Catherine J. Hunter

Enterobacter sakazakii Enhances Epithelial Cell Injury by Inducing Apoptosis in a Rat Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is an inflammatory intestinal disorder that affects 2%-5% of all premature infants. Enterobacter sakazakii, a common contaminant of milk-based powdered infant formula, has been implicated as a causative agent of sepsis, meningitis, and NEC in newborn infants, with high mortality rates. However, the role played by E. sakazakii in the pathogenesis of NEC is, to date, not known. Here, we demonstrate for the first time that E. sakazakii can induce clinical and histological NEC in newborn rats. E. sakazakii was found to bind to enterocytes in rat pups at the tips of villi and to intestinal epithelial cells (IEC-6) in culture, with no significant invasion. Exposure to E. sakazakii induced apoptosis and increased the production of interleukin-6 in IEC-6 cells and in the animal model. These data suggest that E. sakazakii could be a potential pathogen that induces NEC and triggers intestinal disease by modulating enterocyte intracellular signaling pathways.

Cronobacter : an emerging opportunistic pathogen associated with neonatal meningitis, sepsis and necrotizing enterocolitis

Members of the genus Cronobacter are an emerging group of opportunist Gram-negative pathogens. This genus was previously thought to be a single species, called Enterobacter sakazakii. Cronobacter spp. typically affect low-birth-weight neonates, causing life-threatening meningitis, sepsis and necrotizing enterocolitis. Outbreaks of disease have been associated with contaminated infant formula, although the primary environmental source remains elusive. Advanced understanding of these bacteria and better classification has been obtained by improved detection techniques and genomic analysis. Research has begun to characterize the virulence factors and pathogenic potential of Cronobacter. Investigations into sterilization techniques and protocols for minimizing the risk of contamination have been reviewed at national and international forums. In this review, we explore the clinical impact of Cronobacter neonatal and pediatric infections, discuss virulence and pathogenesis, and review prevention and treatment strategies.

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.

Percutaneous liver biopsy: pathologic diagnosis and complications in children.

Objective: The aim of this study was to determine patient factors that predict diagnostic failure or increased risk of bleeding complications following percutaneous liver biopsy in children. Methods: A retrospective review of all children undergoing percutaneous liver biopsy at a single institution between July 2008 and July 2011 was performed. Demographics, comorbid conditions, preprocedural diagnoses/indications, procedural details, laboratory data, pathologic diagnosis, and complications were recorded. Continuous data were analyzed by Wilcoxon test and categorical data by Fisher exact test to determine statistical significance. Results: Two hundred thirteen children (104 girls) with a median age of 7 years (range 1 week–22 years) underwent 328 percutaneous liver biopsies. Nine (4.2%) experienced a decrease in hemoglobin >2 g/dL, 7 required transfusion (3.3%), and 1 patient died (0.5%). Younger age (1.8 vs 84 months, Pu200a=u200a0.05) and lower preprocedural hematocrit (29.3 vs 34.3, Pu200a=u200a0.05) predicted bleeding complications, whereas the number of biopsies, comorbid conditions, and coagulopathy did not. Sixty-three (19.2%) biopsies were insufficient for definitive histologic evaluation on initial biopsy in 57 patients. Twenty-one of 57 patients (37%) underwent repeat percutaneous biopsy and 3 of 57 (8%) underwent surgical biopsy. Biopsy provided definitive diagnosis in 86% of cases when repeat biopsy was performed. Shorter specimen length (1.4 vs 1.7 cm, Pu200a<u200a0.01) and biopsies performed for unexplained elevation of liver function tests (34.9% vs 16.7%, Pu200a<u200a0.01) were predictive of nondiagnosis. Conclusions: Percutaneous liver biopsy is safe with a low rate of bleeding-related complications. Ensuring adequate sample length and careful patient selection may further increase the diagnostic yield.

Inflammatory signaling in NEC: Role of NF-κB, cytokines and other inflammatory mediators.

The pathogenesis of necrotizing enterocolitis (NEC) is complex and the exact etiology remains unknown. Clinically, the presentation and progression of NEC is variable and often difficult to predict. The majority of affected infants (>90%) are premature, and the following factors may play a role in NEC pathogenesis: 1) The immaturity of the intestinal barrier, the mucus layer, decreased Immunoglobulin A (IgA) and defensins; 2) an abnormal intestinal capillary blood flow due do the incomplete development of the intestinal microvasculature or due to the immature regulation of its vascular tone, causing insufficient substrate and O2 delivery to the intestinal epithelial cells; 3) abnormal bacterial colonization of the enteric tract triggering a mucosal pro-inflammatory response; and 4) immaturity of the immune system preventing normal control and killing of microbes, allowing them to penetrate the epithelium. Concomitantly, this immature immune system mounts an excessive production of inflammatory mediators which cause the recruitment of inflammatory cells such as neutrophils and subsequent tissue injury and necrosis. n nIn order to investigate the pathogenesis of NEC, correlative studies have been conducted measuring different inflammatory mediators such as cytokines in the plasma or in the tissues resected from patients with NEC. However, these tissues are obtained at late stages of the disease when these are commonly necrotic, and therefore may not yield information about the early pathogenic events leading to NEC. As mechanistic studies obviously cannot be conducted in humans, animal models have been used. Studies on rats and mice have contributed to the discovery of several potentially important inflammatory mediators in the pathogenesis of NEC. In this chapter, the current evidence for the role of these inflammatory mediators is presented and a current unifying hypothesis regarding NEC pathogenesis is proposed.

The ErbB4 Ligand Neuregulin-4 Protects against Experimental Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) affects up to 10% of premature infants, has a mortality of 30%, and can leave surviving patients with significant morbidity. Neuregulin-4 (NRG4) is an ErbB4-specific ligand that promotes epithelial cell survival. Thus, this pathway could be protective in diseases such as NEC, in which epithelial cell death is a major pathologic feature. We sought to determine whether NRG4-ErbB4 signaling is protective in experimental NEC. NRG4 was used i) in the newborn rat formula feeding/hypoxia model; ii) in a recently developed model in which 14- to 16-day-old mice are injected with dithizone to induce Paneth cell loss, followed by Klebsiella pneumoniae infection to induce intestinal injury; and iii) in bacterially infected IEC-6 cells inxa0vitro. NRG4 reduced NEC incidence and severity in the formula feed/hypoxia rat model. It also reduced Paneth cell ablation-induced NEC and prevented dithizone-induced Paneth cell loss in mice. Inxa0vitro, cultured ErbB4(-/-) ileal epithelial enteroids had reduced Paneth cell markers and were highly sensitive to inflammatory cytokines. Furthermore, NRG4 blocked, through a Src-dependent pathway, Cronobacter muytjensii-induced IEC-6 cell apoptosis. The potential clinical relevance of these findings was demonstrated by the observation that NRG4 and its receptor ErbB4 are present in human breast milk and developing human intestine, respectively. Thus, NRG4-ErbB4 signaling may be a novel pathway for therapeutic intervention or prevention in NEC.

Central venous access in children: indications, devices, and risks

Purpose of review Central venous catheters (CVCs) have a prominent role in the diagnostic and therapy of neonates and children. Herein, we describe the multiple indications for CVC use and the different devices available for central venous access. Given the prevalent use of CVCs, healthcare systems are focused on reducing complications from their use, particularly central line-associated bloodstream infections (CLABSIs). The most up-to-date information available sheds light on best practices and future areas of investigation. Recent findings Large systematic reviews of randomized trials suggest that ultrasound guidance for placement of CVCs in children is safer than using blind technique, at least for internal jugular vein access. Appropriate catheter tip placement is associated with decreased complications. Furthermore, the prophylactic use of ethanol lock between cycles of parenteral nutrition administration has reduced the rates of CLABSI. A recent randomized trial in pediatric CVCs showed a benefit with antibiotic-coated CVCs. Summary Based on the available evidence, multiple techniques for CVC placement are still valid, including the landmark technique based on practitioner experience, but ultrasound guidance has been shown to decrease complications from line placement. Adherence to CVC care protocols is essential in reducing infectious complications.

Probiotic Lactobacillus Species Strengthen Intestinal Barrier Function and Tight Junction Integrity in Experimental Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a serious intestinal disease that occurs in newborn infants. It is associated with major morbidity and affects 5% of all infants admitted to neonatal intensive care units. Probiotics have variable efficacy in preventing necrotizing enterocolitis. Tight junctions (TJ) are protein complexes that maintain epithelial barrier integrity. We hypothesized that the probiotics Lactobacillus rhamnosus and Lactobacillus plantarum strengthen intestinal barrier function, promote TJ integrity, and protect against experimental NEC. Both an in vitro and an in vivo experimental model of NEC were studied. Cultured human intestinal Caco-2 cells were pretreated with L. rhamnosus and L. plantarum probiotics. TJ were then disrupted by EGTA calcium switch or LPS to mimic NEC in vitro. Trans-epithelial resistance (TER) and flux of fluorescein isothiocynate dextran was measured. TJ structure was evaluated by ZO-1 immunofluorescence. In vivo effects of ingested probiotics on intestinal injury and ZO-1 expression were assessed in a rat model of NEC infected with Cronobacter sakazakii (CS). Caco-2 cells treated with individual probiotics demonstrated higher TER and lower permeability compared to untreated cells (p<0.0001). ZO-1 immunofluorescence confirmed TJ stability in treated cells. Rat pups fed probiotics alone had more intestinal injury compared with controls (p=0.0106). Probiotics were protective against injury when given in combination with CS, with no difference in intestinal injury compared to controls (p=0.21). Increased permeability was observed in the probiotic and CS groups (p=0.03, p=0.05), but not in the probiotic plus CS group (p=0.79). Lactobacillus sp. strengthened intestinal barrier function and preserved TJ integrity in an in vitro experimental model of NEC. In vivo, probiotic bacteria were not beneficial when given alone, but were protective in the presence of CS in a rat model of NEC.

Observation for isolated traumatic skull fractures in the pediatric population: unnecessary and costly

BACKGROUNDnBlunt head trauma accounts for a majority of pediatric trauma admissions. There is a growing subset of these patients with isolated skull fractures, but little evidence guiding their management. We hypothesized that inpatient neurological observation for pediatric patients with isolated skull fractures and normal neurological examinations is unnecessary and costly.nnnMETHODSnWe performed a single center 10year retrospective review of all head traumas with isolated traumatic skull fractures and normal neurological examination. Exclusion criteria included: penetrating head trauma, depressed fractures, intracranial hemorrhage, skull base fracture, pneumocephalus, and poly-trauma. In each patient, we analyzed: age, fracture location, loss of consciousness, injury mechanism, Emergency Department (ED) disposition, need for repeat imaging, hospital costs, intracranial hemorrhage, and surgical intervention.nnnRESULTSnSeventy-one patients presented to our ED with acute isolated skull fractures, 56% were male and 44% were female. Their ages ranged from 1week to 12.4years old. The minority (22.5%) of patients were discharged from the ED following evaluation, whereas 77.5% were admitted for neurological observation. None of the patients required neurosurgical intervention. Age was not associated with repeat imaging or inpatient observation (p=0.7474, p=0.9670). No patients underwent repeat head imaging during their index admission. Repeat imaging was obtained in three previously admitted patients who returned to the ED. Cost analysis revealed a significant difference in total hospital costs between the groups, with an average increase in charges of

A Role for cAMP and Protein Kinase A in Experimental Necrotizing Enterocolitis

4,291.50 for admitted patients (p<0.0001).nnnCONCLUSIONnPediatric isolated skull fractures are low risk conditions with a low likelihood of complications. Further studies are necessary to change clinical practice, but our research indicates that these patients can be discharged safely from the ED without inpatient observation. This change in practice, additionally, would allow for huge health care dollar savings.