Donald L. Shapiro

Hydrophobic Surfactant-Associated Protein in Whole Lung Surfactant and Its Importance for Biophysical Activity in Lung Surfactant Extracts Used for Replacement Therapy

ABSTRACT. Hydrophobic protein of 6,000 and 14,000 daltons was isolated from mammalian pulmonary surfactant obtained from canine, human, and bovine alveolar lavage material. Low molecular weight, hydrophobic, surfactant- associated protein (SAP), herein referred to as SAP 6-14, was distinguished from SAP-35, the major glycoprotein in mammalian surfactants (the 35,000 dalton glycoprotein A or apolipoprotein A) by amino acid composition, peptide mapping, and by resistance of SAP 6-14 to digestion by endoglycosidase F, collagenase, trypsin, and other proteases. The amino acid composition of SAP 6-14 was found to be highly enriched in leucine and other hydrophobic amino acids. The characteristics of protein isolated from bovine replacement surfactant extracts utilized for the treatment of hyaline membrane disease in humans were also studied. SAP 6-14 isolated from calf lung surfactant replacement extracts (CLSE) and surfactant- TA were found to be identical to SAP 6-14 isolated from ether/ethanol extracts of various mammalian surfactants. By contrast, SAP-35, the major surfactant-associated glycoprotein of molecular weight=35,000, and other higher molecular weight proteins were not detected in significant quantities in the CLSE or surfactant-TA replacement surfactants, either by highly sensitive silver stain analysis or by immunoblot using monospecific antisera generated against bovine SAP-35. Biophysical studies of the CLSE replacement surfactant containing only SAP 6-14 and native phospholipids demonstrated full surface activity compared to natural lung surfactant. Dynamic surface tension lowering and adsorption properties of CLSE were essentially identical to those of freshly isolated bovine whole surfactant. Thus, hydrophobic SAP 6-14 is the only protein detected in bovine lung extract surfactants with full biophysical activity. The major surfactant associated protein, SAP-35, was not a significant component of either the CLSE or surfactant-TA replacement preparations.

Lung surfactant replacement in premature lambs with extracted lipids from bovine lung lavage: effects of dose, dispersion technique, and gestational age.

ABSTRACT: Extracted bovine calf lung lipids (CLL) with minimal protein (approximately 1 %) were instilled prior to ventilation in groups of premature lambs of average gestational ages of 127 and 133 days. Aqueous dispersions of CLL were prepared by two techniques prior to instillation: sonication in an ice bath (S) and mechanical vortexing at room temperature (V). A low surfactant dose (15 mg CLL/kg animal weight) and a high dose (100 mg/kg) were investigated for each dispersion technique. Following tracheal instillation of surfactant, lambs were ventilated with 100% oxygen for 2 h with umbilical circulation intact, and for up to an additional 10 h after separation. A clear improvement in blood oxygenation and lung compliance was found over controls for lambs given 15 mg/kg and 100 mg/kg CLL(V), and 100 mg/kg CLL(S). Lambs treated with 15 mg/kg CLL(S) failed to improve over controls. Experimental groups treated with equal doses of CLL(V) and CLL(S) had similar amounts of lung lavage phospholipid, with values progressively declining during ventilation. Analyses of in vitro surface properties showed that both vortexed and sonicated CLL dispersions adsorbed to equilibrium surface pressures of 45–47 dynes/cm in seconds at concentrations > 0.25 nig CLL/ml. Both dispersions also lowered surface tension to less than 1 dyne/cm under dynamic compression at 37° C in 100% humidity, although CLL(V) showed some enhancement over CLL(S) in dynamic surface activity at low subphase concentration (0.5 mg/ml). Moreover, CLL(V) and CLL(S) differed markedly in their effects on pressure-volume mechanics in a surfactant-deficient excised rat lung model. Instilled CLL(V) dispersions improved excised lung pressure-volume mechanics at significantly lower concentrations than CLL(S) dispersions.

Newborn tracheal aspirate cytology: Classification during respiratory distress syndrome and bronchopulmonary dysplasia†

Cytopathologic examinations of tracheobronchial aspirates from 108 infants sampled during mechanical ventilation demonstrated a well-defined progression of cytologic changes in bronchial cells that could be divided into three classes. Seventy percent of infants with respiratory distress syndrome who developed bronchopulmonary dysplasia had pulmonary effluent cytology designated Class III; no infants with RDS but without BPD had these cytologic findings. Additionally, a temporal progression of events involving polymorphonuclear leukocyte and macrophage populations occurred in the absence of infection; these events were associated with duration of assisted ventilation and oxygen exposure. The technique described provides a useful way to monitor the progression of lung injury and repair and offers a cytologic method to predict and diagnose the development of bronchopulmonary dysplasia.

Changes in Pulmonary Mechanics after the Administration of Surfactant to Infants with Respiratory Distress Syndrome

We assessed pulmonary mechanics in 35 premature infants with respiratory distress syndrome just before and one hour after the administration of 90 mg of surfactant to each infant. Transpulmonary pressure was measured between the airway opening and an esophageal balloon with use of a differential transducer, and flow rates were measured by a pneumotachometer. Values for pulmonary mechanics were then calculated by microcomputer processing. The administration of surfactant produced a large decrease (56 percent) in the mean (+/- SEM) ratio of alveolar to arterial oxygen, from 7.1 +/- 0.5 to 3.1 +/- 0.2 (P less than 0.0001)--a change that indicates improvement in gas exchange. Associated changes in pulmonary mechanics were not demonstrable when 10 of the infants were studied during continuous mechanical ventilation. However, in the 25 infants examined during spontaneous breathing with continuous positive airway pressures (identical airway pressures before and after treatment), large and consistent improvements in pulmonary mechanics were found after the administration of surfactant. Tidal volume increased by 32 percent (P less than 0.03), minute ventilation by 38 percent (P less than 0.02), dynamic compliance by 29 percent (P less than 0.004), and inspiratory flow rates by 54 percent (P less than 0.01). We conclude that significant improvement in pulmonary mechanics results from surfactant-replacement therapy for respiratory distress syndrome, but that these mechanical changes are apparent only during spontaneous respiration and can be masked if measurements are made during mechanical ventilation.

Early closure of the patent ductus arteriosus in very low-birth-weight infants: A controlled trial

A controlled clinical trial comparing early closure (mean = 48.8 hours) of the patent ductus arteriosus using indomethacin to conventional medical management, with intervention only after cardiopulmonary decompensation (mean = 167.4 hours), was undertaken in 24 preterm infants with severe respiratory distress syndrome and evidence of PDA. An interval analysis of one-half the projected sample revealed that infants undergoing early closure of the PDA had significantly reduced occurrence of BPD or mortality by 6 months of age. A comparison of birth weight, Apgar scores, gestational age, age of initial PDA diagnosis, and fluid therapy during the first seven days of life showed no significant differences between early intervention and control groups. At the time of the interval analysis, there were no differences between the groups in duration of intermittent mandatory ventilation or oxygen exposure. Studies will be required to determine whether these and other variables can be altered by early closure of the PDA.

The early release of surfactant following lung irradiation of alveolar type II cells

Abstract At 1 hour, 24 hours, and l week following irradiation, studies utilizing LAF 1 /J mouse lung showed increase of disaturated alveolar phosphatidylcholine (PC) by radiolabelling and alveolar lavage, thus indicating PC as a nearly immediate post-irradiation biomarker. A corresponding decrease of PC in lung tissue following alveolar lavage correlated with an early decrease of Iamellar bodies in type II pneumocytes after irradiation.

Biophysical activity of synthetic phospholipids combined with purified lung surfactant 6000 Dalton apoprotein

This research studies the biophysical surface activity of synthetic phospholipids combined in vitro with purified lung surfactant apoprotein, having an Mr of 6000. Hydrophobic surfactant-associated protein (SAP-6) was delipidated and purified from both bovine and canine lung lavage, and was combined in vitro with a synthetic phospholipid mixture (SM) of similar composition to natural lung surfactant phospholipids. SM phospholipids were also combined and studied biophysically with another purified surfactant-associated protein, SAP-35. The biophysical activity of synthetic phospholipid-apoprotein combinants was assessed by measurements of adsorption facility and dynamic surface tension lowering ability at 37 degrees C. The SM-SAP-6 combinants had adsorption facility equivalent to natural lung surfactant, and to the surfactant extract preparations CLSE and surfactant-TA used in exogenous surfactant replacement therapy for the neonatal Respiratory Distress Syndrome (RDS). The synthetic phospholipid-SAP-6 combinants also lowered surface tension to less than 1 dyne/cm under dynamic compression in an oscillating bubble apparatus at concentrations as low as 0.5 mg phospholipid/ml. A striking finding was that this excellent dynamic surface activity was preserved as SAP-6 composition was reduced to values as low as 5 micrograms/5 mg SM phospholipid (0.1% SAP-6 protein), an order of magnitude less than the 1% protein content of CLSE and surfactant-TA. Mixtures of SM phospholipids plus SAP-35, the major surfactant glycoprotein, had significantly lower biophysical activity, which did not approach that of a functional lung surfactant. These results suggest that synthetic exogenous surfactants of potential utility for replacement therapy in RDS can be formulated by combining synthetic phospholipids in vitro with specifically purified, hydrophobic surfactant-associated protein, SAP-6.

Isolation of type II alveolar epithelial cells using low protease concentrations

The proteolytic digestion of lung tissue is a necessary prerequisite for the isolation of relatively pure populations of type II alveolar cells. The choice of digestion conditions can have a profound effect on the yield and metabolic integrity of such cells. The specific effects of various proteases on isolation of type II cells from adult rabbits were investigated in a systematic way; maximal numbers of cells were obtained using a mixture of purified elastase (0.3 mg/ml), purified trypsin (0.025 mg/ml) and inflation of intact lung with the protease mixture. Under such conditions, the average yield of cells from an adult rabbit was 10±2.1×107, of which 80–90% were type II cells. Lower yields were obtained by exposing lung tissue to protease after mincing, even if protease concentrations were increased 2–4 fold. By virtue of the lowered protease concentrations, cellular damage as measured by effects on cellular enzyme activities was minimal. NADPH cytochromec reductase activity was not reduced by isolation conditions. In addition, no significant enhanced release of activity into the 150,000 supernatant was observed. The isolated cells were enriched 2–3 fold for the enzymes of phospholipid synthesis compared to whole lung and alveolar macrophages.

Surfactant release as an early measure of radiation pneumonitis

The immediate release of surfactant into lung alveoli following irradiation has been studied as a potential indicator for the later development of radiation pneumonitis. Utilizing single dose radiation exposure to the whole thorax in male LAF1/J mice, steep dose response curves for lavaged alveolar surfactant were identified at 7 and 28 days after exposure. Seven days after irradiation there was no elevation with doses up to and including 12 Gy; above this dose a detectable increase occurred. At 28 days the surfactant recovered by lavage was elevated compared to the levels seen at day 7 for all doses; doses greater than 12 Gy produced surfactant values significantly greater than those found in mice treated with 12 Gy or less. The radiation pulmonary lethality dose response curve assessed four months later indicated an LD50 value of approximately 13 Gy. The early biochemical effect and the later radiation pneumonitis lethalities therefore closely coincided. The evidence strongly indicates that alveolar surfactant release uncovered hours to days after radiation exposure may be an early biochemical marker that predicts for subsequent pneumonitis radiation injury.

Concurrent outbreaks of rhinovirus and respiratory syncytial virus in an intensive care nursery: epidemiology and associated risk factors.

An outbreak of viral respiratory disease occurred in eight infants in a neonatal intensive care unit during the 1980 winter respiratory season. Four infections with respiratory syncytial virus and four infections with rhinovirus were identified. Epidemiologic investigation revealed that viral respiratory infection was significantly associated with intubation with orotracheal tubes (P = 0.001), with the presence of both a nasal feeding tube plus an orotracheal tube together (P = 0.007), and with assisted ventilation (P = 0.009) when compared to uninfected controls. Twenty-seven of 85 (30.6%) personnel working in the unit at the time of the outbreak reported a history of upper respiratory illness during the week prior to the outbreak, and 46 (54.1%) of them had had contact with patients in areas of the hospital where patients infected with RSV and rhinovirus were housed. The data suggest that both viruses were transmitted to the babies by hospital personnel. Rhinoviruses can be nosocomial pathogen in neonates with compromised pulmonary function, and the clinical presentation of rhinovirus infection in neonates may be difficult to distinguish from that produced by RSV.