Search

Immune factors are important antecedents in the pathophysiology of necrotizing enterocolitis (NEC). However, studies on the peripheral blood lymphocyte subsets changes in NEC patients among different Bell stages and in patients requiring surgery are scarce. 34 infants with NEC and 33 age-matched controls were included. Peripheral blood was collected within 48 h after NEC diagnosis. Peripheral blood B and T lymphocytes subsets were detected by 12-color flow cytometry. Cell ratios were calculated, and their relationship to disease severity and their roles as indicators for surgery were assessed. NEC patients showed elevated percentages of unSwB cells (CD27+IgD+ unswitched memory/activated B cells)/B cells, SwB cells (CD27+IgD-switched memory B cells)/B cells, CD8+ T (CD3+CD8+ T cells)/T cells, Tem (CD45RA-CCR7-effector memory T cells)/CD4+ T cells, Tem/CD8+ T cells and decreased Bn (CD27-IgD+ naïve B cells)/B cells, CD4+T (CD3+CD4+ T cells)/T cells, CD45RA+ CCR7+ naïve T cells (CD45RA+CCR7+ naïve T cells)/CD8+T cells. Compared to NEC patients at BELL stage I + II, patients at BELL stage III showed increased percentages of SwB cells/B cells, antibody secreting cell (ASC, CD3-CD20-CD27high CD38high ASCs)/B cells and Tem/CD4+ T cells, and decreased percentages of CD45RA+CCR7+ naïve T cells/CD4+ T cells. The Receiver Operating Characteristic Curve analysis showed that the sensitivity of ASC/B cells ratio (0.52%) is 86.67% and the specificity of Tem/CD4+T ratio (5.22%) is 100%, indicating that NEC patients required surgery. The severity of NEC exhibits codirectional changes with the maturation of B and T lymphocytes, especially CD4+ T cells. The increased ASC/B and Tem/CD4+ T cells could serve as potential indicators for NEC patients requiring surgery. [ABSTRACT FROM AUTHOR]

Multiple studies have documented the safety of intestinal anastomosis after resection for necrotizing enterocolitis (NEC). We sought to evaluate a large population of infants with surgical NEC and assess outcomes after primary anastomosis versus enterostomy. The Pediatric Health Information System database was used to identify infants with Bell Stage 3 NEC who underwent an intestinal resection for acute disease between 2016 and 2021. Demographics and preoperative physiology were assessed, and nutritional, infectious, and surgical outcomes were analyzed. Two hundred twenty-two infants at 38 children's hospitals were included. Thirty-five (15.8%) were managed with a primary anastomosis. Among infants who underwent a resection within 10 d of their first operative intervention and survived for at least 3 d, a primary anastomosis was used in 26 (13.7%). These patients were older but had similar weight and physiological status at the time of resection as those managed with an enterostomy. The incidence of wound and infectious complications, duration of parenteral nutrition and length of stay were similar after anastomosis or enterostomy. In a large, geographically heterogenous population of infants with NEC, only 15.8% were managed with a primary anastomosis after intestinal resection. Survivors who underwent resection within 10 d were demographically and physiologically comparable to those who underwent enterostomy and had similar surgical outcomes. While there are clearly indications for enterostomy in some infants with NEC, these data confirm the conclusions of smaller, single-center studies that a primary anastomosis should be considered more frequently. [ABSTRACT FROM AUTHOR]

Certain congenital cardiac lesions are at increased risk for the development of necrotizing enterocolitis (NEC). These patients are often reliant on pulmonary and systemic vasomodulators to maintain adequate perfusion and oxygenation. This study sought to determine whether pulmonary or systemic vasodilator treatment is protective against the development of NEC in this population. We utilized International Classification of Diseases (ICD) codes to identify high risk congenital cardiac disease patients ≤6 mo of age, cared for at a tertiary children's hospital between January 2011 and January 2021. Cardiac anomalies were stratified into ductal dependent (pulmonary DD-P or systemic DD-S) or independent lesions. The rate of NEC development in those who received vasodilators (inhaled nitric oxide [iNO], pulmonary vasodilators, systemic vasodilators) was compared to controls in a multivariate analysis. Of the 352 patients, who met inclusion criteria, 77.6% had ductal dependent lesions (DD-S 41.9%, DD-P 35.7%), 19.5% received iNO, and 37.5% received other vasodilatory drugs. The overall NEC rate was 15.1%. On univariate analysis, DD-S, iNO use, and systemic vasodilators was associated with a significantly higher risk of NEC, while DD-P was associated with lower NEC risk. On multivariate analysis, only iNO (odds ratio 2.725, confidence interval [1.36-5.44]) and DD-S (odds ratio 2.279, confidence interval [1.02-5.11]) were independent risk factors for NEC. In patients with at-risk congenital cardiac disease lesions, a ductus dependent systemic circulation or iNO treatment is associated with an increased risk of developing NEC. The presence of iNO or DD-S should be utilized as markers of increased risk both in the prevention and workup of suspected NEC. [ABSTRACT FROM AUTHOR]

[Display omitted] • DNA methylation signature of inflammation derived in neonatal saliva samples. • Preterm infants display higher inflammatory-related DNAm than term infants. • DNAm signature associates with postnatal inflammatory conditions such as sepsis. • Inflammatory DNAm associates with alterations in brain structure in preterm infants. • Highlights the potential of immune-DNAm markers for translational medicine. Preterm birth is closely associated with a phenotype that includes brain dysmaturation and neurocognitive impairment, commonly termed Encephalopathy of Prematurity (EoP), of which systemic inflammation is considered a key driver. DNA methylation (DNAm) signatures of inflammation from peripheral blood associate with poor brain imaging outcomes in adult cohorts. However, the robustness of DNAm inflammatory scores in infancy, their relation to comorbidities of preterm birth characterised by inflammation, neonatal neuroimaging metrics of EoP, and saliva cross-tissue applicability are unknown. Using salivary DNAm from 258 neonates (n = 155 preterm, gestational age at birth 23.28 – 34.84 weeks, n = 103 term, gestational age at birth 37.00 – 42.14 weeks), we investigated the impact of a DNAm surrogate for C-reactive protein (DNAm CRP) on brain structure and other clinically defined inflammatory exposures. We assessed i) if DNAm CRP estimates varied between preterm infants at term equivalent age and term infants, ii) how DNAm CRP related to different types of inflammatory exposure (maternal, fetal and postnatal) and iii) whether elevated DNAm CRP associated with poorer measures of neonatal brain volume and white matter connectivity. Higher DNAm CRP was linked to preterm status (-0.0107 ± 0.0008, compared with −0.0118 ± 0.0006 among term infants; p < 0.001), as well as perinatal inflammatory diseases, including histologic chorioamnionitis, sepsis, bronchopulmonary dysplasia, and necrotising enterocolitis (OR range |2.00 | to |4.71|, p < 0.01). Preterm infants with higher DNAm CRP scores had lower brain volume in deep grey matter, white matter, and hippocampi and amygdalae (β range |0.185| to |0.218|). No such associations were observed for term infants. Association magnitudes were largest for measures of white matter microstructure among preterms, where elevated epigenetic inflammation associated with poorer global measures of white matter integrity (β range |0.206| to |0.371|), independent of other confounding exposures. Inflammatory-related DNAm captures the allostatic load of inflammatory burden in preterm infants. Such DNAm measures complement biological and clinical metrics when investigating the determinants of neurodevelopmental differences. [ABSTRACT FROM AUTHOR]

Necrotizing enterocolitis (NEC) is a gastrointestinal disease of premature neonates. We previously validated a NEC enteroid model derived from human infant intestinal tissue. Typical enteroid configuration is basolateral-out (BO) without direct access to the luminal (apical) surface. Apical access is necessary to allow physiologic comparison of pathogen interaction with the intestinal epithelial barrier. We hypothesize that apical-out (AO) enteroids will provide a relevant NEC model to study this relationship. Following the institutional review board approval (#11610-11611), neonatal intestinal tissue was collected from surgical specimens. Stem cells were collected; enteroids were generated and grown to maturity in BO conformation then everted to AO. Enteroids were untreated or treated for 24 h with 100 μg/mL lipopolysaccharide and hypoxia. Protein and gene expression were analyzed for inflammatory markers, tight junction (TJ) proteins and permeability characteristic of NEC. Apical TJ protein zonula occludens-1 and basolateral protein β-catenin immunofluorescence confirmed AO configuration. Treated AO enteroids had significantly increased messenger RNA (P = 0.001) and protein levels (P < 0.0001) of tumor necrosis factor-α compared to controls. Corrected total cell fluorescence of toll-like receptor 4 was significantly increased in treated AO enteroids compared to control (P = 0.002). Occludin was found to have significantly decreased messenger RNA in treated AO enteroids (P = 0.003). Expression of other TJ proteins claudins-1, -4 and zonula occludens-1 was significantly decreased in treated AO enteroids (P < 0.05). AO enteroids present an innovative model for NEC with increased inflammation and gut barrier restructuring. This model allows for a biologically relevant investigation of the interaction between the pathogen and the intestinal epithelial barrier in NEC. • Apical-out (AO) enteroids subjected to lipopolysaccharide and hypoxia show necrotizing enterocolitis characteristics. • Chemically everted enteroids (AO) allow ready access to their luminal surface. • AO enteroids allow study of pathogen-intestinal epithelium interaction. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor (HIF) 1α is essential for the pathogenesis of necrotizing enterocolitis (NEC). HIF-1α is stabilized by glutaredoxin-1 (Grx1) deletion. The precise role of HIF-1α in the intestinal microcirculation in NEC is not well defined. We aimed to determine the role of HIF-1α in the regulation of the intestinal microcirculation during the development of NEC. Experimental NEC was induced in full-term C57BL/6 mice and Grx1−/− pups through the formula gavage and hypoxia technique. HIF-1α signaling was blocked using the HIF-1α inhibitor, YC-1 [3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole]. Intestinal tissues were collected at predetermined time points for the assessment of the intestinal microcirculation and HIF-1α activity and signaling. We found that NEC induction impaired the intestinal microcirculation, but the impairment of the intestinal blood flow and capillary density was ameliorated in Grx1−/− mice. This amelioration was associated with tripeptide glutathione–protein adducts in the intestinal tissue. Grx1 ablation also promoted vascular endothelial growth factor A production in the intestinal tissue. This intestinal microvascular improvement was not found in HIF-1α-inhibited mice, suggesting that HIF-1α was involved in the intestinal microcirculatory perfusion. The current data demonstrated that HIF-1α signaling is involved in the intestinal microvascular modification during the pathogenesis of NEC, suggesting that targeting HIF-1α might be a promising strategy for NEC treatment. [ABSTRACT FROM AUTHOR]

Fecal microbiota transplantation (FMT) is a promising therapy, but it has not been used to treat neonatal necrotizing enterocolitis (NEC) due to reports of adverse side effects. Probiotics are considered relatively safe with practicable administrative procedures; however, no systematic research has compared the results of FMT and probiotic consortium transplantation (PCT) on oxidative stress in the intestines of patients with NEC. We conducted this study to provide a basis for optimizing NEC therapy. Eight-day-old newborn C57BL/6 mice were randomly divided into the following four groups: the dam-fed group (control group); the NEC induction group (NEC group); the NEC induction and transplantation of Lactobacillus reuteri and Bifidobacterium infantis consortium group (NEC + PCT group); and the NEC induction and the FMT group (NEC + FMT). Intestinal injury, oxidative stress indexes, intestinal barrier function, and inflammatory cytokines were assessed in the terminal ileum. FMT more effectively modulates oxidative stress in the intestine than does PCT; however, the difference between the effects of PCT and FMT was not significant. The protective effect was associated with enhanced antioxidant capacity, regulation of the main components of the mucus layer, reduced inflammatory reactions, and improved intestinal integrity. Intestinal dysbiosis affects oxidative stress, inflammatory response, and mucosal integrity. Although FMT is more effective than PCT in regulating oxidative stress, PCT may be preferred in pediatrics because the proportion and dose of transplanted bacteria can be standardized and individualized according to individual conditions. [ABSTRACT FROM AUTHOR]

Necrotizing Enterocolitis (NEC) remains a significant cause of morbidity and mortality. Recently, there has been an increased recognition of the importance of intestinal immunity and the associations with antibiotics and enteral feeds in the pathophysiology of NEC. The primary purpose of this study is to examine the association of enteral feeds on the survival of premature neonates with NEC. A retrospective review using the Vermont Oxford Network for a Level IV NICU from January 1, 2013 through December 31, 2019 was performed. All neonates had a gestational age between 22 to 29 weeks, weighed at least 300 grams (n = 653), had a reported enteral feed status and were treated for NEC (n = 43). Data analysis utilized two-tailed t -tests for NEC and infection rates then Fisher's exact tests for survival status. The incidence of NEC in the population was 6.6% (43/653). Of the 43 neonates treated for NEC, 27 were enterally fed, while the other 16 were not. All 27 neonates with NEC that were able to achieve enteral feeds survived and had an infection rate of 22.2%. Meanwhile, all 16 neonates with NEC that were unable to achieve enteral feeds died and had an infection rate of 62.5%. There is a significant association between enteral feeds and NEC, survival, and infection rates in premature neonates. These findings support the importance of intestinal immunity and the microbiota in NEC. Given the limitations of the retrospective review, the profound survival advantage with enteral feeds reinforces the need for further study. [ABSTRACT FROM AUTHOR]

Surgical simulation-based training (SBT) can increase resident confidence and improve performance. SBT in pediatric surgery is in its infancy and often geared toward training pediatric surgery fellows. Since case volume for various pediatric surgery–specific procedures can be low based on the rarity of the pathology involved and the level of care provided by the institution, our aim was to create a pediatric surgery simulation-based curriculum for general surgery residents to address this need. We performed an institutional needs assessment consisting of 4 pediatric surgeons' and 28 general surgery residents' confidence in resident ability to independently perform pediatric surgery–specific tasks and procedures using a Likert-scaled survey. These included the placement of a silastic silo for gastroschisis, a percutaneous drain for perforated necrotizing enterocolitis, and completion of a laparoscopic pyloromyotomy for pyloric stenosis. Models simulating these pathologies and curriculum for performing each procedure were generated. We successfully created a model and SBT curriculum to teach general surgery residents how to place a silastic silo for patients with gastroschisis, a percutaneous drain for patients with perforated necrotizing enterocolitis, and how to complete a laparoscopic pyloromyotomy for patients with pyloric stenosis. These were deemed high fidelity models based on a survey of our pediatric surgeons. We created a pediatric surgery SBT curriculum for general surgery residents, which can be used to supplement learning of various high-acuity, low-occurrence procedures. Assessment of residents and validation of scores is underway. [ABSTRACT FROM AUTHOR]

There is no clear consensus on the optimal operative management of premature infants with surgical necrotizing enterocolitis (sNEC) or spontaneous intestinal perforation (SIP); thus, a protocol was developed to guide surgical decision making regarding initial peritoneal drainage (PD) versus initial laparotomy (LAP). We sought to evaluate outcomes after implementation of the protocol. Pre-post study including multiple urban hospitals. Premature infants with sNEC/SIP were accrued after implementation of surgical protocol-directed care (June 2014-June 2019). Patients with a birth weight of <750 g and less than 2 wk of age without pneumatosis or portal venous gas were treated with PD on perforation. PD patients received subsequent LAP for clinical deterioration or continued meconium/bilious drainage. Postprotocol characteristics and outcomes were compared with institutional historical controls. Significance set at P < 0.05. Preprotocol and postprotocol cohorts comprise 35 and 73 patients, respectively. There was a statistically significant difference in age at intervention between historical control PD (14 ± 13 d) and postprotocol PD (9 ± 4 d) groups (P = 0.01), PD patient's birth weight (716 ± 212 g versus 610 ± 141 g, P = 0.02) and estimated gestational age of LAP patients (27 ± 1.7 wk versus 31 ± 4 wk, P = 0.002). PD was definitive surgery in 27% (12 of 44) of postprotocol patients compared with 13% (3 of 23) historical controls. A trend in improved survival postprotocol occurred in all PD infants (73% versus 65%), all LAP (75% versus 70%), and for initial PD and subsequent LAP (82% versus 67%). Utilization of a surgical protocol in sNEC/SIP is associated with improved success of PD as definitive surgery and improved survival. [ABSTRACT FROM AUTHOR]

The assessment of fecal volatile organic compounds (VOCs) has emerged as a noninvasive biomarker in many different pathologies. Before assessing whether VOCs can be used to diagnose intestinal diseases, including necrotizing enterocolitis (NEC), it is necessary to measure the impact of variable infant demographic factors on VOC signals. Stool samples were collected from term infants at four hospitals in a large metropolitan area. Samples were heated, and fecal VOCs assessed by the Cyranose 320 Electronic Nose. Twenty-eight sensors were combined into an overall smellprint and were also assessed individually. 16s rRNA gene sequencing was used to categorize infant microbiomes. Smellprints were correlated to feeding type (formula versus breastmilk), sex, hospital of birth, and microbial enterotype. Overall smellprints were assessed by PERMANOVA with Euclidean distances, and individual sensors from each smellprint were assessed by Mann–Whitney U -tests. P < 0.05 was significant. Overall smellprints were significantly different according to diet. Individual sensors were significantly different according to sex and hospital of birth, but overall smellprints were not significantly different. Using a decision tree model, two individual sensors could reliably predict microbial enterotype. Assessment of fecal VOCs with an electronic nose is impacted by several demographic characteristics of infants and can be used to predict microbiome composition. Further studies are needed to design appropriate algorithms that are able to predict NEC based on fecal VOC profiles. [ABSTRACT FROM AUTHOR]

• Prenatal stress disrupts normal fecal IgA increases in pregnant mice. • Prenatal stress does not affect IgA levels in the breast milk. • Prenatal stress alters the fecal microbiome of female offspring. • Prenatal stress leads to increased IgA coating of female offspring microbiota. • Prenatal stress leads to more severe NEC-like injury in female offspring. Necrotizing enterocolitis (NEC) is an intestinal inflammatory disease with high morbidity and mortality that affects almost exclusively premature infants. Breast milk feeding is known to substantially lower NEC incidence, and specific components of breast milk, such as immunoglobulin (Ig) A, have been identified as mediating this protective effect. On the other hand, accumulating evidence suggests dysbiosis of the neonatal intestinal microbiome contributes to NEC pathogenesis. In mice, neonates can inherit a dysbiotic microbiome from dams that experience stress during pregnancy. Here we show that while prenatal stress lowers fecal IgA levels in pregnant mice, it does not result in lower levels of IgA in the breast milk. Nevertheless, coating of female, but not male, offspring microbiota by IgA is increased by prenatal stress. Accordingly, prenatal stress was found to alter the bacterial community composition in female neonates but not male neonates. Furthermore, female, but not male, offspring of prenatally stressed mothers exhibited more severe colonic tissue damage in a NEC-like injury model compared to offspring with non-stressed mothers. Our results point to prenatal stress as a possible novel risk factor for NEC and potentially reveal new avenues in NEC prevention and therapy. [ABSTRACT FROM AUTHOR]

Necrotizing enterocolitis (NEC) is the most severe life threatening gastrointestinal disease among preterm neonates. NEC continues to account for substantial morbidity and mortality in neonatal intensive care units worldwide. Although its pathogenesis remains incompletely elucidated, NEC is recognized as a multifactorial disease involving intestinal unbalanced inflammatory response, feeding strategies, and bacterial colonization. Epidemiological studies, clinical signs, and animal models support the participation of anaerobic bacteria, particularly clostridia species, in NEC development. Colonization by clostridia seems particularly deleterious. The present review is the opportunity to propose an update on the role of clostridia and NEC. • Neonatal necrotizing enterocolitis pathogenesis remains incompletely elucidated. • Culture-independent data link clostridia colonization and necrotizing enterocolitis. • A clostridia phenotypic signature was associated with necrotizing enterocolitis. • The biological role of clostridia in necrotizing enterocolitis is poorly understood. [ABSTRACT FROM AUTHOR]

Abstract Background Necrotizing enterocolitis (NEC) is one of the most devastating diseases in neonates and is characterized by high morbidity and mortality. It has been suggested that neutrophils play a crucial role in NEC pathogenesis and contribute to the hyperinflammatory reaction after bacterial colonization, which ultimately induces NEC. The aim of this study was to investigate whether dissolution of neutrophil extracellular traps (NETs) by systemic DNase1 therapy reduces NEC manifestation and morbidity. Methods NEC was induced in neonatal mice by gavage feeding of lipopolysaccharide mixed in Neocate, followed by hypoxia q12 h for 5d. Inactivated DNase1 and DNase1 were administered intraperitoneally twice daily in the control and treatment groups, respectively, starting on day 5 for 72 h. Survival, NEC score, intestinal damage (Chiu score, malondialdehyde [MDA], glutathione peroxidase [GPx]), inflammation (neutrophil elastase [NE], myeloperoxidase [MPO], toll-like receptor 4 [TLR4]), and NETs markers (SYTOX orange, cell-free DNA [cfDNA], DNase, citrullinated Histone 3 [H3cit]) were then assessed. Results In total, 44 neonatal mice were used in the experiment. Mice in the treatment group demonstrated significantly reduced NEC rates (44 versus 86%, P = 0.029) and improved survival in comparison to controls (65 versus 35%, P = 0.01). Furthermore, mice treated with DNase1 showed significantly less tissue damage (cfDNA, Chiu score), oxidative stress (MDA, GPx), and inflammation (NE, MPO, H3cit, TLR4), which ultimately lead to a significant reduction in mortality. Conclusions The results of the study indicate that systemic DNase1 treatment leads to a significant reduction in tissue damage, NEC severity, and mortality. Therefore, after validation of our findings in human subjects, DNase1 treatment should be considered as a therapeutic option in neonates diagnosed with NEC. [ABSTRACT FROM AUTHOR]

Abstract Background Psychological stress during pregnancy has been shown to cause subsequent harm to the fetus and newborn. Many studies focus on neurodevelopmental outcomes, but little is known about the effect of gestational stress on intestinal immunity and development. The purpose of this study was to determine the effect of psychological stress during pregnancy on intestinal architecture and growth in newborns. Methods Eight-week-old C57BL6 littermates underwent timed breeding. Pregnant dams were subjected to 1 h of daily psychological stress by using a well-established restraint model during days E7-E14. The distal ileum of 2-wk-old offspring of stressed mothers and nonstressed controls was harvested for histologic analysis. Slides were blinded to measure villus height and crypt depth and surface area. Serum was obtained to measure serum corticosterone levels. An explant model was used to measure corticosterone on the intestinal stem cell marker Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) and growth factors epidermal growth factor receptor and insulin-like growth factor-1. Results The villus height, crypt depth, and surface area were significantly decreased in newborn exposed to stress during gestation. In addition, corticosterone levels were elevated in 2-wk-old mice exposed to stress. Real-time polymerase chain reaction revealed that explants exposed to corticosterone had a decrease in LGR5 compared with controls and an increase in epidermal growth factor receptor. Conclusions Here, we establish that neonatal mice from mothers that were subjected to psychological stress during pregnancy have significantly shorter villi and crypts compared with controls. In addition, pups from stressed mothers have decreased expression levels of the intestinal stem cell marker LGR5. These findings will aid in determining the effect of gestational psychological stress on intestinal development and stem cell plasticity. [ABSTRACT FROM AUTHOR]

Abstract Background Necrotizing enterocolitis (NEC) in premature infants is often a devastating surgical condition with poor outcomes. GYY4137 is a long-acting donor of hydrogen sulfide, a gasotransmitter that is protective against intestinal injury in experimental NEC, likely through protection against injury secondary to ischemia. We hypothesized that administration of GYY4137 would improve mesenteric perfusion, reduce intestinal injury, and reduce inflammatory responses in experimental NEC and ischemia-reperfusion injury, and that these benefits would be mediated through endothelial nitric oxide synthase–dependent pathways. Methods NEC was induced in C57BL/6 wild-type (WT) and endothelial nitric oxide synthase (eNOS) knockout (eNOSKO) pups via maternal separation, formula feeding, enteral lipopolysaccharide, and intermittent hypoxic and hypothermic stress. Pups received daily intraperitoneal injections of 50 mg/kg GYY4137 or phosphate buffered saline vehicle. In separate groups, adult male WT and eNOSKO mice underwent superior mesenteric artery occlusion for 60 min. Before abdominal closure, 50 mg/kg GYY4137 or phosphate buffered saline vehicle was administered into the peritoneal cavity. Laser doppler imaging was used to assess mesenteric perfusion of pups at baseline and on postnatal day 9, and the adult mice at baseline and 24 h after ischemic insult. After euthanasia, the terminal ileum of each animal was fixed, paraffin embedded, sectioned, and stained with hematoxylin and eosin. Sections were blindly graded using published injury scores. Intestinal tissue was homogenized and cytokines measured by ELISA. Data were compared using Mann–Whitney U test, and P -values <0.05 were significant. Results After NEC and ischemia reperfusion (I/R) injury, GYY4137 improved perfusion in WT mice compared to vehicle, but this effect was lost in the eNOSKO animals. Histologic injury followed a similar pattern with reduced intestinal injury in WT mice treated with GYY4137, and no significant improvement in the eNOSKO group. Cytokine expression after GYY4137 administration was altered by the ablation of eNOS in both NEC and I/R injury groups, with significant differences noted in Interleukin 6 and vascular endothelial growth factor. Conclusions GYY4137, a long-acting donor of hydrogen sulfide, has potential as a therapeutic compound for NEC. It improves mesenteric perfusion and intestinal injury in experimental NEC and intestinal I/R injury, and these benefits appear to be mediated through eNOS-dependent pathways. [ABSTRACT FROM AUTHOR]

Abstract Necrotizing enterocolitis (NEC) remains one of the leading causes of death in neonatal infants and new therapeutic strategies for NEC are urgently required. The immunomodulatory agent FTY720 has been shown to have protective effects in various inflammatory diseases. In this study, we hypothesized that treatment with FTY720 confers protection against experimental NEC. Experimental NEC was induced in five-day-old C57BL/6 neonatal mice by hyperosmolar formula feeding plus hypoxia and lipopolysaccharide (LPS) challenges. Induction of NEC resulted in substantial weight loss and high mortality compared to the control group, whereas FTY720 treatment significantly attenuated weight loss and improved survival in NEC-challenged neonatal mice. FTY720 treatment strongly ameliorated NEC-induced intestinal injury with reduced apoptosis and up-regulation of intestinal barrier proteins in the ileal tissues. Furthermore, FTY720 treatment abrogated NEC-initiated intestinal and systemic inflammation with markedly diminished inflammatory cytokines and chemokines. Moreover, FTY720 treatment suppressed NEC-activated CXCL5/CXCR2 axis with down-regulated expression of CXCL5 and CXCR2 at both mRNA and protein levels. Thus, we demonstrate that FTY720 protects neonatal mice against NEC-associated lethality by ameliorating intestinal injury and attenuating inflammation, possibly via its down-regulation of NEC-induced activation of intestinal CXCL5/CXCR2 axis. Highlights • FTY720 reduces weight loss and improves survival in experimental NEC. • FTY720 ameliorates intestinal injury in experimental NEC. • FTY720 attenuates inflammatory response in experimental NEC. • FTY720 down-regulates CXCL5/CXCR2 axis in experimental NEC. [ABSTRACT FROM AUTHOR]

Background To evaluate peritoneal drainage (PD) and laparotomy ± resection/ostomy (LAP) as initial approaches to the surgical management of necrotizing enterocolitis (NEC) in premature, extremely low birth weight (ELBW) infants. Methods Kids' Inpatient Database (2003-2012) was searched for cases of NEC ( International Classification of Diseases, ninth revision, Clinical Modification [ICD-9-CM] 777.5x) in premature (<37 weeks), extremely low birth weight (<1000 g) infants. Infants were admitted at <28 days of life. Propensity score (PS)-matched analyses were performed, using end points of hospital mortality, length of stay (LOS), and cost of hospitalization. Cases were matched 1:1 on 48 confounding variables (demographic, clinical, and hospital characteristics and 39 comorbidities). Results On PS-matched comparison, PD had higher survival versus LAP, P = 0.0009. LOS and cost were higher for PD versus LAP, P < 0.003. Survival rates did not differ between PD + LAP and PD-only treatments. LOS and cost were higher for PD + LAP versus PD-only, P < 0.02. PD + LAP infants had higher survival versus LAP, P = 0.0193. LOS and cost were higher for PD + LAP, P < 0.005. Conclusions A risk-adjusted PS-matched analysis of operative management in premature, ELBW infants with NEC found higher survival rates associated with PD placement versus LAP, whether PD was used as definitive treatment or with subsequent LAP even after controlling for potential contributors to selection bias (i.e., stability influencing management preference). [ABSTRACT FROM AUTHOR]

Background Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. Increased intestinal permeability is central to NEC development. We have shown that stem cells (SCs) can reduce the incidence and severity of NEC. Our current goal was to investigate the efficacy of four different types of SC in preservation of gut barrier function during NEC. Materials and methods We compared (1) amniotic fluid–derived mesenchymal SC, (2) bone marrow–derived mesenchymal SC, (3) amniotic fluid–derived neural SC, and (4) enteric neural SC. Premature rat pups received an intraperitoneal injection of 2 × 10 6 SC or phosphate-buffered saline only and were then subjected to experimental NEC. Control pups were breastfed and not subjected to NEC. After 48 h, animals received a single enteral dose of fluorescein isothiocyanate -labeled dextran (FD70), were sacrificed 4 h later, and serum FD70 concentrations determined. Results Compared to breastfed, unstressed pups with intact gut barrier function and normal intestinal permeability (serum FD70 concentration 2.22 ± 0.271 μg/mL), untreated pups exposed to NEC had impaired barrier function with significantly increased permeability (18.6 ± 4.25 μg/mL, P = 0.047). Pups exposed to NEC but treated with SC had significantly reduced intestinal permeability: Amniotic fluid–derived mesenchymal SC (9.45 ± 1.36 μg/mL, P = 0.017), bone marrow–derived mesenchymal SC (6.73 ± 2.74 μg/mL, P = 0.049), amniotic fluid–derived neural SC (8.052 ± 1.31 μg/mL, P = 0.0496), and enteric neural SC (6.60 ± 1.46 μg/mL, P = 0.033). Conclusions SCs improve gut barrier function in experimental NEC. Although all four types of SC reduce permeability equivalently, SC derived from amniotic fluid may be preferable due to availability at delivery and ease of culture, potentially enhancing clinical translation. [ABSTRACT FROM AUTHOR]

Background Necrotizing enterocolitis (NEC) is a severe intestinal disease of premature infants with high mortality. Studies suggest a causative relationship between red blood cell (RBC) transfusion and NEC; however, whether RBC transfusion leads to worse outcomes in NEC is unknown. We sought to determine whether RBC transfusion was associated with an increased risk of surgical NEC and mortality. Methods In this retrospective study, 115 patients were enrolled with NEC Bell's stage 2A or greater from 2010-2015. Patients were classified based on the timing of RBC transfusion before NEC: ≤72 h, >72 h, and no transfusion. Variables including gestational age (GA), birth weight (BW), feedings, and hematocrit levels were analyzed. Outcomes were surgical intervention for NEC following RBC transfusion and mortality. Results Twenty-three (20%) infants developed NEC ≤ 72 h after RBC transfusion, 16 (69.6%) required surgery with a mortality rate of 21.7% ( n = 5). Seventeen (15%) infants developed NEC > 72 h after RBC transfusion, 12 (70.6%) required surgery with a mortality rate of 23.5% ( n = 4). 75 (65%) patients developed NEC without RBC transfusion, 17 (22.7%) required surgery with a mortality rate of 4% ( n = 3). Lower GA and BW were significantly associated with RBC transfusion and the need for surgical intervention. RBC transfusion ≤72 h before NEC was associated with surgical NEC (pairwise adjusted P < 0.001) and mortality (pairwise adjusted P = 0.048). However, multivariable logistic regression analysis revealed RBC transfusion is not an independent risk factor for surgical NEC. Conclusions Infants of lower GA and BW were more likely to receive an RBC transfusion before NEC, which was significantly associated with surgical intervention and an increasing risk of mortality. Judicious use of transfusions in premature infants may improve NEC outcomes. [ABSTRACT FROM AUTHOR]

Necrotizing enterocolitis is a common but serious complication among premature babies. Currently, there are limited treatment options. These include intensive supportive care and surgical intervention. In this study, we hypothesize that erythropoietin (Epo) could be protective against cell necrosis by increasing the levels of glutathione. This can be regulated by increasing the activity of system xC - . This was demonstrated using intestinal epithelial cells (IEC-6) as a model system. S4-CPG and sulfasalazine pharmacologically inhibit xCT, which induced cell death. Our data showed a dose dependent decrease in cell viability when treated with both inhibitors. In addition, the IEC-6 cells displayed a dose dependent increase when treated with Epo. In conclusion, Epo can be protective against cell death and ultimately be considered as a treatment option for intestinal epithelial cell death. [ABSTRACT FROM AUTHOR]

Background Direct peritoneal resuscitation (DPR) has previously been shown to alter blood flow in the small bowel mesenteric vessels in models of intestinal ischemia. However, a survival advantage or its effects on local tissue inflammation have not been previously demonstrated. We hypothesized that DPR would increase survival and decrease intestinal tissue inflammation after intestinal ischemia and reperfusion (I/R) injury. Methods Eight-week-old male C57Bl6J mice were anesthetized and underwent midline laparotomy. I/R and DPR groups were exposed to superior mesenteric artery occlusion for 60 min with a nontraumatic clamp. Immediately after removal of the clamp, 1 mL of phosphate-buffered saline, 1 mL of minimal essential media, or 1 mL of minimal essential media supplemented with fetal bovine serum, penicillin and/or streptomycin, and glutamine were placed into the abdominal cavity of DPR groups. Animals were then closed in two layers and allowed to reperfuse for 6 h (cytokine analysis, n = 6 per group) or 7 d (survival analysis, n = 10 per group). After 6 h of reperfusion, animals were euthanized. Intestines were harvested and homogenized. Extracts were quantified for total protein content (Bradford assay), myeloperoxidase activity, tissue inflammatory cytokine, and growth factor production. P < 0.05 was significant. Results I/R caused marked intestinal ischemia, significant mortality, and a significant increase in tissue cytokine and growth factor levels ( P < 0.05). Seven-day survival was 30% for I/R without treatment and rose to 60% with DPR therapy using phosphate-buffered saline as the dialysate. DPR using plain MEM or MEM with supplements after ischemia increased 7-d survival to 90% ( P < 0.05). DPR also significantly decreased intestinal tissue levels of myeloperoxidase, as well as intestinal tissue levels of multiple growth factors and inflammatory cytokines. Conclusions DPR increases survival and decreases intestinal inflammation after intestinal I/R injury. Translational applications are readily achievable and should be considered for patients with intestinal ischemic pathology. [ABSTRACT FROM AUTHOR]

Background Immunoglobulin A (IgA) plays a key role in coating luminal antigens and preventing translocation of harmful bacteria. The aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix transcription factor that when stimulated activates factors important for barrier function and intestinal homeostasis. We hypothesize that AhR signaling is critical for establishment of intestinal homeostasis in neonates. Material and methods Mice: C57BL/6 (B6) AhR+/+ wild type (WT), B6.AhR−/− Aryl-hydrocarbon receptor knockout (KO), and B6.AhR+/+ raised on an AhR ligand-free diet (AhR LF). Enzyme-linked immunosorbent assay was used to measure fecal and serum IgA levels. Bacterial translocation was measured by culturing the mesenteric lymph nodes. Results Two week old KO mice had significantly less fecal IgA compared with WT (and AhR LF, P value = 0.0393. The amount of IgA from the gastric contents of 2-wk-old mice was not significantly different. At age 8 wk, AhR LF mice had significantly less fecal IgA than WT and KO P value = 0.0077. At 2 wk, KO mice had significantly higher levels of bacterial translocation and at 8 wk AhR LF had significantly higher levels of bacterial translocation compared with WT. Conclusions In neonatal mice, the lack of AhR signaling is associated with loss of intestinal homeostasis, evidenced by decreased levels of IgA and increased bacterial translocation. In adult mice, exogenous AhR ligand and not receptor signaling is necessary for maintenance of intestinal integrity. [ABSTRACT FROM AUTHOR]

Background Necrotizing enterocolitis (NEC) involves impaired ileal blood flow due to alterations in vascular tone control and intestinal angiogenesis. Platelet-derived growth factor (PDGF) is a mediator of normal angiogenesis in intestinal epithelium. We hypothesized that gene dysregulation during experimental NEC results in altered PDGF expression. Methods Sprague–Dawley rats were randomized to groups by litter. Controls were delivered vaginally and dam-fed. NEC groups were delivered prematurely by cesarean section and subjected to an established NEC protocol. Ileum was obtained at 0, 12, 24, 48, 72, and 96 h of life from all animals ( N = 108 animals). Western blot analysis was carried out for every time point, and samples were evaluated by immunohistochemistry. Antibodies against PDGF-A, PDGF-B, and their receptors, PDGFR-α and PDGFR-β, were used. Statistical analysis was performed using two-way analysis of variance with a priori P < 0.05. Results Ileal PDGF-A concentration was higher in controls versus NEC from 24–96 h of life. Its receptor, PDGFR-α, was low in concentration in both groups at all time points. PDGF-B concentration was increased in controls at 24 and 72 h of life but decreased at the 48-h mark. Its receptor, PDGFR-β, was also low in both groups at 12 and 24 h but increased in controls at 48 and 72 h. Conclusions These data support our hypothesis that PDGF and PDGF receptor expression are altered in experimental NEC. Dysregulation of PDGF during intestinal maturation could contribute to the development of NEC. Further investigation into this pathway could yield new therapeutic targets for this devastating disease. [ABSTRACT FROM AUTHOR]

Background Macrophages can be polarized into proinflammatory (M1) and anti-inflammatory (M2) subtypes. However, whether macrophage polarization plays a role in necrotizing enterocolitis (NEC) remains unknown. Materials and methods Macrophages were derived from the THP-1 human monocyte cell line. Apoptosis of human fetal small intestinal epithelial FHs-74 cells was determined by Annexin V/propidium iodide flow cytometry and by Western blotting to detect cleaved caspase-3. The effect of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on macrophage polarization was determined by flow cytometry with M1/M2 markers and real time polymerase chain reaction. In vivo, experimental NEC was induced in mouse pups by repeated exposure to hypoxia, hypothermia, and hypertonic feedings. Intestinal histologic sections were subjected to immunohistochemical staining for the detection of M1 and M2 macrophages. Results In vitro, FHs-74 cell apoptosis was increased after coculture with macrophages and lipopolysaccharide (LPS). This apoptosis was increased by exposure to M1-conditioned medium and suppressed by exposure to M2-conditioned medium. HB-EGF significantly decreased LPS-induced M1 polarization and promoted M2 polarization via signal transducers and activators of transcription 3 activation. Addition of HB-EGF to LPS-stimulated macrophages suppressed the proapoptotic effects of the macrophages on FHs-74 cells. In vivo, we found enhanced intestinal macrophage infiltration in pups subjected to NEC, most of which were M1 macrophages. HB-EGF treatment of pups subjected to experimental NEC significantly reduced M1 and increased M2 polarization and protected the intestines from NEC. Conclusions M1 macrophages promote NEC by increasing intestinal epithelial apoptosis. HB-EGF protects the intestines from NEC by preventing M1 and promoting M2 polarization. [ABSTRACT FROM AUTHOR]

Background Intestinal alkaline phosphatase (IAP) activity is decreased in necrotizing enterocolitis (NEC), and IAP supplementation prevents NEC development. It is not known if IAP given after NEC onset can reverse the course of the disease. We hypothesized that enteral IAP given after NEC induction would not reverse intestinal injury. Materials and methods NEC was induced in Sprague-Dawley pups by delivery preterm followed by formula feedings with lipopolysaccharide (LPS) and hypoxia exposure and continued up to 4 d. IAP was added tofeeds on day 2 until being sacrificed on day 4. NEC severity was scored based on hematoxylin and eosin-stained terminal ileum sections, and AP activity was measured using a colorimetric assay. IAP and interleukin-6 expression were measured using real time polymerase chain reaction. Results: NEC pups' alkaline phosphatase (AP) activity was decreased to 0.18 U/mg compared with controls of 0.57 U/mg (P < 0.01). Discontinuation of LPS and hypoxia after 2 d increased AP activity to 0.36 U/mg (P < 0.01). IAP supplementation in matched groups did not impact total AP activity or expression. Discontinuing LPS and hypoxia after NEC onset improved intestinal injury scores to 1.14 compared with continued stressors, score 2.25 (P < 0.01). IAP supplementation decreased interleukin-6 expression two-fold (P < 0.05), though did not reverse NEC intestinal damage (P = 0.5). Conclusions This is the first work to demonstrate that removing the source of NEC improves intestinal damage and increases AP activity. When used as a rescue treatment, IAP decreased intestinal inflammation though did not impact injury making it likely that IAP is best used preventatively to those neonates at risk. [ABSTRACT FROM AUTHOR]

Background Despite emergence of markers of intestinal mucosal damage such as intestinal fatty-acid binding protein (i-FABP), there are no specific markers of damage extending into the muscle layers. We hypothesized that smooth muscle actin (SMA) released from the intestinal muscularis would be detectable in plasma after severe intestinal injury. Materials and methods Serial blood samples were collected from rats (n = 10) undergoing intestinal ischemia-reperfusion injury (IRI) and controls (n = 5). Additionally, admission and/or preoperative plasma samples were collected from twelve neonates with necrotizing enterocolitis (NEC), and five age- and weight-matched controls. Plasma ileal fatty-acid binding protein (rat) or i-FABP (human) were measured by enzyme-linked immunosorbent assay, and plasma SMA was detected by western blotting. Results Plasma ileal fatty-acid binding protein was low in both the control group and IRI at baseline, but became rapidly elevated in the IRI group even during ischemia. SMA was detected in reperfusion plasma samples of all IRI rats, but in none of the control samples. Plasma i-FABP was higher in infants with NEC than age- and weight-matched controls. Although i-FABP was higher in infants with severe surgical disease compared with focal disease, there was no difference between the operative and nonoperative groups. SMA was detected in the plasma of all four neonates with severe surgical NEC, but not in those with focal disease or those who were successfully conservatively managed. Conclusions SMA is detectable in plasma after severe intestinal injury and maybe a clinically useful maker of intestinal muscle damage. [ABSTRACT FROM AUTHOR]

Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. In order to evaluate the efficacy of probiotics in the prevention of stage II-III NEC, we performed a meta-analysis of clinical studies. We searched in PubMed, Medline and Embase from 1 January 1990 to 16 January 2018. Heterogeneity was examined by Q-test. Publication bias was evaluated by funnel plot and Egger's regression test. 30 articles were identified meeting the inclusion criteria. Data showed that probiotics supplement could significantly reduce the risk of stage II-III NEC (RR = 0.51, 95% CI, 0.38 to 0.67, P < 0.001) and death rate (RR = 0.69, 95% CI, 0.55 to 0.87, P = 0.002). The mixed probiotics and lactobacillus could reduce the risk of stage II-III NEC (for mixed probiotics, RR = 0.39, 95% CI, 0.26 to 0.57; for lactobacillus , RR = 0.53, 95% CI, 0.36 to 0.78), while bifidobacterium or saccharomyces did not have such effect. The results also indicated that only the mixed probiotics could reduce the risk of deaths (RR = 0.52, 95%CI, 0.34 to 0.80). Subgroup analysis for mortality revealed that probiotics had significant effect in Asian region (RR = 0.54, 95% CI, 0.37 to 0.80, P = 0.002) but not in non-Asian region (RR = 0.84, 95% CI, 0.66 to 1.08, P = 0.179). Probiotics could significantly decrease the risk of stage II-III NEC and death. Compared to using single probiotics species, the application of combining different probiotics has a better efficacy in the prevention of stage II-III NEC and death, especially in the Asian population. • Probiotics supplement could significantly reduce the risk of stage II-III NEC and death rate. • The mixed probiotics and lactobacillus rather than bifidobacterium or saccharomyces could reduce the risk of stage II-III NEC. • Only the mixed probiotics could reduce the risk of deaths. [ABSTRACT FROM AUTHOR]