Cause-of-Death Analysis in Rodent Aging Studies.

In research studies using rats or mice, the cause of death is often not evaluated or reported. An analysis of the causes of death is particularly valuable for aging and carcinogenesis studies. Comparing causes of death among the study groups is often an important adjunct to the biochemical, molecular, clinical, and histopathologic findings. The methods for evaluating causes of death, contributing causes of death, and comorbidities have been suggested in several publications. Surprisingly, in important mouse aging studies, causes of death are often not reported. Cause-of-death assignment in preclinical rodent model aging research suffers from a lack of a standardized approach and an understanding of the value that it can add to longevity and interventional studies. While assigning single cause of death may facilitate data analysis, defining and publishing data on contributing causes (comorbidities) provides more information on associated underlying chronic conditions and health span in mouse models. This article reviews factors that affect determination of cause of death and the methods for evaluating causes of death and comorbidities. The proposed systematic pathology analysis includes assigning cause of death and comorbidities to define total disease burden. The combination of pathology with in vivo data will fully characterize the effect of tested interventions on multiple chronic diseases and health span of aging mice with improved translation to human aging and age-associated lesions.

The Vital Role of Pathology in Improving Reproducibility and Translational Relevance of Aging Studies in Rodents.

Pathology is a discipline of medicine that adds great benefit to aging studies of rodents by integrating in vivo, biochemical, and molecular data. It is not possible to diagnose systemic illness, comorbidities, and proximate causes of death in aging studies without the morphologic context provided by histopathology. To date, many rodent aging studies do not utilize end points supported by systematic necropsy and histopathology, which leaves studies incomplete, contradictory, and difficult to interpret. As in traditional toxicity studies, if the effect of a drug, dietary treatment, or altered gene expression on aging is to be studied, systematic pathology analysis must be included to determine the causes of age-related illness, moribundity, and death. In this Commentary, the authors discuss the factors that should be considered in the design of aging studies in mice, with the inclusion of robust pathology practices modified after those developed by toxicologic and discovery research pathologists. Investigators in the field of aging must consider the use of histopathology in their rodent aging studies in this era of integrative and preclinical geriatric science (geroscience).

Early-life co-administration of cockroach allergen and endotoxin augments pulmonary and systemic responses.

BACKGROUND: Environmental exposures to cockroach allergen and endotoxin are recognized epidemiological risk factors for the early development of allergies and asthma in children. Because of this, it is important to examine the role of early-life concurrent inhalation exposures to cockroach allergen and endotoxin in the pathogenesis of allergic airways disease. OBJECTIVE: We examined the effects of repeated concomitant endotoxin and cockroach allergen inhalation on the pulmonary and systemic immune responses of newborn and juvenile mice. METHODS: C3H/HeBFeJ mice were exposed to inhaled endotoxin and cockroach allergen via intranasal instillation from day 2 to 21 after birth, and systemic and pulmonary responses were examined in serum, bronchoalveolar lavage fluid, and lung tissue. RESULTS: Cockroach allergen exposures induced pulmonary eosinophilic inflammation, total and allergen-specific IgE, IgG(1), and IgG(2a) production, and alveolar remodelling. Co-exposures with endotoxin and cockroach allergen significantly increased serum IgE and IgG(1), lung inflammation, and alveolar wall thickness, and decreased airspace volume density. Importantly, compared with exposures with individual substances, the responses to co-exposures were more than additive. CONCLUSIONS: Repeated inhalation exposures of neonatal and juvenile mice to endotoxin and cockroach allergen increased the pulmonary inflammatory and systemic immune responses in a synergistic manner and enhanced alveolar remodelling in the developing lung. These data underscore the importance of evaluating the effect of multiple, concurrent environmental exposures, and of using an experimental model that incorporates clinically relevant timing and route of exposures.

Defective internalization of low density lipoprotein in epidermoid cervical cancer cells.

Cells of an epidermoid cancer cell line of human uterine cervix, which possessed a high-affinity, specific receptor for low density lipoprotein (LDL), internalized and degraded [125I]iodo-LDL at a very low rate. In these cells, LDL did not stimulate cholesteryl ester synthesis, nor did it suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase to the same extent as in the control cells. The binding of [125I]iodo-LDL by these cells was not decreased by preincubation of the cells in medium containing LDL. Using ferritin-labeled LDL (F-LDL) and electron microscopy, it was determined that at 4 degrees C the cells bound F-LDL in the same way as other cancer cell lines that did not have a defect in internalization. When these cells were warmed to 37 degrees C the F-LDL remained on the surface, whereas in cells from control cancer cell lines the F-LDL was internalized and was no longer observed on the cell surface. On the basis of the results of these studies it is concluded that cells of this epidermoid cancer cell line have a defective ability to internalize LDL.

Secondary intracranial neoplasia in the dog: 177 cases (1986-2003).

BACKGROUND: This study investigates the frequency, location, and clinical findings associated with 177 secondary brain tumors in dogs. HYPOTHESIS: Secondary intracranial neoplasia is more common than primary intracranial neoplasia in dogs during the time period studied, and hemangiosarcoma (HSA) is the most common secondary intracranial tumor. ANIMALS: One hundred and seventy-seven client-owned dogs presented to the Matthew J. Ryan Veterinary Hospital between 1986 and 2003. METHODS: Medical records were searched for a diagnosis of intracranial neoplasia in dogs who underwent complete postmortem examination. Of these dogs, those with a diagnosis of primary intracranial neoplasia were excluded. RESULTS: Of the 177 secondary brain tumors, 51 (29%) were HSAs, 44 (25%) were pituitary tumors, 21 (12%) were lymphosarcomas, and 21 (12%) were metastatic carcinomas. The average age at diagnosis was 9.6 +/- 3.0 years. Most tumors were located in the cerebrum, and a mentation change was the most common presenting clinical sign. On postmortem examination, the same tumor that was in the brain was also present in the lung in 84 cases (47%), in the kidney in 62 cases (35%), and in the heart in 55 cases (31%). CONCLUSIONS AND CLINICAL IMPORTANCE: Secondary intracranial neoplasia in dogs was more common than primary intracranial neoplasia during the time period studied. Many of these dogs had related disease in other body systems that was apparent on diagnostic tests such as thoracic radiography.

Localization of cystic fibrosis transmembrane conductance regulator mRNA in human fetal lung tissue by in situ hybridization.

The fetal pulmonary epithelium secretes fluid. Cl transport is presumed to provide the driving force for net fluid secretion, although the cellular mechanisms have not been well identified in the fetus. The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP- and nucleoside triphosphate-regulated Cl channel; mutations in CFTR cause cystic fibrosis. We hypothesized that if CFTR is involved in fetal lung fluid transport, the fetal pulmonary epithelium should express CFTR mRNA. We used the technique of in situ hybridization with 3H-anti-sense and, as a control, 3H-sense CFTR cRNA probes to localize CFTR mRNA in human fetal lung tissue and cultured lung explants and determine when in gestation it is expressed. Epithelial cells of both first and second trimester lung tissues expressed CFTR mRNA. A decreasing gradient of CFTR mRNA expression was present from the proximal to the distal pulmonary epithelium. Cultured second trimester lung tissue explants expressed more CFTR mRNA than the uncultured starting tissue, suggesting CFTR gene expression increased during the five days in culture. Furthermore, alveolar type II cells in cultured explants expressed CFTR mRNA, suggesting that these cells are Cl-secretory and may be involved in lung fluid transport. These data confirm that CFTR mRNA is expressed in the human fetal pulmonary epithelium, consistent with the Cl-secretory properties of the fetal lung.

Induction and characterization of the major surfactant apoprotein during rabbit fetal lung development.

Antibodies directed against the major apoprotein associated with rabbit lung surfactant were used to characterize the induction and cellular localization of this protein during rabbit fetal lung development. In lung tissues from rabbits of 26 days gestational age and older, discrete epithelial type II cells were stained positively using the peroxidase antiperoxidase technique. The content of the major protein in homogenates of fetal lung tissue was analyzed using an immunoblotting technique. A protein of about 29 kDa, pI less than or equal to 5.6, was first detectable in fetal lung tissue on day 24 of gestation. The 29-36 kDa, mature form of the surfactant apoprotein was first detectable in lung homogenates from 30-day gestational age fetal rabbits. Treatment of homogenates of day 26 and 31 fetal lung tissues with endoglycosidase F, yielded, in both cases, an immunoreactive triplet with more neutral isoelectric points than the proteins in the untreated homogenates. By immunoblot analysis, we found that only the 29-36 kDa, mature form of the surfactant apoprotein was present in lamellar bodies purified from lung tissues of fetuses of 28 and 31 days and from day 2 neonates. These findings are suggestive that only the mature, 29-36 kDa form of the surfactant apoprotein is associated with lamellar bodies during fetal lung type II cell differentiation in vivo.

Effect of cortisol on the synthesis of lamellar body glycerophospholipids in fetal rabbit lung tissue in vitro.

The effect of cortisol on the rate of choline incorporation into tissue phosphatidylcholine was investigated in lung explants from fetal rabbits of 19-28 days gestational age. The explants were incubated in medium with or without fetal calf serum for up to 7 days. When lung tissues were incubated in serum-free medium, a stimulatory effect of cortisol on tissue phosphatidylcholine synthesis was found in explants from 21-, 24-, 26- and 28-day fetal rabbits; a stimulatory effect of cortisol was observed in 19-day fetal lung explants only if fetal calf serum was present in the culture medium. To assess directly the effect of cortisol on the synthesis of lamellar body phosphatidylcholine, choline incorporation into phosphatidylcholine associated with a purified lamellar body fraction isolated from lung explants of 21- and 28-day fetal rabbits was also investigated. Cortisol caused a marked stimulation of synthesis and accumulation of lamellar body phosphatidylcholine in lung explants from both 21- and 28-day fetal rabbits. The magnitude of the stimulatory effect of cortisol on the rate of synthesis of lamellar body phosphatidylcholine was always greater than the effect of cortisol on the rate of choline incorporation into lipids of tissue homogenates. The relative rates of synthesis of lamellar body phosphatidylinositol and phosphatidylglycerol were also significantly altered in lung explants from 21- and 28-day fetal rabbits by cortisol treatment. Lamellar bodies that were formed initially in the fetal lung explants were enriched in phosphatidylcholine and phosphatidylinositol and had a relatively low phosphatidylglycerol content. With cortisol treatment there was a decrease in the relative rate of synthesis of lamellar body phosphatidylinositol and an increase in the relative rate of synthesis of phosphatidylglycerol. The stimulatory effect of cortisol on the synthesis of lamellar body phosphatidylcholine was observed at an earlier time-point of incubation than was the effect of cortisol on the relative rates of synthesis of lamellar body phosphatidylinositol and phosphatidylglycerol. The temporal sequence of the cortisol-induced changes in the synthesis of lamellar body glycerophospholipids, therefore, reflects that which occurs with maturation in vivo.

Characterization of rabbit surfactant-associated proteins.

In the present study, the apolipoproteins associated with a purified surfactant fraction isolated from lung lavage of adult rabbits were characterized. Surfactant purity was assessed by the glycerophospholipid composition and by electron microscopic examination. The purified surfactant was delipidated and the apolipoproteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. By use of this technique at least eight proteins or families of proteins were found to be associated with surfactant. Four of these apolipoproteins were families of proteins of 55-70, 29-36, 26-28 and 22-23 kilodaltons (kDa). All of these apolipoprotein families had acidic isoelectric points (pI less than or equal to 5.6), and were specifically bound to a Con A-Sepharose matrix, indicative that these apolipoprotein families are modified by oligosaccharide side-chains. The finding that neuraminidase treatment degraded the 29-36 kDa family is suggestive that this apolipoprotein family contains sialic acid residues. Three major proteins of 66, 43-45 and 35 kDa and a minor protein of 86 kDa were also observed. These proteins had isoelectric points in the more neutral range (pI 6.0-6.5). The 66 kDa protein (pI 6.4) had the same apparent molecular weight and isoelectric point as the major protein of delipidated rabbit serum and as purified rabbit albumin, which suggests that this protein is albumin. These findings are indicative that the apolipoproteins of surfactant are more numerous and complex than previously reported.

Insulin inhibits surfactant protein A and B gene expression in the H441 cell line.

Fetuses of mothers with uncontrolled gestational diabetes have an increased risk of developing neonatal respiratory distress syndrome and are frequently hyperinsulinemic, thus it has been proposed that high levels of insulin delay fetal lung maturation. We have shown previously that insulin inhibits the accumulation of mRNA for the surfactant-associated proteins A and B (SP-A and SP-B) in human fetal lung explants maintained in vitro. To test the hypothesis that the inhibitory effects of insulin on the surfactant proteins are the result of a direct action of insulin on the lung epithelial cell, we evaluated the effects of insulin in the H441 cell line, a human pulmonary adenocarcinoma cell line that expresses SP-A and SP-B mRNA. We observed that insulin treatment for 48 h decreased SP-A mRNA and protein levels in a concentration-dependent manner when compared to controls. The inhibitory effect of insulin on SP-A mRNA levels was apparent as early as after 4 h of exposure. SP-B mRNA levels were also significantly decreased by insulin in a concentration-dependent manner. Insulin, at 2.5 microg/ml, inhibited SP-A gene transcription by approx. 67%, and inhibited SP-B gene transcription by about 32%. There was no significant effect of insulin on SP-A or SP-B mRNA stability. Thus, we have observed a pattern of insulin inhibition of SP-A and SP-B gene expression in the H441 lung epithelial cell line similar to that previously observed in human fetal lung explants, which are comprised of both epithelial and mesenchymal cells. Our findings provide further evidence that insulin may delay fetal lung maturation by inhibiting SP-A and SP-B gene expression. Furthermore, our findings suggest that the inhibitory effects of insulin are, at least partially, the result of a direct action on the lung epithelial cell.

Uptake of the 35 kDa major surfactant apoprotein (SP-A) by neonatal rabbit lung tissue.

Secreted surfactant is made up of both phospholipid and protein components. Therefore, we investigated the possibility that surfactant apoproteins might be taken up by the alveolar type II cell in a manner similar to the uptake of surfactant phosphatidylcholines. Day 2 neonatal rabbits were infused via the trachea with a solution of carrier surfactant and 125I-labelled surfactant apoprotein (SP-A, Mr approx. 35,000). Most of the 125I-SP-A remained within the alveolus; however, a fraction of the 125I-SP-A was taken up by the lung tissue from the alveolus in a time-dependent manner. The small amount of radiolabeled material detected in blood, liver or kidney tissues of 125I-SP-A-infused animals was not trichloroacetic acid (TCA) precipitable, i.e., probably represented degradation products. In contrast, the proportion of TCA-precipitable 125I-SP-A in lung tissue or lavage samples did not change as function of time after tracheal administration. Two-dimensional gel electrophoresis of the 125I-SP-A present in the lavage samples or associated with lung tissue was used to show that a small proportion of the 125I-SP-A was partially degraded in the lung tissue and alveolus. These data are suggestive that the SP-A is taken up by lung tissue, perhaps in a manner similar to the uptake of surfactant phospholipid by the alveolar type II cell.

Insulin inhibits the accumulation of the major lung surfactant apoprotein in human fetal lung explants maintained in vitro.

In the present study, we characterized the proteins associated with a purified lamellar body fraction isolated from human fetal lung explants. We then raised antibodies directed against the major human surfactant apoprotein, a 35-K glycoprotein, and used the technique of immunoblot analysis to evaluate the content of the surfactant apoprotein in human fetal lung explants maintained in serum-free medium in vitro. We found that the 35K surfactant apoprotein was undetectable in homogenates of fetal lung tissue before culture; the surfactant apoprotein was induced in the cultured explants coincident with the appearance of differentiated type II cells. Insulin, at concentrations as low as 2.5 ng/ml, caused marked inhibition of the accumulation of the 35K protein in the cultured fetal lung tissue. The inhibitory effect of insulin was dose dependent and was apparent as early as day 2 of incubation. When explants were cultured in medium containing insulin plus cortisol, the amount of immunoreactive surfactant apoprotein was reduced compared to that of explants cultured in control medium or explants cultured with cortisol alone. On the other hand, as reported previously, insulin and cortisol, in combination, stimulated phosphatidylcholine synthesis. These findings are indicative that the phospholipid and apoprotein components of surfactant are regulated independently. The results of our studies suggest that fetal hyperinsulinemia may cause the production of a surfactant deficient in the 35K apoprotein, and this may provide an explanation for the increased incidence of respiratory distress syndrome in infants of diabetic mothers.

Retinoic acid differentially regulates expression of surfactant-associated proteins in human fetal lung.

Retinoic acid is known to play an essential role in maintaining the differentiation of a wide variety of epithelial cell types. However, its effects on the differentiation of lung alveolar epithelium have not been described. In the present study, we examined the effects of retinoic acid on the differentiation of human fetal lung tissue maintained in vitro. Human fetal lung explants were cultured in serum-free medium for 6 days in the absence or presence of all-trans retinoic acid at concentrations from 0.3 nM to 3 microM. Explant content of the surfactant-associated protein SP-A was measured using a specific enzyme-linked immunosorbent assay. Retinoic acid reduced SP-A protein levels in a concentration-dependent manner [analysis of variance (ANOVA), P < 0.01]. To evaluate possible cytotoxic effects of retinoic acid, culture media were assayed for lactate dehydrogenase (LDH), a cytoplasmic enzyme. LDH levels in media from retinoic acid-treated explants were not significantly different than LDH levels in media from control explants, indicating that retinoic acid is not cytotoxic in human fetal lung explants. Changes in messenger RNA (mRNA) levels for surfactant-associated proteins SP-A, SP-B, and SP-C were measured by Northern blot analysis. Retinoic acid reduced SP-A mRNA levels in a concentration-dependent manner (ANOVA, P < 0.02) and reduced SP-C mRNA levels at 3 microM. In contrast, retinoic acid increased SP-B mRNA levels in a concentration-dependent manner (ANOVA, P < 0.03). Morphometric analysis showed that retinoic acid decreased epithelial volume density in the explants by approximately 17% and increased connective tissue volume density by approximately 20% when compared to dimethyl sulfoxide vehicle controls. These data indicate that retinoic acid regulates type II cell surfactant protein gene expression in human fetal lung tissue.

Insulin regulation of messenger ribonucleic acid for the surfactant-associated proteins in human fetal lung in vitro.

Respiratory distress syndrome (RDS) is primarily caused by an immaturity in the synthesis and secretion of surfactant by the fetal lung type II cell. Fetal hyperinsulinemia associated with maternal diabetes places the newborn at an increased risk of developing RDS, and therefore, it has been hypothesized that insulin inhibits type II cell differentiation. We have previously shown that insulin inhibits the accumulation of surfactant-associated protein A (SP-A), the major surfactant-associated protein, in human fetal lung explants maintained in vitro. In the present study, we used Northern blot analysis to evaluate the effects of insulin on the content of SP-A messenger RNA (mRNA) as well as on the content of mRNA for the hydrophobic surfactant-associated proteins SP-B and SP-C in human fetal lung explants maintained in vitro. Lung explants were maintained in serum-free medium with or without added insulin (0.25-2500 ng/ml) for up to 6 days. We observed that insulin, at concentrations of 25-2500 ng/ml, significantly inhibited the accumulation of SP-A mRNA when compared to controls (P less than 0.01). The inhibitory effect of insulin on SP-A mRNA accumulation was dose dependent with an approximately 75% inhibition observed at 2500 ng/ml. Insulin, at the concentration of 2500 ng/ml, significantly inhibited the accumulation of SP-B mRNA by approximately 30% when compared to control levels (P less than 0.01) but had no effect at lower concentrations. Insulin had no significant effect on SP-C mRNA levels at any concentration tested. Our findings provide evidence that insulin may delay fetal lung development by inhibiting SP-A and SP-B gene expression. A deficiency of these proteins in pulmonary surfactant may account for the increased incidence of RDS in infants of diabetic mothers.

Glucocorticoid regulation of the major surfactant associated protein (SP-A) and its messenger ribonucleic acid and of morphological development of human fetal lung in vitro.

In the present study we investigated the effects of dexamethasone (DEX) on the accumulation of the major pulmonary surfactant-associated protein (SP-A), a glycoprotein of about 35,000 mol wt, and on the levels of mRNA encoding this protein in human fetal lung in organ culture. In addition, the effects of DEX on the structural development of the fetal lung tissue was investigated using morphometric techniques. We observed that DEX had a biphasic effect on the accumulation of SP-A and its mRNA; at concentrations of 10(-10) and 10(-9) M, a stimulatory effect was observed, while at concentrations of 10(-8) M or greater, the glucocorticoid was markedly inhibitory. The inhibitory effect of DEX (10(-7) M) was evident at all time points of incubation and was apparent within 24 h of its addition to the medium at any time during the culture period. In addition, DEX (10(-7) M) antagonized the stimulatory effects of (Bu)2cAMP on the accumulation of SP-A and its mRNA. DEX also had pronounced effects on the morphological development of human fetal lung tissue. At a concentration of 10(-7) M or greater, DEX caused a marked reduction of alveolar lumen size compared to that of fetal lung explants maintained in control medium. A biphasic effect of DEX on the volume density of type II cells in fetal lung explants was observed; at a concentration of 10(-10) M, DEX significantly increased the volume density of type II cells, whereas at a concentration of 10(-7) M or more, the glucocorticoid significantly reduced the volume density of type II cells compared to that of control explants. These findings suggest that synthetic glucocorticoids at concentrations of 10(-10) and 10(-9) M are stimulatory whereas elevated levels are inhibitory of SP-A synthesis and morphological development of the human fetal lung.

Adenosine 3',5'-monophosphate analogs and beta-adrenergic agonists induce the synthesis of the major surfactant apoprotein in human fetal lung in vitro.

The use of beta-adrenergic agonists in the treatment of preterm labor has been found to be associated with a decreased incidence of respiratory distress syndrome (RDS) in premature newborns. beta-Sympathomimetic agents, which activate adenylate cyclase and increase tissue cAMP levels, as well as cAMP analogs stimulate surfactant glycerophospholipid synthesis and secretion by fetal lung tissue. In the present study, we used antibodies directed against the major human pulmonary surfactant apoprotein, a 35,000-dalton glycoprotein, to evaluate the effects of the cAMP analog dibutyryl cAMP (Bt2cAMP) and the beta-adrenergic agonist terbutaline on surfactant apoprotein synthesis in human fetal lung explants in organ culture. By use of immunoblot analysis, we found that Bt2cAMP (1 mM) markedly stimulated accumulation of the major surfactant apoprotein in human fetal lung explants, as did terbutaline. Bt2cAMP treatment also increased the relative rate of incorporation of [35S]methionine into the major surfactant apoprotein. The Bt2cAMP-induced increase in surfactant apoprotein synthesis and accumulation was associated with an increase in the levels of translatable surfactant apoprotein mRNA. Morphometric analysis at both the light and electron microscopic levels was used to evaluate the effects of Bt2cAMP on the morphology of the human fetal lung in vitro. After 48-h incubation with Bt2cAMP, the prealveolar ducts of the fetal lung explants were enlarged greatly, and the relative amount of interalveolar connective tissue was reduced compared to those in control tissues. The volume density of type II cells in the Bt2cAMP-treated explants was significantly greater than that in control explants at this time point; however, after 4 and 6 days of incubation, the volume density of type II cells in control and Bt2cAMP-treated tissues was similar, and the lumina of the prealveolar ducts of control tissues had a volume density similar to that of Bt2cAMP-treated explants. Bt2cAMP also had pronounced effects on the ultrastructural morphology of the human fetal lung explants. Large quantities of secreted lamellar bodies and tubular myelin were observed in the lumina of the prealveolar ducts of the Bt2cAMP-treated tissue. Few lamellar bodies and no tubular myelin were observed in the lumina of the prealveolar ducts of control tissues. These findings suggest that cAMP may serve an important regulatory role in the synthesis and secretion of the major surfactant apoprotein by human fetal lung.

Hormonal regulation of the synthesis of lamellar body phosphatidylglycerol and phosphatidylinositol in fetal lung tissue.

The influence of hormones on the synthesis of phospholipids of lung lamellar bodies was studied using lung tissue explants from 16- to 22-week gestational age human abortuses. Lung explants were incubated in a defined medium containing either no hormonal additions or insulin (2.5 micrograms/ml), cortisol (0.2 microgram/ml), or PRL (2.5 micrograms/ml), added alone or in various combinations. After 7 days of incubation, a purified lamellar body fraction was isolated from the explants. The effects of various hormone combinations on the relative rates of synthesis of lamellar body anionic glycerophospholipids during the last 24 h of culture were analyzed and compared to that of control explants. In control explants, 7.1% of the glycerophospholipid was synthesized as phosphatidylinositol, and 2.2% was synthesized as phosphatidylglycerol. Phosphatidylinositol synthesis was decreased significantly in explants incubated with cortisol alone, insulin plus cortisol, or insulin plus cortisol plus PRL (to 4.4%, 4.3%, and 3.7%, respectively), while phosphatidylglycerol synthesis was increased significantly in lamellar bodies from explants incubated with insulin plus cortisol or insulin plus cortisol plus PRL (to 5.5% and 5.7%, respectively). Although the relative rates of synthesis of lamellar body phosphatidylinositol and phosphatidylglycerol were significantly altered by hormones, the percentages of the total anionic glycerophospholipid were similar in all treatment groups (9.5%). The results of this study, therefore, are suggestive that lamellar body formation and glycerophospholipid composition are under hormonal control.

Prostaglandins regulate surfactant protein A (SP-A) gene expression in human fetal lung in vitro.

We previously have observed that dexamethasone has a biphasic effect on surfactant protein A (SP-A) mRNA levels in human fetal lung in vitro. At concentrations of 10(-10)-10(-9) M, dexamethasone increases the levels of SP-A mRNA, whereas, at concentrations greater than 10(-8) M, the steroid is markedly inhibitory. In studies to define the molecular mechanisms for these effects, we observed that dexamethasone causes a dose-dependent stimulation of SP-A gene transcription, but paradoxically causes a dose-dependent inhibition of SP-A mRNA stability. In light of the well-characterized inhibitory effect of glucocorticoids on prostaglandin (PG) synthesis in a number of tissues, it was our objective in the present study to investigate the role of PGs on SP-A gene expression in human fetal lung in vitro and to determine whether the action of dexamethasone (greater than 10(-8) M) to reduce SP-A mRNA levels could be mediated by its effect to inhibit PG synthesis. We found that dexamethasone 10(-7) M) caused a marked decrease in the secreted levels of the PGE2 and PGF2 alpha, the prostacyclin metabolite, 6-keto-PGF1 alpha, and the thromboxane A2 metabolite, thromboxane B2. Indomethacin, which also caused a pronounced reduction in the levels of these secreted prostanoids, had a marked effect to reduce SP-A mRNA levels in human fetal lung in vitro. The inhibitory effects of indomethacin were associated with an 73% reduction in cAMP formation by the fetal lung in culture, and were prevented by simultaneous incubation with dibutyryl cAMP or with PGE2. PGE2 markedly increased cAMP formation by the human fetal lung tissue incubated in the absence or presence of indomethacin. Inhibitory effects of dexamethasone and indomethacin also were observed on two morphological indices of lung differentiation, alveolar lumenal volume density, and lamellar body volume density. PGE2 significantly increased lumenal volume density of the human fetal lung explants. The finding that the inhibitory action of dexamethasone (10(-7) M) on SP-A mRNA levels could not be prevented by simultaneous incubation with either PGE2 or dibutyryl cAMP and that dexamethasone had no apparent effect on cAMP formation by the fetal lung in vitro is suggestive that the action of dexamethasone (greater than or equal to 10(-8) M) to reduce SP-A mRNA levels is mediated at least in part by actions alternative to its inhibitory effects on PG synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Multihormonal regulation of surfactant synthesis by human fetal lung in vitro.

Explants prepared from the lung tissue of human abortuses of 20-22 weeks gestational age were incubated in defined medium without serum. These tissues developed the capacity for surfactant synthesis within 4 days. The ductular epithelium differentiated into type II pneumonocytes that contained numerous lamellar bodies. These morphological changes were accompanied by an increase in the rate of choline incorporation into phosphatidylcholine as well as an increase in the phosphatidylcholine content of the explants. Cortisol (0.2 micrograms/ml) plus PRL (2.5 micrograms/ml), when added to the medium from the beginning of the culture period, caused a 2- to 3-fold increase in the rate of choline incorporation into phosphatidylcholine, as measured on the second, fourth, sixth, and eighth days of incubation, as well as an increase in the phosphatidylcholine content of the explants. However, when administered alone, neither cortisol nor PRL affected phosphatidylcholine synthesis. In explants incubated with cortisol plus PRL there also was a stimulation of lamellar body secretion into the prealveolar ducts. The lamellar bodies in epithelial cells were larger in cortisol- plus PRL-treated tissue than those in nontreated tissues. Increases in phosphatidylcholine synthesis and lamellar body secretion also were observed in tissues incubated with insulin (2.5 micrograms/ml), cortisol, and PRL in combination or with insulin and cortisol in combination. The stimulatory effect of cortisol plus insulin on phosphatidylcholine synthesis, however, was significantly less than that of cortisol plus PRL or cortisol plus insulin plus PRL. It is suggested that human fetal lung development is under multihormonal control and that PRL and cortisol serve to increase surfactant synthesis and secretion.

Fat content for nutritional labeling by supercritical fluid extraction and an on-line lipase catalyzed reaction.

A method using sequential supercritical fluid extraction (SFE) and enzymatic transesterification has been developed for the rapid determination of total nutritional fat content in meat samples. SFE conditions of 12.16 MPa and 50 degrees C were utilized to extract lipid species from the sample matrix. The enzymatic transesterification of the lipids by methanol was catalyzed by an immobilized lipase isolated from Candida antarctica. Conversion of the triglycerides to fatty acid methyl esters was monitored by supercritical fluid chromatography, while the fatty acid content of the extract was determined by capillary gas chromatography (GC). Total fat, saturated fat and monounsaturated fat contents were calculated from the GC data and compared to values from traditional extraction and lipid determination methods. Both off-line SFE and automated SFE followed by on-line GC analysis using two different instruments were utilized in this study. The enzymatic-based SFE method gave comparable results to the organic solvent extraction-based method followed by conventional BF3-catalyzed esterification.