Harris C. Jacobs

Surfactant function in respiratory distress syndrome

Airway samples from infants with respiratory distress syndrome were recovered by suction immediately after tracheal intubation for respiratory failure. The minimum surface tension of these airway samples was 27.3 +/- 3.0 dynes/cm. Surfactant with low surface tension (1.4 +/- 1.0 dynes/cm) was recovered from these samples by centrifugation; the supernatant fractions from the samples had high minimum surface tensions. The supernatant fractions contained soluble proteins that inhibited the surface tension-lowering properties of natural sheep surfactant. Similar supernatant fractions collected from infants intubated for reasons other than respiratory distress syndrome were much less inhibitory to sheep surfactant. The minimum surface tension of sequential daily airway samples from infants with respiratory distress syndrome fell progressively to 5.7 +/- 2.4 dynes/cm on the day of extubation. These results document the presence of proteins in the airways of infants with respiratory distress syndrome that inhibit the surface tension-lowering properties of surfactant.

Defining the specific physiological requirements for c-Myc in T cell development

c-Myc is associated with cell growth and cycling in many tissues and its deregulated expression is causally implicated in cancer, particularly lymphomagenesis. However, the contribution of c-Myc to lymphocyte development is unresolved. We show here that the formation of normal lymphocytes by c-Myc−/− cells is selectively defective. c-Myc−/− cells are inefficient, in an age-dependent manner, at populating the thymus, and subsequent thymocyte maturation is ineffective: they fail to grow and proliferate normally at the late double-negative (DN) CD4−CD8− stage. Because N-Myc expression in thymocytes usually declines at the late DN stage, these results confirm that the nonredundant contributions of Myc family members to development are related to their distinct patterns of developmental gene expression.

Hex expression suggests a role in the development and function of organs derived from foregut endoderm.

Hex is a divergent homeobox gene expressed as early as E4.5 in the mouse and in a pattern that suggests a role in anterior‐posterior patterning. Later in embryogenesis, Hex is expressed in the developing thyroid, lung, and liver. We now show Hex expression during thymus, gallbladder, and pancreas development and in the adult thyroid, lung, and liver. At E10.0, Hex is expressed in the 3rd pharyngeal pouch, from which the thymus originates, the endodermal cells of liver that are invading the septum transversum, the thyroid, the dorsal pancreatic bud, and gallbladder primoridum. At E13.5, expression is maintained at high levels in the thyroid, liver, epithelial cells lining the pancreatic and extrahepatic biliary ducts and is present in both the epithelial and mesenchymal cells of the lung. Expression in the thymus at this age is less than in the other organs. In the E16.5 embryo, expression persists in the thyroid, pancreatic, and bile duct epithelium, lung, and liver, with thymic expression dropping to barely detectable levels. By E18.5, expression in the thyroid and bile ducts remains high, whereas lung expression is markedly decreased. At this age, expression in the pancreas and thymus is no longer present. Finally, we show the cell types in the adult thyroid, lung, and liver that express Hex in the mature animal. Our results provide more detail on the potential role of Hex in the development of several organs derived from foregut endoderm and in the maintenance of function of several of these organs in the mature animal.

A null mutation of Hhex results in abnormal cardiac development,defective vasculogenesis and elevated Vegfa levels

The homeobox gene Hhex has recently been shown to be essential for normal liver, thyroid and forebrain development. Hhex–/– mice die by mid-gestation (E14.5) and the cause of their early demise remains unclear. Because Hhex is expressed in the developing blood islands at E7.0 in the endothelium of the developing vasculature and heart at E9.0-9.5, and in the ventral foregut endoderm at E8.5-9.0, it has been postulated to play a critical role in heart and vascular development. We show here, for the first time, that a null mutation of Hhex results in striking abnormalities of cardiac and vascular development which include: (1) defective vasculogenesis, (2) hypoplasia of the right ventricle, (3) overabundant endocardial cushions accompanied by ventricular septal defects, outflow tract abnormalities and atrio-ventricular (AV) valve dysplasia and (4) aberrant development of the compact myocardium. The dramatic enlargement of the endocardial cushions in the absence of Hhex is due to decreased apoptosis and dysregulated epithelial-mesenchymal transformation (EMT). Interestingly, vascular endothelial growth factor A (Vegfa) levels in the hearts of Hhex–/– mice were elevated as much as three-fold between E9.5 and E11.5, and treatment of cultured Hhex–/– AV explants with truncated soluble Vegfa receptor 1, sFlt-1, an inhibitor of Vegf signaling, completely abolished the excessive epithelial-mesenchymal transformation seen in the absence of Hhex. Therefore, Hhex expression in the ventral foregut endoderm and/or the endothelium is necessary for normal cardiovascular development in vivo, and one function of Hhex is to repress Vegfa levels during development.

Umbilical venous catheterization and the risk of portal vein thrombosis.

Portal vein thrombosis has been associated with umbilical venous catheterization. We studied the incidence of portal vein thrombosis associated with umbilical venous catheterization with the catheter tip not in the portal venous system. Appropriate placement of an umbilical venous catheter in sick neonates is associated with a low risk of portal vein thrombosis (actual incidence, 1.3%).

HNF3β and GATA-4 transactivate the liver-enriched homeobox gene, Hex

The orphan homeobox gene, Hex, has a limited domain of expression which includes the developing and adult mouse liver. Hex is expressed in the developing liver coincident with the forkhead/winged helix transcription factor, Hepatocyte Nuclear Factor 3beta (HNF3beta). Although preliminary characterization of the mouse Hex promoter has recently been reported, the identity of the molecular regulators that drive liver expression is not known. We hypothesized that putative HNF3beta and GATA-4 elements within the Hex promoter would confer liver-enriched expression. A series of Hex promoter-driven luciferase reporter constructs were transfected in liver-derived HepG2 and fibroblast-like Cos cells+/-HNF3beta or GATA expression plasmids. The Hex promoter region from nt -235/+22 conferred basal activity in both HepG2 and Cos cells, with the region from -103/+22 conferring liver-enriched activity. HNF3beta and GATA-4 transactivated the promoter via response elements located within nt -103/+22, whereas Sp1 activated the -235/+22 construct. Mutation of the HNF3 element significantly reduced promoter activity in HepG2 cells, whereas this element in isolation conferred HNF3beta responsiveness to a heterologous promoter. Electrophoretic mobility shift assays were performed to confirm transcription factor:DNA binding. We conclude that HNF3beta and GATA-4 contribute to liver-enriched expression of Hex.

Reutilization of surfactant phosphatidylcholine in adult rabbits.

32P-saturated phosphatidylcholine was added to [3H]choline-labeled natural surfactant and the mixture was injected intratracheally into 87 adult rabbits. The rabbits were also given [14C]palmitate intravenously at the same time. Rabbits were killed in groups from 10 min to 72 h after injection. In each rabbit we measured the total recovered [3H]phosphatidylcholine (PC) in the alveolar wash, the ratio of [3H]PC to [32P]PC in the alveolar wash, and the specific activity of [14C]PC in the alveolar wash and lamellar bodies. Values were averaged for all rabbits killed at the same times and smooth curves were fit to the data by computer. From the intravenous [14C]palmitate data we calculated a turnover time for alveolar PC of 6.0 h. From the intratracheal labeling data, we calculated a turnover time for alveolar PC of 5.7 h and determined that alveolar PC was reutilized at an efficiency of only 23%. We also concluded that this reutilization occurred as intact molecules.

Cardiovascular effects of surfactant suspensions given by tracheal instillation to premature lambs.

Summary: After delivery by cesarean section at 133–136 days gestational age, 18 lambs were supported with infant ventilators. Peak inspiratory pressure was the only ventilator setting that was changed in an attempt to normalize PCO2. The lambs were retrospectively divided into two groups based on their respiratory status before treatment. Ten lambs with PCO2 values of 38 ± 2 mmHg (mean ± S.E.) at 5 h of age were treated with a 15-ml suspension containing 50 mg natural sheep surfactant lipid/kg body weight. After surfactant instillation, pH fell, PCO2 increased and compliance/kg decreased transiently. Surfactant treatment did not reduce the respiratory support that these lambs required. Eight lambs with PCO2 values of 68 ± 3 mmHg at 3 ± 0.8 h of age responded to surfactant instillation with a decrease in PCO2 and an increase in PO2 and pH. In both groups of lambs heart rates, mean aortic pressures, and mean pulmonary artery pressures changed little after surfactant instillation. Cardiac outputs and regional blood flows measured before and after surfactant treatment were unchanged. Instillation of surfactant suspensions to premature lambs did not adversely affect cardiovascular status; however, some lambs did have a transient deterioration of lung function after surfactant instillation.

Impaired B cell development and function in mice with a targeted disruption of the homeobox gene Hex

Hex is a homeobox gene that is expressed in all stages of B cell development except plasma cells. We studied lymphocyte development in the absence of Hex by using the RAG1-deficient blastocyst complementation system because homozygous disruption of Hex is embryonic lethal. Hex−/−;RAG1−/− chimeric mice had severely reduced numbers of mature B cells, pre-B cells, and CD5+ B cells with a striking 15-fold increase in the percentage of B220−CD19+ cells in the bone marrow. Hex−/−;RAG1−/− chimeric mice failed to generate IgG antibodies to T cell-independent antigens, although their serum IgM levels and antibody responses to T cell-dependent antigens were intact. Therefore, Hex is necessary for B cell development and function and its absence results in a dramatic increase in B220−CD19+ cells.

Reutilization of phosphatidylcholine analogues by the pulmonary surfactant system: the lack of specificity

Five specific 14C-labelled analogues of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, (L-alpha-DPPC) including 1-palmitoyl-sn-glycero-3-phosphocholine, (L-alpha-lysoPC) and 2,3-dipalmitoyl-sn-glycero-1-phosphocholine (the D isomer of DPPC) were individually mixed with L-alpha-[3H]DPPC and unlabelled natural surfactant isolated from 3-day-old rabbits. The mixtures were injected intratracheally into 3-day-old rabbits which were then killed at preset times up to 72 h after injection. Phosphatidylcholine was isolated from the alveolar wash and from lamellar body fraction from each rabbit and was analyzed for the ratio of 3H-to-14C counts/min. These ratios were plotted against the time the rabbits were killed to determine whether a difference existed in the rates of reutilization of the analogues relative to L-alpha-DPPC. By 60 h L-alpha-lysoPC was reutilized at 42% of the efficiency of the L-alpha-[3H]DPPC with which it was injected. That part of the L-alpha-lyso which was reutilized had been converted to [14C]phosphatidylcholine. Each of the other four analogues was reutilized by the surfactant system as efficiently as L-alpha-DPPC. These results are most consistent with a process of bulk uptake of surfactant from the alveolar space by the Type II cell with subsequent processing for resecretion which involves minimal specificity for molecular structure.