conduct normal activities and the extent to which these abilities are improved by effective treatment of DOES.

Night-to-night variability in CPAP use over the first three months of treatment.

The purpose of this study was to examine the relationship between night-to-night variability and nightly duration of continuous positive airway pressure (CPAP) therapy over the first 9 weeks of treatment and to determine when patients begin to establish a nonadherent pattern of use. Data were analyzed from a study of daily CPAP use covertly monitored in 32 diagnosed patients with obstructive sleep apnea (OSA) using a microprocessor monitor encased in a CPAP machine. Patterns of CPAP use were bimodal, based on the frequency of nightly use. Approximately half the subjects were consistent users of CPAP, applying it > 90% of the nights for an average of 6.22 +/- 1.21 hours per night, while the other half comprised intermittent users who had a wide range of daily use averaging 3.45 +/- 1.94 hours per night on the nights CPAP was used. The percent of days skipped was significantly correlated with decreased nightly duration (rho = -0.73, p < 0.0001). Analysis of the night-to-night pattern of use revealed that the two groups differed significantly in the nightly duration of CPAP use by the fourth day of treatment (p = 0.001). Exploration of factors that potentially differentiate the two groups revealed no reliable predictors. However, intermittent users continued to report significantly greater OSA symptoms (snoring, snorting, and apnea) posttreatment, suggesting that they continued to experience sleep disordered breathing.

Comparative inhalation teratogenicity of four glycol ether solvents and an amino derivative in rats.

Previous research demonstrated the inhalation teratogenicity of the solvent 2-ethoxyethanol in rats and rabbits. As this is one of a class of widely used industrial solvents, we investigated the teratogenicity of five structurally related compounds. Each chemical was vaporized and administered to approximately 15 pregnant rats in one to three concentrations for 7 hr/day on gestation days 7 to 15, and dams were sacrificed on day 20. Fetuses were individually weighed, and two-thirds of them were fixed in Bouin's solution and examined for soft-tissue anomalies. The other one-third were fixed in alcohol, stained with Alizarin Red and examined for skeletal defects. Data were analyzed on a litter basis; three solvents were compared with a pooled group (N = 34) of sham-exposed controls, and the remaining two were compared with a group of 15 controls. At concentrations which were apparently not maternally toxic, 2-methoxyethanol was highly embryotoxic, producing complete resorptions at 200 ppm; increased resorptions, reduced fetal weights and skeletal and cardiovascular defects occurred at both 100 and 50 ppm. 2-ethoxyethyl acetate at 600 ppm induced complete resorption of litters; 390 ppm reduced fetal weights and induced skeletal and cardiovascular defects, but only a single defect was observed at 130 ppm. 2-Butoxyethanol evidenced slight maternal toxicity at 200 ppm but produced no increase in congenital defects at that concentration. Neither 2-(2-ethoxyethoxy)ethanol (100 ppm) nor 2-methylaminoethanol (150 ppm) was maternally toxic or embryotoxic. In summary, shorter alkyl chained glycol ethers produced greater embryotoxicity than those having longer chains, and the ester produced effects equivalent to the ether, both patterns predictable from the biochemical literature.

Biosynthetic routing of pulmonary surfactant proteins in alveolar type II cells.

Surfactant proteins A, B, and C (SP-A, SP-B, and SP-C) are synthesized in alveolar type II cells. SP-B and SP-C are both synthesized as large precursor molecules that are proteolytically processed to their mature sizes. In a previous immunoelectron microscopic study, we showed that precursor SP-B is processed to its mature size in multivesicular bodies. In the present study, using a specific antibody against precursor SP-C, we demonstrate that precursor SP-C is present in the same intracellular compartments of the biosynthetic pathway, i.e., endoplasmic reticulum, Golgi complex, and multivesicular bodies, as precursor SP-B. Since mature SP-C is known to be present in multilamellar bodies, this suggests a biosynthetic routing and site of processing of this protein similar to those of SP-B. Double-labeling experiments using antibodies against SP-A, precursor SP-B, precursor SP-C, and an antibody against HA I, an adaptor protein involved in the budding of transport vesicles from the Golgi complex, showed that the different surfactant proteins traverse and exit the Golgi complex via the same route. The surfactant proteins do not exit the Golgi complex via HA I-positive coated buds or vesicles. These data are in accordance with the concept that SP-A, SP-B, and SP-C are transported together through the same biosynthetic pathway via multivesicular bodies to multilamellar bodies.

Identification of canine pulmonary surfactant-associated glycoprotein A precursors.

Surfactant-associated glycoproteins A were identified by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude surfactant from canine alveolar lavage: an unglycosylated form (protein A1), 27,000-28,000 daltons; glycoprotein A2, 32,000-34,000 daltons; and glycoprotein A3, 37,000-38,000 daltons; pH at isoelectric point (pI) 4.5-5.0. Glycoproteins A2 and A3 were electroeluted and used to prepare a monospecific antiserum that identified proteins A1, A2, and A3 in immunoblots of crude surfactant obtained from dog lung lavage. This antiserum precipitated several proteins from in vitro translated canine lung poly(A)+ mRNA; proteins of 27,000 daltons, pI 5.0, and 28,000 daltons, pI 4.8-5.0, which precisely comigrated with proteins A1 from canine surfactant. Cotranslational processing of the primary translation products by canine pancreatic microsomal membranes resulted in larger proteins of 31,000-34,000 daltons, pI 4.8-5.0. Treatment of these processed forms of glycoprotein A with endoglycosidase F, to remove N-linked carbohydrate, resulted in proteins of 27,000-28,000 daltons which precisely comigrated with surfactant protein A1. These observations demonstrate that the polypeptide precursors to the glycoproteins A complex are extensively modified by addition of asparagine N-linked complex carbohydrate and are subsequently secreted as glycoproteins A2 and A3.

Synthesis of surfactant-associated protein, 35,000 daltons, in fetal lung.

The major human pulmonary surfactant-associated protein of 35,000 daltons (Da) (SAP-35), consists of a group of related proteins of 27,000-36,000 Da, with isoelectric points ranging from pH 4.6 to 5.2. SAP-35 precursors were identified by immunoprecipitation of protein products of in vitro translation of normal adult human poly(A)+ mRNA with human SAP-35 antiserum. The translation products nearly comigrated with the most basic components of alveolar SAP-35 (mol mass = 24,500-27,000 Da). Processing of the primary translation products by canine pancreatic microsomal membranes increased their apparent molecular weight to 29,000-30,000-Da forms, which were sensitive to endoglycosidase F, suggesting the addition of asparagine-linked oligosaccharides to the molecules. A smaller protein of 24,500 Da was generated during treatment with canine microsomal membranes likely representing cleavage of a signal peptide. SAP-35 was not detected in explants of [35S]methionine-labeled fetal lung (20-24 wk gestation) after 1 day of culture or immunoprecipitates of in vitro translated poly(A)+ mRNA isolated from fetal human lung. However, after 3-5 days of organ culture, synthesis of SAP-35 was readily detected by immunoprecipitation of [35S] methionine-labeled tissue. Fully sialylated (neuraminidase-sensitive forms) comigrated with fully glycosylated SAP-35 isolated from human surfactant. High mannose (endoglycosidase H-sensitive precursors) were also synthesized by the organ cultures and were distinct from the secreted form in surfactant. Synthesis of surfactant-associated SAP-35 and its precursors was induced in association with morphological maturation of the type II epithelial cell during organ culture of human fetal lung.

Identification of surfactant proteolipid SP-B in human surfactant and fetal lung.

Surfactant proteolipid (SP-B) is one of several hydrophobic peptides detected in organic extracts of pulmonary surfactant and associated with the dramatic surface-active properties of surfactant phospholipids. In the present study human SP-B was identified as a protein with a relative molecular weight (Mr) of 7,500-8,000 under reducing conditions; protein of Mr 18,000 was detected under nonreducing conditions by immunoblot analysis of organic extracts of bovine and human surfactant utilizing an antiserum directed against a 60-amino acid synthetic SP-B peptide. This peptide antiserum was subsequently used to identify SP-B in explant cultures of 18- to 23-wk gestation human fetal lung. Immunoprecipitation of explants labeled with [35S]methionine after 48 h of culture identified proteins of Mr 40,000-42,000, 25,000, and 18,000 after electrophoresis under nonreducing conditions. The Mr 18,000 form was reduced to Mr 7,500-8,000 in the presence of beta-mercaptoethanol. These molecular forms likely represent the SP-B precursor protein, a proteolytic intermediate, and the mature SP-B peptide, respectively. Immunocytochemistry with the peptide antiserum localized SPL(Phe) in granular inclusions in the apical region of type II-like epithelial cells, a pattern of staining similar to that observed for the major surfactant-associated protein of Mr 26,000-38,000 (SP-A). SP-B is a novel pulmonary surfactant-associated protein that is synthesized by the human alveolar type II epithelial cell as an Mr 40,000-42,000 precursor that is subsequently proteolytically processed to Mr 7,500-8,000.

Structure and function of pulmonary surfactant proteins.

In summary, the isolation and characterization of three surfactant proteins have considerably changed our understanding of the nature of pulmonary surfactant and its metabolism. The isolation of the cDNAs and genes encoding the proteins and the elucidation of their structure now makes possible the generation of surfactant proteins for further study and for therapy of surfactant deficient states. Artificial surfactant consisting of appropriately modified human proteins can be produced by recombinant DNA technology. Mixed with appropriate synthetic phospholipids, these proteins may be useful for the treatment of infants with hyaline membrane disease. Significant progress has been made in identifying and characterizing these three more abundant proteins; however, many questions regarding the functions of numerous other proteins present in surfactant (whether produced by Type II cells, Type I cell, Clara cell, or others) remain unanswered. Knowledge of the factors controlling developmental expression of surfactant phospholipids and protein as well as the molecular basis of the interactions among the surfactant proteins and phospholipids may lead to new strategies for therapy of hyaline membrane disease. Major questions regarding the site and nature of posttranslational modification of the surfactant proteins, their sites of assembly with lipids, and their precise roles in the metabolism of surfactant in vitro remain to be clarified. It is hoped that answers to these questions will facilitate the identification and design of appropriate therapy of infants and adults with pulmonary disease associated with surfactant deficiency.