A comparison of albumin-bolus therapy versus normal saline-bolus therapy for hypotension in neonates.

OBJECTIVE: We compared responses to bolus infusion of 5% albumin (ALB) or normal saline (NS) for hypotension in neonates. STUDY DESIGN: Hypotensive infants were given 10 ml kg(-1) of NS or ALB. A second bolus was given for persistent hypotension. Dopamine therapy was started for hypotension after the second bolus. The primary response was increase in arterial blood pressure toward normal range 1 h postinfusion. Secondary measures included duration of normotension, meeting criteria for second bolus, meeting criteria for vasopressor support and cost comparison. RESULT: Those receiving ALB (N=49 ALB and 52 NS) were more likely to achieve a normotensive state (ALB=57.1%, NS=32.1% P=0.01) 1 h following the initial bolus therapy. Subsequently, the NS group was also more likely to qualify for vasopressor infusion (ALB=24.5%, NS=44.2% P=0.02). Overall cost for either therapy was equivalent. CONCLUSION: In hypotensive neonates, ALB results in a greater likelihood of achieving normotension and decreased subsequent use of vasopressors when compared to NS.

Construction and characterization of a recombinant plasminogen activator composed of an anti-fibrin single-chain antibody and low-molecular-weight urokinase.

BACKGROUND: Targeting of plasminogen activators to the fibrin component of a thrombus by antibodies directed against human fibrin can enhance their thrombolytic potency and clot specificity. OBJECTIVES: To overcome the disadvantages of chemical conjugation, we investigated whether the recombinant fusion of a single-chain antibody and a plasminogen activator results in an active bifunctional molecule that might be useful as a therapeutic agent. METHODS: The cDNA of low-molecular-weight single-chain urokinase-type plasminogen activator, comprising amino acids Leu144-Leu411 (scuPA(LMW)), was cloned from human endothelial cells and fused to a single-chain antibody specific for the 7 N-terminal amino acids (beta(15-22)) in the beta-chain of human fibrin (scFv(59D8)). The fusion protein was purified using affinity chromatography with the beta(15-22)-peptide of human fibrin. RESULTS: Purified scFv(59D8)-scuPA(LMW) migrated as a 60-kDa band, which is consistent with a molecule composed of one scFv(59D8) and one scuPA(LMW) moiety. Both functions of the fusion molecule, fibrin-specific binding and plasminogen activation, were fully preserved. In human plasma clots, thrombolysis by scFv(59D8)-scuPA(LMW) is significantly faster and more potent compared with the clinically used urokinase. CONCLUSIONS: ScFv(59D8)-scuPA(LMW) constitutes a new recombinant chimeric plasminogen activator with a significantly enhanced thrombolytic potency and relative fibrin selectivity, that can be produced with modern methods at low cost, large quantities and reproducible activity in Escherichia coli.

Construction and functional evaluation of a single-chain antibody fusion protein with fibrin targeting and thrombin inhibition after activation by factor Xa.

BACKGROUND: Recombinant technology was used to produce a new anticoagulant that is preferentially localized and active at the site of the clot. METHODS AND RESULTS: The variable regions of the heavy and light chains of a fibrin-specific antibody were amplified by polymerase chain reaction (PCR) with hybridoma cDNA. To obtain a functional single-chain antibody (scFv), a linker region consisting of (Gly(4)Ser)(3) was introduced by overlap PCR. After the scFv clones were ligated with DNA encoding the pIII protein of the M13 phage, high-affinity clones were selected by 10 rounds of panning on the Bbeta15-22 peptide of fibrin (beta-peptide). Hirudin was genetically fused to the C-terminus of the variable region of the light chain. To release the functionally essential N-terminus of hirudin at the site of a blood clot, a factor Xa recognition site was introduced between scFv(59D8) and hirudin. The fusion protein was characterized by its size on SDS-PAGE (36 kDa), by Western blotting, by its cleavage into a 29-kDa (single chain alone) and 7-kDa (hirudin) fragment, by its binding to beta-peptide, and by thrombin inhibition after Xa cleavage. Finally, the fusion protein inhibited appositional growth of whole blood clots in vitro more efficiently than native hirudin. CONCLUSIONS: A fusion protein was constructed that binds to a fibrin-specific epitope and inhibits thrombin after its activation by factor Xa. This recombinant anticoagulant effectively inhibits appositional clot growth in vitro. Its efficient and fast production at low cost should facilitate a large-scale evaluation to determine whether an effective localized antithrombin activity can be achieved without systemic bleeding complications.

A multicenter randomized, masked comparison trial of natural versus synthetic surfactant for the treatment of respiratory distress syndrome.

OBJECTIVE: To compare the efficacy and safety of two surfactant preparations in the treatment of respiratory distress syndrome (RDS). METHODS: We conducted a randomized, masked comparison trial at 21 centers. Infants with RDS who were undergoing mechanical ventilation were eligible for treatment with two doses of either a synthetic (Exosurf) or natural (Infasurf) surfactant if the ratio of arterial to alveolar partial pressure of oxygen was less than or equal to 0.22. Crossover treatment was allowed within 96 hours of age if severe respiratory failure (defined as two consecutive arterial/alveolar oxygen tension ratios < or = 0.10) persisted after two doses of the randomly assigned surfactant. Four primary outcome measures of efficacy (the incidence of pulmonary air leak (< or = 7 days); the severity of RDS; the incidence of death from RDS; and the incidence of survival without bronchopulmonary dysplasia (BPD) at 28 days after birth) were compared by means of linear regression techniques. RESULTS: The primary analysis of efficacy was performed in 1033 eligible infants and an analysis of safety outcomes in the 1126 infants who received study surfactant. Preentry demographic characteristics and respiratory status were similar for the two treatment groups, except for a small but significant difference in mean gestational age (0.5 week) that favored the infasurf treatment group. Pulmonary air leak (< or = 7 days) occurred in 21% of Exosurf- and 11% of infasurf-treated infants (adjusted relative risk, 0.53; 95% confidence interval, 0.40 to 0.71; p < or = 0.0001). During the 72 hours after the initial surfactant treatment, the average fraction of inspired oxygen (+/-SEM) was 0.47 +/- 0.01 for Exosurf- and 0.39 +/- 0.01 for infasurf-treated infants (difference, 0.08; 95% confidence interval, 0.06 to 0.10; p < 0.0001); the average mean airway pressure (+/-SEM) was 8.6 +/- 0.1 cm H2O; for Exosurf- and 7.2 +/- 0.1 cm H2O for Infasurf-treated infants (difference, 1.4 cm H2O; 95% confidence interval, 1.0 to 1.8 cm H2O; p < 0.0001). The incidences of RDS-related death, total respiratory death, death to discharge, and survival without bronchopulmonary dysplasia at 28 days after birth did not differ. The number of days of more than 30% inspired oxygen and of assisted ventilation, but not the duration of hospitalization, were significantly lower in Infasurf-treated infants. CONCLUSION: Compared with Exosurf, Infasurf provided more effective therapy for RDS as assessed by significant reductions in the severity of respiratory disease and in the incidence of air leak complications.

Care of the neonate.

Surfactant administration for respiratory distress syndrome continues to make an impact on neonatal care as large controlled trials are published. Although considered safe, synthetic surfactant administration has been associated with a rare complication in the form of pulmonary hemorrhage. Despite this, significant benefits have been shown. With the approval by the FDA of two surfactant preparations, this treatment is now in widespread use. Although the mortality rate from respiratory distress syndrome and the number of ventilator days are generally decreased, surfactant effect on the incidence of bronchopulmonary dysplasia has been disappointing. Studies of steroid administration for bronchopulmonary dysplasia and steroid side effects have been published in the past year. Steroid use has become widespread for this condition, although many details of its administration and side effects have yet to be worked out. A new area of promise is the use of erythropoietin for anemia of prematurity. Natural historic data on the retinopathy of prematurity have added to our understanding of this condition and have raised new questions on its pathogenesis. Review articles and studies in the area of neonatal encephalopathy stress the need for a more accurate definition of asphyxia and discuss possible prenatal causes of this condition. An extensive review of neonatal jaundice and new recommendations for its treatment in healthy term newborns has been published but remains controversial.

Gestational age assessment in preterm neonates weighing less than 1500 grams.

Postnatal assessment of gestational age in preterm neonates traditionally has been performed using the methods of Dubowitz and Ballard. This study was designed to determine the accuracy of these methods in a sample of very low birth weight preterm neonates. Dubowitz and Ballard examinations were done on 110 preterm neonates within the first 72 hours of life by a neonatologist masked to the gestational age assessed antenatally. Mean birth weight was 1066 +/- 256 g (SD). These data were compared with gestational age assessments using last menstrual period and best obstetric estimate calculated by an obstetrician unaware of the neonatal examination. Mean gestational age using last menstrual period was 28.3 +/- 2.9 weeks. Mean differences between last menstrual period and Dubowitz/Ballard were -2.8 +/- 2.1 weeks and -2.6 +/- 2.2 weeks, respectively. Results using best obstetric estimate were similar. An ophthalmologist examined lens vessels of 89 neonates. A similar pattern toward overestimation of gestational age interval by Dubowitz/Ballard was seen at each lens vessel grade. The Dubowitz and Ballard examinations are inaccurate methods of assessing gestational age in preterm neonates with birth weights less than 1500 g.

15-Hydroxyeicosatetraenoic acid stimulates migration of human retinal microvessel endothelium in vitro and neovascularization in vivo.

We evaluated 15-hydroxyeicosatetraenoic acid (15-HETE), a major arachidonic acid product of vascular endothelium and leukocytes, for its effect on neovascularization. In a modified Boyden chamber assay, 15-HETE (10-7 M) stimulated human retinal microvessel endothelial cell migration by 42 +/- 10% (mean +/- S.E.M., p less than 0.01). 12-HETE, a major arachidonic acid metabolite of platelets, had no such effect. Further studies in the rabbit corneal pocket assay revealed that 15-HETE stimulated neovascularization in vivo. Concentrations at which the in vivo effects were observed are within the range generated by several cell types and are achievable in human serum. 15-HETE stimulation of human endothelial cell migration in vitro and neovascularization in vivo suggests that it may play a role in vasoproliferative disorders.

Stimulation of endothelial cell prostacyclin release by retina-derived factors.

An angiogenic extract of bovine retina as well as two purified angiogenic growth factors, acidic and basic fibroblast growth factor, stimulate vascular endothelial cell prostacyclin (PGI2) release in vitro as measured by radioimmunoassay of its stable metabolite 6-keto PGF1 alpha (6kPGF). After incubating fetal bovine aortic endothelial cells with 10% retinal extract (RE) for 24 hr, 6.5 ng of 6-kPGF/10(5) cells were released compared to 0.8 ng of 6kPGF/10(5) cells for unstimulated endothelium. Similar qualitative results were obtained using human retina-derived microvessel endothelium. PGI2 release in response to RE depended on endothelial cell density with subconfluent cultures releasing six-fold more 6kPGF compared to confluent monolayers. Thin layer radiochromatography of endothelial cell conditioned media demonstrated enhanced release of 6-kPGF, PGE2 and arachidonic acid after RE addition. Cycloheximide and actinomycin D inhibited PGI2 release but hydroxyurea had no effect. Most of the PGI2-stimulating activity of RE was adsorbed to heparin-Sepharose and eluted by 2 M NaCl. Purified acidic (10 ng/ml) and basic (1 ng/ml) fibroblast growth factors caused seven-fold and four-fold stimulation of endothelial PGI2 release, respectively. An initial event in the regression of new blood vessels following the removal of an angiogenic stimulus is the formation of intraluminal platelet aggregates. PGI2 is a potent inhibitor of platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

The mitogenic effect of 15- and 12-hydroxyeicosatetraenoic acid on endothelial cells may be mediated via diacylglycerol kinase inhibition.

15-Hydroxyeicosatetraenoic acid (15-HETE), a major lipoxygenase metabolite of arachidonic acid in fetal bovine aortic endothelial cells, was a mitogen for these cells, stimulating both cell proliferation and DNA synthesis in the presence of serum and serum-deprived cells. In [14C]arachidonic acid-labeled confluent endothelial cell monolayers, 15-HETE (30 microM) caused an elevation of [14C]diacylglycerol (DAG) with a concomitant decrease in cellular [14C]phosphatidylinositol (PI) in both unstimulated and stimulated cells. 1-Oleoyl-2-acetylglycerol, a synthetic DAG analog, stimulated endothelial cell DNA synthesis in a concentration-dependent manner. In [3H]inositol-labeled cells, 15-HETE also caused a decrease in cellular PI content under both basal and stimulated conditions. 15-HETE, however, had no effect on either isolated phospholipase C activity or phosphoinositide turnover in lithium chloride-treated cells. In intact cells, 15-HETE (30 microM) inhibited the synthesis of [3H]PI from [3H]inositol (80% inhibition, p less than 0.001). In human red cell membranes, the production of phosphatidic acid from endogenous DAG was inhibited by 15-HETE in a concentration-dependent manner with an IC50 of 41 microM. Although 12-HETE had effects similar to those of 15-HETE, the parent compound arachidonic acid did not affect DNA synthesis or DAG kinase activity. Our study thus demonstrates that the mitogenic activity of 15- and 12-HETE on endothelial cells may be mediated via DAG kinase inhibition with the concomitant accumulation of cellular DAG.

A comparison of insulin receptors in the developing fetal lung in normal and in streptozotocin-induced diabetic pregnancies.

Insulin receptors from fetal rat lungs of normal and streptozotocin-induced diabetic pregnancies were examined for their binding capacities and association constants. Sprague-Dawley rats, given a single dose of streptozotocin at 7 days' gestation, became hypoinsulinemic and hyperglycemic within 48 hours, although they remained healthy enough to carry fetuses to term. The fetuses of the streptozotocin-treated pregnancies had hyperglycemia (3,750 +/- 400 micrograms glucose/ml vs 390 +/- glucose/ml for control subjects), but were not hyperinsulinemic. Insulin receptor binding capacities of fetal lungs from control pregnancies increased as a function of gestational age from 16 to 21 days. Receptor binding capacities of lungs from streptozotocin-treated pregnancies also increased with gestational age until 21 days, when they dropped to 50% of control values. It was demonstrated in organ culture that fetal lung receptors from normal pregnancies of 20 days' gestation could be down-regulated in the presence of insulin and that this down-regulation is coupled to a biologic effect of insulin, hexose transport. It was concluded that fetal lung insulin receptors can be regulated by insulin concentrations in vitro and by experimental diabetic pregnancies in vivo.

Anemia of prematurity: determinants of the erythropoietin response.

This study was undertaken to determine the factors that are important in determining the erythropoietin response in low-birth-weight infants during the period of so-called anemia of prematurity. In the first weeks of life oxygen consumption in a group of 21 infants gradually increased as hemoglobin level fell. The magnitude of the erythropoietin response inversely varied with the central venous oxygen tension (P-vO2) (r = -0.55, P less than 0.001). When the P-vO2 declined to less than 30 torr, erythropoietin values were uniformly increased above the "normal" range (defined as the values associated with P-vO2 greater than 38 torr). Erythropoietin values varied inversely with hemoglobin but in general did not exceed the values observed for normal adult men. The erythropoietin values in the infants were remarkably lower at any given hemoglobin level when compared with those of older children with anemia resulting from bone marrow failure. In general, elevations of erythropoietin were seen when the hemoglobin concentration declined to less than 10.0 gm/dl. Change in heart rate did not appear to be a reliable indicator of the presence of anemia; rather, it correlated best with oxygen consumption.

Effects of hyperoxia and hypoxia on vascular prostacyclin formation in vitro.

Exposure to high oxygen (O2) concentrations, especially in the neonate, is associated with the development of pathologic syndromes characterized by vascular involvement including the retinopathy of prematurity. Some of the initial vascular changes observed appear consistent with a reduction in prostacyclin formation. Exposure of human umbilical arteries to oxygen resulted in more than 30% inhibition in the ability of the vessels to produce prostacyclin either from endogenous stores of arachidonic acid or from exogenously provided substrate. In contrast, hypoxia (which more closely approximates the fetal environment) resulted in more than 30% stimulation in the production of prostacyclin from either endogenous or exogenous arachidonic acid. When microsomes were prepared from treated arterial segments, these effects persisted. In vitro results suggest that neonates exposed to O2 after delivery may experience a marked decrease in vascular prostacyclin formation. Inhibition of the production of this potent vasodilator and antithrombotic metabolite could play an important role in the acute exudative phase of O2 toxicity.

Insulin receptors in the developing fetal lung.

Fetal lung insulin receptor numbers and affinities were studied in rat pregnancies from 15 to 22 days gestation. Insulin receptor binding capacities were found to increase six-fold from approximately 100 fmoles insulin bound/mg lung DNA at 15 days gestation to approximately 600 fmoles bound/mg DNA at 22 days gestation. However, the affinity constants of the receptors were unchanged during this same period (high affinity, 1.9 +/- 0.4 S.E. and low affinity, 0.03 +/- 0.01 S.E.). The results suggest that the lung may become increasingly more sensitive to insulin as development progresses.

Effects of acetylsalicylic-acid ingestion on maternal and neonatal hemostasis.

In a case-control study, we evaluated the effects of maternal ingestion of acetylsalicylic acid (aspirin) within 10 days of delivery on maternal and neonatal hemostasis. Only one of 34 control maternal-neonatal pairs (3 per cent) had hemostatic abnormalities. In 10 pairs, when maternal aspirin ingestion occurred within five days of delivery, 6 of 10 mothers and 9 of the 10 infants had bleeding tendencies. Seven maternal-neonatal pairs in which aspirin was ingested 6 to 10 days before delivery were free of clinical bleeding. Among seven other mothers who ingested aspirin in the immediate post-partum period four of the seven (57 per cent) also had impaired hemostasis. Neonatal hemostatic abnormalities included numerous petechiae over the presenting part, hematuria, a cephalhematoma, subconjunctival hemorrhage, and bleeding from a circumcision. Maternal bleeding was confined to excessive intrapartum or post-partum blood loss. We conclude that aspirin should be avoided during pregnancy. If ingestion has occurred within five days of delivery, the neonate should be evaluated for the presence of bleeding.

Effect of homocysteine and homocystine on platelet and vascular arachidonic acid metabolism.

Normal hemostasis depends in part on the balance achieved between proaggregatory and prothrombotic platelet thromboxane A2, measured as its stable end-product thromboxane B2 (TXB2), and vascular prostacyclin (PGI2), which inhibits platelet aggregation and is antithrombotic. Cystathionine-beta-synthase deficiency is characterized by a high frequency of thromboembolic disease. We therefore studied, in vitro, the effects of homocysteine and related compounds on platelet TXB2 and vascular PGI2 formation. In paired samples of platelet rich plasma, which had been preincubated with L-homocystine (1 mM), mean production of the two platelet cyclooxygenase products, TXB2 and 12-hydroxy-5, 8,10-heptadecatrienoic acid increased significantly from control levels [13.6% +/- 1.9 to 19.8% +/- 2.1 (P less than 0.02) TXB2 and 29.8% +/- 4.2 to 39.4% +/- 4.1 (P less than 0.01) HHT]. In the presence of D,L-homocysteine (1 mM), mean TXB2 and 12-hydroxy-5,8,10-heptadecatrienoic acid production was also significantly increased [12.7% +/- 1.5 to 16.9% +/- 1.5 (P less than 0.01) TXB2 and 27% +/- 4 to 31% +/- 4.1 (P less than 0.02) HHT]. Cystine, cysteine, or methionine (1 mM) did not have similar effects in this test system. Homocysteine and homocystine were without effect on the synthesis of vascular PGI2 by umbilical artery segments [control, 0.22 +/- 0.03 to 0.21 +/- 0.03 ng/mg with D,L-homocysteine and 0.20 +/- 0.04 control to 0.19 +/- 0.04 ng/mg with D,L-homocystine]. A homocyst(e)ine-induced increase in platelet thromboxane production in the absence of an increase in vascular prostacyclin, if present in vivo, may contribute to the vascular thromboses characteristic of human homocystinemias (homocystinurias).