Susan E. Keeney

Comparison of l-Selectin and CD11b on Neutrophils of Adults and Neonates during the First Month of Life

The newborn infant is particularly susceptible to infection in the first weeks of life and this may be, in part, related to functional impairment of neonatal neutrophils in regard to adherence, chemotaxis, and migration. Differences in expression of the neutrophil adherence molecules, l-selectin and CD11b/CD18 (Mac-1), have been previously demonstrated in cord blood and in very young infants (≤ 48 h) compared with adults, but it is unknown for how long these differences persist. We measured surface expression of neutrophil l-selectin and CD11b using flow cytometry in healthy term human newborns from 24 h through 4 wk of age. We also measured levels of soluble l-selectin using an ELISA in neonates though the age of 4 wk. Compared with adults, neonates expressed significantly less l-selectin on resting neutrophils through 4 wk of age, with the lowest levels being at 24 h. Expression of l-selectin on the neutrophil after activation with N-formyl-methionyl-leucyl-phenylalanine was less in the neonate than in the adult (p < 0.05) for the first week of life. Soluble l-selectin showed a steady increase with age in the neonates. Soluble l-selectin was significantly lower in the 24-h neonate compared with the adult and was higher in the 4-wk neonate compared with the adult. CD11b expression was similar between neonates and adults on unstimulated neutrophils, but upon activation, the neonatal neutrophil demonstrated significantly lower up-regulation of CD11b on the neutrophil surface through 4 wk of age compared with adults (p < 0.05 for all ages except 1 wk). These findings that differences in expression of l-selectin and CD11b from that in adults persist throughout the neonatal period provide further evidence that these differences may play a role in the neutrophil defects observed during the neonatal period.

Maternal Factors in Extremely Low Birth Weight Infants Who Develop Spontaneous Intestinal Perforation

BACKGROUND. Spontaneous intestinal perforation of the extremely low birth weight infant (≤1000 g) is associated with a high incidence of Candida and coagulase-negative Staphylococcus sepsis. Little is known about prenatal risk factors, and histopathologic examination of placentas in infants with spontaneous intestinal perforation has not been reported. OBJECTIVES. Our objective was to investigate maternal factors and specific placental findings in a sample of infants with spontaneous intestinal perforation. We compared the maternal factors and clinical outcomes to a matched control group. PATIENTS AND METHODS. This single-center, retrospective cohort study was conducted between January 2001 and December 2005. The records of extremely low birth weight infants with spontaneous intestinal perforation were reviewed (n = 16). Study infants were matched to 2 infants in the control group; any twin of a study patient was also included as a control subject (n = 35). Histopathologic examination of placentas included standard hematoxylin and eosin and methenamine silver stains. RESULTS. Infants with spontaneous intestinal perforation were more likely than control subjects to have severe placental chorioamnionitis with fetal vascular response (40% vs 12%); 2 placentas also tested positive for yeast versus none in the control subjects. Mothers of infants with spontaneous intestinal perforation were more likely than control subjects to have received antibiotics before or at delivery (93% vs 57%). Fifty percent of the infants had Candida, and 31% in the spontaneous intestinal perforation group had coagulase-negative Staphylococcus sepsis versus 6% in the control subjects. Finally, infants with spontaneous intestinal perforation had delayed enteral feeding (64 ± 30 vs 31 ± 10 days) and prolonged hospitalization (155 ± 48 vs 108 ± 36 days). CONCLUSIONS. Spontaneous intestinal perforation in the extremely low birth weight infant is a neonatal disease related to placental inflammation. We alert practitioners to the importance of placental findings, because they may be positive for yeast.

Heat Susceptibility of Interleukin-10 and Other Cytokines in Donor Human Milk

OBJECTIVE Holder pasteurization renders donor human milk safe for consumption. Because human milk reduces the risk of necrotizing enterocolitis in preterm infants, we tested whether Holder pasteurization affects certain factors in human milk that protect the intestines: epidermal growth factor (EGF), transforming growth factor (TGF)-beta1, erythropoietin (EPO), and interleukin (IL)-10. Donor human milk from a milk bank was examined. METHODS The aqueous phase of 17 samples of donor term human milk (mean duration of lactation, 8 +/- 3.5 months) was examined before and after Holder pasteurization. In the case of IL-10, lesser degrees of pasteurization were also evaluated. The agents were quantified using enzyme immunoassays. The function of IL-10 was also tested. RESULTS Concentrations of EGF and IL-10 were markedly lower than previously reported values in human milk from earlier phases of lactation. Holder pasteurization significantly reduced the concentrations of EPO and IL-10, whereas lesser degrees of heating increased the detection of IL-10. The immunosuppression of T-cell proliferation by human milk, thought to be attributed to IL-10 alone, persisted after Holder pasteurization. CONCLUSIONS Holder pasteurization greatly decreased concentrations of EPO and IL-10 in human milk. These decreases may impact the ability of human milk to protect against necrotizing enterocolitis. Evidence of possible binding of IL-10 to other proteins in human milk was also found. Experiments to test whether Holder pasteurization affects the function of IL-10 in human milk produced evidence for an agent in human milk other than IL-10 that inhibits T-cell proliferation and resists Holder pasteurization.

Arteriovenous CO2 Removal Improves Survival Compared to High Frequency Percussive and Low Tidal Volume Ventilation in a Smoke/Burn Sheep Acute Respiratory Distress Syndrome Model

Objectives and Summary Background:Low tidal volume ventilation (LTV) has improved survival with acute respiratory distress syndrome (ARDS) by reducing lung stretch associated with volutrauma and barotrauma. Additional strategies to reduce lung stretch include arteriovenous carbon dioxide removal (AVCO2R), and high frequency percussive ventilation (HFPV). We performed a prospective, randomized study comparing these techniques in our clinically relevant LD100 sheep model of ARDS to compare survival, pathology, and inflammation between the 3 ventilator methods. Methods:Adult sheep (n = 61) received smoke inhalation (48 breaths) and a 40% third-degree burn. After ARDS developed (Pao2/FiO2 <200), animals were randomized. In experiment 1, animals were killed at 48 hours after randomization. Hemodynamics, pulmonary function, injury scores, myeloperoxidase (MPO) in lung tissues and neutrophils, IL-8 in lung tissues, and apoptosis were evaluated. In experiment 2, the end point was survival to 72 hours after onset of ARDS or end-of-life criteria with extension of the same studies performed in experiment 1. Results:There were no differences in hemodynamics, but minute ventilation was lower in the AVCO2R group and Paco2 for the HFPV and AVCO2R animals remained lower than LTV. Airway obstruction and injury scores were not different among the 3 ventilation strategies. In experiment 1, lung tissue MPO and IL-8 were not different among the ventilation strategies. However, in experiment 2, lung tissue MPO was significantly lower for AVCO2R-treated animals (AVCO2R < HFPV < LTV). TUNEL staining showed little DNA breakage in neutrophils from experiment 1, but significantly increased breakage in all 3 ventilator strategies in experiment 2. In contrast, AVCO2R tissue neutrophils showed significant apoptosis at 72 hours post-ARDS criteria as measured by nuclear condensation (P < 0.001). Survival 72 hours post-ARDS criteria was highest for AVCO2R (71%) compared with HFPV (55%) and LTV (33%) (AVCO2R vs. LTV, P = 0.05). Conclusions:Significantly more animals survived AVCO2R than LTV. In experiment 2, Lung MPO was significantly lower for AVCO2R, compared with LTV (P < 0.05). This finding taken together with the TUNEL and neutrophil apoptosis results, suggested that disposition of neutrophils 72 hours post-ARDS criteria was different among the ventilatory strategies with neutrophils from AVCO2R-treated animals removed chiefly through apoptosis, but in the cases of HFPV and LTV, dying by necrosis in lung tissue.

Antioxidant Enzyme Responses to Hyperoxia in Preterm and Term Rats after Prenatal Dexamethasone Administration

ABSTRACT: Although prenatal steroid therapy is known to enhance in utero maturation of the surfactant and antioxidant enzyme systems, little is known about the effects of steroids on the antioxidant system after birth. We measured activities of the antioxidant enzymes, catalase, superoxide dismutase, and glutathione peroxidase, in lung homogenates from both preterm and term rat pups after prenatal dexamethasone treatment. Enzyme activities were measured at birth and after exposure to >98% oxygen. Dexamethasone treatment resulted in significantly higher survival of the preterm pups at 24 h (91.3% for dexamethasone versus 57% for saline). In preterm pups, the activities of catalase and superoxide dismutase at birth were higher after dexamethasone treatment (p < 0.05). However, after 24 h of hyperoxic exposure, there were no differences in activities of any of the antioxidant enzymes between the dexamethasone and control groups of prematurely born pups. In term pups, antioxidant enzyme activities did not differ significantly at birth; nor did they differ after 24 to 72 h of hyperoxic exposure in the dexamethasone and control treatment groups. Our results indicate that although prenatal dexamethasone treatment augments survival and catalase and superoxide dismutase activities at birth in preterm rat pups, dexamethasone does not result in altered early postnatal antioxidant enzyme activities after exposure to hyperoxia.

Comparison of pulmonary neutrophils in the adult and neonatal rat after hyperoxia.

ABSTRACT: The mechanisms of the increased tolerance to hyperoxia of neonatal animals of many species is incompletely understood. To investigate the etiology of this difference we compared neutrophil entry into the lungs of neonatal and adult rats after hyperoxic exposure. Adult rats were studied after exposure to ≥98% O2 for 60 h and neonatal rats after 3 and 7 d. Neonatal survival was prolonged compared with that reported for adult rats (77% after 7 d of exposure). In adult rats, there were significant increases in pulmonary neutrophils after 60 h of O2 exposure. In neonatal rats, these changes were not evident after 72 h of exposure, but pulmonary neutrophils increased after 7 d of hyperoxia. Before mortality, pulmonary neutrophils were distributed differently in the age groups. After 7 d of O2 exposure in the neonates, total neutrophil counts in lung tissue (21.92 ± 7.29 per cm2 grid) and lung myeloperoxidase (0.085 ± 0.02 U/mg protein) remained significantly lower than in adults after 60 h of O2 exposure (41.44 ± 9.08 per cm2 grid and 0.411 ± 0.085 U/mg protein, respectively). However, in histologic specimens, O2-exposed neonates had higher percentages of neutrophils free in the alveolar air space than did adults, corresponding to a trend toward higher neutrophil counts in bronchoalveolar lavage fluid in the neonates. It appears that, in addition to delay in neutrophil influx into the lung, neonatal rats have lowered retention of neutrophils to the alveolar tissue.

The Effect of Hyperoxic Exposure on Antioxidant Enzyme Activities of Alveolar Type II Cells in Neonatal and Adult Rats

ABSTRACT: Neonatal animals of several species are more tolerant of hyperoxic exposure than arc adults, but the mechanisms of increased neonatal tolerance are unknown, as are the cell types, if any, that contribute to oxygen resistance. We studied the effect of in vivo exposure to 85% oxygen for 72 h on the activities of the antioxidant enzymes, glutathionc peroxidase, catalase and superoxide disnwtase (SOD), in alveolar type II cells and whole lung from adult and neonatal rats. Baseline antioxidant enzyme activities were generally lower in neonatal type II cells compared with adults. Baseline enzyme activities did not differ in neonatal type II cells and lung homogenates except for lower catalase activity in type II cells. Hyperoxic exposure resulted in 35–38% increases in antioxidant enzyme activities in neonatal whole lung. In neonatal type II cells, SOD activity increased by 170% after hyperoxia, whereas catalase and glutathione peroxidase were not significantly changed. In the adult whole lung, hypcroxk exposure resulted in increases in only glutathione peroxidase activity, whereas in adnlt type II cells there was a significant decrease in SOD activity after O2 exposure. Therefore, although baseline antioxidant enzyme activities were not higher hi neonatal type II cells compared with whole lung, there were differences in the antioxidant enzyme responses of adult and neonatal type II cells to hyperoxia, particularly with respect to SOD. The ability of the neonatal type II cell to respond to hyperoxia with an early increase in SOD activity may contribute to the enhanced oxygen tolerance of the neonate.

The Association of Early Blood Oxygenation with Child Development in Preterm Infants with Acute Respiratory Disorders

The potential negative impact of early blood oxygenation on development of specific cognitive and motor outcomes in children born at very low birth weight (VLBW; 1000–1500 g) has not been examined even though these infants are exposed to varying durations and amounts of oxygen as part of their neonatal care. While this is the largest group of preterm infants, they receive much less research attention than extremely low birth weight infants (ELBW < 1000 g).

Alpha1-adrenergic and muscarinic receptors in adult and neonatal rat type II pneumocytes

Binding characteristics of the α1-adrenergic radioloigand [3H]prazosin, and the muscarinic cholinergic radioligand, [3H]quinuclidinyl benzilate, were determined both in intact cell preparations of rat alveolar type II pneumocytes (TIIPs) and in membrane preparations of rat lung tissue. Binding in adult and neonatal (<24 h postnatal age) rats was also compared. Binding affinities for both receptor classes on TIIPs and whole lung membrane preparations alike did not vary significantly with age. In lung membrane preparations, the concentrations of both receptor classes were higher in neonates than adults. In TIIPs, the α1-adrenergic receptor concentration was higher in neonates, but muscarinic receptor concentration was higher in adults. To begin investigation of the functional significance of these receptors, the effects of α1-adrenergic and muscarinic agonists on intracellular calcium ion concentration ([Ca2+]i) were also measured. Both agonists induced consistent increases in [Ca2+]i, which were blocked by respective antagonists. These data indicate the presence of receptors on TIIPs for α1-adrenergic and muscarinic agonists that may influence cellular function via modulation of [Ca2+]i.

Effects of postnatal dexamethasone on oxygen toxicity in neonatal rats

We investigated the effect of timing of early postnatal dexamethasone on survival of hyperoxia-exposed neonatal rats. Pups <24 h old were treated with a tapering course of dexamethasone or saline beginning either prior to exposure (day 0), or after 2, 4, or 6 days of ≧98% O2 (n = 11–14) or air (n = 8–11). Exposures were continued for 14 days. By day 14, day 0 pups had poor survival regardless of the exposure (14% in O2, 13% in air). Survival of pups treated with dexamethasone after 2, 4 and 6 days of O2 exposure was significantly higher at 14 days (50, 86 and 79%, respectively) compared to saline O2 controls (9%, p < 0.001 for each). Pulmonary biochemical analyses were conducted after exposure for 7 days in rat pups treated with dexamethasone or saline beginning after 4 days of exposure to air or O2 (n = 11–12 for each group). While pups treated with dexamethasone showed greatly improved survival compared to O2 controls, there was no decrease in neutrophil influx into the lung as measured by lung myeloperoxidase and neutrophil counts in histologic specimens and lavage fluid. Catalase, glutathione peroxidase, total and manganese superoxide dismutase activities as well as manganese superoxide dismutase (MnSOD) mRNA expression were elevated in both O2 groups after 7 days compared to the air groups (p < 0.05) and MnSOD mRNA expression was elevated in the O2/dexamethasone group, but there were no differences between dexamethasone and saline groups in O2. Thus, this study indicates that the timing of dexamethasone administration is crucial. Mechanisms other than increases in antioxidant enzymes or decreases in lung neutrophils underlie the ability of dexamethasone to improve survival of these neonatal rats.