Recruitment of dendritic cells is responsible for intestinal epithelial damage in the pathogenesis of necrotizing enterocolitis by Cronobacter sakazakii.

Cronobacter sakazakii is a Gram-negative pathogen associated with the cases of necrotizing enterocolitis (NEC) that result from formula contamination. In a mouse model of NEC, we demonstrate that C. sakazakii infection results in epithelial damage by recruiting greater numbers of dendritic cells (DCs) than macrophages and neutrophils in the gut and suppresses DC maturation, which requires outer membrane protein A (OmpA) expression in C. sakazakii. Pretreatment of intestinal epithelial cell monolayers with supernatant from OmpA(+) C. sakazakii/DC culture markedly enhanced membrane permeability and enterocyte apoptosis, whereas OmpA(-) C. sakazakii/DC culture supernatant had no effect. Analysis of OmpA(+) C. sakazakii/DC coculture supernatant revealed significantly greater TGF-ß production compared with the levels produced by OmpA(-) C. sakazakii infection. TGF-ß levels were elevated in the intestinal tissue of mice infected with OmpA(+) C. sakazakii. Cocultures of CaCo-2 cells and DCs in a "double-layer" model followed by infection with OmpA(+) C. sakazakii significantly enhanced monolayer leakage by increasing TGF-ß production. Elevated levels of inducible NO synthase (iNOS) were also observed in the double-layer infection model, and abrogation of iNOS expression prevented the C. sakazakii-induced CaCo-2 cell monolayer permeability despite the presence of DCs or OmpA(+) C. sakazakii/DC supernatant. Blocking TGF-ß activity using a neutralizing Ab suppressed iNOS production and prevented apoptosis and monolayer leakage. Depletion of DCs in newborn mice protected against C. sakazakii-induced NEC, whereas adoptive transfer of DCs rendered the animals susceptible to infection. Therefore, C. sakazakii interaction with DCs in intestine enhances the destruction of the intestinal epithelium and the onset of NEC due to increased TGF-ß production.

Role of neutrophils and macrophages in the pathogenesis of necrotizing enterocolitis caused by Cronobacter sakazakii.

BACKGROUND: Cronobacter sakazakii (CS) is a highly virulent gram-negative opportunistic pathogen that has been implicated in clinical outbreaks of necrotizing enterocolitis (NEC). The role of mucosal immune cells in CS infection is not well understood. In this study, we sought to elucidate the role of neutrophils (polymorphonuclear leukocytes; PMNs) and macrophages in the pathogenesis of NEC induced by CS using a novel newborn mouse model. MATERIALS AND METHODS: PMNs and macrophages were depleted in newborn mice using Gr-1 antibody and carrageenan, respectively, and then infected with 10(3) CFU of CS. The development of NEC in these mice was assessed by a pathologist based on the morphologic changes in the intestine. Cytokine production was determined in the serum and intestinal homogenates of infected mice by enzyme-linked immunosorbent assay (ELISA). Inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production was determined by flow cytometry and Greiss method, respectively. RESULTS: Depletion of PMNs and macrophages in newborn mice led to increased recruitment of dendritic cells (DCs) in the intestine compared with wild-type mice upon infection with CS. PMN- and macrophage-depleted mice showed increased bacterial load, production of pro-inflammatory cytokines, iNOS expression, and NO production in the intestines in comparison to wild-type mice fed with CS. In addition, depletion of PMNs and macrophages prior to infection in mice resulted in severe inflammation, villus destruction, and enhanced enterocyte apoptosis in the intestines compared with CS-infected wild-type mice. CONCLUSIONS: Our data suggest that depletion of PMNs and macrophages from the lamina propria (LP) exacerbates experimental NEC, indicating that both of these immunocytes play an important role in the clearance of CS during the initial stages of infection. The increased mucosal cytokine response and NO production in the absence of these immunocytes may be responsible for the observed increase in mucosal injury. Understanding how CS manipulates these cells, employing novel mouse model of NEC reported in this study, will provide significant insights for the development of novel therapeutic and preventive strategies to combat NEC.

Human isolates of Cronobacter sakazakii bind efficiently to intestinal epithelial cells in vitro to induce monolayer permeability and apoptosis.

BACKGROUND: Cronobacter sakazakii (CS) is an emerging opportunistic pathogen that causes life-threatening infections in infants. This pathogen has been implicated in the outbreaks of necrotizing enterocolitis (NEC) with associated rates of high mortality and morbidity. In this study, we compared the abilities of CS strains isolated from human and environmental sources to bind to intestinal epithelial cells and trigger apoptosis. MATERIALS AND METHODS: CS strains were isolated from human and environmental sources and their abilities to bind to intestinal epithelial cells were determined. Monolayer permeability was determined by transepithelial electrical resistance (TEER) and horseradish peroxidase (HRP) leakage. Apoptosis was examined by ApoTag and AnnexinV-7AAD staining. PKC activation was evaluated by non-radioactive PepTag assay. RESULTS: Human isolates of CS bind to rat and human enterocytes more efficiently than environmental strains. Additionally, these strains induced increased enterocyte monolayer permeability as indicated by a decrease in TEER and an increase in transcellular leakage of exogenously added HRP. Human isolates also caused tight junction disruption and significant apoptosis of enterocytes compared with environmental strains due to increased production of inducible nitric oxide. We also observed that human CS isolates caused 2-fold increase in the activation of phosphokinase C (PKC) than environmental strains. Blocking the PKC activity in enterocytes by an inhibitor, Gö 6983, suppressed CS-mediated tight junction disruption, monolayer permeability, and apoptosis of the cells. CONCLUSION: These results suggest that human isolates of CS more efficiently bind to and cause damage to intestinal epithelial cells compared with environmental strains.

Pediatric single incision laparoscopic cholecystectomy: lessons learned in the first 25 cases.

PURPOSE: We are reporting our experience so far with single incision laparoscopic cholecystectomy in children. METHODS: After the approval of the institutional review board, we performed a retrospective chart review of our single port cases from 01/2008 to 10/2009. We used operating room (OR) times, length of stay, as well as IV narcotic use as our outcome measures. Pertinent clinical data were extracted. The single port procedure was performed using a single infra-umbilical incision whereby three 5-mm ports were placed. RESULTS: We identified 25 patients in the single port group (20 females and 5 males). 23 patients in the study group underwent cholecystectomy without intra-operative cholangiogram and one patient had an intra-operative cholangiogram performed. This additional procedure did not add to the overall OR time significantly as compared to simple cholecystectomies. Average OR time was 97.5 min as compared to 71.4 min in the traditional 4-port group. Blood loss was reported as minimal for all cases in both the groups (5-25 ml). There were no intra-operative complications in either group. Mean length of stay was 1.47 days in the study group. All patients in the study group had minimal (1-3 doses) need for intravenous narcotics during their inpatient stay except for one patient, who required more. All patients in the study group had excellent cosmetic results on postoperative follow-up. CONCLUSION: Single incision laparoscopic cholecystectomy is safe and feasible to perform in pediatrics, even in the setting of acute disease.

Single-incision laparoscopic cholecystectomy in a pediatric population: a preliminary report.

Laparoscopic cholecystectomy has become a standard procedure for treatment of gallbladder diseases. The operation is performed through a four-trocar technique. Single-incision laparoscopy (SIL) has recently gained popularity. The purpose of our study was to review our cases of SIL cholecystectomy and to evaluate the safety and feasibility of this technique. After the approval of the Institutional Review Boards, we performed a retrospective chart review of our SIL cholecystectomy cases performed between January 2008 and August 2009. Pertinent clinical data were extracted. The outcomes were reported as operating room time, intraoperative and postoperative complications, length of stay, and intravenous narcotic use. We identified 24 patients (19 females and five males) with a mean age of 15 years. Most patients (67%) had a diagnosis of symptomatic cholelithiasis. Two patients had gallstone pancreatitis, three had acute cholecystitis, and one had a hydropic gallbladder. Two patients had an intraoperative cholangiogram performed. Average operating room time was 97.5 +/- 34.5 minutes (range, 65 to 145 minutes). There were no intraoperative complications. All patients had minimal (one to three doses) need for intravenous narcotics. All patients have had excellent cosmetic results on postoperative follow-up. SIL cholecystectomy in children is safe and feasible, even in the setting of acute cholecystitis and the need for cholangiogram.

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.

A risk-stratified comparison of fascial versus flap closure techniques on the early outcomes of infants with gastroschisis.

BACKGROUND: While fascial closure is traditionally used in gastroschisis (GS), flap closure (skin or umbilical cord) has gained popularity. We evaluated early outcomes and complications of the two techniques. METHODS: A national, population-based gastroschisis data registry was analyzed from 2005 to 2011. We compared fascial to flap closures and stratified patients into low or high-risk groups using the Gastroschisis Prognostic Score (GPS), a validated marker of post-natal bowel injury. Demographic and outcome data, including length of stay, complications, and markers of resource utilization were analyzed using Fisher's exact and Student's t-tests for categorical and continuous variables, respectively (p<0.05 significant). RESULTS: The analyzed dataset included 436 fascial closures (344 [78.8%] low-risk, 92 high-risk) and 129 flap closures (112 [86.7%] low-risk, 17 high-risk; p=0.06). Demographics and birth weight did not differ between groups. In patients with low GPS, flap closure demonstrated significant decreases in resource utilization and failure of closure, without differences in complication rates. Analysis of high-risk patients revealed no statistically significant differences in outcome. CONCLUSION: Flap closure was not associated with an increase in patient morbidity and seemed suitable as a definitive closure method for gastroschisis patients irrespective of disease severity. Furthermore, flap closure reduced several markers of resource utilization in patients with low-risk disease.

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.

Single-incision laparoscopic cholecystectomy in children: a feasible alternative to the standard laparoscopic approach.

PURPOSE: Our aim was to evaluate the outcomes of the single-incision laparoscopic (SIL) cholecystectomy compared with the standard 4-incision laparoscopic (SL) cholecystectomy. METHODS: A retrospective chart review of consecutive patients undergoing cholecystectomy using the SIL approach from January 2008 to September 2010 was performed. These patients were compared with a cohort who underwent an SL cholecystectomy from January 2007 to June 2009. Demographics, operative times, length of stay, blood loss, and intravenous narcotic use was obtained from the charts. A nonpaired Student's t test was used to determine statistical significance. RESULTS: We identified 40 patients in the SIL group and 68 in the SL group. Main diagnosis was cholelithiasis followed by gallstone pancreatitis and cholecystitis. The mean operative time for SIL cholecystectomies was 79.2 minutes vs 63 minutes in the SL group (P < .006). The average length of stay was 1.9 days in the SIL group vs 2.3 days in the SL group (P < .24). The mean intravenous narcotic use was 1 dose in the SIL group vs 2.9 doses in the SL group (P < .007). There were no intraoperative complications. At 1-month postoperative follow-up, all patients had satisfactory recovery. CONCLUSION: Single-incision laparoscopic cholecystectomy is a safe and feasible alternative to the standard laparoscopic approach in children, even in the setting of acute disease.

Inversion herniotomy: a laparoscopic technique for female inguinal hernia repair.

PURPOSE: Laparoscopic inversion herniotomy (LIH) is a method of inguinal hernia repair for female patients. In this article, we report our technique and outcomes for this procedure. We propose that LIH is a valid alternative to conventional open herniorraphy. METHODS: Following institutional review board approval, we reviewed the charts of patients who underwent LIH or open inguinal hernia repair (OIH) from 2004 to 2007. LIH was performed with three instrument sites and no groin incision. The diagnosis, operative time, and complications were reviewed. Follow-up ranged from 5 months to 4 years. RESULTS: We performed 79 LIH repairs (15 bilateral, 23 left, and 26 right) in 63 female children. We also performed 76 contemporaneous OIH repairs in girls. The age range was 1 month to 18 years (median, 3.8 years). Operative times for both the unilateral and bilateral LIH repairs were significantly shorter than those for OIH (P = 0.001). There were 2 recurrences after laparoscopic inguinal hernia repair, both within 5 months following repair early in our experience, compared to no hernia recurrences with OIH. The first recurrence was repaired conventionally via an open technique. The second case was repaired laparoscopically. CONCLUSIONS: LIH is an effective method for the repair of pediatric indirect inguinal hernia in female patients. The procedure allows the evaluation of the asymptomatic contralateral side without the necessity of a second long inguinal incision.

Role of the host defense system and intestinal microbial flora in the pathogenesis of necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating disease that affects primarily the intestine of premature infants. Despite recent advances in neonatology, NEC remains a major cause of morbidity and mortality in neonates. Neonatal mucosal defenses and adherence of bacterial pathogens may play an important role in the pathogenesis of NEC. METHODS: Review and synthesis of pertinent literature. RESULTS: Putative factors that have been implicated in the pathogenesis of NEC include abnormal patterns of gut colonization by bacteria, immaturity of the host immune system and mucosal defense mechanisms, intestinal ischemia, formula feeding, and loss of intestinal epithelial barrier integrity. CONCLUSION: Host defenses and intestinal microbial ecology are believed to play important roles in the pathogenesis of NEC. Commensal bacteria and probiotic therapy may be of therapeutic utility in the maintenance of the gut epithelial barrier.