Programmed cell death in normal fetal rat lung development.

Lung development is a coordinated process regulated by the interactions of extracellular and intracellular factors, yet little is known about the process of programmed cell death during lung development. To study this question, we examined fetal rat lung from the pseudoglandular period (gestational day 15) to the day of birth (gestational day 21) using BrdU incorporation into DNA as a proliferative marker, while in parallel examining several markers of programmed cell death including terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), DNA "laddering, " and expression of programmed cell death pathway proteins. Cell proliferation was ongoing throughout fetal days 15 to 21 with a decrease in proliferation over days 20 and 21. Programmed cell death in fetal lung also appeared to be present at all ages examined, but demonstrated 2 peaks of activity at fetal days 15 and 18 to 20. Bcl-XL expression was detected on fetal days 15 to 21, with diminished expression on days E15 to E18. Cleaved poly(ADP-ribose)polymerase (PARP), activated caspase-3, Bax, and Bad were increased on days 18 to 20. We conclude that proliferation is the primary process driving fetal lung development with programmed cell death occurring throughout the lung developmental process to refine structural remodeling.

Relation between serum insulinlike growth factor-1, insulinlike growth factor binding protein-2, and insulinlike growth factor binding protein-3 and nutritional intake in premature infants with bronchopulmonary dysplasia.

BACKGROUND: The usefulness of serum insulinlike growth factor (IGF)-system-peptide measurement to assess the adequacy of nutritional intake in premature infants with chronic lung disease bronchopulmonary dysplasia (BPD) was assessed. METHODS: Twenty-nine premature infants had serial measurements taken of their serum IGF-1, insulinlike growth factor binding protein (IGFBP)-2, and IGFBP-3 concentrations between 2 and 6 weeks of age. Regression analyses were used to examine the relation between nutritional parameters and IGF-1, IGFBP-2, and IGFBP-3 concentrations in premature infants with and without BPD. RESULTS: The group of infants with BPD (n = 12) did not differ from infants without BPD (n = 17) in gestational age or weight at entry, but gained less weight during the study period. In infants without BPD, IGF-1 correlated positively with protein intake (r = 0.50) and caloric intake (r = 0.41) over the 3 days before sample collection and with weight change over the previous week (r = 0.46). In contrast, infants with BPD showed a significant correlation between IGF-1 and weight change (r = 0.54) only. There was a significant negative correlation between IGFBP-2 and protein intake in infants without BPD (r = -0.50) and in infants with BPD (r = -0.41). Negative correlations between IGFBP-2 and both weight change (r = -0.64) and caloric intake (r = -0.43) over the previous week were found only in the group of infants without BPD. IGFBP-3 correlated positively with weight changes and protein intake in both groups but correlated with caloric intake only in the group without BPD. Multiple regression analyses were used to determine significant independent variables associated with IGF-1, IGFBP-2, and IGFBP-3. In infants without BPD, significant independent predictors of IGFBP-2 were 7-day weight change and 2-day protein intake; 3-day caloric intake was the only significant independent predictor for IGFBP-3. For infants with BPD, 3-day weight gain was the only independent variable associated with serum IGF-1. Protein intake in the week before sample collection was an independent predictor of IGFBP-2 and 3-day weight change and 2-day protein intake were independent predictors of IGFBP-3. CONCLUSIONS: These results confirm that changes in serum IGF-1, IGFBP-2, and IGFBP-3 reflect the nutritional status of premature infants and demonstrate that the relation between these proteins and nutritional intake differs in premature infants with and without BPD. Refinement of these observations by future studies may permit a more accurate determination of the protein and caloric intake sufficient for growth and repair after injury in premature infants with lung disease.

Perinatal regionalization and neonatal mortality in North Carolina, 1968-1994.

OBJECTIVE: Our purpose was to analyze trends across time in the regionalization of low-birth-weight births and time trends for the association between regionalization and decreased neonatal mortality. STUDY DESIGN: Data on 69,452 neonates with birth weights of 500 to 2000 g were obtained from electronic files of birth certificates. Hospitals' perinatal services were classified as level 1, 2, or 3 (level 3 refers to tertiary referral centers). RESULTS: The likelihood of birth outside level 3 hospitals decreased from 1968 to 1994, with an average annual decrease of 24% for infants weighing 500 to 1500 g and 20% for infants weighing 1501 to 2000 g. After 1974, birth in a hospital with level 3 services was associated with a lower risk of dying. The strength of this association increased in the 1990s. CONCLUSIONS: In North Carolina the proportion of infants weighing <2000 g born outside a hospital with level 3 neonatal services declined from 1974 through 1994. After 1974, birth in a hospital with level 3 neonatal services was associated with lower neonatal mortality.

The clinical, morphologic, and molecular changes in the ileum associated with early postnatal dexamethasone administration: from the baby's bowel to the researcher's bench.

Focal small bowel perforation (FSBP) is a life-threatening event that predominantly affects extremely low birth weight (ELBW) infants. Histopathology from surgical specimens of ileum with FSBP shows a healthy mucosa overlying a thinned muscularis with segmental degeneration. Clinical data strongly support an association between early postnatal administration of dexamethasone (EPD) and FSBP. Additional risk factors, including gestational age, administration of prophylactic indomethacin, and severity of illness, may be synergistic with EPD for the pathogenesis of perforations. Animal models of dexamethasone administration show morphologic changes in the ileum, similar to those seen in ELBW infants, including increased mucosal maturation and thinning of the muscularis. These tissue-specific differences may be mediated by a perturbation in growth factor expression or accumulation. In support of this hypothesis, dexamethasone has been associated with increased IGF-I immunolocalization in the mucosa and decreased immunolocalization in the muscularis. The known growth-promoting functions of IGF-I are consistent with the observed dexamethasone-associated changes within both the mucosa and the muscularis. Ongoing studies in this animal model are exploring the potential mechanisms by which dexamethasone might affect IGF-I availability.

Dexamethasone administration to newborn mice alters mucosal and muscular morphology in the ileum and modulates IGF-I localization.

Glucocorticoid exposure accelerates the maturation of small bowel mucosa. We hypothesized that IGF-I, a mitogen and differentiating peptide expressed in small bowel, mediates steroid-induced change within the developing ileum. To investigate this possibility, we intraperitoneally administered 1 microg/gm/d of dexamethasone (DEX) or equal volumes of saline to litter-mate newborn mice. The animals were killed on d 1-3 of life and their ilea were harvested and prepared for microscopy. Tissue sections of ileum were examined for morphologic analyses, mucin staining, immunolocalization of IGF-I and -II, proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and in situ hybridization for IGF-I transcripts. Morphologic comparisons showed increases in goblet cell number, total cell number, and TUNEL-positive cells within the mucosa of DEX-treated animals. In contrast, the number of smooth muscle nuclei per cross-section was unchanged with DEX treatment despite a reduction in the number of PCNA-positive nuclei and an increased bowel circumference. These findings suggest the muscularis stretches to accommodate increasing bowel diameter. IGF-I peptide was localized to the mesenchyme of all control animals. After 48 h of DEX treatment, IGF-I was detected in the epithelia whereas mesenchymal IGF-I localization appeared diminished. In situ hybridization analyses for IGF-I transcripts showed no differences in localization between the groups. We conclude that DEX administration differentially affects adjacent tissues in the newborn ileum and that the associated changes in IGF-I localization are consistent with its participation in this process.

Early postnatal dexamethasone diminishes transforming growth factor alpha localization within the ileal muscularis propria of newborn mice and extremely low-birth-weight infants.

Focal small bowel perforation (FSBP) occurs most commonly in the ileum of extremely low-birth-weight (ELBW) infants. Early postnatal dexamethasone (EPD) administration results in an increased risk for FSBP in this patient population, but the mechanism by which this occurs is unknown. Infants with FSBP have healthy mucosa but thinned smooth muscle, suggesting a mechanism involving the muscularis propria for these perforations. One explanation for these findings would be that dexamethasone alters the tissue availability of pertinent growth factors to the smooth muscle. To explore this possibility, we administered dexamethasone or saline by intraperitoneal injection to newborn mice for 3 days (dosed at 1 microg/g of body weight/day) to simulate EPD protocols. The animals were sacrificed after 72 h of treatment and their ileums harvested and prepared for microscopy. Immunolocalization was performed for three related growth factors (epidermal growth factor [EGF], heparin-binding EGF [h-EGF], and transforming growth factor alpha [TGF-alpha]) and their common receptor. We found TGF-alpha to be abundant and discretely localized in the muscularis propria in control animals but to be diminished in dexamethasone-treated animals. EGF-receptor immunostaining was also decreased with dexamethasone but there was minimal to no detection of EGF or h-EGF in either treatment condition. Surgical and autopsy specimens of the ileum were obtained from seven ELBW infants who either received EPD or not. These tissues were used for immunolocalization of the same growth factors and similar distributions for TGF-alpha were observed in several of these cases. These findings are consistent with an autocrine role for TGF-alpha in ileal smooth muscle proliferation and suggest a mechanism by which EPD might mediate smooth muscle thinning.

Regulation of the rat BB1 RNA during normal rat lung development.

BB1 was recently cloned from the WI-38 human fetal lung cell line. Human BB1 (hBB1) is expressed by multiple tissues, including lung. Because inhibition of BB1 translation using antisense oligodeoxynucleotides resulted in prevention of G1 traversal in cultured cells, we hypothesized that BB1 gene expression would be regulated during lung development with greater expression during periods of active lung growth. To gain insight into the expression of BB1 during lung development, a rat BB1 (rBB1) homologue was cloned and used in Northern hybridization analyses and in situ hybridization histochemistry (ISHH). Northern hybridization analyses of fetal and postnatal rat lung demonstrate that rBB1 RNA abundance is relatively low on fetal days E17 through E19, with a small peak of expression occurring on fetal day E20, then increases at birth with peak expression in adult lung. ISHH correlates with the Northern hybridization data and reveals rBB1 RNA expression throughout lung from E17 to E21 in both epithelium and mesenchyme. In postnatal lung, more intense expression of BB1 was observed than in fetal lung, localizing BB1 transcripts to proximal and distal airways and mesenchymal cells surrounding airways. Proliferating cell nuclear antigen (PCNA) was identified in lung sections adjacent to those used for ISHH and it was found that BB1 expression was present in PCNA-positive cells; however, BB1 expression was not limited to PCNA-positive cells in either the fetal or postnatal periods. This was most apparent in adult (60-day) rat lung where essentially no PCNA-positive cells were detected, but intense BB1 expression was detected in airway epithelium and surrounding mesenchyme. These studies demonstrate developmental regulation of BB1 during lung development. The findings are consistent with BB1 action in cell growth-related processes of fetal and early postnatal lung; however, the distribution of BB1 expression in relation to PCNA localization suggests that BB1 participates in cellular functions in addition to cell proliferation.

Effects of retinoic acid on airspace development and lung collagen in hyperoxia-exposed newborn rats.

Impaired septal formation and decreased alveolarization are often caused by hyperoxic injury to the developing lung and are characteristic features of bronchopulmonary dysplasia. Dexamethasone, frequently administered to infants during oxygen exposure, also inhibits septal formation in the newborn lung. Vitamin A administration reduces the incidence of bronchopulmonary dysplasia in vitamin A-deficient premature infants, and retinoic acid improves alveolarization in newborn rats treated with dexamethasone, indicating that retinoic acid may be useful in preventing hyperoxia-induced impaired septation in bronchopulmonary dysplasia. To investigate whether treatment with retinoic acid during exposure to hyperoxia would improve septal formation, newborn rats exposed to > or =90% O(2) from d 3 of life to d 14 were treated with retinoic acid (d 3-13 of life) and/or dexamethasone (d 4-13 of life). In contrast with the effects of retinoic acid on dexamethasone-induced inhibition of alveolarization, we found that retinoic acid did not improve septal formation or decrease airspace size in animals exposed to hyperoxia alone or to hyperoxia plus dexamethasone. Retinoic acid did, however, increase collagen in airspace walls as demonstrated by staining and immunohistochemistry. There was no increase in procollagen mRNA by Northern hybridization analysis, indicating that retinoic acid-associated increases in lung collagen are likely due to posttranscriptional regulation. There was a trend toward increased survival in hyperoxia in animals treated with retinoic acid to the extent that combined therapy with retinoic acid and dexamethasone resulted in the greatest improvement in animal survival. These results suggest that although retinoic acid may be of benefit in hyperoxia-induced lung injury and may have important effects on lung matrix, it does not prevent impairment of septation or induce alveolar formation during exposure to hyperoxia.

Effect of initial nitric oxide concentration on outcome in infants with persistent pulmonary hypertension of the newborn.

A randomized nonblinded comparison of two treatment groups was performed to determine whether treatment of infants with persistent pulmonary hypertension of the newborn using a continuous 6-ppm dose of inhaled nitric oxide (iNO) changes the likelihood of death or utilization of extracorporeal membrane oxygenation (ECMO) when compared to infants treated with 20 ppm iNO for 4 h followed by 6 ppm. Twenty-nine infants with a gestational age >/=34 weeks and a diagnosis of persistent pulmonary hypertension of the newborn were enrolled during the 3- year study period. The relative risk (20/6 vs. 6 ppm) for treatment with ECMO was 3.11 (p = 0.02), for death it was 2.80 (p = 0.32), and for either death or ECMO it was 3.42 (p = 0. 006). There was no apparent advantage of treatment with a higher dosage of iNO at the initiation of therapy in the reduction of death or utilization of ECMO. These data suggest that a continuous lower dose of iNO results in a comparable improvement in oxygenation as a short exposure of higher dose iNO at the initiation of therapy.

Delivery room resuscitation decisions for extremely premature infants.

BACKGROUND: Neonatologists are criticized for overtreating extremely premature infants who die despite invasive and costly care. Withholding resuscitation at delivery has been recommended as a way to minimize overtreatment. It is not known how decisions to forgo initiating aggressive care are made, or whether this strategy effectively decreases overtreatment. OBJECTIVE: To identify whether physicians' or parents' preferences primarily determine the amount of treatment provided at delivery, to examine factors associated with the provision of resuscitation, and to assess whether resuscitation at delivery significantly postpones death in nonsurvivors. METHODS: We evaluated delivery room resuscitation decisions and mortality for all infants born at 23 to 26 weeks gestation at the University of North Carolina Hospitals from November 1994 to October 1995. On the day of delivery, the attending neonatologist completed a questionnaire regarding discussion with the parents before delivery, the prognosis for survival estimated before delivery, the degree of certainty about the prognosis, parents' preference for the amount of treatment at delivery, and the degree of influence exerted by parents and physicians on the amount of delivery room treatment provided. Medical records were reviewed for demographics and hospital course. RESULTS: Thirty-one of 41 infants were resuscitated (intubation and/or cardiopulmonary resuscitation) at delivery. Resuscitation correlated with increasing gestational age, higher birth weight, estimated prognosis for survival greater than or equal to 10%, and uncertainty about prognostic accuracy. Physicians saw themselves as primarily responsible for delivery room resuscitation decisions when the parents' wishes about initiating care were unknown, and as equal partners with parents when they agreed on the level of care. When disagreement existed, doctors always thought parents preferred more aggressive resuscitation, and identified parents as responsible for the increased amount of treatment at delivery. Twenty-four infants died before hospital discharge. The median age at death was 2 days when physicians primarily determined the amount of treatment at delivery, 1 day when parents primarily determined the amount of treatment, and < 1 day when responsibility was shared equally. The median age at death was < 1 day when physicians and parents agreed about the preferred amount of treatment at delivery and 1.5 days when they disagreed. The median age at death was < 1 day when parents' preferences were known before delivery and 4 days when parents' preferences were unknown. CONCLUSIONS: Physicians resuscitated extremely premature infants at delivery when they were very uncertain about an infant's prognosis or when the parents' desires about treatment were unknown. When parents' preferences were known, parents usually determined the amount of treatment provided at delivery. Resuscitation at delivery usually postponed death by only a few days, decreasing prognostic uncertainty and honoring what physicians perceived were parents' wishes for care, without substantially contributing to overtreatment.

Expression of the insulin-like growth factor system in postpneumonectomy lung growth.

The insulin-like growth factors (IGF-I and IGF-II) may play an important role in postpneumonectomy compensatory lung growth by translating hormonal inputs and mechanical forces into cellular proliferation signals. We examined the mRNA abundance of IGF-I, IGF-II, and IGF binding proteins (IGFBPs) in lungs of rats on postoperative days 1, 2, 3, 5, and 7 following left pneumonectomy (PNX) or shamoperation (SC) and in normal animals (CON). There was no difference in the abundance of lung IGF-I mRNA (measured by Northern analysis) or serum IGF-I (measured by radioimmunoassay (RIA)) between SC and PNX animals. IGF-II mRNA abundance was initially decreased following PNX (73% decrease compared to SC animals on day 1, p < .05) and then rose to approach SC group values on subsequent days. Transcripts for IGFBP-2, -3, -4, -5, and -6 were decreased in both the SC and PNX groups compared to CON animals on the day following pneumonectomy, then rose back to baseline by postoperative day 2-3. Tissue IGFBPs, measured by ligand blot analyses, were not different in either the SC or PNX groups. In contrast, all serum IGFBP bands were increased on postoperative day 1 following either sham or PNX surgery. In addition, serum IGFBP-4 was increased in PNX animals compared to the SC group on days 1 and 2 (increase of 38% and 78%, respectively, p < .05). We conclude that the changes observed in lung IGF and IGFBP expression following pneumonectomy do not represent major.

Comparison of 6-hour infusion versus bolus furosemide in premature infants.

STUDY OBJECTIVE: To compare the renal, hemodynamic, and pulmonary effects of a 6-hour infusion of furosemide versus conventional bolus administration in premature infants. DESIGN: Prospective, blinded, placebo-controlled, randomized study. SETTING: Two level III, university-based neonatal intensive care units. PATIENTS: Thirty premature infants with significant lung disease, requiring furosemide after a red cell infusion. INTERVENTIONS: Infants received furosemide 1 mg/kg over 2 minutes, followed by a 6-hour placebo infusion, or a small loading dose of 0.1 mg/kg, followed by a slow infusion of 0.9 mg/kg over 6 hours. Serum and urine were collected to determine percentage fractional excretion of sodium (FENa). MEASUREMENTS AND MAIN RESULTS: Urine output and blood pressure were measured every 2 hours after furosemide administration. Percentage FENa was measured immediately before furosemide and compared with pooled urine from an 8-hour collection after furosemide administration. Serum sodium, creatinine, and calcium were measured before and 24 hours after drug administration. Mean airway pressure and percentage inspired oxygen were compared before, 1-4 hours after, and 4-12 hours after drug administration. No significant differences were detected between the two methods of drug administration. CONCLUSION: Our data suggest that a 6-hour infusion of furosemide does not offer substantial clinical advantage over conventional bolus administration in premature infants when focusing on urine output, blood pressure, FENa, or pulmonary effect.

Re-emergence of a fetal pattern of insulin-like growth factor expression during hyperoxic rat lung injury.

Chronic injury to the developing lung results in cell proliferation and characteristic architectural changes. It is likely that growth factors produced and acting locally are important to these processes. Insulin-like growth factors I and II (IGF-I and IGF-II) are peptide growth factors expressed by lung cells. Roles for IGF-I and IGF-II in lung injury are suggested by their expression during lung development and by studies showing changes in IGF-I expression by activated alveolar macrophages, and increases in IGF-II peptide in oxidant arrested alveolar epithelial cells. To investigate whether the expression of IGF-I and IGF-II are changed with hyperoxic exposure, newborn rats were exposed to 80-90% oxygen for up to 6 wk and Northern hybridization analyses, in situ hybridization histochemistry, immunohistochemical staining, and reverse transcription-polymerase chain reaction (RT-PCR) studies were performed. Northern hybridization analyses of RNA extracted from whole lung showed increases in IGF-I and IGF-II mRNAs with prolonged hyperoxia. In situ hybridization histochemistry and immunohistochemical staining demonstrated spatial patterns of IGF-I and IGF-II expression similar to those seen during fetal lung development. In addition, alveolar macrophages express IGF-I and type II epithelial cells express IGF-II in control and oxygen-injured lung. These results suggest that in lung injury resident lung cells may re-express IGFs in a manner reminiscent of fetal development, and activated inflammatory cells may contribute to the proliferative response through autocrine and paracrine mechanisms.

Changes in decorin expression with hyperoxic injury to developing rat lung.

Proteoglycans are extracellular matrix components that appear to play important roles in lung development and in the response to injury. Decorin, a small extracellular matrix-associated proteoglycan, is known to be involved in collagen fibrillogenesis and is a likely participant in the pathogenesis of lung injury. We hypothesized that chronic exposure of the developing lung to hyperoxia would result in temporal and spatial changes in decorin expression. To determine the expression of decorin in normal and oxygen-injured lung, newborn rats were exposed to hyperoxia for 6 wk. Decorin mRNA abundance was determined using Northern hybridization analyses, and decorin expression was localized by in situ hybridization and immunohistochemistry. Decorin mRNA expression in type II pneumocytes was studied using reverse transcription-polymerase chain reaction. Oxygen exposure is associated with a 77% reduction in decorin mRNA in whole lung and a decrease in decorin immunoreactivity in connective tissues surrounding large airways and blood vessels, but an increase in decorin mRNA and protein expression at the tips of alveolar septa. Studies using isolated cells indicate that macrophages and polymorphonuclear neutrophils contain decorin core protein but not decorin mRNA. Type II pneumocytes do not contain either decorin mRNA or core protein. These findings demonstrate that hyperoxic lung injury is associated with localized changes in decorin expression, changes that are not reflected in whole lung RNA studies. It is likely that regional changes in lung decorin expression are influenced by factors produced and acting locally, and that such changes may contribute to the morphologic alterations characteristic of oxygen-induced lung injury.

New insights into lung growth and development.

Lung development requires a complex series of developmentally controlled interactions that involve mechanical forces, genetic and endocrine influences, and cell-cell communication. At each step, cell-matrix or cell-cell signaling mediated by peptide growth factors and extracellular matrix components is crucial in directing cell proliferation, differentiation, and migration. Although a comprehensive understanding of how these components interact to guide lung organogenesis has been elusive, the action and control of peptide growth factors in autocrine and paracrine signaling, mesenchymal-epithelial interactions in controlling branching morphogenesis, cell-cell communication in the regulation of cellular differentiation, and factors regulating pattern formation are being clarified.

Cellular localization of messenger RNAs for insulin-like growth factors (IGFs), their receptors and binding proteins during fetal rat lung development.

To gain insight into the role of the insulin-like growth factors (IGFs) in regulating lung development, we have used in situ hybridization histochemistry (ISHH) to examine the ontogeny and sites of expression of IGF-I and IGF-II, IGF binding proteins (IGFBP-1 to IGFBP-6), and IGF cell surface receptors in fetal rat lung from 15 to 21 days of gestation. Both IGF-I and IGF-II mRNAs were expressed throughout the developmental period studied with little change in apparent abundance. IGF-I mRNA localized to mesenchymal cells, especially those surrounding airway epithelium, while IGF-II mRNA, which was somewhat more abundant, localized predominantly to epithelia. The type 1 IGF receptor, the receptor that likely mediates the actions of both IGFs, was expressed widely in virtually all cells, whereas the expression of the type 2 IGF receptor, thought to be involved in IGF internalization and degradation, was confined to the mesenchyme and medial layers of intrapulmonary vessels. As with the IGFs, there was little apparent change in the abundance of IGF receptor mRNAs through fetal development, and the type 2 IGF receptor mRNA was more abundant. The expression of IGFBPs changed significantly during lung development. IGFBP-2, -3, -4, and -5 were expressed from day 15 of gestation, but their sites of expression and ontogeny differed. IGFBP-2 mRNA expression was abundant and constant throughout gestation and was confined to proximal and distal airway epithelia. IGFBP-3 and IGFBP-5 also were expressed by proximal airway epithelia, but also exhibited significant expression in interstitial mesenchyme and in mesenchyme surrounding vessels. The abundance of both increased as gestation progressed (IGFBP-5 greater than IGFBP-3). IGFBP-4 mRNA was confined to interstitial mesenchyme and its abundance peaked at days 16 to 19 of gestation. We found no evidence for expression of either IGFBP-1 or IGFBP-6. We conclude that the expression of IGF-I, IGF-II, and the type 1 IGF receptor throughout gestation in the lung supports a role for the IGFs in lung growth and development. The complex pattern of IGFBP expression (differing sites and ontogeny of expression) suggests that the IGFBPs modulate IGF actions at specific target sites. Furthermore, because there is little change in the expression of IGFs or IGF receptor mRNAs during fetal lung development, regulation of IGFBP expression may be essential to the control of IGF actions during lung development.

Insulin-like growth factor-I (IGF-I) regulates IGFBP-3 and IGFBP-4 by multiple mechanisms in A549 human adenocarcinoma cells.

The insulin-like growth factors (IGF-I and IGF-II) participate in the control of cell proliferation in normal and neoplastic lung cells. To examine the role of IGF binding proteins (IGFBPs) in modulating IGF actions in lung, we examined the production and regulation of IGFBPs from A549 cells, a human adenocarcinoma-derived lung cell line. Ligand blot and immunoblot analysis of conditioned media (CM) from A549 cells demonstrated IGFBP bands of relative molecular mass (M(r)) approximately 39-43,000 (IGFBP-3), 34,000 (IGFBP-2), 30,000 (IGFBP-1), and 24,000 (IGFBP-4). IGFBP-3 abundance in A549 cell CM increased following exposure to IGF-I and IGF-II (3.0- and 1.8-fold, respectively) without a change in IGFBP-3 transcript abundance, suggesting IGFBP-3 is post-transcriptionally regulated. Cycloheximide almost completely abrogated the IGF-I-stimulated increase in CM IGFBP-3, suggesting that ongoing protein synthesis is necessary for the IGF-I-stimulated increase in IGFBP-3 abundance. Increases in IGFBP-3 occurred by at least two mechanisms, through activation of the type 1 IGF receptor and by a type 1 IGF receptor independent mechanism. The increase in IGFBP-3 was due, in part, to activation of the type 1 IGF receptor because blocking type 1 IGF receptor activation with an antibody (alpha IR3) diminished the IGF-I-induced increase in IGFBP-3 and insulin, at doses that stimulate the type 1 IGF receptor, increased IGFBP-3 abundance. The increase in IGFBP-3 was partially independent of type 1 IGF receptor activation because [QAYL]-IGF-I, an analog of IGF-I that binds the type 1 IGF receptor but not IGFBP-3, was less potent than IGF-I in stimulating IGFBP-3 abundance, and IGF-II, which binds IGFBP-3 normally, but binds the type 1 IGF receptor with lower affinity than IGF-I, was nearly equipotent to IGF-I in its stimulation of IGFBP-3 accumulation at low concentrations. These results suggest that ligand binding decreases IGFBP-3 clearance or increases IGFBP-3 accumulation in CM. IGF-I decreased IGFBP-4 abundance in A549 cell CM without decreasing IGFBP-4 mRNA transcripts and without increasing the amount of cell-associated IGFBP-4. To determine whether the decrease in IGFBP-4 was due to increased degradation, cell-free CM was incubated with and without IGF-I, and IGFBP-4 abundance measured by ligand and immunoblot analyses.(ABSTRACT TRUNCATED AT 400 WORDS)

L-glutamine and L-asparagine stimulate ODC activity and proliferation in a porcine jejunal enterocyte line.

We studied the effect of L-glutamine (Gln), the principal intestinal fuel, on proliferation of a porcine jejunal cell line, IPEC-J2. In cells synchronized by serum deprivation for 4 h, Gln stimulated ornithine decarboxylase (ODC; EC 4.1.1.17) in a dose- and time-dependent manner, with maximal effects at 10 mM in 3 h (P < 0.01). Similar effects were seen for the structurally related amino acid L-asparagine and serum. The Gln effect on ODC was specific, as isosmolar mannitol, glucose, methyl-beta-D-glucoside, L-phenylalanine, ammonia, and aminoisobutyric acid were ineffective. The alanine aminotransferase inhibitor aminooxyacetate (AO) inhibited the ODC stimulation by Gln in a dose-dependent manner (half-maximal inhibitory concentration = 0.5 mM). AO was not toxic to cells, as determined by propidium iodide uptake into nuclei. In addition, Gln stimulated a twofold increase of cellular 24-h [3H]thymidine incorporation above rates of control cells bathed in standard media (P < 0.01); this effect was also blocked by AO. Gln and phorbol 12-myristate 13-acetate stimulated ODC in a synergistic manner. The Na+/H+ exchange inhibitor methylisobutyl amiloride blocked the enhancement of ODC by Gln. Gln also induced the mRNA of the immediate-early gene c-jun. Gln stimulates proliferation in a porcine jejunal cell line through a mechanism requiring transamination and intact Na+/H+ exchange. This stimulation of enterocyte proliferation by Gln suggests that therapeutic Gln administration could facilitate epithelial recovery in the injured small intestine.

Southern hybridization analyses of somatic cell hybrids reveal that human BB1 is a member of a multigene family dispersed throughout the human genome and appears to be linked to the human G25K genes.

The hBB1 RNA (2.3 kb in length) encodes a 57-amino-acid protein whose production is essential for cellular transit of G1 phase of the cell cycle (Moats-Staats et al., 1994). Homology searches of GenBank and EMBL revealed that bases 1-234 of the hBB1 cDNA were 97% homologous to the 3' terminal 234 bases of the g25K cDNA (bases 940-1,175, Shinjo et al., 1990) the human homolog of the yeast cdc42 cDNA. We have used the techniques of the long-range polymerase chain reaction (PCR) (Barnes, 1994) and Southern hybridization analyses of a somatic cell hybrid panel to investigate hBB1 gene structure and to determine whether the hBB1 gene(s) overlaps the g25K gene. These studies have demonstrated that the hBB1 RNA is encoded by a gene family composed of eight members that is dispersed throughout the human genome localizing under high-stringency conditions to chromosomes 1, 4, 7, 8, and 20. We have also determined that two hBB1 gene(s) and two g25K gene(s) map to similar-size Bam HI restriction fragments on chromosomes 4 and 7.

Administration of granulocyte colony-stimulating factor to neutropenic low birth weight infants of mothers with preeclampsia.

Nine low birth weight infants with neutropenia born to mothers with preeclampsia were treated with granulocyte-colony stimulating factor, 10 micrograms/kg intravenously, within 24 hours of birth and at 24-hour intervals for a maximum of three doses if neutropenia persisted. The absolute neutrophil count increased significantly in eight of the nine infants within 6 hours, and neutrophilia was sustained for at least 72 hours after administration of a single dose of granulocyte-colony stimulating factor.