Nitrofen induces apoptosis independently of retinaldehyde dehydrogenase (RALDH) inhibition.

BACKGROUND: Nitrofen is a diphenyl ether that induces congenital diaphragmatic hernia (CDH) in rodents. Its mechanism of action has been hypothesized as inhibition of the retinaldehyde dehydrogenase (RALDH) enzymes with consequent reduced retinoic acid signaling. METHODS: To determine if nitrofen inhibits RALDH enzymes, a reporter gene construct containing a retinoic acid response-element (RARE) was transfected into HEK-293 cells and treated with varying concentrations of nitrofen in the presence of retinaldehyde (retinal). Cell death was characterized by caspace-cleavage microplate assays and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assays. Ex vivo analyses of cell viability were characterized in fetal rat lung explants using Live/Dead staining. Cell proliferation and apoptosis were assessed using fluorescent immunohistochemistry with phosphorylated histone and activated caspase antibodies on explant tissues. Nile red staining was used to identify intracellular lipid droplets. RESULTS: Nitrofen-induced dose-dependent declines in RARE-reporter gene expression. However, similar reductions were observed in control-reporter constructs suggesting that nitrofen compromised cell viability. These observed declines in cell viability resulted from increased cell death and were confirmed using two independent assays. Ex vivo analyses showed that mesenchymal cells were particularly susceptible to nitrofen-induced apoptosis while epithelial cell proliferation was dramatically reduced in fetal rat lung explants. Nitrofen treatment of these explants also showed profound lipid redistribution, primarily to phagocytes. CONCLUSIONS: The observed declines in nitrofen-associated retinoic acid signaling appear to be independent of RALDH inhibition and likely result from nitrofen induced cell death/apoptosis. These results support a cellular apoptotic mechanism of CDH development, independent of RALDH inhibition.

Lactic acid is a potential virulence factor for group B Streptococcus.

Group B Streptococcus (GBS) is a Gram-positive bacterium that causes sepsis and meningitis in neonates and infants. Although several GBS-associated virulence factors have been described, the mechanisms of GBS invasive disease are not well understood. To characterize additional virulence factors, a novel in vitro infection assay was developed using rat fetal lung explants. However, application of GBS to the system induced rapid lung tissue destruction associated with increased media acidity. Since lactic acid produced by other streptococci is an important virulence factor, we hypothesized that lactic acid contributed to the virulence of GBS. Spent growth media and neutralized-spent media were applied to explants and results indicated that neutralization of the media completely protected the tissue from degradation. These results were verified using multiple viability assays and with transformed cell lines. Furthermore, comparable spent media from Escherichia coli did not induce tissue cytotoxicity, suggesting that GBS produces organic acids in excess of other potential bacterial pathogens. Analysis of the spent media indicated that l-lactate levels reached approximately 70 mM, indicating that lactic acid is a major constituent of the metabolic acid produced by GBS. Treatment of explants with lactic acid alone produced dose-dependent tissue degradation, indicating that lactic acid is independently sufficient to induce target-tissue cytotoxicity. Finally, both spent media and 23.6 mM lactic acid produced dramatic tissue autofluorescence; the basis for this is currently unknown. These studies demonstrate that GBS-produced lactic acid is a potential virulence factor and may contribute to GBS invasive disease.

Retinoic acid-mediated differentiation protects against nitrofen-induced apoptosis.

BACKGROUND: Nitrofen is a diphenyl ether that induces a spectrum of birth defects subsequent to administration to pregnant rodents, in which the molecular etiology of these defects are poorly characterized. Because previous reports showed that nitrofen induced apoptosis in undifferentiated P19 teratocarcinoma cells, we hypothesized that undifferentiated fetal cells have greater susceptibility to nitrofen-induced apoptosis than their differentiated derivatives. METHODS: To investigate this hypothesis, cell lines including P19 and F9 were differentiated with retinoic acid into neuronal and endodermal derivatives respectively. Apoptosis was characterized by caspase-3 cleavage and Terminal transferase dUTP nick end labeling (TUNEL) assays. RESULTS: Both differentiated cell-types had reduced nitrofen-induced caspase-3 cleavage and DNA fragmentation compared with the naive controls, strongly suggesting that differentiation of these cells protects against nitrofen-induced apoptosis. In addition, resistance to apoptotic induction was proportional to the expression levels of the differentiation marker, p27 (kip1) while direct proportionality was not observed for the antiapoptotic protein Bcl-2. CONCLUSIONS: These studies show that nitrofen may induce its associated birth defects via a mechanism involving apoptosis of undifferentiated fetal cells.

Vitamin A deficiency (VAD), teratogenic, and surgical models of congenital diaphragmatic hernia (CDH).

Congenital diaphragmatic hernia (CDH) is a congenital malformation that occurs with a frequency of 0.08 to 0.45 per 1,000 births. Children with CDH are born with the abdominal contents herniated through the diaphragm and exhibit an associated pulmonary hypoplasia which is frequently accompanied by severe morbidity and mortality. Although the etiology of CDH is largely unknown, considerable progress has been made in understanding its molecular mechanisms through the usage of genetic, teratogenic, and surgical models. The following review focuses on the teratogenic and surgical models of CDH and the possible molecular mechanisms of nitrofen (a diphenyl ether, formerly used as an herbicide) in both induction of CDH and pulmonary hypoplasia. In addition, the mechanisms of other compounds including several anti-inflammatory agents that have been linked to CDH will be discussed. Furthermore, this review will also explore the importance of vitamin A in lung and diaphragm development and the possible mechanisms of teratogen interference in vitamin A homeostasis. Continued exploration of these models will bring forth a clearer understanding of CDH and its molecular underpinnings, which will ultimately facilitate development of therapeutic strategies.

Distribution of ERK1/2 and ERK3 during normal rat fetal lung development.

The extracellular regulated kinases-1 and -2 (ERK1/2) are well-characterized mitogen-activated protein kinases (MAPK) that play critical roles in proliferation and differentiation, whereas the function(s) of MAPK ERK3 are currently unknown. To understand better the roles of these kinases in development, the temporal distribution of ERK1, -2, and -3 proteins were investigated in multiple tissues. The ERK3 protein, in contrast to ERK1/2 varied both between and within individual organs over time. To characterize this variability in greater detail, the temporal and spatial distributions of activated ERK1/2 and ERK3 during rat fetal lung development were investigated. The diphosphorylated (activated) forms of ERK1/2 (dp-ERK1/2), ERK3, and its phosphorylated form (P-ERK3) decreased from embryonic day 17 (E17) through E21 while both ERK1 and ERK2 total proteins remained unchanged, indicating that ERK1/2 and ERK3 proteins are expressed independently during fetal lung development. In addition, characterization of the distribution of these proteins by fluorescent immunohistochemistry indicated that phosphorylated ERK1/2 and total ERK1/2 were distributed throughout multiple cell types, with the phosphorylated ERK1/2 colocalizing with prophase mitotic cells. In contrast, ERK3 was restricted to the distal lung epithelium during the pseudoglandular phase (E17) but shifted to the proximal airways, particularly Clara cells during the saccular stage (E21). The P-ERK3 colocalized with the mitotic marker P-histone H3 in fetal lung and in NIH3T3 and HeLa cells, implicating a potential role for P-ERK3 in mitosis. Thus, expression of ERK1/2 and ERK3 and their phosphorylated forms are expressed independently and are temporally and spatially localized during fetal lung morphogenesis. These observations will facilitate detailed functional analysis of these kinases to assess their roles in pulmonary development and diseases.

Retinoic acid decreases fetal lung mesenchymal cell proliferation in vivo and in vitro.

Retinoic acid (RA) is an important coordinator of mammalian organogenesis. RA is implicated in critical lung developmental events. Cell proliferation is precisely regulated during development. We investigated the effect of RA on proliferating mesenchymal cells in both whole organ lung cultures and cell cultures. The potential pathways required for the response were studied in cultures of lung mesenchymal cells from embryonic day (e) 12. We observed an RA-dependent reduction in proliferation of mesenchymal cells in both whole organ and in cell culture. In mesenchymal cell cultures, RA decreased proliferation in lung mesenchymal cells by 72%. This was associated with a decrease of erk-1/2 activity by 68%. Mesenchymal cell proliferation is erk-1/2 dependent. Erk-1/2 can be activated by G-protein coupled receptors (GPCR) or tyrosine kinase receptors (RTK). RA treatment altered both the RTK and the GPCR pathways in primary lung mesenchymal cells. The Epidermal Growth Factor (EGF) dependent erk-1/2 activation was increased by 35% whereas the G(i)-protein cascade was inhibited by 44% in cells treated with RA. Our results suggest that RA decreases proliferation of lung mesenchyme via a G(i)-protein and the erk-1/2 signaling cascade.

Control and regulation of pulmonary hypoplasia associated with congenital diaphragmatic hernia.

Control of fetal lung growth and development is exquisitely orchestrated and regulated. Branching morphogenesis is carefully choreographed with cell growth, proliferation, differentiation, and apoptosis in a spatially and temporally dependent manner. Some of the signals and pathways mediating these events have recently been uncovered, but much remains unknown. The precise etiologic derangements that give rise to pulmonary hypoplasia in congenital diaphragmatic hernia remain elusive. Some clues have been discovered in developmental and signaling pathways that include receptor tyrosine kinase growth factors, homeobox genes, transcription factors, airway distension, retinoid signaling, and oxidation-reduction.

Open-lung protective ventilation with pressure control ventilation, high-frequency oscillation, and intratracheal pulmonary ventilation results in similar gas exchange, hemodynamics, and lung mechanics.

BACKGROUND: Pressure control ventilation (PCV), high-frequency oscillation (HFO), and intratracheal pulmonary ventilation (ITPV) may all be used to provide lung protective ventilation in acute respiratory distress syndrome, but the specific approach that is optimal remains controversial. METHODS: Saline lavage was used to produce acute respiratory distress syndrome in 21 sheep randomly assigned to receive PCV, HFO, or ITPV as follows: positive end-expiratory pressure (PCV and ITPV) and mean airway pressure (HFO) were set in a pressure-decreasing manner after lung recruitment that achieved a ratio of Pao2/Fio2 > 400 mmHg. Respiratory rates were 30 breaths/min, 120 breaths/min, and 8 Hz, respectively, for PCV, ITPV, and HFO. Eucapnia was targeted with peak carinal pressure of no more than 35 cm H2O. Animals were then ventilated for 4 h. RESULTS: There were no differences among groups in gas exchange, lung mechanics, or hemodynamics. Tidal volume (PCV, 8.9 +/- 2.1 ml/kg; ITPV, 2.7 +/- 0.8 ml/kg; HFO, approximately 2.0 ml/kg) and peak carinal pressure (PCV, 30.6 +/- 2.6 cm H2O; ITPV, 22.3 +/- 4.8 cm H2O; HFO, approximately 24.3 cm H2O) were higher in PCV. Pilot histologic data showed greater interstitial hemorrhage and alveolar septal expansion in PCV than in HFO or ITPV. CONCLUSION: These data indicate that HFO, ITPV, and PCV when applied with an open-lung protective ventilatory strategy results in the same gas exchange, lung mechanics, and hemodynamic response, but pilot data indicate that lung injury may be greater with PCV.

Expression of Netrin-1 and its two receptors DCC and UNC5H2 in the developing mouse lung.

The ligand Netrin-1 and its receptors DCC and UNC5H2 are critical for the regulation of neuronal migration in nervous system development. Here we demonstrate expression of these molecules in lung development. The mRNA expression profiles of Netrin-1, DCC and UNC5H2 are developmentally regulated during embryonic mouse lung formation. Netrin-1 shows a bimodal expression pattern with elevated mRNA levels early followed by a second peak in late gestation. Peak expression of DCC occurs early in development whereas expression of UNC5H2 peaks late in development. We also demonstrate localization of Netrin-1, DCC and UNC5H2 during the stages of lung development. We present evidence that these proteins are modulated spatially in the mesenchyme and epithelium during lung organogenesis.

Slit and robo: expression patterns in lung development.

First described as an axonal guidance cue through its repulsive effect on neurons expressing its receptor Roundabout (Robo), the Slit ligand has effects on cell migration, axon branching and elongation. Indirect evidence implicates Slit and Robo in lung development. We now demonstrate that Slit-2 and Slit-3 are developmentally regulated in embryonic murine lung. Immunohistochemistry demonstrates Slit-2 and Slit-3 expression by the pulmonary mesenchyme and airway epithelium. Robo-1 and Robo-2 are also expressed by the developing mesenchyme and airway epithelium. As lung development progresses, Robo-1 and Robo-2 expression localizes to only the airway epithelium. We conclude Slit/Robo are expressed in temporo-spatially adjacent domains suggesting interactive roles in pulmonary bronchiolar development.

The diagnosis of acute appendicitis in a pediatric population: to CT or not to CT.

PURPOSE: The aim of this study was to determine if focused appendiceal computed tomography with colon contrast (FACT-CC) increases the accuracy of the preoperative diagnosis of acute appendicitis in children. METHODS: A 5-year retrospective review was conducted of a university hospital database of 283 patients (age 0.8 to 19.3 years; mean, 11.3 years) treated with appendectomy for presumed acute appendicitis. RESULTS: Of the 283 patients in whom appendectomies were performed, 268 were confirmed by pathologic analysis of the specimen to have acute appendicitis for a diagnostic accuracy in our institution of 94.7%. Ninety-six patients (34%) underwent FACT-CC scans as part of their preoperative evaluation. The sensitivity of the computed tomography (CT) scan was 94.6%, and the positive predictive value was 95.6%. In girls older than 10 years, CT imaging was not significantly more accurate in predicting appendicitis than examination alone (93.9% v. 87.5%; P =.46). CONCLUSIONS: Preoperative FACT-CC did not increase the accuracy in diagnosing appendicitis when compared with patients diagnosed by history, physical examination and laboratory studies. If there was a strong suspicion of appendicitis, a negative CT scan did not exclude the diagnosis of appendicitis. However, focused appendiceal CT scan is a sensitive test with a high positive predictive value and may be useful in a patient with an atypical history or examination.

Oxidation-reduction (redox) controls fetal hypoplastic lung growth.

INTRODUCTION: The persistent morbidity and mortality of congenital diaphragmatic hernia are largely due to associated pulmonary hypoplasia. We have shown previously that three antioxidants (vitamin C, glutathione, and vitamin E) could accelerate the growth of fetal hypoplastic lungs grown in culture. We hypothesize that this occurs via a reductant mechanism. METHODS: Timed-pregnant rats were gavage-fed nitrofen (100 mg) on day 9.5 of gestation (term = day 22). Fetal lungs were harvested on day 13.5 and placed in organ culture containing serum-free BGJb medium with antibiotics. After randomization, the lung organ cultures were divided into a control group (n = 31) and an experimental group that received the antioxidant N-acetylcysteine (NAC, 100 microM, n = 31). The fetal lung organ cultures were grown for 4 days at 37 degrees C with 5% CO(2). Computer-assisted digital tracings of the airways were performed daily on live, unstained specimens, and lung bud count, perimeter, and area were measured. After 4 days, lungs were pooled, homogenized, and assayed for reduced and oxidized glutathione, normalized to protein, as an estimate of the tissue redox potential. Data were expressed as means +/- SEM, and statistical comparisons were performed using Student's unpaired t test, with P < 0.05 considered significant. RESULTS: Area, perimeter, lung bud count, and complexity (as measured by the perimeter/square root of area) were all significantly increased with NAC treatment from day 2 onward. Reduced glutathione levels were significantly increased following NAC administration (67.1 +/- 5.8 versus 37.5 +/- 4.2 micromol/mg, P = 0.0004). The ratio of reduced to oxidized glutathione was 2.23. CONCLUSIONS: N-Acetylcysteine stimulates nitrofen-induced hypoplastic fetal lung growth in organ culture and increases the ratio of reduced to oxidized glutathione. These data support the concept that oxidation-reduction (redox) may be an important control mechanism for fetal lung growth.

MEK-1/2 inhibition reduces branching morphogenesis and causes mesenchymal cell apoptosis in fetal rat lungs.

The roles of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinases-1 and -2 (ERK-1/2) in fetal lung development have not been extensively characterized. To determine if ERK-1/2 signaling plays a role in fetal lung branching morphogenesis, U-0126, an inhibitor of the upstream kinase MAP ERK kinase (MEK), was added to fetal lung explants in vitro. Morphometry as measured by branching, area, perimeter, and complexity were significantly reduced in U-0126-treated lungs. At the same time, U-0126 treatment reduced ERK-1/2, slightly increased p38 kinase, but did not change c-Jun NH(2)-terminal kinase activities, indicating that U-0126 specifically inhibited the ERK-1/2 enzymes. These changes were associated with increased apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunofluorescent labeling of anti-active caspase-3 in the mesenchyme of explants after U-0126 treatment compared with the control. Mitosis characterized by immunolocalization of proliferating cell nuclear antigen was found predominantly in the epithelium and was reduced in U-0126-treated explants. Thus U-0126 causes specific inhibition of ERK-1/2 signaling, diminished branching morphogenesis, characterized by increased mesenchymal apoptosis, and decreased epithelial proliferation in fetal lung explants.

Long-term consequences of toxic epidermal necrolysis in children.

OBJECTIVE: Toxic epidermal necrolysis (TEN) is an acute inflammatory systemic condition that involves injury not just to the skin. Historically, it has been associated with a high mortality but few long-term consequences among survivors. With improved survival, long-term consequences may be becoming more apparent. The objective of this study was to define these long-term consequences and their frequency. METHODS: From July 1, 1991, to June 30, 2000, 11 children with severe TEN were referred to a regional pediatric burn facility. Wounds were managed with a strategy involving prevention of wound desiccation and superinfection, including the frequent use of biological wound coverings. All children survived and have been followed in the burn clinic. The records of all children were reviewed in detail. RESULTS: Two boys and 9 girls with an average age of 7.2 +/- 1.8 years (range: 6 months-15 years) and sloughed surface area of 76 +/- 6% of the body surface (range: 50%-95%) were admitted to the burn unit for care. Antibiotics (3 children), anticonvulsants (4 children), nonsteroidals (2 children), and viral syndrome or unknown agents (2 children) were believed to have triggered the syndrome. Six (55%) children required intubation for an average of 9.7 +/- 1.8 days (range: 2-14 days). Mucosal involvement occurred in 10 (91%) and ocular involvement in 10 (91%). Lengths of stay averaged 19 +/- 3 days (range: 6-40 days). Overall follow-up averaged 14 +/- 13 months. Three children had no apparent long-term consequences of the disease and were referred to primary care follow-up after the 2-month burn clinic visit. The remaining children had follow-up averaging 23 +/- 13 months. The most common long-term morbidity involved eyes (3 children [27%]), nails (4 children [36%]), and variegated skin depigmentation (all children). One child developed vaginal stenosis from mucosal inflammation. No esophageal strictures or recurrent TEN has been diagnosed. CONCLUSIONS: Survival has improved in children with TEN, but long-term sequelae are not infrequent. The most common long-term consequences involve the eyes, the skin, and the nails.

Young age is not a predictor of mortality in burns.

OBJECTIVE: Conventional wisdom and recently published reports suggest that children <48 months of age have a higher mortality rate after burns than older children and adolescents with similar injuries and that young age is a predictor of mortality. This study was done to validate or refute this impression. DESIGN: Retrospective review. SETTING: Regional pediatric burn center. PATIENTS: All children (n = 1223) managed over a recent 8-yr interval (1991-1998) for acute thermal burns. INTERVENTIONS: The survival rate of children <48 months of age was compared with the survival rate of children >/=48 months of age. MEASUREMENTS AND MAIN RESULTS: Of the 1112 children with burns covering <30% of the body surface, 721 (65%) were <48 months of age. After exclusion of one child who was brain dead and became a solid organ donor, there were no deaths in this burn size group. There were 111 children admitted with burns covering >/=30% of the body surface: 47 (42%) with an average age of 2.0 yrs (range, 4 wks to 3 yrs and 11 months) were <48 months of age, and 64 (58%) with an average age of 10.9 yrs (range, 4 yrs to 17 yrs) were >/=48 months of age. There were no clinically important differences between the two groups in burn size (51.9% +/- 18.1% [range, 30%-90%] vs. 56.9% +/- 19.4% [range, 30%-97%]; p =.18) or need for mechanical ventilatory support (30/47 [63.8%] vs. 44/64 [68.8%]; p =.59). The mortality rate in the young group was 0% (0/47) and 10.9% (7/64) in the older group (p =.04). All children who died had large burns (average burn size, 82.9% +/- 11.5%) with concurrent inhalation injury. CONCLUSION: Young age is not a predictor of mortality in burns.