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.

Family-based transmission disequilibrium test (TDT) and case-control association studies reveal surfactant protein A (SP-A) susceptibility alleles for respiratory distress syndrome (RDS) and possible race differences.

A key cause of respiratory distress syndrome (RDS) in the prematurely born infant is deficiency of pulmonary surfactant, a lipoprotein complex. Both low levels of surfactant protein A (SP-A) and SP-A alleles have been associated with RDS. Using the candidate gene approach, we performed family-based linkage studies to discern linkage of SP-A to RDS and identify SP-A susceptibility or protective alleles. Moreover, we performed case-control studies of whites and blacks to detect association between RDS and SP-A alleles. Transmission disequilibrium test (TDT) analysis revealed that the frequency of transmission (from parent to the offspring with RDS) of alleles 6A(2) and 1A(0) and of 1A(0)/6A(2) haplotype in RDS was increased, whereas transmission of alleles 1A(5) and 6A(4) and of haplotype 1A(5)/6A(4) was decreased. Extended TDT analysis further strengthened the observations made. The case-control studies showed that in whites or blacks with RDS the frequencies of specific genotypes, 1A(0) and 6A(2) or 1A(0), were increased, respectively, but the frequency of specific 6A(3) genotypes was increased in certain white subgroups and decreased in blacks. Regression analysis revealed gestational age (GA) and 6A(3) genotypes are significant factors in blacks with RDS. In whites with RDS, GA and antenatal steroids are important factors. The data together indicate linkage between SP-A and RDS; certain SP-A alleles/haplotypes are susceptibility (1A(0), 6A(2), 1A(0)/6A(2)) or protective (1A(5), 6A(4), 1A(5)/6A(4)) factors for RDS. Some differences between blacks and whites with regard to SP-A alleles may exist.

Abnormal developmental patterns of suck and swallow rhythms during feeding in preterm infants with bronchopulmonary dysplasia.

Infants with bronchopulmonary dysplasia (BFD) often have difficulty achieving coordinated suckle feeding. To analyze rhythmic differences during feeding in infants with BPD we performed weekly studies of 14 infants with BPD (eight male, six female; postmenstrual age [PMA] 32.1 to 39.7 weeks); and a PMA-matched control group without BPD (n=20), from initiation of bottle feeding until discharge, with simultaneous digital recordings of pharyngeal and nipple (teat) pressure. Unlike the control group, there was no significant correlation between PMA and stability of suckle rhythm, aggregation of suckles or swallows into runs, or length of suckle runs. Comparing those infants >35 weeks' PMA, the group with BPD had significantly decreased stability of suckle rhythm (increased coefficient of variation of suckle-suckle intervals: 0.34, SE 0.02 vs 0.254, SE 0.014; p=0.003), decreased aggregation into suckle runs (71.1, SE 3.4% vs 85.4, SE 2%;p=0.001), and decreased length of suckle runs (7.2, SE 0.9 vs 13.1, SE 1.9 suckles/run; p=0.003). Percentage of swallows in runs was also decreased in the cohort with BPD (58, SE 3.8% vs 77.2, SE 3.5%; p<0.001), as was length of swallow run (5.3, SE 0.5 vs 10.7, SE 1.1;p<0.001). Thus, in infants with BPD, anticipated maturational patterns of suckle and swallow rhythms did not occur. Delay in attainment of stable suckle and swallow rhythms in preterm infants, especially after 35 weeks' PMA, may predict subsequent feeding and neurological problems.

Developmental patterns of rhythmic suck and swallow in preterm infants.

Twenty healthy preterm infants (gestational age 26 to 33 weeks, postmenstrual age [PMA] 32.1 to 39.6 weeks, postnatal age [PNA] 2.0 to 11.6 weeks) were studied weekly from initiation of bottle feeding until discharge, with simultaneous digital recordings of pharyngeal and nipple (teat) pressure and nasal thermistor and thoracic strain gauge readings. The percentage of sucks aggregated into 'runs' (defined as > or = 3 sucks with < or = 2 seconds between suck peaks) increased over time and correlated significantly with PMA (r=0.601, p<0.001). The length of the sucking-runs also correlated significantly with PMA (r=0.613, p<0.001). The stability of sucking rhythm, defined as a function of the mean/SD of the suck interval, was also directly correlated with increasing PMA (r=0.503, p=0.002), as was increasing suck rate (r=0.379, p<0.03). None of these measures was correlated with PNA. Similarly, increasing PMA, but not PNA, correlated with a higher percentage of swallows in runs (r=0.364, p<0.03). Stability of swallow rhythm did not change significantly from 32 to 40 weeks' PMA. In low-risk preterm infants, increasing PMA is correlated with a faster and more stable sucking rhythm and with increasing organization into longer suck and swallow runs. Stable swallow rhythm appears to be established earlier than suck rhythm. The fact that PMA is a better predictor than PNA of these patterns lends support to the concept that these patterns are innate rather than learned behaviors. Quantitative assessment of the stability of suck and swallow rhythms in preterm infants may allow prediction of subsequent feeding dysfunction as well as more general underlying neurological impairment. Knowledge of the normal ontogeny of the rhythms of suck and swallow may also enable us to differentiate immature (but normal) feeding patterns in preterm infants from dysmature (abnormal) patterns, allowing more appropriate intervention measures.

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.

Respiratory sinus arrhythmia during feeding: a measure of vagal regulation of metabolism, ingestion, and digestion in preterm infants.

Respiratory sinus arrhythmia (RSA), a non-invasive indicator of vagal regulation of the heart, and heart period (HP) were monitored before, during, and after oral or gastric-tube bolus feedings in 32 preterm infants. Group 1 infants (n=15) were < or =30 weeks gestational age (GA) at birth (mean 28.3 weeks) and group 2 infants (n=17) were > or =31 weeks GA at birth (mean=33.2 weeks). Mean postmenstrual ages at the time of study were 33.5 +/- 2.3 (SD) weeks in group 1 and 33.9 +/- 1.6 (SD) weeks in group 2. RSA and HP decreased in both groups during feeding. However, postfeeding RSA and HP increased toward prefeed levels only for group 2 infants. In addition, RSA and HP changes during feeding were correlated only for group 2 infants. The results suggest that the preterm infant may experience a maturational lag in vagal function and in the influence of vagal activity on metabolic mechanisms (i.e. heart rate) related to ingestive needs. This maturational lag may contribute to continued feeding difficulties and may be a measurable marker of subtle neurodevelopmental problems.

Effect of acute glucose depletion following glucose excess on surfactant phospholipid synthesis in developing fetal lung.

Exposure to high glucose and insulin inhibits surfactant synthesis in vitro. We have demonstrated that fetal lung insulin receptor tyrosine kinase (TK) activity is downregulated after culture in high glucose plus insulin, with a resultant decrease in glucose uptake. To see whether relative substrate depletion following substrate excess would further diminish surfactant synthesis, 20-day fetal rat lung explants were initially cultured for 44 hours in media containing 100 mM glucose with or without 0.1 U/mL insulin, followed by a 4-hour pulse in 10 mM glucose +/- insulin or 100 mM glucose +/- insulin, after which the rate of choline incorporation into phosphatidylcholine (PC) or disaturated PC was measured. Choline incorporation was significantly lower after a 4-hour pulse in low glucose (+/- insulin) than under continuing high glucose (+/- insulin) conditions (P < .01). To determine the time required for reversal of TK downregulation in lung explants, insulin receptor TK activity was assayed after 44 hours in high glucose + insulin, followed by an additional 4, 8, 12, or 24 hours in either 10 mM glucose or continued 100 mM glucose + insulin. After 44 hours in 50 mM or 100 mM glucose + insulin, TK activity was significantly decreased (68 +/- 9% and 57 +/- 9% of control; P < .01). When explants cultured in 100 mM glucose + insulin for 44 hours were subsequently placed in 10 mM glucose for an additional 4 or 8 hours, TK activity remained significantly downregulated (70.2 +/- 7.0% and 84.9 +/- 5.5% of control, respectively, P < .05) compared to explants cultured in low glucose throughout. However, after 12 or 24 hours in low glucose, TK activity was no longer significantly different from control values (106.5 +/- 2.1% and 106.0 +/- 19.6%, respectively). Culture of type II cells for 44 hours in 25 mM glucose + insulin, followed by an additional 4 or 24 hours in either low glucose (5.5 mM) + insulin or high glucose (25 mM) + insulin yielded similar results: TK activity was decreased 20-30% by culture in 25 mM glucose + insulin conditions (P < .05) and this downregulation continued for 4 (but not 24) hours after switching to lower glucose conditions (P < .05). Continued receptor downregulation during a period of relative substrate deprivation may adversely affect surfactant synthesis, as in some infants of diabetic mothers who experience hypoglycemia (following intrauterine hyperglycemia) in the immediate postnatal period.

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.

Glucose decreases steady state mRNA content of hydrophobic surfactant proteins B and C in fetal rat lung explants.

The streptozotocin-induced diabetic (STZ-DB) rat model is associated with fetal hyperglycemia, but with low to normal plasma insulin concentration. Because surfactant protein (SP) mRNA content in fetal rat lung is decreased in STZ-DB pregnancy, we investigated the effect of increasing concentrations of glucose on SP gene expression in lung organ cultures. SP mRNA content (SP-A, SP-B, SP-C) was assessed by Northern blot analysis in fetal day 20 lung explants (term = 22 days) cultured for 44 hours in medium containing 10, 25, 50, or 100 mM glucose. Our findings were (1) No consistent alteration in SP-A mRNA content was observed at different glucose concentrations (P > .05); (2) SP-B and SP-C mRNA content were reduced in a dose-dependent manner when glucose concentration was increased from 10 mM to 100 mM. The mRNA content, compared to 10 mM glucose, decreased to 50-60% at 25 mM glucose, to 20-25% at 50 mM glucose, and to lower than 10% at 100 mM glucose (P < .01). These findings indicate that the decrease in SP-B and SP-C mRNA in fetuses of STZ-DB rats may be, in part, due to a direct effect of hyperglycemia, whereas the decrease in SP-A mRNA content in STZ-DB rats appears to be due to other effects of diabetes in pregnancy.

Hepatitis B vaccination of premature infants: a reassessment of current recommendations for delayed immunization.

OBJECTIVE: Current American Academy of Pediatrics and United States Public Health Service Immunization Practices Advisory Committee recommendations for hepatitis B immunization in premature infants weighing <2 kg at birth born to hepatitis B surface antigen (HBSAg)-negative mothers are to delay the initiation of vaccination until such infants reach 2 kg or until 2 months of age. This proposal to delay vaccination at birth in these low-risk infants was based on limited studies not conducted in the United States. We sought to reassess current recommendations to delay administration of hepatitis B vaccine in low-risk premature infants by determining the immunogenicity of early hepatitis B vaccination in a US population and identifying variables associated with poor immunogenicity. METHODS: A total of 148 infants <37 weeks' gestation born to mothers negative for HBSAg were recruited at birth and stratified to three birth weight groups: <1000 g, 1000 to 1500 g, and >1500 g. Recombinant hepatitis B vaccine was administered within the first week of life, at 1 to 2 months of age, and at 6 to 7 months of age. Serum obtained at birth and after the second and third doses of vaccine was tested for antibody to HBSAg. Variables associated with poor response were sought prospectively by collecting demographic and clinical data. RESULTS: A total of 118 subjects (83%) completed the study. Postsecond dose sera were available for 117 infants and postthird dose sera were available for 112 infants. The seroprotection rate (attaining >/=10 mIU/mL HBS antibody) after two doses was low (25%) regardless of birth weight; infants weighing <1000 g at birth had the poorest response (11%). The seroprotection response rate after three doses of vaccine increased with birth weight; infants weighing 1500 g at birth (group 3; 84% response rate). The seroprotection response rate of group 3 infants after three doses of vaccine, although low, could not be differentiated from the response rates reported for full-term infants using 95% confidence intervals. Of all infants who did not achieve protective levels of antibody after three doses of vaccine, 96% (26/27) weighed <1700 g at birth. The geometric mean HBS antibody levels in responders were 88 and 386 mIU/mL after two and three doses, respectively. Of 36 children with a birth weight >1500 g, 33 (91%) achieved levels of HBS antibody >100 mIU/mL after three doses of vaccine, compared with 25/35 (71%) of infants with birth weight <1500 g. Using logistic regression analysis, nonresponders were more likely than were responders to have been treated with steroids (26% vs 9%) and to have had a low birth weight (1037 g vs 1455 g). In addition, the seroresponse rate of black infants was more likely than that of white infants to be associated with poor weight gain (falling off 2 percentile ranks in weight) in the first 6 months of life: 22% of black and 60% of white children who failed to gain weight adequately responded to vaccination, compared with 92% of black and 70% of white children who were growing adequately. Of interest, the only infant with a birth weight of >1700 g who did not make protective levels of specific antibody after three doses of vaccine was 2300 g at birth, but had inadequate weight gain in the first 6 months of life. CONCLUSIONS: This study supports current recommendations of the American Academy of Pediatrics and the Centers for Disease Control and Prevention for delaying the initiation of hepatitis B immunization beyond the first week of life for premature infants at low risk for hepatitis B infection, particularly in newborns weighing <1700 g at birth. In addition, we have identified variables other than birth weight that were associated with an inadequate immune response to early hepatitis B vaccination in premature infants, such as poor weight gain in the first 6 months of life

Opioids accelerate fetal rat lung maturation in vitro.

Infants born to heroin- and cocaine-addicted mothers have been reported to have a lower incidence of respiratory distress syndrome (RDS) compared with nonaddicted infants. However, it is not known whether these are direct drug-mediated effects or secondary phenomena. We therefore investigated the effect of opioids and cocaine on fetal rat lung maturation in vitro. Using 18- to 20-d fetal rat lung explants and 20-d fetal type II cells, we measured the effect of varying concentrations (1 x 10(-8) to 1 x 10(-3) M) of heroin, morphine, methadone, and the nonopioid cocaine on the rate of choline incorporation into phosphatidylcholine (PC) and disaturated PC. We also analyzed the morphology of 19-d explants after exposure to opioids. Significant increases in rate of choline incorporation were noted in 19- and 20-d explants using 1 x 10(-3) M heroin, 1 x 10(-3) M morphine, and 1 x 10(-4) M methadone (P < 0. 005). No acceleratory effect was seen with cocaine. Morphologic analysis of the three opioid-treated groups revealed a significant (192 to 251%) increase in type II pneumocytes and lamellar bodies per alveolar lining cell (P < 0.01). Choline incorporation into PC by type II cells was also significantly increased by opioids (P < 0. 01); lactate dehydrogenase release and cell viability were not affected by opioid treatment. These data indicate that high-dose opioids have an acceleratory effect on biochemical and morphologic parameters of fetal lung maturation in vitro. The lack of in vitro acceleration with cocaine suggests that any cocaine-related reduction in the incidence of RDS is a secondary effect.

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.

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.

Safety and pharmacokinetics of multiple doses of recombinant human CuZn superoxide dismutase administered intratracheally to premature neonates with respiratory distress syndrome.

OBJECTIVES: To examine the safety and pharmacokinetics of multiple intratracheal (IT) doses of recombinant human CuZn superoxide dismutase (rhSOD) in premature infants with respiratory distress syndrome who are at risk for developing bronchopulmonary dysplasia (BPD). Methods. Thirty-three infants (700 to 1300 g) were randomized and blindly received saline, 2.5 mg/kg or 5 mg/kg rhSOD IT within 2 hours of surfactant administration. Infants were treated every 48 hours (as long as endotracheal intubation was required) up to 7 doses. Serial blood and urine studies, chest radiographs, neurosonograms, SOD concentration and activity measurements, and tracheal aspirate (TA) inflammatory markers were assessed throughout the 28-day study. RESULTS: SOD concentrations in serum (0.1 [0.05/0.15] microg/mL-geometric mean with lower/upper confidence intervals), tracheal aspirates (TA) (0.2 [0.1/0.3] microg/mL) and urine (0.3 [0.2/0.4] microg/mL) were similar at baseline in all 3 groups and did not change significantly in the placebo group. In the rhSOD treatment groups, SOD concentrations were increased on day 3 and did not change significantly thereafter over the 14-day dosing period (also measured on days 5, 7, and 13). SOD concentrations averaged 0.4 [0.3/0.5] microg/mL in serum, 0.8 [0.6/1.2] microg/mL in TA and 1.1 [1.0/1.3] microg/mL in urine for the low-dose group and 0.6 [0.5/0.7] microg/mL in serum, 1.1 [0.9/1.5] microg/mL in TA, and 2.2 [1.6/2.9] microg/mL in urine for the high-dose group over the 14-day dosing period. Enzyme activity directly correlated with SOD concentration and rhSOD was active even when excreted in urine. TA markers of acute lung injury (neutrophil chemotactic activity, albumin concentration) were lower in the rhSOD agroups compared with placebo. No significant differences in any clinical outcome variable were noted between groups. CONCLUSIONS: These data indicate that multiple IT doses of rhSOD increase the concentration and activity of the enzyme in serum, TA and urine, reduce TA lung injury markers and are well-tolerated. Further clinical trials examining the efficacy of rhSOD in the prevention of BPD are warranted.

Surfactant secretion by type II pneumocytes is inhibited by high glucose concentrations.

Delayed fetal lung maturation is observed in poorly controlled diabetic pregnancies. To investigate whether elevated glucose levels inhibit basal surfactant secretion and synthesis in type II cells and whether inhibitory effects on secretion can be reversed by secretagogues, type II cells isolated from 20-day fetal rat lung explants were initially cultured in [H3] choline containing media with glucose concentrations of 5.5, 10, 25, 50, and 100 mM, or in equiosmolar mannitol controls. Further incubation in nonradioactive media containing matched glucose levels with and without 1 x 10(-5) M terbutaline 1 x 10(-6) M and 1 x 10(-8) M 12-O-tetradecanoylphorbol 13-acetate (TPA) allowed assessment of incorporation of choline into phosphatidylcholine (PC) and its subsequent secretion. PC secretion was inhibited by culture in high glucose conditions, resulting in an approximately 30% reduction in secretion under 50 and 100 mM glucose conditions compared to culture at 5.5 or 10 mM glucose (p < .01); this decrease could not be explained by changes in osmolarity or in all viability after culture in high glucose. Insulin (1 unit/mL) had no significant impact on secretion (92 +/- 7% of control). Terbutaline-stimulated cells grown under 50 and 100 mM glucose conditions had significantly lower secretion rates than did terbutaline-stimulated cells cultured in 5.3 mM glucose (p < .05). Exposure to TPA resulted in significant increase in surfactant secretion by cells grown in both 5.5 and 100 mM glucose; however, the percentage increase (39.5 +/- 6.8% and 94.8 +/- 8.0% with 10(-8) M and 10(-6) MTPA, respectively) was significantly lower than in controls (87.8 +/- 8.0% and 152.1 +/- 18.8%, respectively) (p < .001). Choline incorporation into PG was also decreased by 100 mM glucose to 77 +/- 9% of control (p < .01). These data indicate that high glucose levels inhibit both surfactant synthesis and baseline and secretagogue-stimulated surfactant secretion by type II cells. This inhibitory effect on surfactant secretion may further exacerbate the decrease in surfactant synthesis and the pulmonary maturational delay seen in infants of diabetic pregnancies.

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.

Escherichia coli transcytosis in a Caco-2 cell model: implications in neonatal necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a serious gastrointestinal disorder of preterm infants. Other than an association with prematurity and gastrointestinal feeding, no single factor or mechanism has been consistently linked to this disease. We have previously demonstrated that Escherichia coli isolates obtained from the stool of infants with NEC caused NEC-like injury in a weanling rabbit ileal loop model; this injury, in turn, could be blocked by coinfection with selected Gram(+) bacteria (Enterococcus faecium) isolated from asymptomatic controls. Using Caco-2 cells in a trans-well system, we now demonstrate that the same E. coli isolates can cross epithelial cell monolayers in the absence of ultrastructural change or damage. These results with E. coli contrast with those seen with Salmonella typhimurium, which passed through the monolayer at a higher rate and were associated with striking ultrastructural damage. Transcytosis of E. coli was reduced 3-5-fold in the presence of E. faecium previously shown to block NEC-like injury in the loop model. There was a mild increase in the rate of E. coli transcytosis when studies were conducted with younger, undifferentiated cells; these immature cells had no brush border, had decreased production of brush border-specific enzymes, but retained well defined tight junctions, as demonstrated by transepithelial electrical resistance and electron microscopy. A further reduction/ complete blockage of E. coli transcytosis was observed when E. faecium was used as the coinfectant in studies with these undifferentiated cells. We hypothesize that the ability of E. coli to cross epithelial cell layer is a critical initial step in the cascade of events which lead ultimately to NEC; blockage or reduction in E. coli transcytosis in the presence of certain Gram(+) organisms may play a significant role in prevention of NEC.

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.