Jennifer L. Liedel

Intestinal alkaline phosphatase administration in newborns is protective of gut barrier function in a neonatal necrotizing enterocolitis rat model

BACKGROUND Previously, we have shown that supplementation of intestinal alkaline phosphatase (IAP) decreased severity of necrotizing enterocolitis (NEC)-associated intestinal injury. We hypothesized that IAP administration is protective of intestinal epithelial barrier function in a dose-dependent manner. METHODS Control rat pups were vaginally delivered and breast-fed. Premature rats were divided into 4 groups: formula fed with lipopolysaccharide and hypoxia (NEC) or additional daily bovine IAP 40, 4, or 0.4 U/kg (NEC + IAP 40 U, IAP 4 U, or IAP 0.4 U). RESULTS Necrotizing enterocolitis is associated with decreased IAP protein expression and activity. Supplemental IAP increases IAP activity in intestinal homogenates and decreased NEC injury score in a dose-dependent manner. Intestinal injury as measured by fluorescein isothiocyanate-dextran flux from ileal loops showed increased permeability vs control, but supplemental IAP reversed this. Tight junction proteins claudin-1, claudin-3, occludin, and zonula occludin 1 were elevated in the NEC and IAP-treated groups with differences in expression patterns. No differences in messenger RNA levels were observed on postinjury day 3. Intestinal alkaline phosphatase administration decreases intestinal NEC injury in a dose-dependent manner. CONCLUSION Early enteral supplemental IAP may reduce NEC-related injury and may be useful for preserving the intestinal epithelial barrier function.

Transfusion‐related immunomodulation: review of the literature and implications for pediatric critical illness

Transfusion‐related immunomodulation (TRIM) in the intensive care unit (ICU) is difficult to define and likely represents a complicated set of physiologic responses to transfusion, including both proinflammatory and immunosuppressive effects. Similarly, the immunologic response to critical illness in both adults and children is highly complex and is characterized by both acute inflammation and acquired immune suppression. How transfusion may contribute to or perpetuate these phenotypes in the ICU is poorly understood, despite the fact that transfusion is common in critically ill patients. Both hyperinflammation and severe immune suppression are associated with poor outcomes from critical illness, underscoring the need to understand potential immunologic consequences of blood product transfusion. In this review we outline the dynamic immunologic response to critical illness, provide clinical evidence in support of immunomodulatory effects of blood product transfusion, review preclinical and translational studies to date of TRIM, and provide insight into future research directions.

Changes in cerebral oxygen saturation correlate with S100B in infants undergoing cardiac surgery with cardiopulmonary bypass.

Objectives: The relationship of cerebral saturation measured by near-infrared spectroscopy with serum biomarker of brain injury S100B was investigated in infants undergoing cardiac surgery with cardiopulmonary bypass. Design: Prospective cohort study. Setting: Single-center children’s hospital. Patients: Forty infants between 1 and 12 months old weighing greater than or equal to 4 kg with congenital heart disease undergoing cardiac surgery with cardiopulmonary bypass were enrolled. Interventions: None. Measurements and Main Results: Serum S100B was measured at eight time points over 72 hours using enzyme-linked immunosorbent assay. Physiologic data including arterial, cerebral, and somatic regional oxygen saturations measured by near-infrared spectroscopy were synchronously recorded at 1-minute intervals from anesthesia induction through 72 postoperative hours. The arterial-cerebral oxygen saturation difference was calculated as the difference between arterial saturation and cerebral regional saturation. Thirty-eight patients, 5.4 ± 2.5 months old, were included in the analysis; two were excluded due to the use of postoperative extracorporeal membrane oxygenation. Seventeen patients (44.7%) had preoperative cyanosis. S100B increased during cardiopulmonary bypass in all patients, from a median preoperative baseline of mean ± SE: 0.055 ± 0.038 to a peak of 0.610 ± 0.038 ng/mL, p less than 0.0001. Patients without preoperative cyanosis had a higher S100B peak at the end of cardiopulmonary bypass. Although the absolute cerebral regional saturation on cardiopulmonary bypass was not associated with S100B elevation, patients who had arterial-cerebral oxygen saturation difference greater than 50 at any time during cardiopulmonary bypass had a higher S100B peak (mean ± SE: 1.053 ± 0.080 vs 0.504 ± 0.039 ng/mL; p < 0.0001). Conclusions: A wide cerebral arteriovenous difference measured by near-infrared spectroscopy during cardiopulmonary bypass is associated with increased serum S100B in the perioperative period and may be a modifiable risk factor for neurological injury.

Intestinal alkaline phosphatase administration in newborns decreases systemic inflammatory cytokine expression in a neonatal necrotizing enterocolitis rat model

BACKGROUND Supplementation of intestinal alkaline phosphatase (IAP), an endogenous protein expressed in the intestines, decreases the severity of necrotizing enterocolitis (NEC)-associated intestinal injury and permeability. We hypothesized that IAP administration is protective in a dose-dependent manner of the inflammatory response in a neonatal rat model. MATERIALS AND METHODS Pre- and full-term newborn Sprague-Dawley rat pups were sacrificed on day of life 3. Control pups were vaginally delivered and dam fed. Preterm pups were delivered via cesarean section and exposed to intermittent hypoxia and formula feeds containing lipopolysaccharide (NEC) with and without IAP. Three different standardized doses were administered to a group of pups treated with 40, 4, and 0.4U/kg of bovine IAP (NEC+IAP40, IAP4, or IAP0.4U). Reverse transcription-real-time polymerase chain reaction (RT-PCR) for inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α on liver and lung tissues and serum cytokine analysis for interleukin (IL)-1β, IL-6, IL-10, and TNF-α were performed. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests, expressed as mean±standard error of the mean and P≤0.05 considered significant. RESULTS Levels of cytokines IL-1β, IL-6, and TNF-α increased significantly in NEC versus control, returning to control levels with increasing doses of supplemental enteral IAP. Hepatic and pulmonary TNF-α and iNOS messenger ribonucleic acid expressions increased in NEC, and the remaining elevated despite IAP supplementation. CONCLUSIONS Proinflammatory cytokine expression is increased systemically with intestinal NEC injury. Administration of IAP significantly reduces systemic proinflammatory cytokine expression in a dose-dependent manner. Early supplemental enteral IAP may reduce NEC-related injury and be useful for reducing effects caused by a proinflammatory cascade.

Intestinal Alkaline Phosphatase Is Protective to the Preterm Rat Pup Intestine

BACKGROUND Necrotizing enterocolitis (NEC) is the most common surgical emergency in neonates, with a mortality rate between 10 and 50%. The onset of necrotizing enterocolitis is highly variable and associated with numerous risk factors. Prior research has shown that enteral supplementation with intestinal alkaline phosphatase (IAP) decreases the severity of NEC. The aim of this study is to investigate whether IAP is protective to the preterm intestine in the presence of formula feeding and in the absence of NEC. METHODS Preterm rat pups were fed formula with or without supplementation with IAP, and intestine was obtained on day of life 3 for analysis of IAP activity, mRNA expression of TNFα, IL-6 and iNOS and permeability and cytokine expression after LPS exposure. RESULTS There was no difference in the absolute and intestine specific alkaline phosphatase activity in both groups. Rat pups fed IAP had decreased mRNA expression of the inflammatory cytokines TNFα, IL-6 and iNOS. Pups supplemented with IAP had decreased permeability and inflammatory cytokine expression after exposure to LPS ex vivo when compared to formula fed controls. CONCLUSIONS Our results support that IAP is beneficial to preterm intestine and decreases intestinal injury and inflammation caused by LPS.

Enteral intestinal alkaline phosphatase administration in newborns decreases iNOS expression in a neonatal necrotizing enterocolitis rat model.

PURPOSE To determine if intestinal alkaline phosphatase (IAP) decreases intestinal injury resulting from experimentally induced necrotizing enterocolitis (NEC). We hypothesized that IAP administration prevents the initial development of NEC related intestinal inflammation. METHODS Pre- and full-term newborn Sprague-Dawley rat pups were sacrificed on day 1 of life. Pre-term pups were exposed to intermittent hypoxia and formula containing LPS to induce NEC. Select NEC pups were given 40, 4 or 0.4 units/kg of bovine IAP (NEC+IAP40u, IAP4u or IAP0.4u) enterally, once daily. Ileal sections were evaluated by real-time PCR (qRT-PCR) for IAP, iNOS, IL-1β, IL-6, and TNF-α mRNA and immunofluorescence for 3-nitrotyrosine (3-NT). RESULTS Experimentally induced NEC decreased IAP mRNA expression by 66% (p ≤ 0.001). IAP supplementation increased IAP mRNA expression to control. Supplemental enteral IAP decreased nitrosative stress as measured by iNOS mRNA expression and 3-NT staining in the NEC stressed pups (p ≤ 0.01), as well as decreased intestinal TNF-α mRNA expression. In addition, IAP decreased LSP translocation into the serum in the treated pups. CONCLUSIONS We conclude that enterally administered IAP prevents NEC-related intestinal injury and inflammation. Enteral IAP may prove a useful strategy in the prevention of NEC in preterm neonates.

Early Enteral Stressors in Newborns Increase Inflammatory Cytokine Expression in a Neonatal Necrotizing Enterocolitis Rat Model

INTRODUCTION Inflammation in the premature intestine is a key factor that leads to the development of necrotizing enterocolitis (NEC). Activation of nuclear factor kappa B (NF-κB) and subsequent inflammation increases the severity of NEC. The aim of this study was to investigate the early temporal expression of inflammatory markers and activation of NF-κB in a neonatal rat model of NEC. METHODS Pre- and full-term newborn Sprague-Dawley rat pups were sacrificed at birth, 1.5, 4, 8, and 24 hours after receiving their first feed. Control pups were vaginally delivered and mother fed; NEC was induced by a combination of gavage feeding formula, hypoxia, and enteral lipopolysaccharide (LPS); and formula fed pups were fed every 4 hours with infant formula. Ileal tissue was collected for immunohistochemistry, real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay. Serum was collected for cytokine content. Fold change of expression of inducible nitric oxide synthase (iNOS), interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), IL-10, NF-κB p65, and IκBα used RT-PCR. Data were analyzed by paired two-tailed t test, expressed as mean ± standard error of the mean, and p ≤ 0.05 considered significant. RESULTS No histologic injury was evident in ileal sections. At 1.5 h, iNOS expression increased twofold over control in NEC pups (2.1 vs. 1.0, p ≤ 0.05) and remained elevated at 24 h (0.7 vs. 9.4, p ≤ 0.05). IL-1β and IL-6 reached a peak at 24 h in NEC tissue compared with control. IL-10 expression rose in NEC pups after 4 h of insult and remained elevated in formula and NEC stressed pups. Coincident with an increase in p65 translocation into the nucleus and a reduction of IκBα detected in the cytoplasm, increased transcription of IκBα occurs. CONCLUSION These findings suggest that NF-κB activation initiates inflammation early in the course of NEC resulting in increased proinflammatory protein expression, underscoring the importance of the inflammatory response in this NEC model, which precedes evidence of histological injury.

Intestinal NADPH Oxidase 2 Activity Increases in a Neonatal Rat Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a complication of prematurity. The etiology is unknown, but is related to enteral feeding, ischemia, infection, and inflammation. Reactive oxygen species production, most notably superoxide, increases in NEC. NADPH oxidase (NOX) generates superoxide, but its activity in NEC remains unknown. We hypothesize that NOX-derived superoxide production increases in NEC. Newborn Sprague-Dawley rats were divided into control, formula-fed, formula/LPS, formula/hypoxia, and NEC (formula, hypoxia, and LPS). Intestinal homogenates were analyzed for NADPH-dependent superoxide production. Changes in superoxide levels on days 0-4 were measured. Inhibitors for nitric oxide synthase (L-NAME) and NOX2 (GP91-ds-tat) were utilized. RT-PCR for eNOS, NOX1, GP91phox expression was performed. Immunofluorescence studies estimated the co-localization of p47phox and GP91phox in control and NEC animals on D1, D2, and D4. NEC pups generated more superoxide than controls on D4, while all other groups were unchanged. NADPH-dependent superoxide production was greater in NEC on days 0, 3, and 4. GP91-ds-tat decreased superoxide production in both groups, with greater inhibition in NEC. L-NAME did not alter superoxide production. Temporally, superoxide production varied minimally in controls. In NEC, superoxide generation was decreased on day 1, but increased on days 3-4. GP91phox expression was higher in NEC on days 2 and 4. NOX1 and eNOS expression were unchanged from controls. GP91phox and p47phox had minimal co-localization in all control samples and NEC samples on D1 and D2, but had increased co-localization on D4. In conclusion, this study proves that experimentally-induced NEC increases small intestinal NOX activity. All components of NEC model are necessary for increased NOX activity. NOX2 is the major source, especially as the disease progresses.

A toll-like receptor 9 (rs352140) variant is associated with placental inflammation in newborn infants

Abstract Objective: Chorioamnionitis contributes to premature birth and associated postnatal morbidity. The genetic basis of altered immune responses underlying placental inflammation (PI) remains understudied. The aim of this study was to evaluate the relationship among TLR signaling pathway polymorphisms and different patterns of PI. Methods: Prospective cohort study in infants involving cord blood collection and placental examination for PI. One hundred and fifty-nine infants enrolled in study out of which 28 were term (eight with PI) and 131 preterm (47 with PI). DNA from blood was genotyped for SNPs in TLR2, 4, 5, 9, NFKBI, NFKBIA, TIRAP, and IRAK1 genes using multiplexed single base extension assay. Results: While there were no differences in BW, GA, gender, race, and SPL among infants with or without PI, there was a higher incidence of PPROM, maternal smoking, drug use, and clinical chorioamnionitis among infants with PI. Out of nine TLR variants, only CT and/or TT genotypes of the TLR9 variant (rs352140) were significantly associated (p = 0.004) with any PI and maternal pattern of inflammation (p = 0.012) both by univariate analysis and logistic regression. Conclusions: The presence of a variant T allele in a common SNP (rs352140) in the TLR9 gene whose product recognizes bacterial DNA is associated with increased PI.

A Case Series of Patients with Cor Triatriatum Dexter: Unique Cause of Neonatal Cyanosis

Cor triatriatum dexter is a rare congenital heart defect that can lead to cyanosis in a newborn with an otherwise normal exam. The initial evaluation of these patients typically focuses on searching for a pulmonary etiology for arterial desaturation, which often leads to a negative work up. When cardiac evaluation is performed, it may be challenging because the heart lesion can be difficult to visualize on an echocardiogram. The diagnosis requires a high index of suspicion and thorough echocardiographic imaging. Once diagnosed, surgical repair can alleviate the shunt created by the defect. This case series describes all patients (3) with cor triatriatum dexter seen at Children’s Hospital of Wisconsin from 2000 to 2013.