Yigit S. Guner

The human milk oligosaccharide disialyllacto-N-tetraose prevents necrotising enterocolitis in neonatal rats

Background Necrotising enterocolitis (NEC) is one of the most common and fatal intestinal disorders in preterm infants. Breast-fed infants are at lower risk for NEC than formula-fed infants, but the protective components in human milk have not been identified. In contrast to formula, human milk contains high amounts of complex glycans. Objective To test the hypothesis that human milk oligosaccharides (HMO) contribute to the protection from NEC. Methods Since human intervention studies are unfeasible due to limited availability of HMO, a neonatal rat NEC model was used. Pups were orally gavaged with formula without and with HMO and exposed to hypoxia episodes. Ileum sections were scored blindly for signs of NEC. Two-dimensional chromatography was used to determine the most effective HMO, and sequential exoglycosidase digestions and linkage analysis was used to determine its structure. Results Compared to formula alone, pooled HMO significantly improved 96-hour survival from 73.1% to 95.0% and reduced pathology scores from 1.98±1.11 to 0.44±0.30 (p<0.001). Within the pooled HMO, a specific isomer of disialyllacto-N-tetraose (DSLNT) was identified to be protective. Galacto-oligosaccharides, currently added to formula to mimic some of the effects of HMO, had no effect. Conclusion HMO reduce NEC in neonatal rats and the effects are highly structure specific. If these results translate to NEC in humans, DSLNT could be used to prevent or treat NEC in formula-fed infants, and its concentration in the mothers milk could serve as a biomarker to identify breast-fed infants at risk of developing this disorder.

Current concepts regarding the pathogenesis of necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a devastating disease that predominantly affects premature neonates. The mortality associated with NEC has not changed appreciably over the past several decades. The underlying etiology of NEC remains elusive, although bacterial colonization of the gut, formula feeding, and perinatal stress have been implicated as putative risk factors. The disease is characterized by massive epithelial destruction, which results in gut barrier failure. The exact molecular and cellular mechanisms involved in this complex disease are poorly understood. Recent studies have provided significant insight into our understanding of the pathogenesis of NEC. Endogenous mediators such as prostanoids, cyclooxygenases, and nitric oxide may play a role in the development of gut barrier failure. Understanding the structural architecture of the gut barrier and the cellular mechanisms that are responsible for gut epithelial damage could lead to the development of novel diagnostic, prophylactic and therapeutic strategies in NEC.

The Role of Nitric Oxide in Intestinal Epithelial Injury and Restitution in Neonatal Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most common life-threatening gastrointestinal disease encountered in the premature infant. Although the inciting events leading to NEC remain elusive, various risk factors, including prematurity, hypoxemia, formula feeding, and intestinal ischemia, have been implicated in the pathogenesis of NEC. Data from our laboratory and others suggest that NEC evolves from disruption of the intestinal epithelial barrier, as a result of a combination of local and systemic insults. We postulate that nitric oxide (NO), an important second messenger and inflammatory mediator, plays a key role in intestinal barrier failure seen in NEC. Nitric oxide and its reactive nitrogen derivative, peroxynitrite, may affect gut barrier permeability by inducing enterocyte apoptosis (programmed cell death) and necrosis, or by altering tight junctions or gap junctions that normally play a key role in maintaining epithelial monolayer integrity. Intrinsic mechanisms that serve to restore monolayer integrity following epithelial injury include enterocyte proliferation, epithelial restitution via enterocyte migration, and re-establishment of cell contacts. This review focuses on the biology of NO and the mechanisms by which it promotes epithelial injury while concurrently disrupting the intrinsic repair mechanisms.

Ubiquitin-Editing Enzyme A20 Promotes Tolerance to Lipopolysaccharide in Enterocytes

Although enterocytes are capable of innate immune responses, the intestinal epithelium is normally tolerant to commensal bacteria. To elucidate the mechanisms of tolerance, we examined the effect of preexposure to LPS on activation of p38, c-Jun, and NF-κB in enterocytes by several inflammatory and stress stimuli. Shortly after the initial LPS challenge, enterocytes become tolerant to restimulation with LPS or CpG DNA, but not with IL-17 or UV. The state of tolerance, which lasts 20–26 h, temporally coincides with LPS-induced expression of the anti-inflammatory ubiquitin-editing enzyme A20. Small interfering RNA silencing of A20 prevents tolerance, whereas ectopic expression of A20 blocks responses to LPS and CpG DNA, but not to IL-17 or UV. A20 levels in the epithelium of the small intestine are low at birth and following gut decontamination with antibiotics, but high under conditions of bacterial colonization. In the small intestine of adult rodents, A20 prominently localizes to the luminal interface of villus enterocytes. Lower parts of the crypts display relatively low levels of A20, but relatively high levels of phospho-p38. Gut decontamination with antibiotics reduces the levels of both A20 and phospho-p38. Along with the fact that A20-deficient mice develop severe intestinal inflammation, our results indicate that induction of A20 plays a key role in the tolerance of the intestinal epithelium to TLR ligands and bacteria.

Disparities in the delivery of pediatric trauma care.

BACKGROUND Trauma is the leading cause of morbidity and mortality in children. During the last few decades, trauma systems have evolved to improve the care of the injured with an ultimate goal of saving lives. As a result, pediatric trauma centers (PTC) have been established to optimize outcomes for injured children. We sought to determine whether injured children treated at PTC or adult trauma centers (ATC) with added qualifications to treat injured children receive better trauma care than those treated at other hospitals or trauma centers. METHODS We reviewed more than 60 published studies on pediatric trauma outcomes. The studies included registry analysis: single and multihospital experience; abdominal, head and neck, and thoracic trauma; as well as functional outcomes. RESULTS The data show that most injured children are not treated at PTC due to the geographically limited distribution of such specialized care, lack of pediatric surgeons, and other specialists. These limitations create persistent disparities in outcomes for injured children depending on where they are treated. Some of the larger database analyses suggest lower mortality rate, better outcome for nonoperative treatment of blunt abdominal injuries, and improved overall functional outcome for those children treated at PTC. However, others fail to demonstrate differences for children treated at ATC or ATC with added qualifications. CONCLUSION Although this analysis does not provide a definitive answer to the question as to which type of trauma center provides better care for injured pediatric patients, it identifies current gaps and disparities in the care of injured children that can be remedied through education and training.

Outcome analysis of neonates with congenital diaphragmatic hernia treated with venovenous vs venoarterial extracorporeal membrane oxygenation.

PURPOSE Venoarterial extracorporeal membrane oxygenation (ECMO) (VA) is used more commonly in neonates with congenital diaphragmatic hernia (CDH) than venovenous ECMO (VV). We hypothesized that VV may result in comparable outcomes in infants with CDH requiring ECMO. METHODS We retrospectively analyzed the Extracorporeal Life Support Organization (ELSO) database (1991-2006). Multivariate logistic regression analyses were used to compare VV- and VA-associated mortality. RESULTS Four thousand one hundred fifteen neonates required ECMO, with an overall mortality rate of 49.6%. Venoarterial ECMO was used in 82% and VV in 18% of neonates. Pre-ECMO inotrope use and complications were equivalent between VA and VV. The mortality rate for VA and VV was 50% and 46%, respectively. After adjusting for birth weight, gestational age, prenatal diagnosis, ethnicity, Apgar scores, pH less than 7.20, Paco(2) greater than 50, requiring high-frequency ventilation, and year of ECMO, there was no difference in mortality between VV vs VA. Renal complications and on-ECMO inotrope use were more common with VV, whereas neurologic complications were more common with VA. The conversion rate from VV to VA was 18%; conversion was associated with a 56% mortality rate. CONCLUSION The short-term outcomes of VV and VA are comparable. Patients with CDH who fail VV may be predisposed to a worse outcome. Nevertheless, VV offers equal benefit to patients with CDH requiring ECMO while preserving the native carotid.

Lactobacillus bulgaricus Prevents Intestinal Epithelial Cell Injury Caused by Enterobacter sakazakii-Induced Nitric Oxide both In Vitro and in the Newborn Rat Model of Necrotizing Enterocolitis

ABSTRACT Enterobacter sakazakii is an emerging pathogen that has been associated with outbreaks of necrotizing enterocolitis (NEC) as well as infant sepsis and meningitis. Our previous studies demonstrated that E. sakazakii induces NEC in a newborn rat model by inducing enterocyte apoptosis. However, the mechanisms responsible for enterocyte apoptosis are not known. Here we demonstrate that E. sakazakii induces significant production of nitric oxide (NO) in rat intestinal epithelial cells (IEC-6) upon infection. The elevated production of NO, which is due to increased expression of inducible NO synthase, is responsible for apoptosis of IEC-6 cells. Notably, pretreatment of IEC-6 cells with Lactobacillus bulgaricus (ATCC 12278) attenuated the upregulation of NO production and thereby protected the cells from E. sakazakii-induced apoptosis. Furthermore, pretreatment with L. bulgaricus promoted the integrity of enterocytes both in vitro and in the infant rat model of NEC, even after challenge with E. sakazakii. Infection of IEC-6 cells with E. sakazakii upregulated several genes related to apoptosis, cytokine production, and various signaling pathways, as demonstrated by rat gene array analysis, and this upregulation was subdued by pretreatment with L. bulgaricus. In agreement with these data, L. bulgaricus pretreatment protected newborn rats infected with E. sakazakii from developing NEC, resulting in improved survival.

Role of interleukin-10 in the pathogenesis of necrotizing enterocolitis

BACKGROUND Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in premature neonates. The pathogenesis of NEC is characterized by an intestinal epithelial injury caused by perinatal insults, leading to the activation of the mucosal innate immune system and exacerbation of the epithelial barrier damage. Cytokines play an important role in mucosal immunity. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that has been shown to play a role in epithelial integrity and modulation of the mucosal immune system. We hypothesized that IL-10 may protect against the development of experimental NEC by blunting the inflammatory response in the intestine. METHODS Wild-type and IL-10 -/- mice underwent a NEC-inducing regimen of formula feeding in combination with hypoxia and hypothermia (FF+HH). Integrity of the gut barrier was assessed through measurement of epithelial apoptosis, tight junction disruption, and inducible nitric oxide synthase. A total of 5 μg of exogenous IL-10 was administered intraperitoneally to IL-10-/-mouse pups before the initiation of FF+HH to test dependence of gene knockout phenotype on IL-10. RESULTS IL-10 -/- FF+HH showed more severe morphologic and histologic changes compared with controls as evidenced by increased epithelial apoptosis, decreased junctional adhesion molecule-1 localization, and increased intestinal inducible nitric oxide synthase expression. Administration of exogenous IL-10 alleviated the mucosal injury. CONCLUSIONS We conclude that IL-10 plays a protective role in the pathogenesis of NEC by attenuating the degree of intestinal inflammation.

Necrotizing enterocolitis--bench to bedside: novel and emerging strategies.

Necrotizing enterocolitis (NEC) is a devastating illness that predominantly affects premature neonates. The mortality associated with this disease has changed very little during the last two decades. Neonates with NEC fall into two categories: those who respond to medical management alone and those who require surgical treatment. The disease distribution may be focal, multifocal, or panintestinal. Surgical treatment should therefore be based on disease presentation. Recent studies have added significant insight into our understanding of the pathogenesis of NEC. Several groups have shown that upregulation of nitric oxide plays an integral role in the development of epithelial injury in NEC. As a result, some treatment strategies have been aimed at abrogating the toxic effects of nitric oxide. In addition, several investigators have reported the cytoprotective effect of epidermal growth factor, which is found in high levels in breast milk, on the intestinal epithelium. Thus, fortification of infant formula with specific growth factors could soon become a preferred strategy to accelerate intestinal maturation in the premature neonate to prevent the development of NEC. One of the most devastating complications of NEC is the development of short bowel syndrome (SBS). The current treatment of SBS involves intestinal lengthening procedures or bowel transplantation. A novel emerging method for treating SBS involves the use of tissue-engineered intestine. In laboratory animals, tissue-engineered small intestine has been shown to be successful in treating intestinal failure. This article examines recent data regarding surgical treatment options for NEC as well as emerging treatment modalities.

Laparoscopic Choledochal Cyst Excision: Lessons Learned in Our Experience

BACKGROUND Choledochal cyst (CDC) is a rare biliary disorder. Surgical treatment consists of CDC excision and biliary-enteric reconstruction. Recently, some institutions have reported successful CDC excision by using minimally invasive techniques. In this study, we report our experience with the laparoscopic management of CDC, with a focus on key operative maneuvers that enhance the likelihood of successful excision. METHODS Following institutional review board approval, we performed a retrospective review of patients who underwent the laparoscopic excision of CDC and Roux-en-Y hepaticojejunostomy. Between October 2003 and November 2007, we performed laparoscopic CDC excision in 9 patients (8 female and 1 male). Median age was 4 years (range, 8 months to 16 years). There were 7 type I and 2 type IV cysts, according to Todanis classification. Average cyst size was 4.4 cm (range, 1.3-8.5). The procedures were performed by utilizing four or five trochars. RESULTS Six of 9 children presented with preoperative pancreatitis, 1 with abdominal pain, 1 with jaundice, and 1 was found incidentally. Three patients required the conversion to laparotomy due to dense adhesions, secondary to pancreatitis. Six patients underwent successful laparoscopic procedures, 5 had complete cyst excisions, and 1 underwent a proximal excision with distal mucosectomy. Of the 3 patients who required conversion, 2 underwent complete excisions; the other underwent a proximal excision, distal mucosectomy. There were no intraoperative complications. One patient had a postoperative bile leak that required an open hepaticojejunostomy revision. Eight patients had an uneventful recovery. Oral feedings were resumed within an average of 3.4 days (range, 2-9). Average time to discharge was 6.1 days (range, 5-12). Average follow-up time was 18 months (range, 4-48). No further laboratory abnormalities were detected in any of the patients. CONCLUSIONS Laparoscopic resection of CDC and Roux-en-Y hepaticojejunostomy in children is an excellent treatment option. Preoperative pancreatitis may cause increased technical difficulty, necessitating a conversion. Proximal excision with distal mucosectomy