Antenatal infection in the rabbit impairs post-natal growth and lung alveolarisation.

Clinical and experimental studies indicate an association between chorioamnionitis and bronchopulmonary dysplasia in preterm infants. The present authors hypothesised that, in the rabbit, antenatal infection may impair lung development after birth, despite effective maternal antibiotic therapy. Pregnant rabbits received an intra-uterine inoculation of 10(3) Escherichia coli colony forming units or vehicle at the end of gestation (day 29). Intravenous ceftriaxone therapy was initiated 8 h after inoculation for a period of 8 days. Pups born between 60 and 84 h after inoculation were kept with their mother until sacrifice on days 0, 1, 5, 8 and 15. Blood cultures from antenatally infected animals were sterile at birth. Postnatal growth was significantly impaired by day 8. Lung morphometry showed a significant decrease of alveolar surface density and interstitial density, with a significant increase of alveolar airspace volume, indicating impaired alveolarisation for the first 2 weeks of postnatal life. Inflammatory and apoptotic processes were not detected in the lung at birth or subsequently. Intra-uterine infection in rabbits is, therefore, responsible for concomitant postnatal growth retardation and abnormal pulmonary development despite early and effective antenatal antibiotic therapy. This may constitute an alternative model to study the consequences of antenatal infection on postnatal growth and lung development.

Fetal lung surfactant lipid synthesis from glycogen during organ culture.

The role of fetal lung glycogen as a precursor for lipids during late gestational development was explored by a combination of in vivo labeling with [U-14C]glucose, administered directly to rat fetuses at 18.5 days, and in vitro assessment using an organ explant culture system. Our major objectives were to demonstrate that radioactivity was transferred specifically and preferentially to surfactant lipids, as glycogenolysis occurred, and to determine the molecular distribution of 14C labeling in newly synthesized phosphatidylcholine (PC). Surfactant and residual (non-surfactant) lipids were separated by sucrose density gradient centrifugation, and other subcellular fractions such as microsomes were isolated by subsequent centrifugations. After 72 h of culture, there was a 5.7-fold increase in the concentration of PC in the surfactant fraction, which contributed 8.8% of total PC at the beginning and 29.6% (P less than 0.001) at the end of the 72 h period. The labeling of PC in the surfactant fraction increased markedly during culture, but there was no significant change in the residual fraction or microsomal PC. Hydrolysis of surfactant PC indicated that the radioactivity was predominantly located in the fatty acyl portion of the molecule, both before and after culture; however, PC glycerol labeling also increased significantly during culture. The distribution of PC radioactivity was similar in the residual fraction and microsomes, with the majority of 14C in the fatty acids. Neutral lipid radioactivity also increased significantly in both the surfactant (240%) and residual (136%) fractions. Quantitation of the changes in radioactivity among subcellular components during lung explant culture indicated that the greatest decrease occurred in glycogen, whereas only lipids, particularly those of the surfactant fraction, were found to show significant increases. These results support the hypothesis that glycogen, which accumulates in fetal lung prior to augmented surfactant production, can supply precursors for synthesis of functionally essential pulmonary phospholipids.

Comparison of effects of epidermal and insulin-like growth factors, gastrin releasing peptide and retinoic acid on fetal lung cell growth and maturation in vitro.

The role in cell multiplication and maturation of several factors present in the late fetal lung was explored on isolated fetal rat pulmonary fibroblasts and alveolar epithelial type II cells cultivated in serum-free medium. The low degree of reciprocal contamination of each cell population was assessed by immunocytochemistry. Epidermal Growth Factor (EGF) stimulated thymidine incorporation and DNA accumulation in both cell types. In type II cells, it increased labeled-choline incorporation into surfactant phosphatidylcholine (PC), consistently with previous data obtained with lung explant cultures, but not into non-surfactant PC. Insulin-like growth factor (IGF)-I slightly stimulated DNA accumulation in fibroblasts although it did not significantly stimulate thymidine incorporation, contrary to IGF-II which presented a dose-dependent stimulating activity of thymidine incorporation. Neither IGF-I nor IGF-II stimulated type II cell growth. IGFs thus appear to primarily control the growth of lung mesenchyme. In type II cells, they stimulated the most non-surfactant PC biosynthesis. Gastrin releasing peptide (GRP) which was recently reported to promote fetal lung growth in vivo and to stimulate surfactant biosynthesis in lung organ culture revealed as a growth factor for type II cells only, at concentrations below 10(-9) M. At concentration 10(-8) M, although it did not affect DNA synthesis, GRP tended to increase surfactant and non-surfactant-PC biosynthesis. Retinoic acid inhibited thymidine incorporation into type II cells on a dose-dependent manner but nevertheless enhanced surfactant-PC biosynthesis to a similar extent as EGF. It is suggested that retinoic acid may represent a differentiation or maturation factor for the alveolar epithelium.

Role of alpha-glucosidase in fetal lung maturation.

The role of lysosomal enzyme acid alpha-glucosidase in fetal lung development was investigated with the aid of a specific inhibitor, the pseudosaccharide acarbose. The drug was added to a Waymouth culture medium of fetal rat lung explants cultivated for 48 h from gestational stage 19.5 days, an in vitro system previously shown to allow morphological and biochemical maturation of alveolar epithelium. Glycogenolysis was reduced by 40% as compared with tissue cultivated on control medium, which means that alpha-glucosidase could account for as much as 40% of fetal lung glycogenolysis, the remaining 60% being presumably achieved by cytosolic phosphorylase and by a microsomal neutral alpha-glucosidase. By the same time, the increase of phospholipids of surfactant fraction extracted from cultivated explants was partially inhibited: total and saturated phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol were about 30-40% lower than in lungs cultivated on control medium. It should be emphasized that DNA concentration and increases in non-surfactant phospholipids were unchanged by the drug. alpha-Glucosidase activity was evidenced in the lysosomal fraction, in the microsomal fraction and, although in lower amounts, in the surfactant fraction extracted from term fetal lung. The results suggest that lysosomal alpha-glucosidase plays a major role in lung maturation and could facilitate glycogenolysis for the specific use of glycogen stores in providing substrates for surfactant phospholipid biosynthesis.

Relationships among surfactant fraction lipids, proteins and biophysical properties in the developing rat lung.

Lung development is associated with increases in specific phospholipids and proteins that function as critical pulmonary surfactant components. Attempts to characterize the pattern of surfactant development in fetal rat lungs have been hampered by the lack of a micromethod which will permit quantitative isolation of surface active components from small tissue specimens. As part of studies designed to elucidate the metabolic regulation of lung development in the rat, we developed sucrose density gradient centrifugation procedures to separate pulmonary phospholipids and proteins into a presumed surfactant (S) fraction and a residual (R) fraction. Electron microscopy of S pellets from mature fetuses identified predominant lamellar bodies and minimal contamination; incubation with 5 mM CaCl2 induced the appearance of tubular myelin figures, implying functional potential. This was confirmed by demonstrating low surface tension (less than 1 dyn/cm) in S, but not R, fractions at term gestation (21.5 days) and in 1-day-old neonatal lung isolates, based on dynamic measurements using the oscillating bubble technique. Surface activity was also high in the S pellets from fetuses at 20.5 days of gestation; however, at 19.5 days, minimum surface tension values of at least 19 dyne/cm were seen. These results correlated directly with biochemical analyses which indicated striking increases in three surfactant-associated proteins (SP-A, SP-B, and SP-C) after 19.5 days of gestation; a finding in agreement with previously reported data on the developmental increase of disaturated phosphatidylcholine in fetal rat lung. We conclude that isolation of S fraction components is valuable for demonstrating maturation of the fetal rat lung and may provide a useful tool for the study of regulatory mechanisms influencing surfactant production and function.

Effects of acute variation of fetal glycemia on glycogen storage and on glycogen synthase and phosphorylase activities in the liver of the rat fetus.

The effects of variations of glycemia from 1.7 to 35 mM on the activity of glycogen synthase and phosphorylase, on glycogen content, and on U-14C-glucose incorporation into glycogen in the liver of the near-term rat fetus were investigated. Hypoglycemia did not affect the activities of phosphorylase and synthase; total glycogen content was not modified, but incorporation of labeled glucose was markedly decreased. This is consistent with a decreased glycogen synthesis. A slight hyperglycemia (about 5.5 mM) sharply decreased phosphorylase a (active) activity but increased slightly glycogen synthase a activity; liver glycogen content and labeled glucose incorporation were both enhanced. Higher levels of glycemia induced a decrease of phosphorylase a activity of the same order, but by contrast, glycogen synthase a activity increased progressively with increasing glycemia. Sequential study showed that hyperglycemia first induced the decrease of phosphorylase activity, then increased synthase activity. Marked hyperglycemia strongly enhanced liver glycogen content and labeled glucose incorporation. The fetal liver appears very responsive to acute variations of glycemia. The mechanisms seem to be oriented toward maximal glycogen accumulation.

Maturation of fetal rat lung in diabetic pregnancies of graduated severity.

Biochemical and morphologic maturation of fetal rat lung was studied in pregnant, diabetic rats with different levels of glucose intolerance (sub-, mildly, and severely diabetic). Fetuses were almost normoglycemic and hyperinsulinemic in subdiabetic rats, both hyperglycemic and hyperinsulinemic in mildly diabetic rats, and hyperglycemic but hypoinsulinemic in severely diabetic rats. A similar delay in type II pneumocyte differentiation and a similar decrease of disaturated phosphatidylcholine (DSPC) content in lung tissue and broncho-alveolar material recovered by lung lavage occurred in the fetuses of the three diabetic groups, independently of the severity of diabetes. Phosphatidylglycerol (PG) was decreased in fetuses from severely diabetic rats only. DSPC appeared specifically affected in fetuses of sub- and mildly diabetic groups, whereas in those of severely diabetic groups, DSPC alterations accompanied a variety of abnormalities including whole lung hypoplasia and hypotrophy and decreases of sphingomyelin, unsaturated PC, and lysoPC. The mechanisms leading to abnormal lung development could therefore be different in fetuses of sub- and mildly diabetic rats on one hand and fetuses of severely diabetic rats on the other. Since hyperinsulinemia was the prominent feature of fetal "milieu intérieur" in subdiabetic rats, this study presents arguments gained from in vivo experiments for an implication of hyperinsulinemia in lung developmental retardation due to maternal diabetes. However, the decrease of PG seems to depend on increased blood glucose level in itself. Diminished lung glycogen breakdown and decreased lung triglyceride content, more pronounced in fetuses of sub- and mildly diabetic rats than in those of severely diabetic rats, suggest that in the former, the decrease of DSPC biosynthesis could be due to decreased availability of substrates because of abnormal glycogen utilization. Fetuses from sub- and mildly diabetic rats constitute experimental models most closely resembling the human fetus of the diabetic mother with respect to circulating glucose and insulin. They appear therefore more adequate for elucidating the mechanisms of abnormal lung development in the diabetic pregnancy. In contrast, fetuses from severely diabetic rats associate very high blood glucose levels and hypoinsulinemia, which are features closer to those of adult diabetic subjects than to those of the human fetus of the diabetic mother.

Role of keratinocyte growth factor in the control of surfactant synthesis by fetal lung mesenchyme.

Fetal lung maturation is regulated by mesenchymal-epithelial cell communication, which plays a major role in the control of surfactant synthesis by alveolar type II cells. We have recently shown that keratinocyte growth factor (KGF), also called fibroblast growth factor-7, enhances the maturation of fetal alveolar epithelial type II cells. Here, we investigated, among the factors produced by lung mesenchyme, the part attributable to KGF in the control of surfactant synthesis. Using a KGF-neutralizing antibody, we assessed surfactant phospholipid synthesis by measuring choline incorporation into disaturated phosphatidylcholine of isolated fetal type II cells. We found that KGF accounts for about half of the stimulating activity present in fetal lung fibroblast-conditioned medium (FCM). By contrast, the use of an epidermal growth factor-neutralizing antibody did not alter the FCM-stimulating activity. To further delineate KGF properties as a mesenchymal mediator, we wondered about its possibility to relay glucocorticoid-stimulating activity on the synthesis of the phospholipid moiety of surfactant in fetal lung fibroblasts. A 24-h exposure to dexamethasone led us to detect a 50% increase in the level of KGF messenger RNA (mRNA) in isolated fetal lung fibroblasts. Moreover, anti-KGF antibody totally abolished the further increase of FCM-stimulating activity induced by dexamethasone. Thus, KGF seems to be a major player in mediating glucocorticoid stimulation of fetal lung maturation.

Glucocorticosteroid receptors in the liver of normal and decapitated rabbit foetuses.

The affinity and the number of receptor sites for the synthetic steroid triamcinolone acetonide were studied in the livers of foetal rabbits. Twenty-two-, 24- and 29-day-old control and 29-day-old foetuses decapitated on day 22 were used to determine whether a change in steroid receptors occurs in the liver when it accumulates glycogen, and whether decapitation impairs such a change. Steroid receptors were found as early as day 22, when they were as numerous (0.091 pmol/mg protein) as on days 24 (0.098 pmol/mg) and 29 (0.104 pmol/mg). The binding affinity was found to be slightly higher on day 24 than on day 22 and it remained the same on days 24 and 29. The binding affinity of the receptors was the same in decapitated foetuses as in 29-day-old controls but the number of sites was slightly lower. The labelled steroid was transferred to the nucleus of the liver cells in vivo. It seems unlikely that steroid receptors are the limiting factor preventing the accumulation of glycogen in the liver of the rabbit foetus before day 26 or after decapitation.

Changes in phospholipid composition of tracheal aspirates from newborns with hyaline membrane disease or transient tachypnoea.

Phospholipid analysis of tracheal aspirates obtained from 37 newborn infants, all intubated for respiratory diseases, was performed in order to compare infants having hyaline membrane disease (HMD) (n = 11), to those presenting with transient tachypnoea (TT) (n = 16) or another respiratory disorder (n = 10) and to determine if distinguishing features could be discovered for HMD or TT. In the HMD group, a significantly lower amount (about 20%) of recoverable phospholipid material was observed. Furthermore, the groups differed in their phospholipid profile: infants with HMD presented with a deficiency in saturated phosphatidylcholine, but had a related increase in unsaturated phosphatidylcholine, and an increased proportion of phosphatidylethanolamine (about 2.5 times more) as compared with both other groups. In infants suffering HMD and TT, phosphatidylglycerol was lower and phosphatidylinositol was higher than in infants with other diseases. This change was the only one displayed in infants with TT. We speculate that the observed changes reflect changes in amount and composition of surfactant and are involved in the etiology of HMD and TT.

Development of the surfactant system.

Pulmonary surfactant is a lipoprotein complex produced by type II pneumocytes that lines and stabilizes alveoli. Surfactant-specific proteins are early markers of the embryonic lung. Precursors of type II pneumocytes appear to be committed early and are able to pursue their differentiation program in a relatively autonomous way. Despite this potential, surfactant ontogeny and storage actually take place relatively late in the course of fetal development. Surfactant accumulation is, therefore, delayed until a certain stage after which it is stimulated by a number of factors. Recent investigations have pointed out the possible role of paracrine mechanisms in the determinism of this control, especially with regard to retinoids and TGF-beta.

[Surfactants in the digestive tube]. / Les surfactants dans le tube digestif.

Epithelia of the gastrointestinal tract both synthesize and secrete surfactant-like materials. Like the pulmonary surfactant, these materials contain not only phospholipids, but also surfactant apoproteins, regarded until recently as specific products of the bronchoalveolar epithelium. Presented here are the various roles, most of them still speculative, of these phospholipids-containing products in the gastro-intestinal tract.

[Normal and abnormal alveolar development]. / Développement alvéolaire normal et pathologique.

INTRODUCTION: Alveolar growth predominantly occurs post-natally and is characterised by the multiplication of alveoli, the thinning of inter-alveolar walls, and the maturation of capillary vessels. STATE OF THE ART: Alveolar growth is controlled by numerous factors whose interactions remain poorly understood. Many phenomena can interfere with normal alveolar growth resulting in abnormal development and a reduction in alveolar surface. This is especially the case in premature human neonates, whose lungs are structurally and functionally immature. Oxygen therapy, mechanical ventilation, and airway inflammation may induce alveolar growth disorders in these children, resulting in the development of bronchopulmonary dysplasia. PERSPECTIVES AND CONCLUSIONS: Further studies are needed to improve our knowledge about alveolar growth regulatory mechanisms so that better strategies can be developed to prevent these respiratory complications in premature neonates.

Dihydrofolate reductase and thymidylate synthetase activities in the liver of rabbit foetuses during the haemapoietic period.

Dihydrofolate reductase activity did not change with variations of mitotic rate in foetal rabbit haemopoietic tissue, whereas the activity of thymidylate synthetase declined with decrease in cell divisions. Both the activities were normal in the brachydactylia strain, in which a failure in folate metabolism was previously assumed.

Role of fetal insulin in glycogen metabolism in the liver of the rat fetus.

In the near-term rat fetus liver, insulin decreased phosphorylase a activity, slightly increased synthase a activity and increased both glycogen content and 14C-glucose incorporation into glycogen; anti-insulin serum increased phosphorylase a activity and decreased glucose incorporation but did not modify synthase a activity. When pregnant rats were previously rendered hypoglycemia, insulin injection to the fetus enhanced glycogen accumulation. It is concluded that insulin is a regulatory factor of secondary importance for liver glycogen storage through its action on phosphorylase activity.