Protection of Meconium-Induced Lung Epithelial Injury by Protease Inhibitors.

Earlier work form this laboratory showed that exposure of alveolar epithelial cells (AECs) to meconium caused significant cell detachment and that meconium-induced detachment of cells was prevented by a protease inhibitor cocktail. Therefore, it was hypothesized that protease inhibitors might protect AEC monolayers against meconium-induced collapse of epithelial barrier function both in vitro and in vivo. To investigate this theory in vitro, albumin flux was measured across cultured, confluent monolayers of human type II derived cell line A549 on microporous filter inserts. Human meconium was collected from seven healthy full-term neonates and the samples were pooled and diluted prior to analysis. Exposure of AECs to 5% human meconium increased albumin flux across the cultured AEC monolayers, but the increase was significantly blocked by protease inhibitors (P<0.001). In C57/BL6 mice, intratracheal instillation of 5% human meconium increased the passage of Evans Blue Dye (EBD) from the vascular compartment into the alveolar spaces, measured in bronchoalveolar lavage (BAL) fluid after intravenous injection of EBD. Moreover, intratrachial coinstillation of protease inhibitors prevented the meconium-induced increase in EBD passage into BAL fluid (P<0.01). The data presented herein clearly demonstrate that protease inhibitors protect AEC barrier function against meconium-induced injury, and suggest the future possibility of using protease inhibitors in the treatment of meconium aspiration syndrome.

Neonatal sepsis in rural India: timing, microbiology and antibiotic resistance in a population-based prospective study in the community setting.

OBJECTIVE: To examine the timing and microbiology of neonatal sepsis in a population-based surveillance in the Indian community setting. STUDY DESIGN: All live born infants in 223 villages of Odisha state were followed at home for 60 days. Suspect sepsis cases were referred to study hospitals for further evaluation including blood culture. RESULTS: Of 12 622 births, 842 were admitted with suspected sepsis of whom 95% were 4 to 60 days old. Culture-confirmed incidence of sepsis was 6.7/1000 births with 51% Gram negatives (Klebsiella predominating) and 26% Gram positives (mostly Staphylococcus aureus). A very high level of resistance to penicillin and ampicillin, moderate resistance to cephalosporins and extremely low resistance to Gentamicin and Amikacin was observed. CONCLUSION: The bacterial burden of sepsis in the Indian community is not high. Judicious choice of empiric antibiotics, antibiotic stewardship and alternate modalities should be considered for the management or prevention of neonatal sepsis in India.

Fine structural abnormalities of the placenta in diabetic rats.

In the streptozocin-induced diabetic rat, the placenta is larger and the fetus is smaller than normal. To study cellular differences that might contribute to the size and functional disparity between diabetic and control placentas, a light- and electron-microscopic analysis was performed on 14-, 18-, and 22-day (term) control and diabetic placentas. Diabetic placentas, especially later in gestation, were marked by the presence of large numbers of glycogen-distended cells in the basal zone. Within the placental labyrinth, the trophoblastic layers of the interhemal membrane were significantly thicker in the diabetic placentas on days 18 and 22, and large accumulations of liid droplets were present, especially in the inner two trophoblastic layers. In normal placentas there is a marked thinning of the placental barrier in the labyrinth by day 22 of gestation, making the thickness of the labyrinthine layers in age-matched diabetic placentas even more impressive. Finally, the labyrinth of 22-day diabetic placentas contained more glycogen and rough endoplasmic reticulum in the inner trophoblastic layer, a feature that is found in less-mature (18-day) control placentas. Thus, the diabetic placentas have a number of features that are consistent with functional immaturity/dysmaturity. Cytologic evidence confirms the presence of increased glycogen and lipid reserves in both the junctional zone and the cellular area between maternal and fetal blood.(ABSTRACT TRUNCATED AT 250 WORDS)

Unconjugated cortisol in human amniotic fluid: relationship to lecithin/sphingomyelin ratio.

A specific and thoroughly validated competitive protein binding radioassay has been developed for the measurement of unconjugated cortisol in human amniotic fluid. Cortisol levels rose throughout normal gestation from 8.6 +/- 0.8 ng/ml at less than 20 weeks to 19.8 +/- 1.5 ng/ml at 30-40 weeks. Levels were significantly higher in amniotic fluid samples having mature lecithin/sphingomyelin (L/S) ratios (greater than 2), and good correlation with individual cortisol values was obtained (r = 0.79; P less than 0.001). These results support the hypothesis that cortisol plays a role in fetal lung maturation.

Creatine kinase brain isoenzyme: relationship of cerebrospinal fluid concentration to the neurologic condition of newborns and cellular localization in the human brain.

Immunocytochemical study of human brain showed creatine kinase brain isoenzyme (CKBB) present in both neurons and astrocytes. Because creatine kinase brain isoenzyme is an intracellular enzyme that might be released with brain injury, its concentration in the CSF of newborns was measured using a radioimmunoassay. Infants who suffered a documented neurologic insult (a cerebroventricular hemorrhage or a CNS infection) were found to have a greater mean CSF creatine kinase brain isoenzyme concentration than those without a history of neurologic insult. Infants with a high concentration had a poor short-term outcome (death or neurologic abnormality when discharged) more frequently than did those with a lower concentration. Infants with a grade 3 or 4 cerebroventricular hemorrhage had a higher mean concentration than did those with a grade 1 or 2 hemorrhage. These data are consistent with the hypothesis that CSF creatine kinase brain isoenzyme is a metabolic indicator of brain damage in newborns.

Endemic necrotizing enterocolitis: lack of association with a specific infectious agent.

We conducted a comprehensive analysis of bacterial, parasitic and viral agents present in stool samples of 23 necrotizing enterocolitis cases and 23 matched and 10 random controls. Enterococcus spp., Staphylococcus epidermidis, and Escherichia coli were the most common aerobic bacterial species isolated. Astrovirus was identified in a stool sample from one control. Eight infants were bacteremic; in 7 of 8 the same organism was also present in the stool. No one bacterial species or strain (as identified by plasmid profile analysis) was associated with occurrence of illness. Plasmid analysis further suggested that each infant was colonized with his or her own distinctive aerobic bacterial flora. With the exception of isolates from two control patients which hybridized with a probe for diffuse adherence, no diarrheagenic E. coli was identified. Five (45%) of 11 case infants were colonized with coagulase-negative staphylococci (all S. epidermidis) that produced delta-hemolysin in vitro, as compared with 13 (87%) of 15 control infants. Necrotizing enterocolitis was not associated with an increased ability to ferment carbohydrate, as measured by in vitro beta-galactosidase activity. Our data do not support the hypothesis that endemic necrotizing enterocolitis in our institution is caused by a single infectious agent, nor was there evidence that previously proposed virulence mechanisms such as production of delta-hemolysin or increased in vitro carbohydrate fermentation play a critical role in disease occurrence.

Amniotic fluid cortisol in high-risk human pregnancies.

Amniotic fluid cortisol (AFC) levels were measured in 76 normal and 67 high-risk human pregnancies. When compared to normal gestations, diabetic pregnancies were found to be associated with lower AFC levels, while toxemic, early Rh-sensitized, and other stressed pregnancies had higher mean cortisol values. Serially followed normal and high-risk pregnancies showed a progressive rise in AFC with increasing gestational age. Significant correlation between AFC concentrations and lecithin/sphingomyelin (L/S) ratios was noted in normal, diabetic, and toxemic gestations. These results are concordant with the hypothesis that cortisol may be involved in fetal lung development.

Amniotic fluid cortisol as an index of fetal lung maturity.

Amniotic fluid cortisol concentration was compared with lecithin/sphingomyelin ratio, gestational age, and eventual birth-weight in a large series of normal and high-risk human pregnancies in an effort to determine whether cortisol could be used as a reliable indicator of fetal maturity. It appears that when used alone amniotic fluid cortisol is of limited clinical value as an antepartum predictor of fetal respiratory status because of unacceptable percentages of false positive and false negative predictions. Whether amniotic fluid cortisol can be used as an adjunct in the evaluation of high-risk pregnancies with "transitional" L/S ratios remains to be explored.

Prenatal diagnosis of a human pseudoacardiac anomaly.

A unique case is presented of dichorionic-diamnionic twins, one of which contained a primitive heart arrested at an early stage of development and which resembled in many other respects a classic case of acardia. Lack of vascular anastomoses, the dichorionic nature of the twins, and ultrasonic evidence of independent heart rates suggest that the primary pathogenesis in acardia may be cardiac dysmorphogenesis rather than reversal of vascular flow. Ultrasonography and fetal echocardiography proved useful in delineating many of the features of the amorphous twin before delivery.

Stool microflora in extremely low birthweight infants.

AIM: To serially characterise aerobic and anaerobic stool microflora in extremely low birthweight infants and to correlate colonisation patterns with clinical risk factors. METHODS: Stool specimens from 29 infants of birthweight <1000 g were collected on days 10, 20, and 30 after birth. Quantitative aerobic and anaerobic cultures were performed. RESULTS: By day 30, predominant species were Enterococcus faecalis, Escherichia coli, Staphylococcus epidermidis, Enterbacter cloacae, Klebsiella pneumoniae, and Staphylococcus haemolyticus. Lactobacillus and Bifidobacteria spp were identified in only one infant. In breast milk fed (but not in formula fed) infants, the total number of bacterial species/stool specimen increased significantly with time (2.50 (SE 0.34) on day 10; 3.13 (0.38) on day 20; 4.27 (0.45) on day 30) as did quantitative bacterial counts; Gram negative species accounted for most of the increase. On day 30, significant inverse correlations were found between days of previous antibiotic treatment and number of bacterial species (r=0.491) and total organisms/g of stool (r=0.482). Gestational age, birthweight, maternal antibiotic or steroid treatment, prolonged rupture of the membranes, and mode of delivery did not seem to affect colonisation patterns. CONCLUSIONS: The gut of extremely low birthweight infants is colonised by a paucity of bacterial species. Breast milking and reduction of antibiotic exposure are critical to increasing fecal microbial diversity.

Role of glutamine in bacterial transcytosis and epithelial cell injury.

BACKGROUND: L-Glutamine is the principal energy source for small intestinal enterocytes. Diminution of intestinal function, mucosal atrophy, and increased bacterial translocation have been noted during total parenteral nutrition (TPN). In a rat model of glutamine starvation, we previously showed that luminal glutamine is essential for optimal intestinal function. In this study, we examined the effect of apical vs basolateral glutamine on bacterial translocation in a Caco-2 cell culture system and bacteria-induced tissue injury in a weanling rabbit ileal loop model. METHODS: Caco-2 cells were grown in a transwell system. After confluence, apical and basolateral chambers received defined media, and glutamine deprivation was carried out over a 4- to 48-hour period. Escherichia coli transcytosis and structure/function studies were then performed. In a second series of experiments, the effect of intraluminal glutamine supplementation was evaluated in an E. coli-induced tissue injury model in weanling rabbit ileal loops. RESULTS: Expression of disaccharidases, glucoamylase, and Na+/K(+)-adenosine 5'-triphosphatase (ATPase) were significantly reduced when cells were deprived of glutamine from the apical side, and there was increased bacterial translocation across the monolayer. Transepithelial epithelial resistance (TEER) across the monolayer was also reduced in the glutamine-free cultures. Glutamine replenishment over 24 to 48 hours restored the original functions. Basolateral deprivation had a smaller effect on the Caco-2 cells. Typical necrotic mucosal injury caused by E. coli in the ileal loops was blocked by co-infiltration of the loops with glutamine. CONCLUSIONS: This study demonstrates for the first time that the supply of glutamine from the apical side is of critical importance for maintaining optimal structure and function of the enterocytes. The effects are not acute or energy related. These observations have important clinical implications in the management of patients under critical care, including premature infants and patients receiving TPN, for whom lack of glutamine from the luminal side could produce mucosal dysfunction, resulting ultimately in severe atrophic/necrotic complications.

Insulin-like growth factor-I receptor signal transduction: at the interface between physiology and cell biology.

The insulin-like growth factor-I receptor (IGF-IR) mediates the biological actions of IGF-I and IGF-II. The IGFs play a critical role in promoting development, stimulating growth and organogenesis via mitogenic, antiapoptotic and chemotactic activity. Recent research has focused on the events that occur intracellularly upon receptor activation. Several pathways have been shown to be important. The insulin-receptor substrate (IRS), SHC, GRB2, CRKII and CRKL adaptor proteins have all been implicated in transmitting signals to the nucleus of the cell. This review outlines some of the signalling pathways believed to be important in converting IGF-IR activation into changes in cell behavior and metabolism.

Fluorescence anisotropy of tracheal and oropharyngeal aspirates as an indicator of lung disease in neonates.

Fluorescence anisotropy was assessed in tracheal and oropharyngeal aspirates to evaluate its use in the diagnosis of respiratory distress syndrome. Samples from 31 neonates at birth were purified by using Sephacryl S-300 column chromatography. After the addition of the molecular probe, 1,6-diphenyl 1,3,5 hexatriene fluorescence was measured and anisotropy calculated. Ten neonates with normal respiratory function had a mean anisotropy (+/- SD) of 0.226 +/- 0.023. Fourteen infants with a diagnosis of respiratory distress syndrome had a mean anisotropy of 0.260 +/- 0.014 (p < 0.001). Seven neonates with respiratory problems other than respiratory distress syndrome had a mean anisotropy of 0.211 +/- 0.027 (p < 0.001 vs respiratory distress syndrome). A diagnosis of respiratory distress syndrome was associated with higher tracheal fluid anisotropy, indicating a qualitative difference in the characteristics of the pulmonary surfactant. We conclude that this 30-minute test could help identify neonates who benefit from surfactant replacement therapy.

Evolution and variability of pulmonary mechanics during postnatal transition in term infants.

This study was designed to assess whether progressive changes related to the transition to extrauterine life explain in part the interassay variability of pulmonary mechanics in the immediate postnatal period. We performed sequential pulmonary function tests in 21 normal term infants with the use of a pneumotachometer and an esophageal balloon. The average percent increase in values from the first period (2 to 10 hours) to the last period of study (66 to 78 hours) was 27% for tidal volume, 24% for minute ventilation, and 42% for dynamic compliance. The maximum difference between two pulmonary function measurements obtained within 30 minutes of each other ranged between 10% for dynamic compliance and 27% for minute ventilation. The coefficient of variation for dynamic compliance over a 2-hour period (12%) was less than half the 3-day coefficient of variation (31%). The reliability coefficient ranged between 0.72 for tidal volume and 0.92 for dynamic compliance. We conclude that dynamic compliance in term infants increases progressively during the first 3 days of life, whereas tidal volume and minute ventilation increase mostly during the first day. The variability of pulmonary mechanics reflects in part those progressive physiologic changes during postnatal transition.

The effect of application of aquaphor on skin condition, fluid requirements, and bacterial colonization in very low birth weight infants.

OBJECTIVE: To determine the effects of repeated application of an occlusive ointment on the skin of very low birth weight infants. STUDY DESIGN: Nineteen neonates of 26 to 30 weeks gestational age were randomly assigned to receive topical Aquaphor ointment twice daily for 2 weeks or to receive standard skin care. Skin quality, fluid requirements, and skin bacterial colonization counts were assessed. RESULTS: Infants treated with Aquaphor had significantly improved skin condition scores versus controls (p = 0.002). Aquaphor improved skin scores over time (p = 0.012) in treated infants, whereas skin scores of untreated infants worsened before eventually healing. There were no significant differences in total fluid requirements, urine output, serum sodium concentrations, skin bacterial counts, fungal counts, or colonization patterns between treated and control infants in either gestational age cohort. CONCLUSION: Aquaphor ointment, used during the first two postnatal weeks, improved skin condition in infants of 26 to 30 weeks' gestation without changing skin bacterial flora. We speculate that improved skin condition may limit transepidermal water loss and decrease portals of entry for pathogens, thereby potentially decreasing fluid and electrolyte imbalances and sepsis in very low birth weight infants.

Long-term follow-up of premature infants treated with prophylactic, intratracheal recombinant human CuZn superoxide dismutase.

OBJECTIVE: To examine the long-term effects of treatment with recombinant human CuZn superoxide dismutase (rhSOD) in infants enrolled previously in two placebo-controlled trials. STUDY DESIGN: Records for 46 (88%) infants were examined, with 19 infants having received either single or multiple intratracheal (i.t.) doses of placebo, 12 having received a single i.t. dose of rhSOD, and 15 having received multiple i.t. doses of rhSOD. Mean age at follow-up was 28 months corrected age. Records were examined for neurologic dysfunction, developmental delay, and any significant medical disorders. RESULTS: Four placebo infants (21%) had evidence of neurodevelopmental abnormalities and four infants developed asthma. Four single-dose rhSOD infants (33%) had neurodevelopmental abnormalities and two infants developed asthma. One multiple-dose rhSOD infant had evidence of neurodevelopmental abnormalities and one developed asthma. No other differences were found between the placebo and rhSOD groups. CONCLUSION: Preliminary data suggest that rhSOD is safe and not associated with any long-term adverse effects. Further results will depend on the results of multicenter trials of rhSOD in preterm infants.

High glucose causes delayed fetal lung maturation in vitro.

The diabetic pregnancy is characterized by a delay in the process of fetal lung maturation. To determine whether high glucose conditions per se have an adverse impact on fetal lung phospholipid metabolism, 20-day fetal rat lung explants were cultured in F-12 medium (10 mM glucose) to which glucose was added to achieve final concentrations of 10, 25, 50, and 100 mM. Significant dose-dependent decreases in the rate of incorporation of [H3]choline into PC were noted at high glucose concentrations: Rate of choline incorporation was decreased by 19 and 28% under 50 and 100 mM glucose conditions, respectively (p < .001). Similar results were obtained when choline incorporation into DSPC was assayed. Equiosmolar mannitol concentrations did not result in comparable decreases, indicating a specific adverse effect attributable to glucose. Total PC and DSPC contents were also decreased by approximately 25% under high glucose conditions (p < .001). Glycogen concentrations were increased two- to threefold in the explants grown under high glucose conditions, consistent with a less mature lung. The addition of 1 unit/mL of insulin to F-12 medium did not result in decreases in choline incorporation into PC or DSPC; the addition of this amount of insulin to medium containing 100 mM glucose did not result in further decreases in choline incorporation rate. These results indicate that high glucose alone can have an adverse effect on fetal lung maturation in vitro and may be involved in the pathogenesis of the delay in fetal lung development observed in the diabetic pregnancy.

Effect of high glucose on fetal lung maturation at different times in gestation.

Hyperglycemia has been implicated as a cause of delayed fetal lung maturation in the diabetic pregnancy. It has been previously demonstrated that high glucose levels inhibit biochemical and morphological maturation of fetal rat lung in vitro. To explore whether there is a critical period for maximal effects of high glucose, fetal rat lung explants from day 18-22 (term) were examined after culture for 44 h in media containing 10 or 100 mM glucose. Rate of choline incorporation into PC and DSPC was significantly decreased in explants derived from day 20-22 fetuses after culture with high glucose. Paradoxically, in explants derived from day 18-19 fetuses choline incorporation rate was increased. Significant decreases in total PC and DSPC were seen in the high glucose group on days 20-21; however, earlier in gestation no differences in PC and DSPC were noted, despite the higher rate of choline incorporation into PC and DSPC, suggesting an increased turnover rate. High glucose levels also resulted in significantly decreased incorporation of labeled glycerol and palmitate into PC. Morphologic analysis of right upper lobe explants revealed significant decreases in the number of type II pneumocytes and lamellar bodies per alveolar lining cell in high glucose treated explants derived from days 19-20, but not before or after that time in gestation. Prior to day 20 there were few luminal lamellar bodies in potential airspaces. By day 20, the high glucose group had significantly fewer luminal lamellar bodies, although by day 21 the difference was no longer significant. These results suggest that high glucose may have a greater inhibitory effect late in gestation and that there may be critical periods in lung development when differences in substrate availability and utilization have differing effects.

Fetal and maternal corticosterone and corticosteroid binding globulin in the diabetic rat gestation.

Delayed fetal lung development is a feature of the diabetic pregnancy. Since fetal glucocorticoids are important in the regulation of lung maturation, we measured corticosterone and corticosteroid-binding globulin binding capacity in streptozotocin-diabetic pregnant rats and their fetuses. Previous studies have demonstrated delayed fetal lung maturation in this animal model. In control fetuses, total corticosterone concentration increased through day 20 of gestation, then declined until day 22 (term). The unbound steroid, which accounted for 5-10% of the total, increased approximately 3-fold from day 18 to term. Corticosteroid-binding globulin binding capacity peaked on day 19 after which it decreased. Maternal total and unbound corticosterone levels and corticosteroid-binding globulin binding capacity remained relatively constant throughout the final week of normal gestation. When compared to controls, fetuses from diabetic pregnancies had significantly lower total corticosterone from day 19 through 22. Corticosteroid-binding globulin binding capacity was also significantly decreased in these fetuses for the last 4 days of gestation. Similar differences were noted in maternal samples. However, no significant differences in unbound, biologically active, corticosterone were seen when diabetic and control groups were compared. Thus, delayed fetal lung maturation observed in fetuses of streptozotocin-diabetic rats is associated with a decrease in total circulating corticosteroid levels late in gestation. However, since unbound corticosteroid levels were similar in fetuses of control and diabetic animals, it is likely that other mechanisms may be responsible for the observed delay in lung development in fetuses of diabetic pregnancies.