Surfactant lipid component and its uptake by alveolar monocytes.

<p>(A) Saturated phosphatidylcholine (Sat PC µmol/kg) in CHF5633 (Input), Survanta (Input), lamb lung, and BALF were analyzed. Two fold higher Sat PC was instilled to CHF5633 group than Survanta group and Sat PC in the lung was higher for CHF5633 group than Survanta group. *p<0.001 vs. Survanta In contrast, Sat PC in BALF was similar between the 2 groups, suggesting increased CHF5633 catabolism compared with Survanta. (B) Microphotograph of alveolar cells. Phagocytosis of surfactant by monocytes was higher for the CHF5633 group and monocytes contained lipid droplets. Scale bar: 10 µm. (C) Phosphatidylcholine (PC) and phosphatidylglycerol (PG) were analyzed by thin layer chromatography. There were similar amounts of PC and PG in CHF5633 and the major component of Survanta was PC. The ratio of PC to PG did not change in the ventilated preterm lamb lung and was similar to Input samples.</p

Lung inflammation.

<p>Before surfactant treatment, lambs were ventilated for 15 min with limited PIP to 40 cmH₂O that resulted in low V_T of 5.3 ml/kg. Thus, extremely immature lungs with low compliance were not overstretched and lung inflammation was minimized. (A) Number of total cells, neutrophils, and monocytes in BALF were low for both groups. Although the inflammatory cells tended to be lower for CHF5633, they were not statistically different. (B) Expression of IL-1β, IL-6, IL-8, and TNFα mRNA were analyzed by RT-PCR. Although the mean value of IL-1β tended to be lower for the CHF5633 group, there were no statistical differences in IL-1β, IL-6, IL-8, and TNFα mRNA between CHF5633 and Survanta groups.</p

Surfactant ultrastructure.

<p>(A) CHF5633. Ultrastructure of CHF5633 Input was extremely large with ball bearing like form. In the supernatant of the BALF, ultrastructure of small aggregate of CHF5633 was lipid layers with various forms. The recovered CHF5633 in the pellet from BALF were large, softly aggregated lipid particles. (B.) Ultrastructure of Survanta was simple lipid layers that were changed to small lipid vesicle or lamellated structure in the preterm lamb lung. (C.) Air group or Endogenous Surfactant. In the extremely preterm lamb lung airways, small amounts of surfactant with normal ultrastructure including lamellar bodies and tubular myelin were present. Scale bar: 2 µm: Scale bar in inset: 0.5 µm.</p

Oxygenation.

<p>These preterm newborn lambs required high Fio₂ (A) to maintain Po₂ (B) at targeted level. Ratio of Po₂ to Fio₂ (C) was improved soon after surfactant treatment. Although not significant, the increase in Po₂/Fio₂ after treatment tends to be faster by CHF5633 than Survanta (p = 0.052 at 20 min).</p

pH and modified ventilation index (MVI).

<p>(A) In spite of ventilation with high PIP for 15 min after birth, pH was decreased in association with high Pco₂. pH was improved after CHF5633 or Survanta treatment. (B) MVI was calculated as peak inspiratory pressure x Pco₂ x respiratory rate/1,000. MVI was similarly improved by CHF5633 and Survanta treatment. Some error bars are within symbols.</p

Increased Uptake of CHF5633 by Alveolar Monocyte.

<p>(A) Cell pellet from BALF isolated by 10 min centrifugation at 284× g. The microphotographs were without fixation or staining. Extremely large and soft aggregate of CHF5633 was recovered in the pellet together with cells. Detected Survanta in the cell pellet was minimum after short and low-speed centrifugation and only the cells were detected. Scale bar: 50 µm. (B) Sat PC in pellet and supernatant of input surfactants and BALFs were analyzed. Over 40% of CHF5633 were large and heavy aggregates and were recovered in the pellet. *p<0.001 vs. Survanta. The percent Sat PC in the pellet from the CHF5633 group BALF was higher than that of Input CHF5633. tp<0.05 vs. input CHF5633 pellet. Some of the standard error bars are within the mean value bars. (C) SP-C in pellet and supernatant samples of BALF from CHF5633 group, containing 6.5nmol Sat PC were analyzed by Western blot. SP-C in CHF5633 input sample was given a value of 1. SP-C relative to Sat PC was decreased in supernatant or smaller form surfactant to half of that in input sample and BALF pellet samples. *p<0.05 vs. others. (D) In the surfactant pellet of BALF from the CHF5633 group, a large number of immunogold-labeled SP-C particles (arrow) were associated with surfactant lipid vesicles. Image is representative of findings in n = 3 lambs. Scale bar: 0.1 µm.</p

Lung Histology.

<p>Lung histology of peripheral area assessed after staining with hematoxylin and eosin. Both CHF5633 (A) and Survanta (B) groups showed various size alveoli with patchy atelectasis. The lung of the Air group (C) was atelectatic. Scale bar: 100 µm.</p

Lung appearance.

<p>Representative appearance of the lung for CHF5633 group (A), Survanta group (B), and Air group (C). CHF5633 expanded lungs uniformly for the most part. Half of the Survanta treated lambs had dark colored injured and small hemorrhagic areas in the lung. The lungs of the Air group were atelectatic with bullas.</p

Peak inspiratory pressure (PIP), tidal volume (V_T), and pCO₂.

<p>Preterm newborn lambs were resuscitated after birth with (A) high peak inspiratory pressure (PIP) to regulate (C) tidal volume (V_T) at 6 ml/kg. CHF5633, Survanta, or Air was given at 15 min of age and ventilation was changed to regulate (B) Pco₂ at 50–60 mmHg. Lung immaturity was comparable between the groups and all the control (Air) lambs died within 90 min of age. PIP and Pco₂ were similarly improved by treatment with CHF5633 and Survanta. Initial response of V_T to surfactant treatment was faster by CHF5633 than Survanta. *p<0.03. Some error bars are within symbols.</p

Lung compliance.

<p>(A) Lung compliance of the preterm newborn lamb was improved by both CHF5633 and Survanta treatment. Overall compliance after treatment was not significantly different between CHF5633 and Survanta group (p>0.05 by two-way repeated measures ANOVA). Compliance was higher for CHF5633 than Survanta group at 20 min and 300 min of age (*p<0.04 by t-test). (B) The overall deflation limbs of pressure-volume curves were similar between CHF5633 group and Survanta group (p>0.05 by two-way repeated measures ANOVA). The volume at 10 cmH₂O was higher for CHF5633 group than Survanta group (*p<0.05 by t-test).</p