Henri R. Ford

Enterocyte TLR4 Mediates Phagocytosis and Translocation of Bacteria Across the Intestinal Barrier

Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis, although the mechanisms by which bacterial translocation occurs remain largely unknown. We hypothesized that bacterial translocation across the intact barrier occurs after internalization of the bacteria by enterocytes in a process resembling phagocytosis and that TLR4 is required for this process. We now show that FcγRIIa-transfected enterocytes can internalize IgG-opsonized erythrocytes into actin-rich cups, confirming that these enterocytes have the molecular machinery required for phagocytosis. We further show that enterocytes can internalize Escherichia coli into phagosomes, that the bacteria remain viable intracellularly, and that TLR4 is required for this process to occur. TLR4 signaling was found to be necessary and sufficient for phagocytosis by epithelial cells, because IEC-6 intestinal epithelial cells were able to internalize LPS-coated, but not uncoated, latex particles and because MD2/TLR4-transfected human endothelial kidney (HEK)-293 cells acquired the capacity to internalize E. coli, whereas nontransfected HEK-293 cells and HEK-293 cells transfected with dominant-negative TLR4 bearing a P712H mutation did not. LPS did not induce membrane ruffling or macropinocytosis in enterocytes, excluding their role in bacterial internalization. Strikingly, the internalization of Gram-negative bacteria into enterocytes in vivo and the translocation of bacteria across the intestinal epithelium to mesenteric lymph nodes were significantly greater in wild-type mice as compared with mice having mutations in TLR4. These data suggest a novel mechanism by which bacterial translocation occurs and suggest a critical role for TLR4 in the phagocytosis of bacteria by enterocytes in this process.

Impact of pediatric trauma centers on mortality in a statewide system.

Background: Regional pediatric trauma centers (PTC) were established to optimize the care of injured children. However, because of the relative shortage of PTC, many injured children continue to be treated at adult trauma centers (ATC). As a result, a growing controversy has evolved regarding the impact of PTC and ATC on outcome for injured children. Methods: A retrospective analysis of 13,351 injured children entered in the Pennsylvania Trauma Outcome Study between 1993 and 1997 was conducted. Patients were stratified according to mechanism of injury, injury severity, specific organ injury, and type of trauma center: PTC; Level I ATC (ATC I); Level II ATC (ATC II); or ATC with added qualifications to treat children (ATC AQ). Mortality was the major outcome variable measured. Results: Most injured children were treated at a PTC or ATC AQ. The majority of children below 10 years of age were admitted to PTC. Patients treated at PTC and ATC had similar injury severity as determined by median Injury Severity Score, mean Revised Trauma Score, and Glasgow Coma Scale. Overall survival was significantly better at PTC and ATC AQ compared with ATC I and ATC II. Survival for head, spleen, and liver injuries was significantly better at PTC compared with ATC AQ, ATC I, or ATC II. Children who sustained moderate or severe head injuries were more likely to undergo neurosurgical intervention and have a better outcome when treated at a PTC. Despite similar mean Abbreviated Injury Scores for spleen and liver, significantly more children underwent surgical exploration (especially splenectomy) for spleen and liver injuries at ATC compared with PTC. Conclusion: Children treated at PTC or ATC AQ have significantly better outcome compared with those treated at ATC. Severely injured children (Injury Severity Score > 15) with head, spleen, or liver injuries had the best overall outcome when treated at PTC. This difference in outcome may be attributable to the approach to operative and nonoperative management of head, liver, and spleen injuries at PTC.

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.

Understanding the Susceptibility of the Premature Infant to Necrotizing Enterocolitis (NEC)

Necrotizing enterocolitis (NEC) is the most common life-threatening gastrointestinal emergency encountered in the neonatal intensive care unit. Despite advances in neonatal care, NEC remains a leading cause of morbidity and mortality among premature infants. Epidemiologic studies have identified multiple factors that increase an infants risk for the development of NEC, although premature birth, bacterial colonization, and enteral feeding are thought to play central roles in disease pathogenesis. Appreciating factors that underlie the susceptibility of prematurely born infants to NEC is important for the development of new strategies aimed at the prevention and treatment of disease. In this review, we discuss defense mechanisms in the intestine and discuss how these systems may be insufficient in the prematurely born infant and thereby further contribute to initiation of NEC. In addition, based on a review of the literature, we suggest that, although numerous bacterial and viral pathogens have been associated with NEC, no individual organism is known to be responsible for disease. Finally, we comment on the possible role for probiotics in promoting maturation of intestinal defense mechanisms thereby attenuating or preventing the sequence of events that lead to NEC.

Improved functional outcome for severely injured children treated at pediatric trauma centers.

BACKGROUND Controversy exists regarding the impact of pediatric trauma centers (PTC) on survival for injured children. However, functional outcome for children treated at PTC compared with adult trauma centers (ATC) has not been evaluated. METHODS An analysis of children entered in the Pennsylvania Trauma Outcome Study between 1993 and 1997 was conducted. Patients were stratified according to type of trauma center: PTC; Level I ATC; Level II ATC; or ATC with added qualifications (AQ). Functional outcome at discharge was analyzed. RESULTS For severely injured children, there was an overall trend toward improved functional outcome at PTC compared with ATC AQ and ATC I, but no difference compared with ATC II. PTC showed improved functional outcome at discharge for head injury compared with ATC AQ and ATC I. CONCLUSION There is an overall trend toward improved functional outcome at discharge for children treated at PTC compared with those treated at ATC AQ and ATC I. Improved outcome for head injury may be a key factor contributing to improved outcome at PTC.

Recovery after open versus laparoscopic pyloromyotomy for pyloric stenosis: a double-blind multicentre randomised controlled trial

BACKGROUND A laparoscopic approach to pyloromyotomy for infantile pyloric stenosis has gained popularity but its effectiveness remains unproven. We aimed to compare outcomes after open or laparoscopic pyloromyotomy for the treatment of pyloric stenosis. METHODS We did a multicentre international, double-blind, randomised, controlled trial between June, 2004, and May, 2007, across six tertiary paediatric surgical centres. 180 infants were randomly assigned to open (n=93) or laparoscopic pyloromyotomy (n=87) with minimisation for age, weight, gestational age at birth, bicarbonate at initial presentation, feeding type, preoperative duration of symptoms, and trial centre. Infants with a diagnosis of pyloric stenosis were eligible. Primary outcomes were time to achieve full enteral feed and duration of postoperative recovery. We aimed to recruit 200 infants (100 per group); however, the data monitoring and ethics committee recommended halting the trial before full recruitment because of significant treatment benefit in one group at interim analysis. Participants, parents, and nursing staff were unaware of treatment. Data were analysed on an intention-to-treat basis with regression analysis. The trial is registered with ClinicalTrials.gov, number NCT00144924. FINDINGS Time to achieve full enteral feeding in the open pyloromyotomy group was (median [IQR]) 23.9 h (16.0-41.0) versus 18.5 h (12.3-24.0; p=0.002) in the laparoscopic group; postoperative length of stay was 43.8 h (25.3-55.6) versus 33.6 h (22.9-48.1; p=0.027). Postoperative vomiting, and intra-operative and postoperative complications were similar between the two groups. INTERPRETATION Both open and laparoscopic pyloromyotomy are safe procedures for the management of pyloric stenosis. However, laparoscopy has advantages over open pyloromyotomy, and we recommend its use in centres with suitable laparoscopic experience.

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.

Immunoglobulin A supplementation abrogates bacterial translocation and preserves the architecture of the intestinal epithelium.

BACKGROUND Breast milk has been shown to prevent gut-origin infections in neonates through undefined mechanisms. Putative protective factors in breast milk include immunoglobulin (Ig)A, IgG, and lactoferrin. We examined their role in bacterial translocation in neonatal rabbits. METHODS IgA, IgG, and lactoferrin were isolated from rabbit breast milk through gel filtration and ion-exchange chromatography. Neonates were randomized to receive breast milk, formula alone, or formula supplemented with IgA, IgG, or lactoferrin. Quantitative cultures were performed on day 7 for bacterial translocation. Hematoxylin-eosin-stained sections of distal ileum were examined by light microscopy. Transmucosal bacterial passage was determined in vitro, and the ileal mucosal membranes were examined by confocal microscopy. RESULTS IgA supplementation abrogated bacterial translocation. IgG and lactoferrin had no significant effect. Neonates that received IgA or breast milk gained more weight than those in the other groups. IgA reduced transmucosal bacterial passage in vitro. In contrast to the normal-appearing distal ileum of neonates fed breast milk, intestinal epithelium from neonates that received formula or formula with IgG or IgA demonstrated prominent vacuoles by light microscopy. Those fed formula alone or formula with lactoferrin had slightly shortened villi. CONCLUSIONS IgA supplementation prevents bacterial translocation by enhancing gut mucosal barrier function.

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.

Abdominal and pelvic trauma in children.

Trauma is the leading cause of death and disability in children. More than 90% of pediatric trauma admissions are the result of a blunt mechanism. Although injury to the abdomen and pelvis account for only 10% of injuries sustained by victims of pediatric trauma, they can be potentially life threatening. Optimal evaluation of the injured child may require the use of multiple diagnostic modalities. The spleen is the most frequently injured intra-abdominal organ, followed by the liver, intestine, and pancreas. Fortunately, the majority of injuries to the spleen and liver can be treated nonoperatively. Conversely, injuries involving the intestine and pancreas often require operative intervention.