Intra-Amniotic Sildenafil Treatment Promotes Lung Growth and Attenuates Vascular Remodeling in an Experimental Model of Congenital Diaphragmatic Hernia.

BACKGROUND: Defective lung development resulting in lung hypoplasia and an attenuated and hypermuscularized pulmonary vasculature contributes to significant postnatal mortality in congenital diaphragmatic hernia (CDH). We hypothesize that deficient embryonic pulmonary blood flow contributes to defective lung development in CDH, which may therefore be ameliorated via enhancement of embryonic pulmonary blood flow. METHODS: The mouse nitrofen model of CDH was utilized to measure embryonic pulmonary blood flow by in utero intracardiac injection of FITC-labeled tomato lectin and color-flow Doppler ultrasound. The effect of prenatal intra-amniotic treatment with sildenafil on survival, lung growth, and vascular morphology in the nitrofen model was determined. RESULTS: Nitrofen-treated embryos exhibited decreased blood flow in the lung periphery compared to controls, and intra-amniotic sildenafil significantly improved embryonic pulmonary blood flow. Similar to nitrofen alone, pups delivered after nitrofen treatment and intra-amniotic injection of dextrose control exhibited respiratory distress and never survived beyond 6 h. Intra-amniotic sildenafil ameliorated respiratory distress in nitrofen-treated pups and improved postnatal survival to 82%. Following intra-amniotic sildenafil treatment at embryonic day (E)10.5, nitrofen-treated P0 lungs were larger with increased left lobe weight, reduced small pulmonary arterial wall muscularization, and increased airway branching complexity compared to controls. Intra-amniotic sildenafil treatment later at E15.5 also resulted in improved survival, lung growth, and attenuation of vascular remodeling in nitrofen-treated embryos. CONCLUSIONS: Defective embryonic pulmonary blood flow may contribute to lung maldevelopment in CDH. Enhancement of embryonic pulmonary blood flow via intra-amniotic sildenafil results in lung growth and attenuation of pulmonary vascular remodeling and may have therapeutic potential for CDH.

Defective parasympathetic innervation is associated with airway branching abnormalities in experimental CDH.

Developmental mechanisms leading to lung hypoplasia in congenital diaphragmatic hernia (CDH) remain poorly defined. Pulmonary innervation is defective in the human disease and in the rodent models of CDH. We hypothesize that defective parasympathetic innervation may contribute to airway branching abnormalities and, therefore, lung hypoplasia, during lung development in CDH. The murine nitrofen model of CDH was utilized to study the effect of the cholinergic agonist carbachol on embryonic day 11.5 (E11.5) lung explant cultures. Airway branching and contractions were quantified. In a subset of experiments, verapamil was added to inhibit airway contractions. Sox9 immunostaining and 5-bromo-2-deoxyuridine incorporation were used to identify and quantify the number and proliferation of distal airway epithelial progenitor cells. Intra-amniotic injections were used to determine the in vivo effect of carbachol. Airway branching and airway contractions were significantly decreased in nitrofen-treated lungs compared with controls. Carbachol resulted in increased airway contractions and branching in nitrofen-treated lungs. Nitrofen-treated lungs exhibited an increased number of proliferating Sox9-positive distal epithelial progenitor cells, which were decreased and normalized by treatment with carbachol. Verapamil inhibited the carbachol-induced airway contractions in nitrofen-treated lungs but had no effect on the carbachol-induced increase in airway branching, suggesting a direct carbachol effect independent of airway contractions. In vivo treatment of nitrofen-treated embryos via amniotic injection of carbachol at E10.5 resulted in modest increases in lung size and branching at E17.5. These results suggest that defective parasympathetic innervation may contribute to airway branching abnormalities in CDH.

Embryonic mouse blood flow and oxygen correlate with early pancreatic differentiation.

The mammalian embryo represents a fundamental paradox in biology. Its location within the uterus, especially early during development when embryonic cardiovascular development and placental blood flow are not well-established, leads to an obligate hypoxic environment. Despite this hypoxia, the embryonic cells are able to undergo remarkable growth, morphogenesis, and differentiation. Recent evidence suggests that embryonic organ differentiation, including pancreatic ß-cells, is tightly regulated by oxygen levels. Since a major determinant of oxygen tension in mammalian embryos after implantation is embryonic blood flow, here we used a novel survivable in utero intracardiac injection technique to deliver a vascular tracer to living mouse embryos. Once injected, the embryonic heart could be visualized to continue contracting normally, thereby distributing the tracer specifically only to those regions where embryonic blood was flowing. We found that the embryonic pancreas early in development shows a remarkable paucity of blood flow and that the presence of blood flow correlates with the differentiation state of the developing pancreatic epithelial cells in the region of the blood flow.

Defective pulmonary innervation and autonomic imbalance in congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is associated with significant mortality due to lung hypoplasia and pulmonary hypertension. The role of embryonic pulmonary innervation in normal lung development and lung maldevelopment in CDH has not been defined. We hypothesize that developmental defects of intrapulmonary innervation, in particular autonomic innervation, occur in CDH. This abnormal embryonic pulmonary innervation may contribute to lung developmental defects and postnatal physiological derangement in CDH. To define patterns of pulmonary innervation in CDH, human CDH and control lung autopsy specimens were stained with the pan-neural marker S-100. To further characterize patterns of overall and autonomic pulmonary innervation during lung development in CDH, the murine nitrofen model of CDH was utilized. Immunostaining for protein gene product 9.5 (a pan-neuronal marker), tyrosine hydroxylase (a sympathetic marker), vesicular acetylcholine transporter (a parasympathetic marker), or VIP (a parasympathetic marker) was performed on lung whole mounts and analyzed via confocal microscopy and three-dimensional reconstruction. Peribronchial and perivascular neuronal staining pattern is less complex in human CDH than control lung. In mice, protein gene product 9.5 staining reveals less complex neuronal branching and decreased neural tissue in nitrofen-treated lungs from embryonic day 12.5 to 16.5 compared with controls. Furthermore, nitrofen-treated embryonic lungs exhibited altered autonomic innervation, with a relative increase in sympathetic nerve staining and a decrease in parasympathetic nerve staining compared with controls. These results suggest a primary defect in pulmonary neural developmental in CDH, resulting in less complex neural innervation and autonomic imbalance. Defective embryonic pulmonary innervation may contribute to lung developmental defects and postnatal physiological derangement in CDH.

Feasibility of laparoscopic Nissen fundoplication after pediatric lung or heart-lung transplantation: should this be the standard?

BACKGROUND: Five-year graft survival in the pediatric lung transplant (LTxp) population is less than 50%, with obliterative bronchiolitis (OB) the leading cause of death at 1, 3, and 5 years post-transplant. Bronchiolitis obliterans syndrome (BOS), defined using spirometry values, is the clinical surrogate for the histological diagnosis of obliterative bronchiolitis. Surgical correction of documented gastroesophageal reflux disease (GERD) has been proposed as a means to potentially delay the onset of BOS and prolong allograft survival in adults before or after lung transplantation but only one such study exists in children. We have examined the safety and possible benefits of laparoscopic antireflux surgery in pediatric patients following lung (LTxp) and heart-lung transplantation (HLTxp). METHODS: An Institutional Review Board (IRB)-approved retrospective chart review was performed to evaluate the outcomes and complications of laparoscopic antireflux surgery in pediatric lung and heart-lung transplant patients. Spirometry data were collected for BOS staging using BOS criteria for children. RESULTS: Twenty-five lung and heart-lung transplants were performed between January 2003 and July 2009. Eleven transplant recipients, including six double-lung and five heart-lung (HLTxp), with a median age of 11.7 years (range 5.1-18.4 years), underwent a total of 12 laparoscopic Nissen fundoplications at a median of 427 days after transplant (range 51-2310 days). GERD was determined based upon clinical impression, pH probe study, gastric emptying study, and/or esophagram in all patients. Three patients already had a gastrostomy tube in place and two had one placed at the time of fundoplication. There were no conversions to open surgery, 30-day readmissions, or 30-day mortalities. Complications included one exploratory laparoscopy for free air 6 days after laparoscopic Nissen fundoplication for a gastric perforation that had spontaneously sealed. Another patient required a revision laparoscopic Nissen 822 days following the initial fundoplication for a paraesophageal hernia and recurrent GERD. The average length of hospital stay was 4.4 ± 1.7 days. Nine of the 12 fundoplications were performed in patients with baseline spirometry values prior to fundoplication and who could also complete spirometry reliably. One of these nine operations was associated with improvement in BOS stage 6 months after fundoplication; seven were associated with no change in BOS stage; and one was associated with a decline in BOS stage. CONCLUSION: It is feasible to perform laparoscopic Nissen fundoplication in pediatric lung and heart-lung transplant recipients without mortality or significant morbidity for the treatment of GERD. The real effect on pulmonary function cannot be assessed due to our small sample size and lack of reproducible spirometry in our younger patients. Additional studies are needed to elucidate the relationship between antireflux surgery and the potential for improving pulmonary allograft function and survival in children which has been previously observed in adult patients.

Minimally invasive congenital diaphragmatic hernia repair: a 7-year review of one institution's experience.

BACKGROUND: Minimally invasive surgery (MIS) has been described for the repair of congenital diaphragmatic hernias (CDH) in neonates, infants, and children. This report evaluates patient selection, operative technique, and clinical outcomes for MIS repair of CDHs from a single center's experience. METHODS: All cases of CDH at a tertiary care pediatric hospital with an initial attempt at MIS repair from January 2001 to December 2007 were reviewed. RESULTS: A total of 22 children underwent an initial attempt at MIS repair of their CDH (5 Morgagni and 17 Bochdalek hernias). The children ranged in age from 1 day to 6 years (mean, 13.9 +/- 23 months) and weighed 2.2 to 21 kg (mean, 7.4 +/- 5.50 kg) at the time of the operation. All five Morgagni hernias were managed successfully with laparoscopic primary repair. Six of the Bochdalek hernias were found in infants and children (age range, 6-71 months). All these were managed successfully with primary repair by an MIS approach (2 by laparoscopy and 4 by thoracoscopy). The remaining 11 Bochdalek hernias were found in neonates (age range, 1 day to 8 weeks). Four of the Bochdalek hernias were right-sided. Nine of the Bochdalek hernias in neonates were repaired thoracoscopically. One neonate required conversion to laparotomy, and another underwent conversion to thoracotomy. Four of the neonates with Bochdalek hernias required a prosthetic patch. Two of the neonates also had significant associated congenital cardiac defects. Overall, there were two recurrences involving one 3-day-old who underwent a primary thoracoscopic repair and another 3-day-old who underwent a thoracoscopic patch repair. The follow-up period ranged from 5 months to 5 years. CONCLUSIONS: Morgagni hernias can be managed successfully by laparoscopy, whereas thoracoscopy is preferred for neonatal Bochdalek hernias. Either approach can be successful for infants and children with Bochdalek hernias. Additionally, patients with congenital cardiac defects and those requiring prosthetic patches can undergo a MIS CDH repair with a successful outcome.

Thoracoscopic aortopexy for vascular compression of the trachea: approach from the right.

Tracheomalacia resulting from vascular compression of the trachea may require aortopexy for symptomatic relief. Several operative approaches have been described for infants and children. The authors describe the technique of aortopexy by means of a right-sided thoracoscopic method as the initial approach to relieve tracheal compression in 2 children. Intraoperative bronchoscopy is mandatory to assess the adequacy of aortopexy prior to the completion of suture placement. This procedure was very successful in relieving tracheal compression in a 17-month-old boy with an aberrant innominate artery take-off and in a 2-year and 7-month-old boy with a history of esophageal atresia/tracheoesophageal fistula repair and severe tracheomalacia. A right-sided thoracoscopic approach to aortopexy in infants with severe tracheomalacia may be successfully performed as the initial operative intervention in infants with vascular compression of the trachea.

Intra-amniotic Sildenafil Treatment Modulates Vascular Smooth Muscle Cell Phenotype in the Nitrofen Model of Congenital Diaphragmatic Hernia.

The etiology of pulmonary vascular abnormalities in CDH is incompletely understood. Studies have demonstrated improvement in pulmonary vasculature with prenatal therapy in animal models. We hypothesize that prenatal sildenafil may attenuate defective pulmonary vascular development via modulation of vSMC phenotype from undifferentiated, proliferative phenotype to differentiated, contractile phenotype. We utilized the nitrofen model of CDH to examine the effect of IA sildenafil on pulmonary vSMC phenotype during lung development. Timed-pregnant CD-1 mice were gavage fed 25 mg nitrofen or olive oil (control) at E8.5 of gestation. Single IA injections of Sildenafil (Revatio; 10 µL of 4 mg/4 ml solution) or dextrose control were performed at E12.5. Mice were sacrificed on various gestational days for embryonic lung harvest. Markers of vSMC development of undifferentiated and differentiated phenotypes were analyzed by immunostaining and western blot. Across all time points in gestation, nitrofen-treated embryonic lungs demonstrated increased vSMC expression of NOTCH3, Hes-5, PDGFR-ß, desmin and α-SMA and decreased expression of calponin and SMMHC, compared to oil controls. IA dextrose treatment had no effect on expression levels. However, IA Sildenafil treatment resulted in down-regulation of NOTCH3, Hes-5, PDGFR-ß, desmin and α-SMA and upregulation of calponin and SMMHC, comparable to oil controls. In the nitrofen model, vSMC express markers consistent with more undifferentiated proliferative phenotype, resulting in hypermuscularization of intrapulmonary arterioles in CDH. A single dose of IA Sildenafil treatment early in gestation, results in sustained normalization of vSMC phenotype. Pharmacologic modulation of the vSMC phenotype at key gestational points may have therapeutic potential.

Laparoscopic versus open Nissen fundoplication in infants after neonatal laparotomy.

BACKGROUND: Nissen fundoplication is an effective treatment of gastroesophageal reflux in infants. Laparoscopic procedures after previous laparotomy are technically more challenging. The role of laparoscopic Nissen fundoplication after neonatal laparotomy for diseases unrelated to reflux is poorly described. METHODS: This was a retrospective review of open vs laparoscopic Nissen fundoplication in infants after neonatal laparotomy. Of 32 infants who underwent neonatal laparotomy, 26 required a surgical antireflux operation within the first year of life. Twelve infants underwent laparoscopic Nissen fundoplication versus 14 infants who underwent open Nissen fundoplication. Parameters like age, weight, operative time, number of previous operations, length of stay following fundoplication, time to feedings, and complications were compared between the 2 groups. RESULTS: No statistically significant differences existed between most of the parameters compared following laparoscopic vs open Nissen fundoplication. No conversions to open procedures were necessary in infants undergoing laparoscopic fundoplication, and these infants resumed enteral feeds earlier than those who underwent the open procedure. CONCLUSION: Laparoscopic compared with open Nissen fundoplication performed in infants after a neonatal laparotomy were comparable procedures across most data points studied. However, a laparoscopic fundoplication did allow for earlier return to enteral feeds compared with the open approach. Laparoscopic Nissen fundoplication is technically feasible, safe, and effective in the treatment of gastroesophageal reflux in infants with a previous neonatal laparotomy.

Aminoguanidine renders inducible nitric oxide synthase knockout mice more susceptible to Salmonella typhimurium infection.

Aminoguanidine (AG), a nitric oxide synthase (NOS) inhibitor, has been widely used to study the role of inducible NOS (iNOS) in host defense against infections caused by various pathogens including Salmonella typhimurium. iNOS has been reported to play an important role in host defense against S. typhimurium infection both in vitro and in vivo. In this report we show those AG treatment lead to weight loss in both wild-type and iNOS knockout mice, and rendered them more susceptible to Salmonella infection. These results suggest that AG may have side effects other than the inhibition of iNOS, and that data obtained from studies using AG should be interpreted with caution.

Triple fistula: management of a double tracheoesophageal fistula with a third H-type proximal fistula.

Esophageal atresia (EA) with or without tracheoesophageal fistula (TEF) is a relatively common congenital condition in which there have been several described anatomical variants. The most common type, EA with distal TEF, comprises more than 75% of cases in many reports. Less commonly, a smaller proximal pouch fistula (H-type) will be associated with this most common variant in 1.4% of these cases. Only 2% of all cases of EA/TEF will have 2 large fistulas between the trachea and esophagus in which the end of the upper esophageal pouch connects terminally to the midtrachea and the distal esophagus arises from the trachea near the carina. Here we describe the management of an infant with this type of EA/TEF who was also found to have an H-type TEF of the proximal trachea. The combination of this type of EA/TEF with an associated H-type TEF or "triple fistula" has been previously described in the literature in only 1 other patient.

Lipopolysaccharide induces cyclooxygenase-2 in intestinal epithelium via a noncanonical p38 MAPK pathway.

Necrotizing enterocolitis (NEC), a severe intestinal inflammation in neonates, occurs following bacterial colonization of the gut. LPS-induced production of inflammatory factors in immature enterocytes may be a factor in NEC. Previously, we described LPS-induced p38 MAPK-dependent expression of cyclooxygenase-2 (COX-2) in rat IEC-6 cells. In this study, we examine COX-2 expression in newborn rat intestinal epithelium and further characterize the mechanisms of COX-2 regulation in enterocytes. Induction of NEC by formula feeding/hypoxia increased phospho-p38 and COX-2 levels in the intestinal mucosa. Celecoxib, a selective COX-2 inhibitor, exacerbated the disease, suggesting a protective role for COX-2. COX-2 was induced in the intestinal epithelium by LPS in vivo and ex vivo. The latter response was attenuated by the p38 inhibitor SB202190, but not by inhibitors of ERK, JNK, or NF-kappaB. In IEC-6 enterocytes, COX-2 was induced by the expression of MAPK kinase 3 EE (MKK3EE), a constitutive activator of p38, but not of activators of ERK or JNK pathways. However, neither MKK3/6 nor MKK4, the known p38 upstream kinases, were activated by LPS. Dominant-negative MKK3 or MKK4 or SB202190 failed to prevent LPS-induced, p38-activating phosphorylation, ruling out important roles of these kinases or p38 autophosphorylation. LPS increased COX-2 and activating phosphorylation of p38 with similar dose-response. Blockade of LPS-induced expression of COX-2-luciferase reporter and destabilization of COX-2 message by SB202190 indicate that p38 regulates COX-2 at transcription and mRNA stability levels. Our data indicate that p38-mediated expression of COX-2 proceeds through a novel upstream pathway and support the role of the neonate's enterocytes as bacterial sensors.

The high morbidity associated with handlebar injuries in children.

BACKGROUND: Although injury prevention strategies for bicyclists have focused on legislation requiring helmet use to prevent head trauma, direct impact handlebar injuries account for a significant proportion of bicycle-related injuries. Little attention, however, has been paid to strategies that prevent direct impact handlebar injuries. We reviewed our experience with bicycle-related injuries and compared outcome for children who flipped over the handlebars to those for children who sustained direct impact from the handlebars. METHODS: We queried our prospective trauma database for all bicycle injuries from 1998 to 2003. All patients with the descriptor "handlebar" in the subtext were selected. Patients were divided into two groups: those who flipped over the handlebars (n = 160) and those who sustained direct impact from the handlebars (n = 61). We examined age, gender, helmet use, injury severity score (ISS), Glasgow Coma Score (GCS), length of stay (LOS) and the need for operation. The Student's t test was used to compare continuous variables when the data were normally distributed and the Mann-Whitney was used when the data were skewed. Chi-square analysis or Fisher's exact test was used to compare categorical data. RESULTS: There was no difference between the two groups with respect to age, gender, helmet use, ISS, and GCS. However, children who suffered from handlebar injuries were more likely to require operative intervention (19/61 versus 28/160, p = 0.04) and had a significantly longer LOS (3 days versus 1 day, p < 0.001). Children who sustained direct impact from the handlebars and required operative intervention were statistically more likely to suffer from abdominal or soft tissue injuries, while those who flipped over the handlebars were statistically more likely to suffer from facial or skeletal injuries. CONCLUSIONS: Children who suffer from direct impact of the handlebars are more likely to require operative intervention and have a longer LOS than those who flip over the handlebars. While helmet utilization by bicyclists may have reduced the number of serious head injuries, direct impact from the handlebars remains a major source of bicycle-related morbidity since nearly one third of these patients required surgery. Future injury prevention strategies for bicyclists should be aimed at reducing the incidence of direct impact handlebar-related injuries.

Risk factors for splenectomy in children with blunt splenic trauma.

BACKGROUND/PURPOSE: Nonoperative management and splenic preservation have become standards of care for management of pediatric blunt splenic trauma. However, review of the Pennsylvania Trauma Outcome Study (PTOS) registry found that 15% of children with blunt splenic injury still underwent splenectomy. The authors sought to determine the factors that predisposed to splenectomy in this population. METHODS: Between 1993 and 1997, 754 children, ages 0 to 16 years, who sustained blunt splenic trauma were entered in the PTOS database. These patients were stratified into groups according to the mode of management: nonoperative, splenorrhaphy, or splenectomy. Logistic regression was performed to determine factors associated with splenectomy. RESULTS: Overall, 15.1% of patients underwent splenectomy, 7.4% underwent splenorrhaphy, and 77.5% were treated nonoperatively. Spleen injury grade, nonspleen abdominal injuries, Glasgow Coma Scale 3 to 8, and age 15 to 16 years were significant determinants of splenectomy by multivariate analysis. Children treated at pediatric trauma centers (PTC) underwent significantly fewer splenectomies. CONCLUSIONS: Injury grade, but not hemodynamic instability, was a significant independent determinant of splenectomy in children with blunt splenic trauma. Children treated at PTC are less likely to undergo splenectomy. Ongoing analysis of the management of blunt pediatric splenic injury and reduction of unnecessary splenectomies are needed to optimize care for injured children.

A new method for estimating probability of survival in pediatric patients using revised TRISS methodology based on age-adjusted weights.

BACKGROUND: TRISS methodology estimates probability of survival (P(s)) based on coefficients derived largely from adult data. We developed a novel pediatric age-specific method to estimate P(s). METHODS: The Pennsylvania Trauma Outcome Study database was queried for pediatric patients injured between 1993 and 1996 (n = 9730). P(s) derived from the Pediatric Age-Adjusted TRISS (PAAT) methodology was generated using our Age-Specific Pediatric Trauma Score and Injury Severity Score with corresponding weights. A test data set of 7138 pediatric patients entered in the Pennsylvania Trauma Outcome Study from 1997 to 1999 was used to compute an expected number of survivors for PAAT, TRISS, and ASCOT (A Severity Characteristic of Trauma). Observed and expected survival were compared for blunt injured patients, for head injured patients, and by age category. RESULTS: PAAT showed no significant difference between observed and expected survival. TRISS and ASCOT significantly underestimated overall survival: across age groups, for blunt injuries, for head injuries, and for patients whose P(s) was less than 91%. CONCLUSION: PAAT offers a more reliable methodology than TRISS and ASCOT for comparing pediatric trauma outcomes.

Development of a novel method to predict disability after head trauma in children.

BACKGROUND/PURPOSE: Although analysis of functional independence measures (FIM) at discharge are useful for assessing extent of disability in head-injured children, there is no reliable method to predict the severity of disability at the time of admission. The authors developed a novel method to predict severe disability after head trauma on admission. METHODS: Head-injured patients, 2 to 16 years old, with FIM recorded at discharge (n = 3,491) were identified in our state trauma database for the period from 1993 through 1996. Patients categorized as completely dependent by one or more of the FIM (Feeding, Locomotion, Expression, Transfer Mobility, Social Interaction) were classified as disabled. Probability of disability (P(D)) was estimated based on regression weights for Glasgow Coma Scale (GCS), Injury Severity Score (ISS), age, and number of anatomic regions injured. Observed to expected disability rates were compared using a test data set of 2,553 patients entered in the database between 1997 through 1999. RESULTS: There was no statistically significant difference between observed and expected disability across all P(D) intervals, which suggests that the P(D) accurately predicted disability. CONCLUSIONS: P(D) offers a novel and reliable method for early prediction of likelihood of disability in children who sustain head trauma. Routine use of the P(D) may lead to earlier intervention to improve long-term results in head-injured children.

Role of nitric oxide and peroxynitrite in gut barrier failure.

Bacterial translocation (BT) may be a normal physiologic process that is important for mucosal antigen sampling in the gut. However, physiologic insults such as endotoxemia, hemorrhagic shock, or necrotizing enterocolitis (NEC) may lead to pathologic BT and thus contribute to the pathogenesis of nosocomial infection. The mechanism may involve accelerated enterocyte apoptosis at the intestinal villus apex resulting, at least transiently, in a "bare area" at the villus tip where bacteria can attach and traverse the epithelium. Evidence suggests that sustained upregulation of the inducible isoform of nitric oxide synthase (NOS-2) co-localizes with enterocyte apoptosis and immunoreactivity to 3-nitrotyrosine, the footprint of peroxynitrite (ONOO-), a potent oxidant formed by the reaction of nitric oxide (NO) with superoxide. We propose that the bare area at the villus apex is caused by apoptosis of enterocytes that have migrated from the base of the crypts to the villus apex and are shed into the intestinal lumen. These bare areas, and thus the degree of BT, may be the result of an imbalance between enterocyte proliferation and apoptosis. We postulate that normal enterocyte apoptosis is mediated by the caspase cascade, whereas enterocyte proliferation and differentiation in the crypt may be regulated by tyrosine kinase-dependent signaling pathways. Both of these cellular pathways may be influenced by overproduction of NO and its metabolite ONOO-. Therefore, sustained NO production and ONOO- formation occurring in inflammatory states may differentially accelerate apoptosis in the villus apex and/or inhibit proliferation at the base of the crypts resulting in expanded extrusion zones at the villus tip and accelerated BT.

Mechanism of intestinal-derived fungal sepsis by gliotoxin, a fungal metabolite.

BACKGROUND/PURPOSE: Gut barrier dysfunction resulting from fungal overgrowth may be caused by the interaction of gliotoxin (GT), a fungal metabolite, with enterocytes. The goal of this study was to determine the mechanisms by which gliotoxin (GT), a fungal metabolite, causes enterocyte apoptosis. METHODS: The authors measured enterocyte apoptosis, caspase-3 activity, pro-caspase-3, and poly (ADP-ribose) polymerase (PARP) cleavage in GT-exposed IEC-6 cells, a rat intestinal cell line. RESULTS: GT induced apoptosis in IEC-6 cells. The pan-caspase inhibitor ZVAD suppressed this GT-mediated apoptosis. GT induced a 15-fold increase in caspase-3 activity over media control. The authors detected PARP cleavage by after GT exposure. DTT pretreatment decreased apoptosis compared with GT alone. CONCLUSIONS: This study supports the concept that fungal overgrowth may lead to gut barrier dysfunction by the local release of gliotoxin and the induction enterocyte apoptosis.

Peroxynitrite inhibits enterocyte proliferation and modulates Src kinase activity in vitro.

Overproduction of nitric oxide (NO) or its toxic metabolite, peroxynitrite (ONOO-), after endotoxemia promotes gut barrier failure, in part, by inducing enterocyte apoptosis. We hypothesized that ONOO- may also inhibit enterocyte proliferation by disrupting the Src tyrosine kinase signaling pathway, thereby blunting repair of the damaged mucosa. We examined the effect of ONOO- on enterocyte proliferation and Src kinase activity. Sprague-Dawley rats were challenged with LPS or saline, whereas intestinal epithelial cell line cells were treated with ONOO- or decomposed ONOO- in vitro. Enterocyte proliferation in vivo and in vitro was measured by 5-bromo-2'-deoxyuridine (BrdU) or [3H]thymidine incorporation. Src kinase activity in cell lysates was determined at various times. LPS challenge in vivo and ONOO- treatment in vitro inhibited enterocyte proliferation. ONOO- treatment blunted the activity of Src and its downstream target, focal adhesion kinase, in a time-dependent manner. ONOO- blocked mitogen (FBS, EGF)-induced enterocyte proliferation and Src phosphorylation while increasing Src nitration. Thus ONOO- may promote gut barrier failure not only by inducing enterocyte apoptosis but also by disrupting signaling pathways involved in enterocyte proliferation.

NF-kappaB inhibition enhances peroxynitrite-induced enterocyte apoptosis.

BACKGROUND: Sustained overproduction of nitric oxide and peroxynitrite (ONOO(-)) in conditions such as necrotizing enterocolitis and inflammatory bowel disease may promote gut barrier failure by inducing enterocyte apoptosis. NF-kappaB is upregulated in the gut during inflammation and, in addition to its proinflammatory effects, may upregulate protective or antiapoptotic factors such as inhibitor of apoptosis proteins (IAPs). We have previously demonstrated that NF-kappaB inhibition increases cytokine-induced enterocyte apoptosis; however, the effect of NF-kappaB on ONOO(-)-induced enterocyte apoptosis is unknown. MATERIALS AND METHODS: Rat intestinal epithelial cells (IEC-6) were transfected with the adenoviral vector AdIkappaB or AdlacZ. AdIkappaB contains a mutated form of IkappaB which functions as a superrepressor of NF-kappaB. Cells were then treated with 50 microM ONOO(-) or decomposed ONOO(-). Apoptosis was then determined by flow cytometry with annexin V-FITC and propidium iodide staining. Caspase activation and IAP, Bcl-2, Bad, and Bax expression were examined using Western blot analysis, and NF-kappaB activation was determined via electrophoretic mobility shift assay (EMSA). RESULTS: Inhibition of NF-kappaB with AdIkappaB significantly enhanced ONOO(-)-induced apoptosis in IEC-6 cells. ONOO(-) treatment did not activate NF-kappaB in IEC-6 cells as determined by EMSA. There was no difference in IAP, Bcl-2, Bad, and Bax expression between nontransfected, AdlacZ-transfected, and AdIkappaB-transfected cells. Baseline procaspase 3 activation was increased in AdIkappaB-transfected cells. CONCLUSIONS: NF-kappaB inhibition enhances ONOO(-)-induced enterocyte apoptosis, suggesting that NF-kappaB upregulates a protective factor. This protective factor does not appear to be an IAP or Bcl-2 family member and may be expressed constitutively, since ONOO(-) did not activate NF-kappaB over baseline levels of activation.