AAP-Section on Perinatal Pediatrics

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AAP-Section on Perinatal Pediatrics Abstracts &&&&&&&&&&&&&& AAP-Section on Perinatal Pediatrics 1. INTRACELLULAR ADHESION MOLECULE ONE (ICAM-1) IS expression of the inflammatory adhesion molecule, ICAM-1. This INCREASED IN THE CLARA CELL SECONDARY TO EXPOSURE TO supports the premise that the Clara cell may function to modulate LPS, TNF , AND HYPEROXIA pulmonary inflammation by either the secretion of the anti- W.D. Clark1, S.E. Welty2 and P.L. Ramsay3; 1Pediatrics, inflammatory protein, CCSP, or by the expression of the pro- Baylor College of Medicine, Houston, TX, 2Pediatrics, inflammatory protein, ICAM-1. These data further suggest that Children’s Research Institute, Columbus, OH, potential therapeutic interventions that modulate the gene expression 3Pediatrics and Molecular and Cell Biology, Baylor within the Clara cell may provide a method to regulate pulmonary College of Medicine, Houston, TX inflammation. Purpose. Early lung inflammation in prematurely born newborns 2. GENETIC ENHANCEMENT OF GLUTATHIONE REDUCTASE results from a variety of exposures, including cytokines and hyperoxia. Previously, we have shown that serum levels of the PROTECTS CHINESE HAMSTER OVARY CELLS FROM HYPOXANTHINE/ inflammatory adhesion molecule, ICAM-1, are higher in infants XANTHINE OXIDASE–MEDIATED OXIDANT STRESS that develop BPD than in infants that do not. In addition, the D.J. O’Donovan, R.C. Husser, T. Tamura and C.J. content of an anti-inflammatory tracheal fluid protein secreted by Fernandes; Pediatrics, Baylor College of Medicine, the Clara cell, the Clara cell secretory protein (CCSP), is decreased Houston, TX in infants that develop BPD, whereas others have shown that the level of tracheal fluid ICAM-1 is increased. Therefore, this study was Purpose. Reactive oxygen species (ROS) have been implicated in designed to test the hypothesis that the Clara cell in response to the causation of diseases related to prematurity, such as known pro-inflammatory mediators will increase the expression of bronchopulmonary dysplasia and necrotizing enterocolitis. Inad- ICAM-1. equate and immature antioxidant defense function predisposes the premature infant to injury from ROS. The glutathione (GSH)- Methods. We utilized a mouse transformed Clara cell (mtCC) line to dependent antioxidant system protects against the damaging test whether the pro-inflammatory mediators lipopolysaccharide effects of ROS in the cytoplasm and mitochondria, and (LPS), TNF- , and hyperoxia would increase ICAM-1 mRNA. regeneration of GSH from glutathione disulfide by GR is vital for Following 0 to 24 hours of exposure to either LPS, TNF- ,or the optimal function of this system. Previously, we reported hyperoxia, the cells were harvested, ICAM-1 mRNA was assayed by resistance to t-BuOOH–induced cell injury in H441 cells Northern blot, and ICAM-1 protein was assayed immunohisto- following augmentation of mitochondrial GR activities by chemistry. Further analyses with variable length of the 50 upstream adenoviral-mediated transfer of GR cDNA with a mitochondrial region of the ICAM-1 promoter ligated to a luciferase reporter gene targeting sequence (MTS). The purpose of these studies was to were analyzed to identify the regions of the ICAM-1 promoter that determine whether CHO cells with stably enhanced cytoplasmic or were responsive to these pro-inflammatory stimuli. mitochondrial GR activities are resistant to injury from ROS enzymatically generated by HX/XO, and whether greater protection Results. We observed an increase in ICAM-1 mRNA by both LPS and against oxidant stress is conferred on cells with enhanced TNF- by 2 hours, which peaked at 4 hours and returned to baseline mitochondrial GR activities. by 8 to 12 hours. The immunohistochemistry demonstrated an increase in ICAM-1 protein secondary to LPS and TNF- at 4 hours. Methods. Transformed CHO cells with increased cytoplasmic (CHO- Hyperoxia increased ICAM-1 mRNA at 12 hours and increased GR) (17.0±2.0 vs 6.1±2.9 mU/mg Pr in native CHO cells) or ICAM-1 protein at 24 hours. The transient transfection studies mitochondrial (CHO-LGR) (77.5±3.9 vs 7.9±0.4 mU/mg Pr) GR demonstrated a several-fold greater induction of reporter gene activities were generated by liposomal transfer of GR cDNA±MTS activities with the 1520 and 1462 bp ICAM-1 promoter segments and growth in selection media. Native and transformed CHO cells than in the longer 2513 bp region. were grown for 24 hours, and exposed to media with 1 mM HX and XO (0, 5, 7.5, and 10 mU/ml). Percentage cellular lactate Conclusion. These results suggest that the Clara cell is capable of dehydrogenase (LDH) release (12 hours) and LDH activities in the responding to pro-inflammatory stimuli with an increase in media (0, 4, 8, 10, 12 hours) were determined as indices of Journal of Perinatology 2001; 21:482 – 509 # 2001 Nature Publishing Group All rights reserved. 0743-8346/01 $17 482 www.nature.com/jp Abstracts AAP-Section on Perinatal Pediatrics cytotoxicity. Data were analyzed by ANOVA with appropriate post-hoc radioimmunoassay. We observed a time-dependent decrease in tests (n=3, mean±SD). serum levels of hGH as a function of hyperoxia exposure. Further, our observed decrease in hGH occurred prior to the measurable onset of Results. Dose- and time-dependent increases in media LDH were lung injury. Next, we collected the cord blood of intubated premature observed in all cells exposed to XO of 7.5 and 10 mU/ml ( p<0.05). infants 29 weeks’ gestation and measured cord blood levels of Cells with increased CHO-GR and CHO-LGR activities exposed to 7.5 CCSP by Western blot and quantified by densitometry. and 10 mU/ml of XO released less LDH than did native CHO cells (CHO-GR: 16.1±2.9 and 39.2±3.1, CHO-LGR: 15.3±1.4 and Results. We detected soluble CCSP in 100% of the infants that 58.1±6.7, CHO: 22.8±1.6 and 66.7±3.8, respectively). Cells with developed CLD, whereas we detected soluble cord CCSP in only 66% of enhanced CHO-GR activities exposed to the 10 mU/ml XO released infants that did not develop CLD. The level of cord CCSP measured in less LDH than did cells with enhanced CHO-LGR activities similarly the infants that developed CLD was 80% greater that the level of cord exposed. CCSP that was detectable in infants that did not develop CLD. ( p<0.05). Conclusion. Stable enhancement of cytoplasmic or mitochondrial GR activities in CHO cells provides measurable protection against low Conclusion. These results suggest that soluble secreted lung levels of HX/XO-induced oxidant stress; however, at higher levels of proteins may serve as markers of intrapulmonary injury. Moreover, HX/XO-induced oxidant stress, enhancement of cytoplasmic GR that CCSP in the cord blood of extremely premature infants may activities provided greater cellular protection. reflect a degree of pulmonary immaturity that predisposes a subset of infants at greatest risk of developing CLD. Further investigations are underway to determine whether serum CCSP levels postnatally are 3. SOLUBLE CLARA CELL SECRETORY PROTEIN IS DECREASED IN associated with ongoing intrapulmonary inflammation in CLD. HYPEROXIA EXPOSURE AND CORD BLOOD LEVELS ARE PREDICTIVE OF THE DEVELOPMENT OF CHRONIC LUNG DISEASE A.C. Kanu1, P.L. Ramsay3,4, S.E. Hegemier3, S.E. Welty2 4. COMPARISON OF CYTOKINE RESPONSE BETWEEN PRETERM AND and F.J. DeMayo3,4; 1Pulmonary Medicine, Baylor TERM INFANTS IN NEONATAL SEPSIS College of Medicine, Houston, TX, 2Pediatrics, G. Laborada1, F. Cruz1, M. Rego1, J. DePaz1, Children’s Research Institute, Columbus, OH, M. Giulano2, J. Stavola1,A.Jain1, A. Krauss1 and 3Pediatrics, Baylor College of Medicine, Houston, TX, M. Nesin1; 1Pediatrics, New York Presbyterian Hospital- 4Molecular and Cellular Biology, Baylor College of Weill Medical College of Cornell University, New York, Medicine, Houston, TX NY, 2Pediatrics, Lenox Hill Hospital, New York, NY Purpose. Advancements in neonatal care have improved survival for Purpose. Neonatal sepsis ranges from 5 to 10 per 1000 live births infants born prematurely. However, there is a growing population of and remains a major cause of mortality and morbidity. Recent extremely premature infants that develop chronic lung disease studies showed increased production of various cytokines such as (CLD). The underlying mechanisms that are responsible for the interleukin-8 (IL8), interleukin-6 (IL6), and tumor necrosis development of CLD remain ill defined. Early pulmonary factor-alpha (TNA) during sepsis. These cytokines have high inflammation in association with exposure to supplemental oxygen sensitivity and specificity, and are potentially useful diagnostic tests to and mechanical ventilation is recognized to play a critical role in the differentiate infected from non-infected neonates. However, there is development of CLD. We have previously shown that there are specific little data comparing the cytokine levels in preterm and term infants. oxidative changes and alterations in expression of the Clara cell This is particularly important if these cytokines become standard secretory protein (CCSP) in the airways of premature infants that inflammatory markers in neonatal sepsis. The objectives of the study subsequently develop CLD. This study was designed to test the were: (1) to determine if preterm infants could mount similar hypothesis that serum levels of proteins secreted by the Clara cell will cytokine response (IL8, IL6, TNA) when compared to term infants reflect lung injury secondary to hyperoxia exposure and be predictive during neonatal sepsis; and (2) to determine if these cytokines are of the development of CLD in prematurely born infants. significantly elevated in infected compared to non-infected infants. Methods. To test these hypotheses, we utilized transgenic mice Methods. One hundred and nine neonates (59 preterm and 50 previously generated by ligating the CCSP promoter to hGH reporter term) suspected for sepsis were enrolled. Blood was drawn during the gene. These transgenic mice model have been shown to express hGH first 6 hours of sepsis evaluation.
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