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This paper reports the chemical synthesis and purification of a novel phospholipase-resistant C16:0, C16:1 diether phosphonoglycerol with structural analogy to ester-linked anionic phosphatidylglycerol (PG) in endogenous pulmonary surfactant. This diether phosphonoglycerol (PG 1) is studied for phospholipase A(2) (PLA(2)) resistance and for surface activity in synthetic exogenous surfactants combined with Super Mini-B (S-MB) peptide and DEPN-8, a previously-reported diether phosphonolipid analog of dipalmitoyl phosphatidylcholine (DPPC, the major zwitterionic phospholipid in native lung surfactant). Activity experiments measured both adsorption and dynamic surface tension lowering due to the known importance of these surface behaviors in lung surfactant function in vivo. Synthetic surfactants containing 9 : 1 DEPN-8:PG 1 + 3% S-MB were resistant to degradation by PLA(2) in chromatographic studies, while calf lung surfactant extract (CLSE, the substance of the bovine clinical surfactant Infasurf®) was significantly degraded by PLA(2). The 9 : 1 DEPN-8:PG 1 + 3% S-MB mixture also had small but consistent increases in both adsorption and dynamic surface tension lowering ability compared to DEPN-8 + 3% S-MB. Consistent with these surface activity increases, molecular dynamics simulations using Protein Modeller, GROMACS force-field, and PyMOL showed that bilayers containing DPPC and palmitoyl-oleoyl-PC (POPC) as surrogates of DEPN-8 and PG 1 were penetrated to a greater extent by S-MB peptide than bilayers of DPPC alone. These results suggest that PG 1 or related anionic phosphono-PG analogs may have functional utility in phospholipase-resistant synthetic surfactants targeting forms of acute pulmonary injury where endogenous surfactant becomes dysfunctional due to phospholipase activity in the innate inflammatory response.

Dynamic surface pressure-area (π-A) isotherms were measured for dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylethanolamine (DPPE), and 13 chain-equivalent synthetic phospholipid and phosphonolipid diether and etheramide analog compounds structurally related to these glycerophospholipids. Spread interfacial films at a high initial concentration of 15 A2/molecule were studied under continuous cycling to emphasize maximum surface pressure and dynamic respreading behaviors relevant for pulmonary surfactant. Wilhelmy balance subphases containing NaCl and total ionic strength ∼0.15 were used at pH 2.6, 5.6, and 11.5 to give different headgroup ionization states without significantly affecting compound steric bulk. Intracompound changes in isotherm behavior with pH were correlated with variations in headgroup hydrogen bonding and charge for four groups of molecules: (1) DPPC and analog compounds with choline or similar headgroups (Group I) had π-A isotherms with low minimum surface tensions 70 mN/m) at all three pH values, with dynamic respreading on successive cycles that was lowest at pH 2.6 and increased at pH 5.6 and 11.5; (2) DPPE and related compounds with primary amine headgroups (Group II) had higher minimum surface tensions ranging from 5 to 20 mN/m, with respreading that was greater than that of DPPC-related compounds at pH 2.6 and 5.6, and which decreased dramatically at pH 11.5; (3) secondary amine/ammonium compounds with one N-substitution (Group IIIa) behaved similarly to DPPE and its analogs, with higher minimum surface tensions than DPPC-related molecules and respreading that was maximal at low pH and decreased substantially at pH 11.5; and (4) tertiary amine/ammonium compounds with two N-substitutions (Group IIIb) respread better at pH 11.5 than at 5.6 and 2.6, with strikingly altered isotherms and elevated minimum surface tensions (∼30 mN/m) at high pH. The dynamic π-A isotherm characteristics of the majority of phospholipids and phosphonolipids indicated that increased intermolecular hydrogen bonding between headgroups led to increased dynamic respreading in interfacial films. Additionally, in the absence of substantial intermolecular hydrogen bonding, a cationic lipid headgroup charge (neutral phosphate/phosphonate group) correlated with decreased respreading facility for the surfactant molecules studied.

Increased phospholipase A2 activity demonstrated in some forms of lung injury may contribute to surfactant dysfunction. Phospholipase A2-resistant analogs of dipalmitoylphosphatidylcholine (DPPC) with surfactant properties might therefore be useful lipid components of treatment surfactants for certain lung injuries. The in vivo function of surfactants containing DPPC or the phospholipase-resistant analogs dihexadecylphosphatidylcholine (DEPC) or dihexadecylphosphonotidylcholine (DEPnC), with or without surfactant proteins B and C (SP-B+C), was thus evaluated in preterm rabbits (27 days' gestation). Rabbits randomly received one of seven surfactants (DPPC, DEPC, DEPnC, DPPC+SP-B+C, DEPC+SP-B+C, DEPnC+SP-B+C, or lipid extract surfactant [LES]) or 0.45% NaCl (control) and were ventilated for 30 min. Lipid-only surfactants decreased ventilatory pressures (peak inspiratory pressures minus positive end-expiratory pressure) relative to control (p0.05). Addition of SP-B+C further decreased ventilatory pressures to levels similar to LES (p0.01 versus control, lipid-only surfactants). Lung dynamic compliances and postventilation pressure-volume curves improved in the following order: LES, SP-B+C lipid surfactantslipid-only surfactantscontrol (p0.05). All surfactant preparations decreased intravascular 125I-albumin recoveries in the lungs relative to control (p0.01 for all surfactants versus control). These results indicate that DEPC and DEPnC were as effective as DPPC as lipid components of synthetic surfactants. And like DPPC, the analogs interacted with isolated SP-B+C and improved in vivo function to levels comparable to LES.

This study reports the biochemical separation of the hydrophobic constituents of calf lung surfactant into separate fractions from which specific components are excluded. Gel permeation chromatography on LH-20 with acidified chloroform-methanol separated the constituents of calf lung surfactant extract (CLSE) into fractions containing purified phospholipids (PPL), the neutral lipids and phospholipids (N&PL), or the hydrophobic surfactant proteins (SP)-B and -C together with the phospholipids (SP&PL). Extraction of acid to prevent phospholipid degradation after separation reduced recovery of the apoproteins in SP&PL. This fraction was therefore supplemented with protein purified separately to attain the initial levels present in CLSE. Biochemical analyses confirmed that the resulting preparations had the expected composition not only of protein, neutral lipids and phospholipids, but also of the phospholipid head groups. In addition to these fractions obtained with acidified solvent, elution of CLSE with chloroform-methanol without acid yielded the zwitterionic phospholipids substantially depleted of anionic phosphatidylglycerol and phosphatidylinositol. Limited interfacial measurements also demonstrated that the process of separation did not alter the fundamental surface characteristics of the surfactant constituents. Recombined CLSE (rCLSE) reconstituted from all of the separated components had surface activity indistinguishable from the original CLSE. The individual fractions of surfactant components also had average molecular areas at the air-liquid interface which agreed with predictions based on their biochemical composition. These well defined preparations of the hydrophobic constituents of pulmonary surfactant provide the basis for future studies to establish the role of individual components in the function of this complex surface active material.

We investigated the effects of KL4, a 21-residue amphipathic peptide approximating the overall ratio of positively charged to hydrophobic amino acids in surfactant protein B (SP-B), on the structure and collapse of dipalmitoylphosphatidylcholine and palmitoyl-oleoyl-phosphatidylglycerol monolayers. As reported in prior work on model lung surfactant phospholipid films containing SP-B and SP-B peptides, our experiments show that KL4 improves surfactant film reversibility during repetitive interfacial cycling in association with the formation of reversible collapse structures on multiple length scales. Emphasis is on exploring a general mechanistic connection between peptide-induced nano- and microscale reversible collapse structures (silos and folds).

Background Surfactant protein B (SP-B; 79 residues) belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., approximately residues 8-25 and 63-78), confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1-7) attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity. Methodology/results FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary alpha-helix and secondary beta-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR), predictive aggregation algorithms, and molecular dynamics (MD) and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a "saposin-like" fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B. Conclusion Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B.

A series of synthetic analogs of naturally occurring glycerophospholipids has been studied for gel-to-liquid transition temperature (Tc) by using differential scanning calorimetry. The compounds investigated were dihexadecyl phosphatidylcholine (DHPC) plus 11 newly synthesized phosphonolipids. Six were racemates and have ether-ether fatty chains and 5 were chiral (R) with ether-amide fatty chains bonded to the glycerol moiety; each series also contained a methylene isosteric substitution for one of the oxygen atoms in naturally occurring glycerophospholipid phosphate esters. All analogs have fatty chains of equivalent length in saturated carbon atoms C2C16, but varied in N-headgroup structures. The thermal behaviors of these novel molecules were found to depend upon the degree of alkylation of the headgroups and upon whether the hydrocarbon chains are bonded as ethers or amides. The ether-ether (diether) phosphonolipid (DEPN) analogs of dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE) had increased gel-to-liquid crystal transition temperatures (Tc) compared to their naturally occurring (fatty acyl ester) glycerophospholipid counterparts. In general, DEPN analogs had Tc values 3.7−5.8°C higher than ether-amide phosophonolipid (EAPN) compounds of equivalent headgroup, although EAPN-13 was an exception with the highest Tc of any compound studied. In both the DEPN and EAPN series, analogs with hydrogen atom(s) covalently bonded to the headgroup nitrogen atom showed higher Tc values, possibly related to their increased potential for hydrogen bonding. Interfacial property studies (adsorption, Wilhelmy balance, oscillating bubble) showed that several analogs with higher transitions than DPPC, the principal lung surfactant glycerophospholipid, had improved adsorption and respreading, with equivalent surface tension lowering in compressed spread films on the balance and with minimum surface tensions

Despite its potentially adverse effects on lung development and function, supplemental oxygen is often used to treat premature infants in respiratory distress. To understand how neonatal hyperoxia can permanently disrupt lung development, we previously reported increased lung compliance, greater alveolar simplification, and disrupted epithelial development in adult mice exposed to 100% inspired oxygen fraction between postnatal days 1 and 4. Here, we investigate whether oxygen-induced changes in lung function are attributable to defects in surfactant composition and activity, structural changes in alveolar development, or both. Newborn mice were exposed to room air or 40%, 60%, 80%, or 100% oxygen between postnatal days 1 and 4 and allowed to recover in room air until 8 wk of age. Lung compliance and alveolar size increased, and airway resistance, airway elastance, tissue elastance, and tissue damping decreased, in mice exposed to 60–80% oxygen; changes were even greater in mice exposed to 100% oxygen. These alterations in lung function were not associated with changes in total protein content or surfactant phospholipid composition in bronchoalveolar lavage. Moreover, surface activity and total and hydrophobic protein content were unchanged in large surfactant aggregates centrifuged from bronchoalveolar lavage compared with control. Instead, the number of type II cells progressively declined in 60–100% oxygen, whereas levels of T1α, a protein expressed by type I cells, were comparably increased in mice exposed to 40–100% oxygen. Thickened bundles of elastin fibers were also detected in alveolar walls of mice exposed to ≥60% oxygen. These findings support the hypothesis that changes in lung development, rather than surfactant activity, are the primary causes of oxygen-altered lung function in children who were exposed to oxygen as neonates. Furthermore, the disruptive effects of oxygen on epithelial development and lung mechanics are not equivalently dose dependent.

This study uses microarray analyses to examine gene expression profiles for Mycobacterium tuberculosis (Mtb) induced by exposure in vitro to bovine lung surfactant preparations that vary in apoprotein content: (i) whole lung surfactant (WLS) containing the complete mixture of endogenous lipids and surfactant proteins (SP)-A, -B, -C, and -D; (ii) extracted lung surfactant (CLSE) containing lipids plus SP-B and -C; (iii) column-purified surfactant lipids (PPL) containing no apoproteins, and (iv) purified human SP-A. Exposure to WLS evoked a multitude of transcriptional responses in Mtb, with 52 genes up-regulated and 23 genes down-regulated at 30min exposure, plus 146 genes up-regulated and 27 genes down-regulated at 2h. Notably, WLS rapidly induced several membrane-associated lipases that presumptively act on surfactant lipids as substrates, and a large number of genes involved in the synthesis of phthiocerol dimycocerosate (PDIM), a cell wall component known to be important in macrophage interactions and Mtb virulence. Exposure of Mtb to CLSE, PPL, or purified SP-A caused a substantially weaker transcriptional response (

A homologous series of chiral (R) ether-amide phosphonolipid analogs of naturally occurring (R) glycerophospholipids were synthesized and characterized for their interfacial behaviors. The phosphonolipids possess isoteric ether, amide, and phosphonate functions at positions corresponding to the sn-1, sn-2, and sn-3 ester functions, respectively, of naturally occurring glycerophospholipids. All compounds were synthesized with disaturated C16:0 alkyl/acyl moieties to give structural analogy with dipalmitoyl phosphatidylcholine (DPPC), the major glycerophospholipid component of lung surfactant. Further substitutions at the headgroup nitrogen were also used to generate differences in headgroup size and polarity in the synthetic compounds. The surface activity of the ether-amide phospholipids was investigated in terms of adsorption to the air-water interface, together with studies of dynamic respreading after monolayer collapse and surface tension lowering in dynamically compressed spread films and dispersions. Results showed that several ether-amide phosphonolipids had more rapid adsorption and improved dynamic respreading behavior compared to DPPC, plus the ability to lower surface tension into the range of less than 1 to 4 mN/m in spread films and in dispersions under dynamic conditions. In combination with a series of diether phosphonolipids synthetized in a companion study [1], these ether-amide compounds are useful in the development of molecular structure-surface activity correlates for lung surfactant-related materials, and should assist in investigating the specificity of interactions between phospholipids and other pulmonary biological molecules.

Background. Exogenous pulmonary surfactants are administered into the trachea either to prevent respiratory distress syndrome in premature infants or to treat it. In a randomized, multicenter trial, we compared the results of surfactant therapy initiated as prophylaxis with the results of rescue therapy with surfactant. Methods. Before birth, 479 infants with an estimated gestational age of less than 30 weeks were randomly assigned to receive surfactant as prophylaxis (n = 235) or rescue therapy (n = 244). The infants in the prophylaxis group received a 90-mg intratracheal dose of an exogenous calf-lung surfactant extract at the time of delivery, whereas the infants in the rescue-therapy group received 90 mg of the surfactant several hours after delivery if the fractional inspiratory oxygen concentration was at least 0.40 or if the mean airway pressure was at least 0.686 kPa (7 cm of water), or both. Infants in both groups received additional doses of surfactant at intervals of 12 to 24 hours if ...

Surfactant inactivation has been shown to be a significant factor in animal models of lung injury and may also be important in some forms of respiratory failure in full-term newborns. Fourteen full-term newborns with respiratory failure associated with pneumonia (7 patients) and meconium aspiration syndrome (7 patients) were treated with 90 mg/kg of a calf lung surfactant extract, given intratracheally up to every 6 hours for a maximum of four doses. The group mean fraction of inspired oxygen (Fi02) before treatment was 0.99 ± 0.01 SEM, and the mean airway pressure (MAP) was 14.6 ± 1.0 cm H2O. Patients showed significant improvement in oxygenation after initial surfactant treatment, with the arterial-alveolar oxygenation ratio (a/A ratio) rising from 0.09 ± 0.01 before surfactant treatment to 0.22 ± 0.05 by 15 minutes (P = .03) and remaining improved for 6 hours. The oxygenation index, incorporating MAP as well as oxygen variables, also improved significantly from 26.2 ± 3.1 to 11.2 ± 1.7 at 15 minutes (P < .001), with improvement sustained for more than 6 hours. Chest radiographs were blindly scored from 0 (normal) to 5 (severe opacification), and these improved with marginal significance after initial surfactant treatment (from 2.9 ± 0.2 to 2.5 ± 0.2, P = .05). Eight patients subsequently met retreatment criteria (Fi02 ≥ 0.5 and MAP ≥ 7 cm H2O) and received a second surfactant dose, with a/A ratio rising from 0.124 ± 0.02 before treatment to 0.26 ± 0.07 after 45 minutes (P = .03) and remaining improved for more than 4 hours; the oxygenation index was also significantly improved over similar times by the second surfactant dose. Six patients received a third surfactant dose, with no increase in a/A ratio and a less prominent improvement in oxygenation index. There were no significant improvements in oxygenation in the three patients who received a fourth surfactant dose. Of the 14 patients treated, none died, required extracorporeal membrane oxygenation, had tension pneumothorax after study entry, required oxygen supplementation for more than 14 days, or required oxygen supplementation at discharge. These findings suggest that surfactant supplementation may provide therapeutic benefits for newborns with respiratory failure due to pneumonia or meconium aspiration and that expanded controlled trials of this therapy are indicated.

The inclusion of one or more sulfur atoms at the backbone of glycerophospholipids or phosphonolipids has created a number of new non-natural substrates for investigation in medicinal chemistry and enzymology. This review, containing 138 references, summarizes research contributions to date, with an emphasis on the synthetic organic chemistry that provides the lipids.

Acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) are characterized by rapid-onset respiratory failure following a variety of direct and indirect insults to the parenchyma or vasculature of the lungs. Mortality from ALI/ARDS is substantial, and current therapy primarily emphasizes mechanical ventilation and judicial fluid management plus standard treatment of the initiating insult and any known underlying disease. Current pharmacotherapy for ALI/ARDS is not optimal, and there is a significant need for more effective medicinal chemical agents for use in these severe and lethal lung injury syndromes. To facilitate future chemical-based drug discovery research on new agent development, this paper reviews present pharmacotherapy for ALI/ARDS in the context of biological and biochemical drug activities. The complex lung injury pathophysiology of ALI/ARDS offers an array of possible targets for drug therapy, including inflammation, cell and tissue injury, vascular dysfunction, surfactant dysfunction, and oxidant injury. Added targets for pharmacotherapy outside the lungs may also be present, since multiorgan or systemic pathology is common in ALI/ARDS. The biological and physiological complexity of ALI/ARDS requires the consideration of combined-agent treatments in addition to single-agent therapies. A number of pharmacologic agents have been studied individually in ALI/ARDS, with limited or minimal success in improving survival. However, many of these agents have complementary biological/biochemical activities with the potential for synergy or additivity in combination therapy as discussed in this article.

The effects of surfactant protein (SP)-A on the dynamic surface tension lowering and resistance to inhibition of dispersions of calf lung surfactant extract (CLSE) and mixtures of synthetic phospholipids combined with SP-B,C hydrophobic apoproteins were studied at 37 degrees C and rapid cycling rate (20 cycles/min). Addition of SP-A to CLSE, which already contains SP-B and -C, gave a slight improvement in the time course of surface tension lowering on an oscillating bubble apparatus in the absence of inhibitory protein molecules such as albumin or hemoglobin. However, when these proteins were present at concentrations of 10-50 mg/ml, SP-A substantially improved the resistance of CLSE to their inhibitory effects. The beneficial effect of SP-A required the presence of Ca2+ ions, and disappeared when EDTA was substituted for this divalent cation in the subphase. The effect was also retained when SP-A was heated to 50 degrees C prior to addition to CLSE, but was abolished by heating SP-A to 99 degrees C. Additional studies showed that similar improvements in resistance to inhibition were found when SP-A was added to synthetic mixtures of dipalmitoyl phosphatidylcholine (DPPC):egg phosphatidylglycerol (PG) (80:20 by weight) reconstituted with 1% SP-B or SP-B and -C, but not to phospholipid mixtures containing only SP-C. The requirements for SP-B and calcium for the beneficial effects of SP-A on surface activity suggest that the formation of ordered, larger phospholipid-apoprotein aggregates may be involved in the process. The finding that SP-A enhances the ability of CLSE and other surfactant mixtures containing SP-B to resist inhibition is an advantage that will need to be weighed against other factors such as increased antigenicity and heat sensitivity in therapeutic applications in surfactant replacement therapy.

To assess the short-term effects of high-frequency jet ventilation (HFJV) on the neonatal lung, 28 newborn piglets were studied. Nine piglets were unventilated except during brief pulmonary measurements, nine animals were conventionally ventilated (arterial CO2 tension 35-45 torr, arterial O2 tension 70-80 torr) for 4 h, and 10 piglets were ventilated with HFJV for the same period. Pulmonary function was analyzed using a computerized technique and tracheobronchial aspirates were examined for biochemical indicators of lung injury; after 4 h, bronchoalveolar lavage was obtained for surfactant composition and activity, and lung sections were examined by light and electron microscopy. Results showed that HFJV provided adequate ventilation at lower inspiratory pressure compared with conventional ventilation (8.6 +/- 0.3 versus 13.8 +/- 1.3 cm H2O; p less than 0.01), while pulmonary mechanics did not vary significantly among the three animal groups. Tracheobronchial aspirates from HFJV animals had higher elastase activity versus unventilated piglets (118.5 +/- 14.1 versus 57.7 +/- 8.4 micrograms/mL; p less than 0.01), as well as higher albumin concentration versus unventilated animals (94.2 +/- 18.7 versus 23.2 +/- 6.5 micrograms/mL; p less than 0.01). In addition, there were small but statistically significant differences between all three groups in the distribution of surfactant phospholipids in bronchoalveolar lavage, although biophysical activity was normal. Scanning electron microscopy revealed flattening of Clara cells in the terminal bronchioles of HFJV animals due to loss of glycogen and secretory granules. These data indicate that despite lower peak inspiratory pressures, HFJV can cause subtle biochemical changes in lungs. Further studies are indicated to determine if these changes precede significant lung injury.

The biophysical activity and inhibition of a series of synthetic surfactant mixtures was studied and correlated with physiological effectiveness in restoring pressure-volume (P-V) mechanics of excised lungs. Results showed that several simple mixtures of dipalmitoyl phosphatidylcholine (DPPC) with fatty acids or diacylglycerols could be formulated to give good adsorption facility and dynamic surface tension lowering to less than 1 mN/m in pulsating bubble measurements at 37 degrees C. However, although biophysical activity approached that of natural lung surfactant (LS) and a related surfactant extract (CLSE) under normal conditions, surface properties were sharply inhibited by relatively small amounts of the plasma protein albumin (2 mg/ml) with minimum surface tensions greater than 30 nM/m even at high surfactant concentrations (5-20 mg lipids/ml). This sensitivity to biophysical inhibition was markedly increased compared to LS and CLSE, and had direct consequences for physiological efficacy: in spite of initially high activity, synthetic surfactants did not exert beneficial effects on P-V mechanics when instilled into surfactant-deficient excised rat lungs. Endogenous protein material was shown to be present upon surfactant recovery by lavage, and bubble measurements confirmed surface activity well below pre-instillation levels. Moreover, full biophysical activity was restored when lavage fluid was extracted to separate the synthetic surfactants from endogenous inhibitors. These results show that it is important to define relative sensitivity to biophysical inhibition in the development of effective lung surfactant substitutes. In addition, the existence of inhibition effects can generate an apparent lack of correspondence between initial biophysical activity and ultimate physiological actions of exogenous surfactant mixtures.

Background. This study examines the surface activity and resistance to phospholipase degradation of a fully-synthetic lung surfactant containing a novel diether phosphonolipid (DEPN-8) plus a 34 amino acid peptide (Mini-B) related to native surfactant protein (SP)-B. Activity studies used adsorption, pulsating bubble, and captive bubble methods to assess a range of surface behaviors, supplemented by molecular studies using Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD), and plasmon resonance. Calf lung surfactant extract (CLSE) was used as a positive control. Results. DEPN-8+1.5% (by wt.) Mini-B was fully resistant to degradation by phospholipase A2 (PLA2) in vitro, while CLSE was severely degraded by this enzyme. Mini-B interacted with DEPN-8 at the molecular level based on FTIR spectroscopy, and had significant plasmon resonance binding affinity for DEPN-8. DEPN-8+1.5% Mini-B had greatly increased adsorption compared to DEPN-8 alone, but did not fully equal the very high adsorption of CLSE. In pulsating bubble studies at a low phospholipid concentration of 0.5 mg/ml, DEPN-8+1.5% Mini-B and CLSE both reached minimum surface tensions ,1 mN/m after 10 min of cycling. DEPN-8 (2.5 mg/ml)+1.5% Mini-B and CLSE (2.5 mg/ml) also reached minimum surface tensions ,1 mN/m at 10 min of pulsation in the presence of serum albumin (3 mg/ml) on the pulsating bubble. In captive bubble studies, DEPN-8+1.5% Mini-B and CLSE both generated minimum surface tensions ,1 mN/m on 10 successive cycles of compression/expansion at quasi-static and dynamic rates. Conclusions. These results show that DEPN-8 and 1.5% Mini-B form an interactive binary molecular mixture with very high surface activity and the ability to resist degradation by phospholipases in inflammatory lung injury. These characteristics are promising for the development of related fully-synthetic lipid/peptide exogenous surfactants for treating diseases of surfactant deficiency or dysfunction.

Gram-negative pneumonia results in significant morbidity, mortality, and cost to the healthcare system. Previously the authors demonstrated that capsule and O-antigen, virulence factors of the extraintestinal Escherichia coli isolate CP9, modulate pulmonary neutrophil influx in a rat pneumonia model. In this report, the authors utilized CP9 and mutants deficient in O-antigen (CP921), capsule (CP9.137), or both (CP923) to test the hypothesis that modulation of cytokine levels by capsule and/or O-antigen may be a contributory mechanism. Effects of capsule and O-antigen on cytokine levels in rats in vivo and in isolated pulmonary macrophages in vitro were assessed. In vivo, capsule and O-antigen had no significant effect on tumor necrosis factor (TNF)-alpha levels in bronchoalveolar lavage fluid (BALF), but both were associated with significant increases in the levels of interleukin (IL)-1beta and Cytokine-induced neutrophil Chemoattractant-1 (CINC-1). However, potential difficulties in interpreting data occurred because challenge bacterial strains exhibited differential growth, and clearance characteristics and mixed cell populations were present. Therefore, added mechanistic studies investigated specific interactions of capsule and O-antigen with pulmonary macrophages purified from normal rats and exposed to CP9, CP921, CP9.137, or CP923 in vitro. Results indicated that the presence of capsule led to significantly increased levels of TNF-alpha, IL-1beta, and CINC-1, whereas O-antigen significantly decreased macrophage-associated levels of these mediators. These findings support the hypothesis that CP9 capsule is proinflammatory for macrophage-induced neutrophil recruitment, whereas O-antigen attenuates macrophage production of proinflammatory mediators in pneumonia. These results expand our understanding on the mechanisms by which these virulence traits may contribute to the inflammatory pathogenesis of pneumonia.

This study investigates the activity and inhibition resistance in excised rat lungs of a novel synthetic surfactant containing the phospholipase-resistant diether phosphonolipid DEPN-8 plus 1.5% bovine surfactant protein (SP)-B/C compared to calf lung surfactant extract (CLSE). DEPN-8 + 1.5% SP-B/C surpassed CLSE in normalizing surfactant-deficient pressure-volume (P-V) deflation mechanics in lavaged excised lungs in the presence of phospholipase A(2) (PLA(2)) or C18:1 lyso-phosphatidylcholine (LPC). DEPN-8 + 1.5% SP-B/C had activity equal to CLSE in normalizing P-V mechanics in the absence of inhibitors or in the presence of serum albumin. These physiologic activity findings were directly consistent with surface activity measurements on the pulsating bubble surfactometer. In the absence of inhibitors, DEPN-8 + 1.5% SP-B/C and CLSE rapidly reached minimum surface tensions1 mN/m (0.5 and 2.5 mg surfactant phospholipid/ml). DEPN-8 + 1.5% SP-B/C maintained its high surface activity in the presence of PLA(2), while the surface activity of CLSE was significantly inhibited by exposure to this enzyme. DEPN-8 + 1.5% SP-B/C also had greater surface activity than CLSE in the presence of LPC, and the two surfactants had equivalent surface activity in the presence of albumin. DEPN-8 + 1.5% SP-B/C also had slightly greater surface activity than CLSE when exposed to peroxynitrite in pulsating bubble studies. These results support the potential of developing highly active and inhibition-resistant synthetic exogenous surfactants containing DEPN-8 + apoprotein/peptide constituents for use in treating direct pulmonary forms of clinical acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS).

This study tests the hypothesis that the virulence factor hemolysin (Hly) expressed by extraintestinal pathogenic Escherichia coli contributes to surfactant dysfunction and lung injury in a rat model of gram-negative pneumonia. Rats were instilled intratracheally with CP9 (wild type, Hly-positive), CP9 hlyA (Hly-minus), CP9 /pEK50 (supraphysiological Hly), or purified LPS. At 6 h postinfection, rats given CP9 had a decreased percentage content of large surfactant aggregates in cell-free bronchoalveolar lavage (BAL), decreased large aggregate surface activity, decreased PaO2/FiO2ratio, increased BAL albumin/protein levels, and increased histological evidence of lung injury compared with rats given CP9 hlyA or LPS. In addition, rats given CP9/ pEK50 or CP9 had decreased large aggregate surface activity, decreased PaO2/FiO2ratios, and increased BAL albumin/protein levels at 2 h postinfection compared with rats given CP9 hlyA. The severity of permeability lung injury based on albumin/protein levels in BAL at 2 h was ordered as CP9 /pEK50 > CP9 > CP9 hlyA > normal saline controls. Total lung titers of bacteria were increased at 6 h in rats given CP9 vs. CP9 hlyA, but bacterial titers were not significantly different at 2 h, indicating that increased surfactant dysfunction and lung injury were associated with Hly as opposed to bacterial numbers per se. Further studies in vitro showed that CP9 could directly lyse transformed pulmonary epithelial cells (H441 cells) but that indirect lysis of H441 cells secondary to Hly-induced neutrophil lysis did not occur. Together, these data demonstrate that Hly is an important direct mediator of surfactant dysfunction and lung injury in gram-negative pneumonia.

Surfactant dysfunction was studied in C57BL/6 (B6), B6.SP-A(-/-), and B6.iNOS(-/-) mice with pulmonary mycoplasma infection (10(7) colony-forming units). Cell-free bronchoalveolar lavage (BAL) from uninfected B6.SP-A(-/-) versus B6 mice had a reduced content of very large aggregates (VLA) and an increase in intermediate large aggregates (ILA), with no difference in total large aggregates (LA = VLA + ILA). However, LA from uninfected B6.SP-A(-/-) versus B6 mice contained less protein and were more sensitive to inhibition by serum albumin and lysophosphatidylcholine in pulsating bubble studies in vitro. Infection with Mycoplasma pulmonis caused significant lung injury and surfactant abnormalities in B6.SP-A(-/-), B6.iNOS(-/-), and B6 mice at 24, 48, 72 h after infection compared with uninfected mice of the same strain. Analyses of time-pooled data indicated that mycoplasma-infected B6.SP-A(-/-) and B6.iNOS(-/-) mice had significantly lower levels of LA and higher protein/phospholipid ratios in BAL compared with infected B6 mice. Infected B6.iNOS(-/-) versus B6 mice also had increased minimum surface tensions on the pulsating bubble and decreased levels of surfactant protein (SP)-B in BAL. These results indicate that pulmonary mycoplasma infection in vivo causes lung injury and surfactant abnormalities that are dependent in part on iNOS and SP-A. In addition, SP-A deficiency modifies surfactant aggregate content and lowers the inhibition resistance of LA surfactant in vitro compared with congenic normal mice.

Lung contusion is the leading cause of death from blunt thoracic trauma in adults, but its mechanistic pathophysiology remains unclear. This study uses a recently developed rat model to investigate the evolution of inflammation and injury in isolated lung contusion. Bilateral lung contusion with minimal cardiac trauma was induced in 54 anesthetized rats by dropping a 0.3-kg hollow cylindrical weight onto a precordial shield (impact energy, 2.45 Joules). Arterial oxygenation, pressure-volume (P-V) mechanics, histology, and levels of erythrocytes, leukocytes, albumin, and inflammatory mediators in bronchoalveolar lavage (BAL) were assessed at 8 min, at 4, 12, 24, and 48 h, and at 7 days after injury. The role of neutrophils in the evolution of inflammatory injury was also specifically studied by depleting these cells with intravenous vinblastine before lung contusion. Arterial oxygenation was severely reduced at 8 min to 24 h postcontusion, but became almost normal by 48 h. Levels of erythrocytes, leukocytes, and albumin in BAL were increased ator=24 h, and returned toward normal by 48 h. Deficits in P-V mechanics were most apparent at 24 h postcontusion. Levels of macrophage inflammatory polypeptide-2, cytokine-induced neutrophil chemoattractant-1, and interleukin 6 in BAL peaked at 24 h, whereas monocyte chemoattractant protein-1 and interleukin 1beta peaked at 24 to 48 h postcontusion. Histology showed early hemorrhagic injury (8 min-12 h), with neutrophilic infiltration at 24 h and areas of bronchiolitis obliterans organizing pneumonia-associated fibrosis at 7 days. Vinblastine-treated neutropenic rats had significantly reduced lung injury based on total lung volume at 4 h and on BAL albumin levels at 24 h postcontusion. Inflammatory injury from isolated bilateral lung contusion in rats is most severe in the acute period (8 min-24 h) after initial blunt trauma, and includes a component of neutrophil-dependent pathology.

Introduction. Principles of Lung Development, Growth, and Repair. Acute Lung Injury: Etiologies and Basic Features. Cellular and Animal Models of Lung. Transgenic Models of Lung Injury and Repair. Inhalation Toxicology: Methods and Models. Inflammatory Cells and Mediators in Acute Lung Injury. Mechanisms of Chronic Lung Injury and Fibrosis. Roles of Oxidants and Antioxidants in Lung. Vascular Dysfunction in Lung Injury. Surfactant Dysfunction in Lung Injury. Ventilation Therapies and Strategies for Lung Injury. Anti-Inflammatory Therapies for Lung Injury. Surfactant-Based Therapies for Lung Injury.

A model of aspiration lung injury was developed in WT C57BL/6 mice to exploit genetically modified animals on this background, i.e., MCP-1(−/−) mice. Mice were given intratracheal hydrochloric acid (ACID, pH 1.25), small nonacidified gastric particles (SNAP), or combined acid plus small gastric particles (CASP). As reported previously in rats, lung injury in WT mice was most severe for “two-hit” aspiration from CASP (40 mg/ml particulates) based on the levels of albumin, leukocytes, TNF-α, IL-1β, IL-6, MCP-1, KC, and MIP-2 in bronchoalveolar lavage (BAL) at 5, 24, and 48 h. MCP-1(−/−) mice given 40 mg/ml CASP had significantly decreased survival compared with WT mice (32% vs. 80% survival at 24 h and 0% vs. 72% survival at 48 h). MCP-1(−/−) mice also had decreased survival compared with WT mice for CASP aspirates containing reduced particulate doses of 10–20 mg/ml. MCP-1(−/−) mice given 5 mg/ml CASP had survival similar to WT mice given 40 mg/ml CASP. MCP-1(−/−) mice also had differing responses from WT mice for several inflammatory mediators in BAL (KC or IL-6 depending on the particle dose of CASP and time of injury). Histopathology of WT mice with CASP (40 mg particles/ml) showed microscopic areas of compartmentalization with prominent granuloma formation by 24 h, whereas lung tissue from MCP-1(−/−) mice had severe diffuse pneumonia without granulomas. These results indicate that MCP-1 is important for survival in murine aspiration pneumonitis and appears to act partly to protect uninjured lung regions by promoting isolation and compartmentalization of tissue with active inflammation.

This study examines lung injury and inflammation over 24 hours following intratracheal instillation of hydrochloric acid (acid), small nonacidic gastric particles (SNAP), or combined acid and small particles (CASP) in adult rats. The severity and duration of injury was significantly greater for CASP compared to acid or SNAP based on PaO2/FiO2, bronchoalveolar lavage (BAL) albumin, and BAL cell numbers. The inflammatory response associated with aspiration injury from CASP was distinct in several respects. Tumor necrosis factor (TNF)-alpha was greatly reduced in CASP compared to SNAP or acid, whereas interleukin (IL)-1beta was increased. Levels of cytokine-induced neutrophil chemoattractant (CINC)-1, monocyte chemoattractant protein (MCP)-1, and IL-10 in lavage were also significantly increased in animals injured with CASP compared to other forms of aspiration. Statistical analysis showed that BAL levels of IL-10 correlated most strongly with albumin leakage in aspiration-injured animals at 6 and 24 hours, followed by BAL levels of MCP-1. Additional cytokine cluster analyses indicated that levels of MCP-1 and CINC-1 in BAL from all injured animals were strongly correlated with inflammatory neutrophil numbers at 6 and 24 hours post aspiration, and that IL-10 levels in BAL were strongly correlated with inflammatory cell numbers at 24 hours. Preliminary blocking experiments showed that administration of anti-IL-10 antibody increased the albumin permeability index at 6 hours in SNAP and CASP animals, but anti-MCP-1 antibody did not affect the severity of injury. The results of this study support the possibility that different forms of aspiration are associated with identifiable cytokine profiles, and that specific cytokines, including IL-10 and MCP-1, may have utility as diagnostic or prognostic markers in clinical applications.

Synthesis methods and initial surface property characterizations are reported for two sulfur-containing phosphonolipids related structurally to dipalmitoyl phosphatidylcholine (DPPC), the major lung surfactant glycerophospholipid. Sulfur linkages in these compounds affect molecular interactions relative to ester linkages, and are structurally resistant to cleavage by phospholipases. The SO2-linked analog synthesized here had increased adsorption and improved film respreading compared to DPPC, while reaching very low surface tensions (1 N/m) in cycled interfacial films on both the Wilhelmy balance and the pulsating bubble surfactometer. This compound appears to have potential utility as a component in future phospholipase-resistant synthetic exogenous surfactants for treating clinical forms of inflammatory lung injury.

The conformation and orientation of synthetic monomeric human sequence SP-B(1-25) (mSP-B(1-25)) was studied in films with phospholipids at the air-water (A/W) interface by polarization modulation infrared reflectance absorption spectroscopy (PM-IRRAS). Modified two-dimensional infrared (2D IR) correlation analysis was applied to PM-IRRAS spectra to define changes in the secondary structure and rates of reorientation of mSP-B(1-25) in the monolayer during compression. PM-IRRAS spectra and 2D IR correlation analysis showed that, in pure films, mSP-B(1-25) had a major alpha-helical conformation plus regions of beta-sheet structure. These alpha-helical regions reoriented later during film compression than beta structural regions, and became oriented normal to the A/W interface as surface pressure increased. In mixed films with 4:1 mol:mol acyl chain perdeuterated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DPPC-d(62):DOPG), the IR spectra of mSP-B(1-25) showed that a significant, concentration-dependent conformational change occurred when mSP-B(1-25) was incorporated into a DPPC-d(62):DOPG monolayer. At an mSP-B(1-25) concentration of 10 wt.%, the peptide assumed a predominantly beta-sheet conformation with no contribution from alpha-helical structures. At lower, more physiological peptide concentrations, 2D IR correlation analysis showed that the propensity of mSP-B(1-25) to form alpha-helical structures was increased. In phospholipid films containing 5 wt.% mSP-B(1-25), a substantial alpha-helical peptide structural component was observed, but regions of alpha and beta structure reoriented together rather than independently during compression. In films containing 1 wt.% mSP-B(1-25), peptide conformation was predominantly alpha-helical and the helical regions reoriented later during compression than the remaining beta structural components. The increased alpha-helical structure of mSP-B(1-25) demonstrated here by PM-IRRAS and 2D IR correlation analysis in monolayers of 4:1 DPPC:DOPG containing 1 wt.% (and, to a lesser extent, 5 wt.%) peptide may be relevant for the formation of the intermediate order 'dendritic' surface phase observed in similar surface films by epi-fluorescence.

Epifluorescence microscopy was used to study the structure and phase behavior of phospholipid films containing a human-sequence monomeric SP-B(1-25) synthetic peptide (mSP-B(1-25)). Measurements were done directly at the air-water (A/W) interface on films in a Langmuir-Whilhelmy balance coupled to a fluorescence microscope and real-time detection system to yield an approximate optical resolution of 1 mum. Fluorescence was achieved by laser excitation of 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-PC (BODIPY-PC, concentration/=1 mol%). The presence of mSP-B(1-25) in films of 4:1 (mol/mol) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) had a substantial effect on lipid morphology and phase behavior that depended on both surface pressure and peptide concentration (10, 5, and 1 wt.%). The mSP-B(1-25) peptide tended to fluidize phospholipid monolayers based on expanded molecular areas and reduced collapse pressures. In addition, epifluorescence measurements revealed the formation of solid-phase domains apparent as three-armed counterclockwise spirals separated from regions of fluid liquid-expanded phase domains in compressed phospholipid-peptide films. The appearance of these separated solid-phase domains resembled pure L-DPPC rather than the ensemble-type solid domains found in films of DPPC/DOPG alone and were most apparent when 10 wt.% mSP-B(1-25) was present. In contrast, films containing lower, more physiological mSP-B(1-25) contents of 5 and 1 wt.% exhibited a prominent intermediate 'dendritic' phase that increased in extent as surface pressure was raised. This phase was characterized by branching structures that formed a lattice-like mesh network with fluorescence intensities between a dye-depleted solid domain and a dye-enriched liquid phase. These results indicate that mSP-B(1-25) at near-physiological levels produces morphological changes in phospholipid monolayers analogous to those observed for native SP-B(1-79).

Surface activity and sensitivity to inhibition from phospholipase A2(PLA2), lysophosphatidylcholine (LPC), and serum albumin were studied for a synthetic C16:0 diether phosphonolipid (DEPN-8) combined with 1.5% by weight of mixed hydrophobic surfactant proteins (SP)-B/C purified from calf lung surfactant extract (CLSE). Pure DEPN-8 had better adsorption and film respreading than the major lung surfactant phospholipid dipalmitoyl phosphatidylcholine and reached minimum surface tensions 2, and DEPN-8 + 1.5% SP-B/C maintained high adsorption and dynamic surface activity in the presence of this enzyme. Incubation of CLSE with PLA2led to chemical degradation, generation of LPC, and reduced surface activity. DEPN-8 + 1.5% SP-B/C was also more resistant than CLSE to direct biophysical inhibition by LPC, and the two were similar in their sensitivity to biophysical inhibition by serum albumin. These findings indicate that synthetic surfactants containing DEPN-8 combined with surfactant proteins or related synthetic peptides have potential utility for treating surfactant dysfunction in inflammatory lung injury.

OBJECTIVE To report cases of neonates successfully treated with both exogenous surfactant and inhaled nitric oxide (INO). DESIGN Retrospective chart review of full term infants treated between January and May 1999 in the neonatal intensive care unit of The Children's Hospital at Strong, University of Rochester, Rochester, New York. PATIENTS Three full-term infants treated with surfactant and INO were identified. Each infant had severe acute respiratory failure (as a result of severe aspiration syndromes) and a clinical diagnosis of pulmonary hypertension and parenchymal lung disease in the absence of congenital malformations. INTERVENTIONS One infant received INO (20-40 ppm) followed by exogenous surfactant (100mg/kg); the other two received surfactant followed by INO. MAIN RESULTS All three infants exhibited a favorable response to treatment with these agents in terms of improved arterial oxygenation as summarized by oxygenation index and all survived to discharge home without referral for extracorporeal membrane oxygenation. CONCLUSIONS No adverse interactions were observed related to INO plus surfactant therapy. The responses of these critically ill infants were consistent with the hypothesis that the actions of INO in dilating the pulmonary microvasculature and of exogenous surfactant in stabilizing and recruiting alveoli are complementary and may lead to additive clinical benefits. These case results suggest that more extensive clinical studies are warranted for combined-modality therapy with INO and exogenous surfactant in patients with the acute respiratory distress syndrome.

The content-dependent activity of surfactant protein (SP)-B was studied in mixtures with dipalmitoyl phosphatidylcholine (DPPC), synthetic lipids (SL), and purified phospholipids (PPL) from calf lung surfactant extract (CLSE). At fixed SP-B content, adsorption and dynamic surface tension lowering were ordered as PPL/SP-B ≈ SL/SP-B > DPPC/SP-B. All mixtures were similar in having increased surface activity as SP-B content was incrementally raised from 0.05 to 0.75% by weight. SP-B had small but measurable effects on interfacial properties even at very low levels ≤0.1% by weight. PPL/SP-B (0.75%) had the highest adsorption and dynamic surface activity, approaching the behavior of CLSE. All mixtures containing 0.75% SP-B reached minimum surface tensions

Composition, surface activity and effects on pressure–volume (P–V) mechanics are examined for lavaged calf lung surfactant (LS) and the clinical exogenous surfactants Infasurf and Survanta. Lavaged LS and Infasurf had closely-matching compositions of phospholipids and neutral lipids. Survanta had higher levels of free fatty acids and triglycerides consistent with its content of added synthetic palmitic acid and tripalmitin. Infasurf and Survanta both contained less total protein than LS because of extraction with hydrophobic solvents, but the total protein content relative to phospholipid in Survanta was about 45% lower than in Infasurf. This difference was primarily due to surfactant protein (SP)-B, which was present by ELISA at a mean weight percent relative to phospholipid of 1.04% in LS, 0.90% in Infasurf, and 0.044% in Survanta. Studies on component fractions separated by gel permeation chromatography showed that SP-B was a major contributor to the adsorption, dynamic surface activity, and P–V mechanical effects of Infasurf, which approached whole LS in magnitude. Survanta had lower adsorption, higher minimum surface tension, and a smaller effect on surfactant-deficient P–V mechanics consistent with minimal contributions from SP-B. Addition of 0.05% by weight of purified bovine SP-B to Survanta did not improve surface or physiological activity, but added 0.7% SP-B improved adsorption, dynamic surface tension lowering, and P–V activity to levels similar to Infasurf. The SP-B content of lung surfactants appears to be a crucial factor in their surface activity and efficacy in improving surfactant-deficient pulmonary P–V mechanics.

Several protocols for purification of milligram quantities of lung surfactant proteins (SP)-B and SP-C were studied for separation efficiency and surface activity of the isolated proteins recombined with synthetic phospholipids (SPL). SP-B and SP-C were obtained from calf lung surfactant extract by C8 chromatography with isocratic elution by either of three solvent systems: 7:1:0.4 MeOH/CHCl(3)/5% 0.1 M HCl (solvent A), 7:1 MeOH/CHCl(3)+ 0.1% TFA (solvent B), and 7:1:0.4 MeOH/CHCl(3)/H(2)O + 0.1% TFA (solvent C). Solvents A and C yielded pure apoprotein in a single pass, with estimated total protein recoveries of85 and90%, respectively. Solvent B was less effective in purifying SP-B and SP-C, had a lower recovery efficiency, and gave isolates with less surface activity. Mixtures of SPL plus SP-B eluted with solvents A and C adsorbed to equilibrium surface tensions of 21-22 mN/m and reached minimum surface tensions1 mN/m during dynamic cycling. Mixtures of SPL with SP-C obtained with solvents A and C had equilibrium surface tensions of 26-27 mN/m and minimum dynamic values of 2-7 mN/m. The ability to obtain milligrams of virtually lipid-free SP-B and SP-C in a single column pass will facilitate research on their biological, structural, and biophysical properties.

Introduction. Previous trials of surfactant therapy in premature infants have demonstrated a survival advantage associated with prophylactic therapy as an immediate bolus, compared with the rescue treatment of established respiratory distress syndrome. The optimal strategy for prophylactic therapy, however, remains controversial. When administered as an endotracheal bolus immediately after delivery, surfactant mixes with the absorbing fetal lung fluid and may reach the alveoli before the onset of lung injury. This approach, however, causes a brief delay in the initiation of standard neonatal resuscitation, including positive pressure ventilation, and is associated with a risk for surfactant delivery into the right main stem bronchus or esophagus. As an alternative approach, surfactant prophylaxis may be administered in small aliquots soon after resuscitation and confirmation of endotracheal tube position. Although this strategy has substantial logistical advantages in clinical practice, its efficacy has not been established. Objective. The purpose of this study was to determine whether the established benefits of the immediate bolus strategy for surfactant prophylaxis could still be achieved using a postventilatory aliquot strategy after initial standard resuscitation and stabilization. Design. Multicenter randomized clinical trial with patients randomized before delivery to immediate bolus or postventilatory aliquot therapy. Participants. Inborn premature infants delivered to mothers at an estimated gestational age of 24[07] to 28[67] weeks. Interventions. Those infants who were randomized to the immediate bolus strategy were intubated as rapidly as possible after birth, and a 3-mL intratracheal bolus of calf lung surfactant extract (Infasurf) was administered before the initiation of positive pressure ventilation. Those infants who were randomized to the postventilatory aliquot strategy received standard resuscitation measures with intubation by 5 minutes of age, if not required earlier. At 10 minutes after birth, 3 mL of surfactant was administered in 4 divided aliquots of 0.75 mL each. Patients in both groups were eligible to receive up to three additional doses of surfactant as rescue therapy in the neonatal intensive care unit, if needed. Outcome Measures. The primary outcome variable was survival to discharge to home. Secondary variables included neonatal complications and requirement for oxygen therapy at 36 weeks' postmenstrual age. Results. Among three centers, 651 infants were enrolled and randomized before delivery. Survival to discharge to home was similar for the two strategies for surfactant therapy as prophylaxis: 76% for the immediate bolus group and 80% for the postventilatory aliquot group. In a secondary analysis, the rate of supplemental oxygen administration at 36 weeks' postmenstrual age was 18% for the immediate bolus group and 13% for the postventilatory aliquot group. Conclusions. Survival to discharge to home was similar with immediate bolus and postventilatory aliquot strategies for surfactant prophylaxis. Because of its logistical advantages in the delivery room and its beneficial effects on prolonged oxygen requirements, we recommend the postventilatory aliquot strategy for surfactant prophylaxis of premature infants delivered before 29 weeks' gestation.

Lung injuries, including bronchopulmonary dysplasia, alter the surfactant system. We developed a newborn rabbit model of acute, followed by chronic, hyperoxic injury to study surfactant protein (SP) gene expression. Initial litters were exposed to >95% O2 until 50% died (LD50; 7-11 days old). Subsequent litters were exposed to >95% O2 for 8 days, followed by 60% O2 until 22-36 days. Controls were exposed to room air. LD50 animals displayed acute pulmonary inflammation, edema, protein leak, and surfactant dysfunction. These changes resolved, and fibrosis developed by 22 days. Whole lung SP-A mRNA expression (measured by membrane hybridization) was twice control levels at 4 days of >95% O2, with specific elevations in terminal bronchioles and type II cells at 4 days and the LD50 by in situ hybridization. Whole lung SP-B and SP-C mRNA were unchanged from control throughout exposure. However, in situ hybridization showed elevations in SP-B and SP-C mRNA in type II cells in inflamed areas at the LD50. SP mRNA alterations resolved by 22-36 days. The surfactant system recovers from acute hyperoxic injury, despite continued 60% O2 exposure.

The content of dipalmitoyl phosphatidylcholine (DPPC) in the phosphatidylcholine (PC) fraction of calf lung surfactant extract (CLSE) is measured by gas chromatography (GC) and estimated from the widely used osmium tetroxide assay for disaturated phosphatidylcholine (DSPC). The surface-active properties of model phospholipid/apoprotein surfactants with varying DPPC content are also defined and compared relative to CLSE. GC analysis of fatty acids in PC isolated from CLSE indicated a possible range of 30 to 65% for DPPC content depending on C16:0 fatty chain mismatching, and further studies using phospholipase A2 treatment indicated an actual DPPC content≤41%. The osmium tetroxide assay gave a very high value of 70% for the DSPC content of surfactant PC, and experiments with synthetic phospholipids demonstrated that this assay responded inappropriately in the presence of monounsaturated PC, leading to falsely elevated DSPC values. The influence of DPPC content on adsorption and film behavior was investigated in model surfactants containing 40, 60, and 80% DPPC (DPPC/egg PC/egg PG, 40:50:10, 60:30:10, and 80:10:10 by mol) combined with 1.3% hydrophobic surfactant protein (SP)-B and -C. The biophysical properties of the model surfactant with 40% DPPC were found to be closer to CLSE than those of mixtures with 60 or 80% DPPC. The adsorption of dispersions containing 40% DPPC with 1.3% SP-B, C was almost identical to CLSE and was improved in rate and magnitude compared with the mixtures with higher DPPC content (60 or 80%). In Wilhelmy balance studies of cycled films, respreading was increased and maximum surface pressure was decreased for the 40% versus higher DPPC content mixtures, again approaching CLSE in behavior. All synthetic phospholipid (SPL):SP mixtures lowered surface tension to

Rabbits treated with intravenous free fatty acid suffer an acute lung injury. Material obtained from these lungs by bronchoalveolar lavage (BAL) has dramatically impaired ability to lower surface tension in vitro despite normal levels of surfactant phospholipids. Although large quantities of surface-active inhibitors are present in BAL, their effects are not sufficient to explain the magnitude of surfactant inactivation. This study determines if alterations in the surfactant aggregates can explain the loss of surfactant function in fatty acid lung injury. In injured animals, the larger, most active surfactant particles recovered by centrifugal pelleting were found to be decreased in amount. The remaining large particles had reduced surface activity compared with control aggregates. In addition, large particles in injured animals had a higher density than control animals on sucrose gradients following equilibrium centrifugation. Interaction with serum components present in the injured BAL could explain these higher densities. The ability of the injured BAL to lower surface tension was improved by supplementation with normal levels of particles from injured lungs. Supplementation of injured BAL with control large aggregates improved activity further and restored the ability to lower surface tension to1 mN/m. Therefore both the decreased amount and activity of large surfactant aggregates in injured animals contributed significantly to the observed inactivation of surfactant. Diminished surfactant function from alteration in surfactant aggregates is a mechanism common to other forms of acute lung injury, and the design of therapies with exogenous surfactants in injured lungs will need to consider strategies that restore surfactant function towards normal.

This paper examines two factors, shape deformation and surface viscosity, that affect measurements of surface tension of lung surfactants with the oscillating bubble surfactometer. At lower surface tensions, the compressed bubble in this apparatus becomes deformed to an oblate ellipsoid that cannot be analyzed rigorously using the simplified (spherical) Laplace equation to calculate surface tension from interfacial pressure drop. However, for the small air bubbles present in this apparatus, analysis with more general equations for ellipsoids of revolution shows that deformation effects are limited to extremely low surface tensions, and the absolute error from the spherical approximation is minimal in practice. In contrast, this was not the case for the effects of surface dilational viscosity in oscillating bubble calculations. Direct measurements and values from the literature indicated that the surface dilational viscosities of lung surfactant, dipalmitoyl phosphatidylcholine, and palmitic acid were sufficient to give substantial errors if their effects on interfacial pressure drop were neglected during dynamic cycling. Surface tension calculations at maximum and minimum radii on the oscillating bubble apparatus remain accurate, because the time derivative of radius becomes zero and viscous effects vanish. However, surface tensions determined at points other than these extremes of bubble size should be interpreted with caution.

Twenty-eight newborn infants (birthweight, 2.4 ± 1.1 kg; gestational age, 34.6 ± 6.1 weeks) with respiratory distress syndrome (RDS), meconium aspiration syndrome, or pneumonia who deteriorated in spite of optimal conventional mechanical ventilation (CMV) and exogenous surfactant therapy were treated with high-frequency jet ventilation (HFJV) and continued surfactant therapy. For enrollment, infants had to have a limited response to surfactant therapy and conventional ventilation, and meet clinical criteria that confirmed clinical deterioration and severity of illness. Study infants had received exogenous calf lung surfactant extract (CLSE) and conventional ventilation prior to the start of HFJV at 46.3 ± 8.2 hours of age. Patients initially responded to HFJV alone with significant improvement in several respiratory variables, but deteriorated subsequently and received additional doses of exogenous surfactant on HFJV. Exogenous surfactant and HFJV resulted in significant and sustained improvement in several respiratory variables. Only ten patients deteriorated to meet criteria for a second surfactant dose on HFJV, and two patients received a third dose. Twenty-five of the 28 patients studied survived (89%). No patients received extracorporeal membrane oxygenation or were discharged home on oxygen. The results of this pilot study suggest that the combination of HFJV and exogenous surfactant replacement may be effective in treating infants with more severe respiratory failure, and indicate the need for more extensive controlled investigations. © 1992 Wiley-Liss, Inc.

The inhibitory effects of oleic acid (OA) on the surface activity of pulmonary surfactant were characterized by use of the oscillating bubble surfactometer, the Wilhelmy balance, and excised rat lungs. Oscillating bubble studies showed that OA prevented lavaged calf surfactant [0.5 mM phospholipid (PL)] from lowering surface tension below 15 mN/m at or above a molar ratio of OA/PL = 0.5. In contrast to inhibition of surfactant by plasma proteins, increasing the surfactant concentration did not eliminate inhibition by oleic acid, which occurred at OA/PL greater than 0.67 on the oscillating bubble even at surfactant concentrations of 1.5 and 12 mM PL. Studies of surfactant adsorption showed that preformed films of OA had little effect on the adsorption of pulmonary surfactant. Wilhelmy balance studies showed that OA did interfere with the ability of spread films of surfactant to reach low surface tensions during dynamic compression. Further balance experiments with binary films of OA and dipalmitoyl phosphatidylcholine showed that these compounds were miscible in surface films. Together these findings suggested that OA inhibited pulmonary surfactant activity by disrupting the rigid interfacial film responsible for the generation of very low surface tension during dynamic compression. Mechanical studies in excised rat lungs showed that instillation of OA gave altered deflation pressure-volume characteristics with decreased quasi-static compliance, indicating disruption of pulmonary surfactant function in situ. This alteration of mechanics occurred without major changes in the composition of lavaged PLs or in the tissue compliance of the lungs defined by mechanical measurements during inflation-deflation with saline.(ABSTRACT TRUNCATED AT 250 WORDS)

The biophysical properties and physiologic effects of a series of clinical exogenous pulmonary surfactants was compared to determine the importance of the hydrophobic apoproteins (SP-B and C) as constituents of these preparations. The three exogenous surfactants studied, calf lung surfactant extract (CLSE), Survanta (Surfactant-TA), and Exosurf, all contain dipalmitoyl phosphatidylcholine (DPPC) as their major constituent. CLSE and Survanta also contain 1 to 2% of SP-B,C but Exosurf has the additives hexadecanol and tyloxapol instead to enhance the activity of DPPC. In adsorption experiments, CLSE reached a final surface tension of 22 mN/m, and Survanta and Exosurf reached 28 and 38 mN/m, respectively. Addition of 1% by weight of an apoprotein isolate containing both SP-B and C to Exosurf slightly improved its adsorption. In oscillating bubble studies, CLSE and Survanta decreased surface tension to low values of less than 1 and 2 mN/m, respectively, but Exosurf achieved a minimum value of only 29 mN/m. Addition of SP-B,C to Exosurf improved this minimum to 1 mN/m and approached the behavior of mixtures of synthetic DPPC with SP-B,C. In both adsorption and pulsating bubble experiments, the minimum surface tensions found for Exosurf were almost identical to those generated by tyloxapol alone. In studies of physiologic activity, 20 mg of CLSE or Survanta restored the pressure-volume mechanics of lavaged, surfactant-deficient excised rat lungs to 95 and 50%, respectively, of normal prelavage levels. Instillation of Exosurf (37.5 mg) produced a minimal improvement of only 10% compared to 70% for mixtures containing 1% SP-B,C with either Exosurf or DPPC.(ABSTRACT TRUNCATED AT 250 WORDS)