INTRODUCTION

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Surfactants are routinely used to treat preterm infants with respiratory distress syndrome, and are being evaluated for the treatment of other lung diseases, such as meconium aspiration in infants and adults with acute respiratory distress syndrome (ARDS) (1). Synthetic surfactants that contain lipids only are useful in preterm infants with respiratory distress syndrome, although natural surfactants that contain the hydrophobic surfactant proteins B and C (SP-B and SP-C) are more effective at decreasing pneumothorax and mortality (4). Surfactants that contain the hydrophobic surfactant proteins have better surface biophysical properties (5), are more resistant to inhibition by edema fluid (6), and cause larger increases in compliance in animal models of surfactant deficiency (7, 8). Although SP-A is the most abundant surfactant protein in native surfactant, its effects on biophysical properties are less striking than are the effects of SP-B and SP-C (8). SP-A is not present in the lung-derived surfactants used clinically, and mice that lack SP-A have normal lung function and surfactant metabolism (11, 12).

The synthetic and recombinant production of the hydrophobic surfactant proteins SP-B and SP-C has been the focus for the development of synthetic surfactants (9, 13). There is no consensus about the relative importance of SP-B or SP-C or both proteins for optimal function of surfactants for clinical use. SP-B is an essential component of endogenously produced surfactant, because its genetic absence results in lethal respiratory failure in full-term infants, and anti-SP-B antibodies cause respiratory failure in adult animals (16, 17). The genetic absence of SP-B results also in a deficiency in SP-C, because the proprotein is not appropriately processed (18). Several reports have described mixtures of SP-B and lipids as yielding surfactants that are more effective at improving compliance and are more resistant to inactivation by plasma proteins than are SP-C-based surfactants (5, 7, 19). Although SP-C has less effect than SP-B on the adsorption rate of phospholipids, it enchances film stability and respreading after film collapse (20). Concerns about previous observations made with native SP-C are the purity of the preparations and the tendency of SP-C to aggregate (20). In recent work, synthetic analogues of SP-C mixed with phospholipids have resulted in surfactants with good biophysical properties that can increase the lung volumes of preterm rabbits comparably with natural surfactant (15, 21). Recombinant SP-C (rSP-C)-based surfactants also were effective in surfactant deficiency created by saline lung lavage in adult animals (9, 22). The goals of the present study were to evaluate, in two models of surfactant deficiencyventilated preterm rabbits and lambsa new analogue of human SP-C that contains phenylalanine instead of cysteine in positions 4 and 5, and isoleucine instead of methionine in position 32 of the SP-C protein. This analogue was chosen because the amino-acid substitutions prevented aggregation and yielded a protein with biophysical properties similar to those of native palmitoylated SP-C.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Surfactant

Natural surfactant was isolated from alveolar lavages of adult sheep lungs through a three-step centrifugation procedure that included a 0.8 M sucrose gradient (7). The surfactant was extracted with chloroform/methanol (2:1) (vol/vol). The lipid extract of sheep surfactant was dried on a round-bottom flask by flash evaporation at 50° C, and was resuspended with glass beads in 0.9% NaCl to a concentration of 25 mg/ml. This organic solvent extract of sheep surfactant contains SP-B and SP-C but no SP-A, is similar to the surfactants used clinically, and will be called natural surfactant. rSP-C surfactant was provided by Byk Gulden (Konstanz, Germany). A recombinant 34-amino-acid human SP-C sequence, altered by replacement of cysteine in positions 4 and 5 with phenylalanine, and of methionine in position 32 by isoleucine, was expressed in bacteria and purified. The surfactant contained 2% rSP-C in phospholipids (dipalmitoylphosphatidylcholine [DPPC] and palmitoyloleoylphosphatidylglycerol in a 70:30 [wt/ wt] ratio), plus 5% palmitic acid. Palmitic acid was added to facilitate the preparation of this surfactant, and is a component of other surfactants used clinically. The rSP-C surfactant was suspended in 0.9% NaCl to a concentration of 25 mg phospholipid/ml with a Pasteur pipette.

Surface-tension Measurements

A Wilhelmy balance with a platinum dipping plate was used to measure minimum surface tensions after the fourth cycle from a maximum area of 64 cm² to a minimum area of 12.8 cm² at a temperature of 37° C and a rate of 3 mm/cycle (23). Surface tensions for natural and rSP-C surfactants were measured at dilutions ranging from 10 to 75 µg/ml lipid in 35 ml saline. Plasma was then added to 75 µg/ml surfactant and mixed with it by hand with a glass rod, and surface-tension measurements were repeated (23).

Preterm Lambs

Delivery and ventilation of preterm lambs. Pregnant ewes at 128 d gestation were preanesthetized with ketamine (20 mg/kg intramuscularly) and given spinal-epidural anesthesia (10 ml 2% lidocaine-0.5% marcaine [1:1, wt/wt]). After its head had been mobilized, the fetal lamb was anesthetized with ketamine (10 mg/kg intramuscularly) plus acepromazine (0.1 mg/kg intramuscularly). Following local anesthesia to the anterior neck with 2% lidocaine, a 4.5-mm ID tracheal tube was tied into the trachea, freely flowing fluid from fetal airways was removed by syringe, and the endotracheal tube was clamped (23). The umbilical cord was then cut and the fetus was delivered and weighed. Immediately after birth and before breathing, each preterm lamb was randomly treated with natural surfactant or rSP-C surfactant (100 mg lipid/kg body weight). The lambs were then ventilated for 5 h with time-cycled, pressure-limited ventilators (Sechrist Industries, Anaheim, CA), with initial settings of FI_O2 = 1; rate = 40 breaths/min; PIP = 35 cm H₂O; PEEP = 3 cm H₂O; flow = 10 L/min; and inspiratory time = 0.7 s. Subsequently, only FI_O2 and PIP were changed, in an attempt to maintain the arterial PO₂ in the range of 100 to 200 mm Hg and the arterial PCO₂ at 50 mm Hg by 1 h of age. Over the first hour, tidal volumes were limited to 6 to 8 ml/kg in order to minimize injury to the lungs, using a neonatal pulmonary monitor (Bicore, Irvine, CA) to measure tidal volumes (23). A catheter was placed in the aorta via an umbilical artery, and 5% dextrose was infused at 100 ml/kg/d. Each lamb received a transfusion of 10 ml/kg cord blood soon after birth. The arterial line was used for blood pressure monitoring and intermittent blood gas analysis. To assess the intravascular-to-interstitial alveolar albumin leak, 10 µCi of [¹³¹I]albumin was infused at 4 h of age (24).

VT was measured every hour with a pneumotachometer, and dynamic compliance was calculated as VT normalized to body weight and divided by the ventilatory pressure (positive inspiratory pressure [PIP]  PEEP). The ventilatory efficiency index (VEI) was calculated according to Notter and colleagues (25). This measurement evaluates the overall ventilation efficiency of mechanically ventilated animals, and compensates for differences resulting from the combined effects of changing ventilatory pressures, rates, and PCO₂. It is calculated from the following equation: VEI = 3,800  (P × R × Pa_CO2), where 3,800 is a constant for CO₂ production (ml/mm Hg/kg), P is PIP  PEEP (cm H₂O), R is respiratory rate (breaths/min), and Pa_CO2 is in mm Hg. The functional residual capacity (FRC) was measured by the helium dilution technique (Equilibrated Bio Systems Inc., Melville, NY) (26).

Body temperature was maintained in a physiologic range for lambs (between 38° C and 39° C) with radiant warmers and heating pads. Supplemental ketamine (10 mg/kg intramuscularly) and acepromazine (0.1 mg/kg intramuscularly) was given as necessary to suppress spontaneous ventilation. After 5 h of ventilation, each animal was deeply anesthetized with sodium pentobarbital (25 mg/kg intravenously) and ventilated briefly with 100% oxygen, and the endotracheal tube was clamped for 3 min to permit oxygen absorption. The animal was exsanguinated by cutting the abdominal aorta. The thorax was opened and the lungs were filled with air to 35 cm H₂O pressure for 1 min and maximal lung volume was recorded (26).

Processing of lungs. The lungs were removed from the thorax and an alveolar wash was performed by filling the lungs by gravity with 0.9% NaCl at 4° C until they were visually distended, and the alveolar wash was recovered with a syringe (24). The procedure was repeated five times; the five washes were pooled and aliquots were saved for determinations of saturated phosphatidylcholine (Sat-PC), total proteins, and [¹³¹I]albumin. The lungs were homogenized in 250 ml 0.9% NaCl, and aliquots of homogenates were used for measurements of Sat-PC, [¹³¹I]albumin, and total blood content.

The alveolar wash was centrifuged at 140 × g for 10 min, and the pellet of cellular debris was discarded. The supernatant was centrifuged at 40,000 × g for 15 min and the pellet containing the large- aggregate surfactant was resuspended in 0.9% NaCl, layered over 0.8 M sucrose in 0.9% NaCl, and centrifuged again at 40,000 × g for 15 min (23). The interface was recovered, diluted with 0.9% NaCl, and recovered as a pellet after centrifugation at 40,000 × g for 15 min. The large-aggregate surfactant was suspended in a small volume of 0.9% NaCl and frozen for testing in preterm rabbits.

Analytic techniques. Samples for Sat-PC measurement were extracted with chloroform-methanol (2:1, vol/vol) and treated with osmium tetroxide (27). Sat-PC was isolated by column chromatography with alumina, and was quantified with a phosphorus assay (28). Total protein in alveolar washes was measured with the Lowry assay (29). Radiolabeled albumin was made from monomer standard bovine serum albumin (BSA), using carrier-free ¹³¹I (ICN Pharmaceuticals, Irvine, CA). The [¹³¹I]albumin was extensively dialyzed before use, and a 10% tetrachloroacetic acid precipitation verified > 97% label incorporation. The intravascular [¹³¹I]albumin in the lung tissue was estimated from the amount of hemoglobin present in lung homogenates and the amount of [¹³¹I]albumin present in blood (24).

Preterm Rabbits

Preterm rabbits were used to evaluate the natural surfactant and the rSP-C surfactant at treatment doses of 100 mg/kg by ventilation with and without 3 cm H₂O PEEP (7). Other rabbits were used to evaluate the function of the large-aggregate surfactants recovered from the preterm lambs at a dose of 50 mg/kg, using ventilation with 3 cm H₂O PEEP. A 50-mg/kg dose had previously been used to evaluate surfactant recovered from preterm lambs (23). New Zealand White rabbits at 27 d ± 2 h gestation were lightly anesthetized with pentobarbital and then given spinal anesthesia, using 1.5 ml of 2% lidocaine-0.5% bupivacaine (1:1, vol/vol). The does received oxygen, and the preterm rabbits were sequentially delivered by cesarean section. The newborns were given acepromazine (0.1 mg/kg) and ketamine (10 mg/kg) by intraperitoneal injection. A tube made from an 18-gauge stainless-steel needle was secured into the trachea of each rabbit. Controls for each litter received no treatment, and the treated rabbits were randomized to receive surfactant by intratracheal instillation before lung inflation in a continuous sequence across each litter. Initial lung inflation was achieved with five breaths of 100% oxygen, using an anesthesia bag with just enough pressure to visibly move the chest. The rabbits were then transferred to a 37 ° C temperature-controlled ventilator- plethysmography system on initial settings of PIP = 35 cm H₂O; rate = 30 breaths/min; inspiratory time = 1 s; FI_O2 = 1; and PEEP = 0 or 3 cm H₂O. The PIP was individually adjusted for each rabbit to achieve tidal volumes of 8 ml/kg as measured with a pneumotachometer. At the end of 15 min of ventilation, the endotracheal tube was obstructed for 5 min to allow complete atelectasis to occur by oxygen absorption. Each rabbit was killed with intrathecal lidocaine. Quasistatic pressure-volume curve measurements were made at 37° C by inflating and deflating the lungs in increments of 5 cm H₂O pressure from 0 to 35 cm H₂O. Lung volumes were corrected for the compression volumes of the system, and expressed as ml/kg body weight (7).

Data analysis. Results are given as means ± SEM. Two-way analysis of variance (ANOVA) was used for comparison of differences in physiologic measurements during the 5-h ventilation period. All other comparisons were made with one-way ANOVA, and the Student- Newman-Keuls test was used as the discriminating post test. Comparisons of two groups were made with two-tailed t tests. Significance was accepted at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Surface-tension Measurements

Minimum surface tensions were lower at all dilutions for the rSP-C surfactant than for natural surfactant (Figure 1A). The curves for the inhibition of minimum surface tension by plasma were similar for the two surfactants (Figure 1B).

View larger version (23K): [in this window] [in a new window]   Figure 1.   Minimum surface tensions (A) and sensitivity to inhibition by plasma (B) for surfactants. Each figure gives the mean ± SEM of five measurements for each group at each dilution. The minimum surface tensions with rSP-C surfactant were significantly less than with natural surfactant at all concentrations to 75 mg/ml. The rSP-C and natural surfactants had similar resistance patterns to plasma protein. *p < 0.05.

Preterm Lambs

Nine lambs (2.5 ± 0.1 kg, five females and four males) were treated with natural surfactant, and nine lambs (2.5 ± 0.1 kg, seven females and two males) were treated with rSP-C surfactant. The cord pH values for the two groups were similar (7.37 ± 0.02 and 7.35 ± 0.02 for natural- and rSP-C-surfactant-treated animals, respectively). The ventilatory pressures and Pa_CO2 values were similar for the 5-h study period (Figure 2A and B). The mean VEI values for the two groups did not differ (0.107 ± 0.078 for natural-surfactant-treated and 0.105 ± 0.079 for rSP-C-surfactant-treated lambs). The compliance measurements were not different between the groups (Figure 3A). Similarly, FRC values were equivalent for the groups of lambs at each time of measurement (Figure 3B). Although there were no changes in PO₂ or FI_O2 with time over the 5-h study period for the two treatment groups, the mean PO₂ values were significantly higher for natural-surfactant-treated lambs (201 ± 10 mm Hg) than for rSP-C-surfactant-treated lambs (146 ± 8 mm Hg). The mean FI_O2 of 0.85 ± 0.02 for the natural-surfactant-treated lambs was significantly lower than the mean FI_O2 of 0.92 ± 0.03 for the rSP-C-surfactant-treated lambs. Therefore, there was a modest increase in oxygenation in the natural-surfactant-treated lambs relative to rSP-C-surfactant-treated lambs. Maximal lung volumes measured with 35 cm H₂O in the open-chest state were 52 ± 4 ml/kg for natural-surfactant-treated and 48 ± 4 ml/kg for rSP-C-surfactant-treated lambs, which were not different from one another.

View larger version (22K): [in this window] [in a new window]   Figure 2.   Sequential measurements of ventilatory pressure (A) and PaCO2 (B) in preterm lambs ventilated for 5 h and treated with either natural or rSP-C surfactant. There were no differences in the measurements.

View larger version (18K): [in this window] [in a new window]   Figure 3.   Sequential dynamic compliance (A) and FRC values (B). Compliances and FRCs measured at 1.5 h, 3.5 h, and 5 h were similar for lambs treated with natural or rSP-C surfactants.

The amounts of Sat-PC in the treatment doses (100 mg/kg) of the surfactants were 60 µmol/kg for natural- and 68 µmol/ kg for rSP-C-surfactant-treated lambs (Table 1). The amount of Sat-PC recovered by alveolar washing after 5 h ventilation was 14 ± 2 µmol/kg for natural-surfactant-treated lambs, which was 23% of the amount given. The recovery for the rSP-C-surfactant-treated lambs was 19 ± 3 µmol/kg, or 28% of the amount given. There was more Sat PC in the total lungs from the rSP-C-surfactant-treated lambs than from the natural-surfactant-treated lambs. The amounts of protein and the recoveries of intravascular [¹³¹I]albumin in alveolar washes were measured as indicators of lung injury. There were no differences in recoveries of [¹³¹I]albumin in the alveolar washes or total lungs (Table 1). Total protein in alveolar washes was not different in the two study groups.

Preterm Rabbits

Evaluation of treatment surfactants. The preterm rabbits that were treated with 100 mg/kg of natural or rSP-C surfactants had similar body weights and tidal volumes (Table 2). Ventilation with 3 cm H₂O PEEP resulted in large decreases in ventilatory pressures and increases in compliances that were similar for the two surfactants (Figure 4A). The natural surfactant decreased the peak pressures needed to ventilate the rabbits and increased compliance for rabbits ventilated without PEEP, although the effect was small compared with the improved compliance with PEEP. The rSP-C surfactant showed a trend toward improving the compliance of rabbits when ventilated without PEEP. The pressure-volume curves for rabbits within each group did not differ for the two PEEP groups. Therefore, curves for rabbits ventilated with and without PEEP and treated with natural and rSP-C surfactants were compared and contrasted with the pressure-volume curves for control rabbits. The lungs of the natural-surfactant-treated rabbits opened at a lower pressure than did those of the other groups (Figure 4B). Maximal lung volumes and the deflation limbs for the natural- and rSP-C-surfactant-treated lungs were similar and improved over control values. The maximal lung volumes for the natural-surfactant-treated lungs were slightly improved relative to the rSP-C surfactant-treated lungs (p < 0.05).

View larger version (30K): [in this window] [in a new window]   Figure 4.   (A) Dynamic compliances of preterm rabbits ventilated with and without 3 cm H2O PEEP and treated with 100 mg/kg of either natural or rSP-C surfactants. At 0 PEEP the natural-surfactant-treated group had improved compliance as compared with the control group. *p < 0.05. A PEEP of 3 cm H2O (3 PEEP) improved compliances equivalently in both the natural- and rSP-C-surfactant-treated groups. *p < 0.05. (B) Mean pressure-volume curves for preterm rabbits ventilated with and without PEEP. The curves for both surfactant-treated groups were improved when compared with the curve for the control group. *p < 0.05. The natural-surfactant-treated group had a slightly improved maximal lung volume and lower opening pressure versus the rSP-C-surfactant-treated group. p < 0.05.

Evaluation of surfactants recovered from preterm lambs. The surfactants used to treat the preterm lambs, and the large-aggregate surfactants recovered by alveolar washing after 5 h ventilation, were tested in preterm rabbits at a dose of 50 mg/ kg (Table 3). All surfactants tested with 3 cm H₂O PEEP and at tidal volumes of about 8 ml/kg produced large decreases in ventilatory pressures from the control value of 22 cm H₂O to about 11 cm H₂O. There were similar increases in compliance for the surfactants used to treat the lambs and for the surfactants recovered from the lambs (Figure 5A). There were no differences in the lung volumes for pressure-volume curves measured at 35 cm H₂O (Figure 5B). At 5 cm H₂O on deflation, the natural surfactant resulted in higher retained lung volumes than did the rSP-C surfactant, and the same result occurred for the surfactants recovered from the lambs (Figure 5C).

View larger version (27K): [in this window] [in a new window]   Figure 5.   Dynamic compliances (A) and lung volumes at 35 and 5 cm H2O (B and C ) in preterm ventilated rabbits given 50 mg/kg of the surfactants used to treat lambs and the large-aggregate (LA) surfactants recovered from lambs. There were no differences between the surfactants used to treat the lambs and the large-aggregate surfactants recovered from the lambs. *p < 0.05 versus control; tp < 0.05, natural > rSP-C > control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The recombinant SP-C used in these experiments was remarkably effective in two standard preterm animal models of surfactant deficiency (30). Lung function of preterm lambs was equivalent after treatment with either the rSP-C or natural surfactant, except for a small difference in oxygenation. We did not include untreated lambs because preterm lambs delivered at 128 d gestation and not treated with surfactant will die of severe respiratory failure despite the use of ventilatory pressures of 40 cm H₂O (31). We also did not evaluate the lipid mixtures that did not contain rSP-C, because such mixtures do not perform well in preterm rabbits or lambs (1, 7, 9). The treated lambs in our study required ventilatory pressures of less than 20 cm H₂O to normalize PCO₂ values. The small difference in oxygenation is not explained by a difference in FRC, which was more than 20 ml/kg for both groups of lambs. Because tidal volumes and compliances also were similar, the differential oxygenation is probably explained by differences in ventilation-perfusion matching as a result of subtle differences in the uniformity of inflation of alveoli. A major effect of surfactant treatment in preterm lambs is to make alveolar inflation more uniform (1). The lungs of preterm rabbits treated with 100 mg/kg of rSP-C surfactant had higher opening pressures, and the retained lung volumes at 5 cm H₂O on deflation were lower for rabbits treated with the rSP-C surfactant recovered from preterm lambs than for rabbits treated with natural surfactant. These are subtle differences in performance that might result in differences in the uniformity of alveolar inflation in lung regions receiving less surfactant. Although we used a treatment technique to try to optimize surfactant distribution, some regions of lung will receive low amounts of surfactant (26). Oxygenation in preterm lambs depends on shunts as well as lung function, and the cardiovascular status of the lambs was not evaluated. Overall, the lambs had very similar responses to the two surfactants.

The responses of the preterm rabbits to treatment with the rSP-C surfactant when the rabbits were ventilated with 3 cm H₂O PEEP were equivalent to those of the natural-surfactant-treated rabbits. However, the rSP-C surfactant was not effective in the absence of PEEP. Similar results were reported by other groups (7, 8). Rider and colleagues (7) and Ogawa and associates (8) showed that neither natural nor synthetic lipid mixtures lacking SP-A, SP-B, and SP-C improved compliances of preterm rabbits unless PEEP was used. Hawgood and colleagues (9) found that surfactant containing another recombinant SP-C analogue did not improve compliance unless PEEP was used. There also are differences in the effects of PEEP on surfactant function that depend on the model used. For example, Häfner and coworkers (22) reported no response to treatment of rat lungs injured by saline lavage with natural or synthetic surfactant. The organic-solvent-extracted natural surfactant used for these experiments was not as effective in the absence of PEEP in preterm rabbits as were natural surfactants containing SP-A (7). The testing of surfactants in animal models without PEEP can reveal differences in surfactants that are not apparent otherwise. However, from the practical perspective, surfactants are used clinically with mechanical ventilation and PEEP, or with continuous positive airway pressure, and any differences between natural and rSP-C surfactant would not be evident.

In previous reports, SP-B was considered to be the most essential protein component for surfactant function (16). In congenital SP-B deficiency, infants die of severe respiratory failure (16). Disruption of the SP-B gene results in abnormal processing of SP-C and a lack of lamellar bodies in type II cells (18). Therefore, the absence of SP-B also causes a deficiency in the mature form of SP-C. Rider and colleagues (7) found that surfactants that contain native SP-B only were more effective at improving the compliance of preterm rabbits than were surfactants containing only SP-C. However, a caveat with testing an SP-B-based surfactant in the immature lung is that SP-C is present, although the quantities have not been measured. The messenger RNA (mRNA) levels for SP-C are relatively high even at early gestation in the rabbit and lamb (32, 33).

An rSP-C analogue containing serine in positions 5 and 6 in place of cysteine was formulated into a surfactant that improved lung volumes and compliances in preterm rabbits to a similar extent as natural surfactant (9). Our results with the rSP-C surfactant tested in this study also show that SP-C and lipids alone can yield an effective surfactant. Native SP-C is very difficult to purify, to measure, and to prevent from aggregating (20). Therefore, previous results with native SP-C were probably less positive because the quality of the SP-C used in those experiments was poor. The rSP-C used in the present study does not aggregate in organic solvents and is stable when associated with lipids in saline suspensions. The phenylalanine substitutions confer characteristics on the peptide that are similar to those of the normally dipalmitoylated cysteines in native SP-C. Palmitoylation is thought to improve the interactions of SP-C with phospholipid and to increase film stability (34). The importance of palmitoylation of SP-C has not been evaluated in vivo. Although evaluated in different models, the rSP-C tested in the present study performed similarly to a dipalmitoylated rSP-C that was evaluated in saline-lavaged rats (22).

The minimal surface tensions measured for the rSP-C-based surfactant were remarkably low, even at high dilution. Of particular note was the equivalent sensitivity of the natural and rSP-C surfactants to inhibition by plasma. SP-C-based surfactants have been reported to be more sensitive to inhibition by plasma than SP-B-based surfactants or natural surfactants (19). The difference between the present results and those in the previous reports may be due either to a unique property of the rSP-C used in the present study or to the state of the protein having minimized its aggregation.

The phospholipids and protein components of surfactant normally become integrated into the recycling pathways of the lung. These metabolic effects explain the prolonged effects and enhanced properties of surfactants after treatment (35). The metabolic fates of analogues of surfactant proteins remain to be evaluated. When tested in the same models, the rSP-C-based surfactant used in the present study compares favorably with modified natural surfactants that are used clinically, and is superior to synthetic surfactants containing lipids and no surfactant protein (7, 30, 35, 36). In conclusion, this study demonstrates that rSP-C can be formulated into a surfactant that should be very effective as therapy for respiratory distress syndrome.