interleukin-8 and Enterocolitis--Necrotizing

Necrotizing enterocolitis (NEC) often leads to gastrointestinal emergency resulting high mortality in very low birth weight infants (VLBWIs) requiring surgery. To date, few studies have explored the role of serum cytokines in the development of feeding intolerance (FI) or NEC outcomes in VLBWIs. Infants born weighing <1500 g or of 32 weeks of gestational age were prospectively enrolled from May 2018 to Dec 2019. We measured several cytokines routinely within 72 h of life, even before NEC-like symptoms developed. NEC or FI group comprised 17 (27.4%) infants, and 6 (9.7%) infants had surgical NEC. The gestational age and birth weight were significantly lower in the NEC or FI group with more prematurity-related complications. The surgical NEC group also demonstrated significantly lower gestational age and birth weight along with more infants experiencing refractory hypotension within a 1 week of life, pulmonary hypertension, and patent ductus arteriosus. IL-10 levels were significantly higher in the NEC or FI group, whereas IL-8 levels were significantly higher in the infants with surgical NEC. Our findings indicated to IL-8 can predict surgical NEC while increased IL-10 can predict NEC development in VLBWIs.

Topics: Biomarkers; Cytokines; Enterocolitis, Necrotizing; Gestational Age; Humans; Infant; Infant, Newborn; Infant, Premature; Infant, Premature, Diseases; Infant, Very Low Birth Weight; Inflammation Mediators; Interleukin-10; Interleukin-8; Prognosis; Prospective Studies

Fetal swallowing of human amniotic fluid (hAF) containing trophic factors (TFs) promotes gastrointestinal tract (GIT) development. Preterm birth interrupts hAF swallowing, which may increase the risk of necrotizing enterocolitis (NEC). Postnatally, it is difficult to replicate fetal swallowing of hAF due to volume. We aimed to evaluate whether hAF lyophilization is feasible and its effect on hAF-borne TFs.. We collected hAF (n = 16) from uncomplicated pregnancies. hAF was divided into three groups: unprocessed control (C), concentration by microfiltration (F), and by dialysis and lyophilization (L). EGF, HGF, GM-CSF, and TGF-α were measured in each group by multiplex assay. Bioavailability of TFs was measured by proliferation and LPS-induced IL-8 production by intestinal epithelial cells FHs74.. After dialysis/lyophilization, GM-CSF and TGF-α were preserved with partial loss of EGF and HGF. hAF increased cell proliferation and reduced LPS-induced IL-8 production compared to medium alone. Compared to control, dialysis/lyophilization and filtration of hAF increased FHs74 cell proliferation (p < 0.001) and decreased LPS-induced IL-8 production (p < 0.01).. Lyophilization and filtration of hAF is feasible with partial loss of TFs but maintains and even improves bioavailability of TFs measured by proliferation and LPS-induced IL-8 production by FHs74.

Topics: Amniotic Fluid; Cell Proliferation; Cryopreservation; Deglutition; Enterocolitis, Necrotizing; Female; Freeze Drying; Gastrointestinal Tract; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation; Interleukin-8; Pregnancy; Transforming Growth Factor alpha

Necrotizing enterocolitis (NEC), a necrotic inflammation of the intestine, represents a major health problem in the very premature infant. Although prevention is difficult, the combination of ingestion of maternal-expressed breastmilk in conjunction with a probiotic provides the best protection. In this study, we establish a mechanism for breastmilk/probiotic protection.. Ultra-high-performance liquid chromatography-tandem mass spectrometry of Bifidobacterium longum subsp. infantis (B. infantis) secretions was used to identify an anti-inflammatory molecule. Indole-3-lactic acid (ILA) was then tested in an established human immature small intestinal cell line, necrotizing colitis enterocytes, and other immature human enteroids for anti-inflammatory effects and to establish developmental function. ILA was also examined in immature and mature enterocytes.. We have identified ILA, a metabolite of breastmilk tryptophan, as the anti-inflammatory molecule. This molecule is developmentally functional in immature but not mature intestinal enterocytes; ILA reduces the interleukin-8 (IL-8) response after IL-1β stimulus. It interacts with the transcription factor aryl hydrocarbon receptor (AHR) and prevents transcription of the inflammatory cytokine IL-8.. This molecule produced by B. infantis (ATCC No. 15697) interaction with ingested breastmilk functions in a complementary manner and could become useful in the treatment of all at-risk premature infants for NEC if safety and clinical studies are performed.

Topics: Animals; Anti-Inflammatory Agents; Bifidobacterium longum; Chromatography, High Pressure Liquid; Chromatography, Liquid; Cytokines; Enterocolitis, Necrotizing; Enterocytes; Humans; Hydrocortisone; Indoles; Infant, Newborn; Inflammation; Interleukin-1beta; Interleukin-8; Intestines; Male; Mice; Mice, Inbred C57BL; Milk, Human; Organ Culture Techniques; Probiotics; Receptors, Aryl Hydrocarbon; Tandem Mass Spectrometry; Tryptophan

BackgroundExcess vernix caseosa produced by the fetal skin appears as particles suspended in the amniotic fluid in late gestation, is swallowed by the fetus, and is found throughout the newborn gastrointestinal tract as the first organisms are arriving to colonize the gut. Lipid-rich vernix contains an unusually high 29% branched chain fatty acids (BCFA). BCFAs reduce the incidence of necrotizing enterocolitis in an animal model, and were recently found predominantly in the sn-2 position of human milk triacylglycerols. Nothing is known about the influence of vernix BCFA on proinflammatory markers in human enterocytes.MethodsWe investigated the effect of vernix-monoacylglycerides (MAGs) (enriched with 30% BCFA) on interleukin (IL)-8 and NF-κB production in a human intestinal epithelial cell line (Caco-2). Caco-2 cells were pretreated with vernix-MAG or vernix-free fatty acid (FFA) prior to lipopolysaccharide (LPS) activation.ResultsBoth vernix-MAG and vernix-FFA increased cell BCFA and eliminated an LPS-induced 20% reduction in cell viability. In stimulated Caco-2 cells, vernix-MAG was more effective than vernix-FFA in suppressing IL-8 and NF-κB. Activated vernix-MAG-treated cells expressed less of the cell-surface Toll-like receptor4 (TLR-4) compared with controls.ConclusionThis is the first study to show the reduction of proinflammatory markers in human cells mediated by BCFA-MAG.

Topics: Amniotic Fluid; Biomarkers; Caco-2 Cells; Cell Survival; Enterocolitis, Necrotizing; Enterocytes; Fatty Acids; Female; Gastrointestinal Tract; Gene Expression Profiling; Humans; Infant, Newborn; Inflammation; Interleukin-8; Lipids; Lipopolysaccharides; Milk, Human; NF-kappa B; Pregnancy; Skin; Triglycerides; Vernix Caseosa

Topics: Enterocolitis, Necrotizing; Feeding and Eating Disorders of Childhood; Female; Humans; Infant; Infant, Newborn; Infant, Premature; Intensive Care Units, Neonatal; Interleukin-6; Interleukin-8; Prospective Studies; Tumor Necrosis Factor-alpha

To what extent the risks of neonatal morbidities are directly related to premature birth or to biological mechanisms of preterm birth remains uncertain. We aimed to examine the effect of exposure to amniotic fluid (AF) infection and elevated cytokine levels on the mortality and pulmonary, intestinal, and neurologic outcomes of preterm infants, and whether these associations persist after adjustment for gestational age at birth. This retrospective cohort study included 152 premature singleton infants who were born at ≤ 32 weeks. AF obtained by amniocentesis was cultured; and interleukin-6 (IL-6) and IL-8 levels in AF were determined. The primary outcome was adverse perinatal outcome defined as the presence of one or more of the followings: stillbirth, neonatal death, bronchopulmonary dysplasia, necrotizing enterocolitis, intraventricular hemorrhage, and periventricular leukomalacia. Logistic regression analysis was adjusted for gestational age at birth and other potential confounders. In bivariate analyses, elevated AF IL-6 and IL-8 levels were significantly associated with adverse perinatal outcome. These results were not changed after adjusting for potential confounders, such as low Apgar scores, mechanical ventilation, and surfactant application. However, the independent effect of elevated cytokine levels in AF disappeared when additionally adjusted for low gestational age at birth; consequently, low gestational age remained strongly associated with the risk of adverse perinatal outcome. In conclusion, elevated levels of pro-inflammatory cytokines in AF are associated with increased risk of adverse perinatal outcomes, but this risk is not independent of low gestational age at birth. Culture-proven AF infection is not associated with this risk.

Topics: Adult; Amniotic Fluid; Area Under Curve; Chorioamnionitis; Enterocolitis, Necrotizing; Enzyme-Linked Immunosorbent Assay; Female; Gestational Age; Humans; Infant, Newborn; Intensive Care Units, Neonatal; Interleukin-6; Interleukin-8; Leukomalacia, Periventricular; Logistic Models; Lung Diseases; Male; Multivariate Analysis; Odds Ratio; Perinatal Mortality; Pregnancy; Premature Birth; Retrospective Studies; Risk Factors; ROC Curve; Young Adult

Colonizing bacteria interacting with the immature, unlike the mature, human intestine favors inflammation over immune homeostasis. As a result, ten percent of premature infants under 1500 grams weight develop an inflammatory necrosis of the intestine after birth, e.g., necrotizing enterocolitis (NEC). NEC is a major health problem in this population causing extensive morbidity and mortality and an enormous expenditure of health care dollars. NEC can be prevented by giving preterm infants their mother's expressed breast milk or ingesting selective probiotic organisms. Vaginally delivered, breast fed newborns develop health promoting bacteria ("pioneer" bacteria) which preferentially stimulate intestinal host defense and anti-inflammation. One such "pioneer" organism is Bacteroides fragilis with a polysaccharide (PSA) on its capsule. B. fragilis has been shown developmentally in intestinal lymphocytes and dendritic cells to produce a balanced T-helper cell (TH1/TH2) response and to reduce intestinal inflammation by activity through the TLR2 receptor stimulating IL-10 which inhibits IL-17 causing inflammation. No studies have been done on the role of B. fragilis PSA on fetal enterocytes and its increased inflammation. Accordingly, using human and mouse fetal intestinal models, we have shown that B. fragilis with PSA and PSA alone inhibits IL-1β-induced IL-8 inflammation in fetal and NEC intestine. We have also begun to define the mechanism for this unique inflammation noted in fetal intestine. We have shown that B. fragilis PSA anti-inflammation requires both the TLR2 and TLR4 receptor and is in part mediated by the AP1 transcription factor (TLR2) which is developmentally regulated. These observations may help to devise future preventative treatments of premature infants against NEC.

Topics: Animals; Bacteroides fragilis; Cells, Cultured; Disease Models, Animal; Enterocolitis, Necrotizing; Enterocytes; Fetus; Humans; Inflammation; Interleukin-10; Interleukin-17; Interleukin-1beta; Interleukin-8; Mice; Mice, Inbred C57BL; Mice, Knockout; Polysaccharides; RNA Interference; Th1 Cells; Th2 Cells; Toll-Like Receptor 2; Toll-Like Receptor 4

Weight gain before the clinical diagnosis of necrotising enterocolitis (NEC) is described as a predictive factor.. Weight gain of more than 5% one day prior to clinical suspicion plus increase of plasma Iinterleukin-8 (IL-8) are predictive for NEC.. 48 infants with diagnosis of NEC stage II and III were enrolled in a case-control study. Oral and parenteral nutrition, diuresis and kinetics of weight and of IL-8 were documented.. 31 infants with NEC-II and 17 infants with NEC-III were enrolled. Weight gain>5% occurred in 35.3% of NEC-III, in 0% of NEC-II and in 4.2% of the control group. IL-8 increased significantly [NEC-III (6 561.4 pg/mL) vs. NEC-II: (326.7 pg/mL) vs. control group (38.9 pg/mL); p<0.05]. Sensitivity of IL-8 in NEC-II was 87.10% (70.15-96.25) and in NEC-III 100.00% (80.33-100.00). Sensitivity of weight gain was 0.00% (0.00-11.32) in NEC-II and 35.29% (14.30-61.65) in NEC-III.. Weight gain>5% was found in only 35.3% of the cases with NEC-III. Combination of weight gain and IL-8 did not improve the diagnosis of NEC.

Topics: Biomarkers; Enterocolitis, Necrotizing; Female; Humans; Infant, Newborn; Infant, Premature; Interleukin-8; Male; Prognosis; Reproducibility of Results; Sensitivity and Specificity; Weight Gain

To analyze how enteral food introduction affects intestinal gene regulation and chromatin structure in preterm pigs.. Preterm pigs were fed parenteral nutrition plus/minus slowly increasing volumes of enteral nutrition. Intestinal gene-expression and chromatin structure were analyzed 5 days after birth.. Enteral feeding led to differential upregulation of inflammatory and pattern recognition receptor genes, including IL8 (median: 5.8, 95% CI: 3.9-7.8 for formula; median: 2.2, 95% CI: 1.3-3.3 for colostrum) and TLR4 (median: 3.7, 95% CI: 2.6-4.8 for formula; no significant differences for colostrum) with corresponding decondensed chromatin configurations. On histology this correlated with mild mucosal lesions, particularly in formula-fed pigs. In CaCo-2 cells, histone hyperacetylation led to a marked increase in TLR4 mRNA and increased IL8 expression upon stimulation with lipopolysaccharide (median: 7.0; interquartile range: 5.63-8.85) compared with naive cells (median 4.2; interquartile range: 2.45-6.33; p = 0.03).. Enteral feeding, particular with formula, induces subclinical inflammation in the premature intestine and more open chromatin structure in key inflammatory genes. This may increase the susceptibility for necrotizing enterocolitis.

Topics: Animals; Animals, Newborn; Caco-2 Cells; Chromatin; Enteral Nutrition; Enterocolitis, Necrotizing; Humans; Interleukin-8; Swine; Toll-Like Receptor 4

Bacterial involvement is believed to play a pivotal role in the development and disease outcome of NEC. However, whether a bloodstream infection (BSI) predisposes to NEC (e.g. by activating the pro-inflammatory response) or result from the loss of gut wall integrity during NEC development is a longstanding question.. We hypothesize that the occurrence of a BSI plays a complementary role in the pathogenesis of NEC. The first aim of the study was to correlate the occurrence of a BSI during the early phase of NEC with intestinal fatty acid-binding protein (I-FABP) levels, as a marker for loss of gut wall integrity owing to mucosal damage, and Interleukin (IL)-8 levels, as a biomarker for the pro-inflammatory cascade in NEC. The second aim of the study was to investigate the relation between the occurrence of a BSI and disease outcome.. We combined data from prospective trials from two large academic pediatric surgical centers. Thirty-eight neonates with NEC, 5 neonates with bacterial sepsis, and 14 controls were included.. BSIs occurred in 10/38 (26%) neonates at NEC onset. No association between the occurrence of BSIs and I-FABP levels in plasma (cohort 1: median 11ng/mL (range 0.8-298), cohort 2: median 6.8ng/mL (range 1.3-15)) was found in NEC patients (cohort 1: p=0.41; cohort 2: p=0.90). In addition, the occurrence of BSIs did not correlate with IL-8 (median 1562pg/mL (range 150-7,500); p=0.99). While the occurrence of a BSI was not correlated with Bell's stage (p=0.85), mortality was higher in patients with a BSI (p=0.005).. The low incidence of BSIs and the absent association of both the markers for loss of gut wall integrity and the pro-inflammatory response during the early phase of NEC, support the hypothesis that the presence of a BSI does not precede NEC.

Topics: Bacteremia; Biomarkers; Enterocolitis, Necrotizing; Fatty Acid-Binding Proteins; Female; Humans; Incidence; Infant, Newborn; Interleukin-8; Male; Prospective Studies

Evidence suggests that excessive inflammation of the immature intestine may predispose premature infants to necrotizing enterocolitis (NEC). We investigated the anti-inflammatory effects of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) in human fetal and adult intestinal epithelial cells (IEC) in primary culture.. Human fetal IEC in culture were derived from a healthy fetal small intestine (H4) or resected small intestine of a neonate with NEC (NEC-IEC). Intestinal cell lines Caco2 and NCM460 in culture were used as models for mature IEC. IEC in culture were pretreated with 100 µmol/l palmitic acid (PAL), DHA, EPA, ARA, or ARA+DHA for 48 h and then stimulated with proinflammatory IL-1β.. DHA significantly attenuated IL-1β induced proinflammatory IL-8 and IL-6 protein and mRNA in fetal H4, NEC-IEC, and mature Caco2, NCM460 IEC, compared to control and PAL treatment. DHA downregulated IL-1R1 (IL-1β receptor) and NFk β1 mRNA expression in fetal and adult IEC. ARA had potent anti-inflammatory effects with lower IL-8 and IL-6 (protein and mRNA) in fetal H4 but not in NEC-IEC or adult IEC.. The present study provides evidence that DHA and ARA may have important anti-inflammatory functions for prevention of NEC in premature infants.

Topics: Anti-Inflammatory Agents; Arachidonic Acid; Caco-2 Cells; Cytoprotection; Docosahexaenoic Acids; Eicosapentaenoic Acid; Enterocolitis, Necrotizing; Epithelial Cells; Gene Expression Regulation; Humans; Ileum; Infant, Newborn; Inflammation Mediators; Interleukin-1beta; Interleukin-6; Interleukin-8; Intestinal Mucosa; NF-kappa B p50 Subunit; Palmitic Acid; Receptors, Interleukin-1 Type I

Necrotizing enterocolitis (NEC) is a severe inflammatory disorder, associated with the difficult transition from parenteral to enteral feeding after preterm birth. We hypothesized that minimal enteral nutrition (MEN) with amniotic fluid (AF), prior to enteral formula feeding, would improve resistance to NEC in preterm pigs.. Experiment 1: IEC-6 cells were incubated with porcine (pAF) and human AF (hAF) to test AF-stimulated enterocyte proliferation and migration in vitro. Experiment 2: Cesarean-delivered, preterm pigs were fed parenteral nutrition and MEN with pAF, hAF, or control fluid (MEN-pAF, MEN-hAF, or MEN-CTRL; all n = 9) for 2 days before tissue collection. Experiment 3: Preterm pigs were fed MEN diets as in experiment 2, but followed by 2 days of enteral formula feeding, which predisposes to NEC (NEC-pAF, NEC-hAF, or NEC-CTRL; n = 10-12).. Both pAF and hAF stimulated enterocyte proliferation and migration in vitro. In experiment 2, MEN-pAF and MEN-hAF pigs showed increased body weight gain and reduced intestinal interleukin (IL)-8 and colonic IL-6 levels, indicating reduced inflammatory response. In experiment 3, body weight gain was highest in the 2 groups fed AF as MEN, but NEC incidences were similar (NEC-pAF) or increased (NEC-hAF) compared with controls.. Intake of pAF or hAF improved body growth and modulated intestinal inflammatory cytokines during a period of parenteral nutrition, but did not protect against later formula-induced NEC in preterm pigs. Further studies are required to show if MEN feeding with species-specific AF, combined with an optimal enteral diet (eg, human milk), will improve adaptation during the transition from parenteral to enteral feeding in preterm neonates.

Topics: Amniotic Fluid; Animals; Animals, Newborn; Body Weight; Cell Line; Cell Movement; Cell Proliferation; Enteral Nutrition; Enterocolitis, Necrotizing; Enterocytes; Female; Humans; Inflammation; Interleukin-6; Interleukin-8; Male; Pregnancy; Premature Birth; Swine; Treatment Outcome

Only few hours of formula feeding may induce proinflammatory responses and predispose to necrotizing enterocolitis (NEC) in preterm pigs. We hypothesized that bovine colostrum, rich in bioactive factors, would improve intestinal function in preterm pigs following an initial exposure to formula feeding after some days of total parenteral nutrition (TPN).. After receiving TPN for 2 days, preterm pigs were fed formula (FORM, n = 14), bovine colostrum (COLOS, n = 6), or formula (6 h) followed by bovine colostrum (FCOLOS, n = 14). Intestinal lesions, function, and structure, abundance and location of bacteria, and inflammation markers were investigated.. NEC severity and interleukins (IL)-1β and -8 protein concentrations were lower, while villus height, galactose absorption, and brush-border enzyme activities were increased in the distal small intestine in COLOS and FCOLOS pigs, relative to FORM pigs. Intestinal gene expression of serum amyloid A, IL-1β, -6 and -8, and bacterial abundance, correlated positively with NEC severity of the distal small intestine.. Bovine colostrum restores intestinal function after initial formula-induced inflammation in preterm pigs. Further studies are required to test if bovine colostrum may also benefit preterm infants during the challenging transition from total parenteral nutrition to enteral nutrition, when human milk is unavailable.

Topics: Animals; Animals, Newborn; Cattle; Colostrum; Enterocolitis, Necrotizing; Gastrointestinal Tract; Inflammation; Interleukin-1beta; Interleukin-8; Intestinal Mucosa; Intestines; Milk Substitutes; Parenteral Nutrition, Total; Serum Amyloid A Protein; Sus scrofa

The purpose of this study is to characterize the cytokine response of preterm newborns with surgical necrotizing enterocolitis (NEC) or spontaneous intestinal perforation (SIP) before surgical treatment and to relate these finding to intestinal disease (NEC vs. SIP).. The study was a 14-month prospective, cohort study of neonates undergoing surgery or drainage for NEC or SIP or surgical ligation of patent ductus arteriosus (PDA). Multiplex cytokine detection technology was used to analyze six inflammatory markers: interleukin-2, interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1 β (IL-1β), interferon-gamma, and tumor necrosis factor-α (TNF-α).. Patients with NEC had much higher median preoperative levels of IL-6 (NEC: 8,381 pg/mL; SIP: 36 pg/mL; PDA: 25 pg/mL, p < 0.001), IL-8 (NEC: 18,438 pg/mL; SIP: 2,473 pg/mL; PDA: 1,110 pg/mL, p = 0.001), TNF-α (NEC: 161 pg/mL; SIP: 77 pg/mL; PDA: 71 pg/mL, p < 0.001), and IL-1β (NEC: 85 pg/mL; SIP: 31 pg/mL; PDA: 24 pg/mL, p = 0.001). Patients with NEC totalis (NEC-totalis had the highest levels of IL-8 and were significantly different from infants with limited NEC (28,141 vs. 11,429 pg/mL, p = 0.03).. Surgical NEC is a profoundly more proinflammatory disease than SIP. The cytokine profiles of patients with SIP are closer to those of a nonseptic surgical neonate.

Topics: Biomarkers; Cytokines; Ductus Arteriosus, Patent; Enterocolitis, Necrotizing; Female; Humans; Infant, Newborn; Interferon-gamma; Interleukin-1beta; Interleukin-6; Interleukin-8; Intestinal Perforation; Male; Premature Birth; Prospective Studies; Severity of Illness Index; Tumor Necrosis Factor-alpha

For preterm neonates, the quality of the first milk is crucial for intestinal maturation and resistance to necrotizing enterocolitis (NEC). Among other factors, milk quality is determined by the stage of lactation and processing. We hypothesized that unprocessed mature bovine milk (BM; raw bovine milk) would have less bioactivity than corresponding bovine colostrum (BC) in a preterm pig model, but have improved bioactivity relative to its homogenized, pasteurized, spray-dried equivalent, whole milk powder (WMP), or a bovine milk protein-based infant formula (IF). For 5 days, newborn preterm pigs received parenteral and enteral nutrition consisting of IF (n = 13), BM (n = 13), or BC (n = 14). In a second study, WMP (n = 15) was compared with IF (n = 10) and BM (n = 9). Compared with pigs fed IF, pigs that were fed BM had significantly improved intestinal structure (mucosal weight, villus height) and function (increased nutrient absorption and enzyme activities, decreased gut permeability, nutrient fermentation, and NEC severity). BC further improved these effects relative to BM (lactase activity, lactose absorption, plasma citrulline, and tissue interleukin-8). WMP induced similar effects as BM, except for lactase activity and lactose absorption. In conclusion, the maturational and protective effects on the immature intestine decreased in the order BC>BM>WMP, but all three intact bovine milk diets were markedly better than IF. The stage of lactation (colostrum vs. mature milk) and milk processing (e.g., homogenization, fractionation, pasteurization, spray-drying) are important factors in determining milk quality during the early postnatal period of preterm neonates.

Topics: Animals; Animals, Newborn; Cattle; Citrulline; Colostrum; Enterocolitis, Necrotizing; Feeding Methods; Female; Food Analysis; Food Quality; Interleukin-8; Intestinal Absorption; Intestinal Mucosa; Intestines; Lactase; Lactose; Milk; Models, Animal; Obstetric Labor, Premature; Permeability; Pregnancy; Swine

The purpose of this study was to evaluate the predictiveness of circulating interleukin (IL)-8 for 60-day mortality in premature infants with necrotizing enterocolitis (NEC).. NEC affects up to 5% of premature infants and remains a leading cause of mortality among neonates.. A total of 113 infants with surgically (n=50) or medically (n=63) treated NEC were retrospectively analyzed. Laboratory parameters including serum IL-8 were assessed at the diagnosis of NEC and during the preoperative workup.. The 60-day mortality was 19% (22/113), 10% (6/63) in medical and 33% (16/50) in surgical NEC. IL-8 levels significantly correlated with 60-day mortality (odds ratio: 1.38; CI 1.14-1.67; p=0.001). Median IL-8 levels at diagnosis were significantly higher in neonates who were later treated surgically (median=2625 pg/ml; range: 27-7500) compared with those treated medically (median=156 pg/ml; range: 5-7500; p<0.001). The AUC to discriminate between medical and surgical NEC was 0.82 (CI, 0.74-0.90), and an exploratory IL-8 cutoff point could be established at 1783 pg/ml (sensitivity of 90.5%; specificity of 59.2%).. Our findings that serum IL-8 (i) correlates directly with 60-day mortality and (ii) differs significantly between medically and surgically treated infants may change the process of therapeutic decision making in NEC.

Topics: Biomarkers; Diseases in Twins; Enterocolitis, Necrotizing; Female; Gestational Age; Hospital Mortality; Humans; Infant, Newborn; Infant, Premature; Infant, Premature, Diseases; Interleukin-8; Kaplan-Meier Estimate; Male; Odds Ratio; Platelet Count; Predictive Value of Tests; Prognosis; Vasoconstrictor Agents

The goal was to identify cytokines associated with necrotizing enterocolitis (NEC). Based on our earlier reports of decreased tissue expression of transforming growth factor (TGF)-β, we hypothesized that infants with NEC also have low blood TGF-β levels. We further hypothesized that because fetal inflammation increases the risk of NEC, infants who develop NEC have elevated blood cytokine levels in early neonatal period.. Data on 104 extremely-low-birth-weight infants with NEC and 893 without NEC from 17 centers were analyzed. Clinical information was correlated with blood cytokine levels on postnatal day 1 (D1), D3, D7, D14, and D21.. Male gender, non-Caucasian/non-African American ethnicity, sepsis, lower blood TGF-β and interleukin (IL)-2 levels, and higher IL-8 levels were associated with NEC. The NEC group had lower TGF-β levels than controls since D1. The diagnosis of NEC was associated with elevated IL-1β, IL-6, IL-8, IL-10, monocyte chemoattractant protein-1/CC-motif ligand-2, macrophage inflammatory protein-1β/CC-motif ligand-3, and C-reactive protein.. Clinical characteristics, such as gender and ethnicity, and low blood TGF-β levels are associated with higher risk of NEC. Infants who developed NEC did not start with high blood levels of inflammatory cytokines, but these rose mainly after the onset of NEC.

Topics: Biomarkers; Cytokines; Enterocolitis, Necrotizing; False Positive Reactions; Female; Humans; Infant, Extremely Low Birth Weight; Infant, Newborn; Infant, Premature; Inflammation; Interleukin-2; Interleukin-8; Male; Reproducibility of Results; Risk; Transforming Growth Factor beta

A balance between pro- and anti-inflammatory signals from milk and microbiota controls intestinal homeostasis just after birth, and an optimal balance is particularly important for preterm neonates that are sensitive to necrotizing enterocolitis (NEC). We suggest that the intestinal cytokine IL-8 plays an important role and hypothesize that transforming growth factor-β2 (TGF-β2) acts in synergy with bacterial lipopolysaccharide (LPS) to control IL-8 levels, thereby supporting intestinal homeostasis. Preterm pigs were fed colostrum (containing TGF-β2) or infant formula (IF) with or without antibiotics (COLOS, n = 27; ANTI, n = 11; IF, n = 40). Intestinal IL-8 levels and NEC incidence were much higher in IF than in COLOS and ANTI pigs (P < 0.001), but IL-8 levels did not correlate with NEC severity. Intestinal TGF-β2 levels were high in COLOS but low in IF and ANTI pigs. Based on these observations, the interplay among IL-8, TGF-β2, and LPS was investigated in a porcine intestinal epithelial cell line. TGF-β2 attenuated LPS-induced IL-6, IL-1β, and TNF-α release by reducing early ERK activation, whereas IL-8 secretion was synergistically induced by LPS and TGF-β2 via NF-κB. The TGF-β2/LPS-induced IL-8 levels stimulated cell proliferation and migration following epithelial injury, without continuous NF-κB activation and cyclooxygenase-2 expression. We suggest that a combined TGF-β2-LPS induction of IL-8 stimulates epithelial repair just after birth when the intestine is first exposed to colonizing bacteria and TGF-β2-containing milk. Moderate IL-8 levels may act to control intestinal inflammation, whereas excessive IL-8 production may enhance the damaging proinflammatory cascade leading to NEC.

Topics: Animals; Anti-Bacterial Agents; Cell Line; Cell Movement; Cell Proliferation; Colostrum; Disease Models, Animal; Enterocolitis, Necrotizing; Extracellular Signal-Regulated MAP Kinases; Gestational Age; Homeostasis; Humans; Infant Formula; Infant, Newborn; Interleukin-8; Intestine, Small; Lipopolysaccharides; NF-kappa B; Premature Birth; Signal Transduction; Swine; Time Factors; Transforming Growth Factor beta2

In recent years several potential biochemical markers have been evaluated to facilitate a reliable diagnosis of necrotizing enterocolitis (NEC), but none have made progress to clinical routine. We performed a comparative assessment in premature infants to evaluate the diagnostic value of the routinely available cytokine interleukin (IL)-8, and two promising experimental biomarkers, the gut barrier proteins liver fatty acid binding protein (L-FABP) and intestinal fatty acid binding protein (I-FABP), respectively, for the diagnosis of NEC.. IL-8, L-FABP, and I-FABP concentrations were analyzed in the serum of 15 infants with NEC and compared with 14 gestational age-matched infants serving as a control group.. Serum concentrations of I-FABP, L-FABP and IL-8 were significantly higher in infants with NEC compared with controls. IL-8 showed the highest diagnostic value with an area under the curve of 0.99, followed by L-FABP and I-FABP. In addition we found a significant correlation between IL-8 and both FABPs in infants with NEC.. Our results further advocate the possible role of IL-8 as a specific marker for NEC. The diagnostic value of IL-8 seems to be superior to I-FABP, and similar to L-FABP. The routinely availability facilitates IL-8 as a possible candidate for further clinical investigations.

Topics: Biomarkers; Enterocolitis, Necrotizing; Fatty Acid-Binding Proteins; Female; Humans; Infant; Infant, Newborn; Infant, Premature; Interleukin-8; Male; Prospective Studies

Preterm birth and formula feeding predispose to necrotizing enterocolitis (NEC) in infants. As mother's milk is often absent following preterm delivery, infant formula (IF) and human donor milk (HM) are frequently used as alternatives. We have previously shown that porcine and bovine colostrum (BC) provide similar NEC protection in preterm piglets relative to IF. We hypothesized that HM exerts similar effects and that this effect is partly species-independent. Preterm piglets (n = 40) received 2 days of total parenteral nutrition, followed by a rapid transition to full enteral feeding (15 ml·kg(-1)·2 h(-1)) for 2 days using BC (n = 13), HM (n = 13), or IF (n = 14). Intestinal passage time and hexose absorption were tested in vivo. Body and organ weights were recorded on day 5, and macroscopic NEC lesions in the gastrointestinal tract were assessed. Intestinal samples were collected for determination of histomorphology, histopathology, tissue IL-6 and IL-8, organic acids, bacterial adherence by fluorescence in situ hybridization score, and digestive enzyme activities. Relative to IF, pigs from BC and HM showed longer intestinal passage time; higher weight gain, hexose absorptive capacity, mucosal proportion, and enzyme activities; lower NEC incidence, organic acid concentration, and IL-8 concentration; and reduced histopathology lesions. Tissue IL-6 concentration and bacterial adherence score were lower for HM, relative to both BC and IF groups. We conclude that BC and HM are both superior to IF in stimulating gut structure, function, and NEC resistance in preterm piglets. BC may be a relevant alternative to HM when mother's milk is unavailable during the first week after preterm birth.

Topics: Animals; Animals, Newborn; Cattle; Colostrum; Disease Models, Animal; Enteral Nutrition; Enterocolitis, Necrotizing; Female; Humans; Incidence; Interleukin-6; Interleukin-8; Intestinal Mucosa; Intestines; Milk Banks; Milk, Human; Parenteral Nutrition, Total; Pregnancy; Pregnancy, Animal; Premature Birth; Swine; Swine Diseases

Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) play important roles in chronic intestinal inflammation. Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in preterm infants and is characterized by acute intestinal inflammation and necrosis. The objective of the study is to investigate the role of ER stress and the UPR in NEC patients.. Ileal tissues from NEC and control patients were obtained during surgical resection and/or at stoma closure. Splicing of XBP1 was detected using PCR, and gene expression was quantified using qPCR and Western blot.. Splicing of XBP1 was only detected in a subset of acute NEC (A-NEC) patients, and not in NEC patients who had undergone reanastomosis (R-NEC). The other ER stress and the UPR pathways, PERK and ATF6, were not activated in NEC patients. A-NEC patients showing XBP1 splicing (A-NEC-XBP1s) had increased mucosal expression of GRP78, CHOP, IL6 and IL8. Similar results were obtained by inducing ER stress and the UPR in vitro. A-NEC-XBP1s patients showed altered T cell differentiation indicated by decreased mucosal expression of RORC, IL17A and FOXP3. A-NEC-XBP1s patients additionally showed more severe morphological damage and a worse surgical outcome. Compared with A-NEC patients, R-NEC patients showed lower mucosal IL6 and IL8 expression and higher mucosal FOXP3 expression.. XBP1 splicing, ER stress and the UPR in NEC are associated with increased IL6 and IL8 expression levels, altered T cell differentiation and severe epithelial injury.

Topics: Blotting, Western; Cell Differentiation; DNA Primers; DNA-Binding Proteins; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Enterocolitis, Necrotizing; Female; Fluorescent Antibody Technique; Gene Expression Regulation; Humans; Immunohistochemistry; Infant, Newborn; Infant, Premature; Interleukin-6; Interleukin-8; Male; Polymerase Chain Reaction; Regulatory Factor X Transcription Factors; Statistics, Nonparametric; T-Lymphocytes; Transcription Factors; Unfolded Protein Response; X-Box Binding Protein 1

The aim of our study is to establish a reliable neonatal rat model by formula feeding only for evaluation of early surgical intervention on the course of experimental necrotizing enterocolitis (NEC).. Newborn Sprague-Dawley rats were divided into 50 breast-fed (group 1) and 38 formula fed (Similac/Esbilac, group 2) animals. The pups were sacrificed on the 4th, 5th, and 6th day of life and the terminal intestine examined for macroscopic and histologic changes as well as cytokine expression.. The histological mucosal damage was significantly higher of group 2 compared to group 1. The area of the vital mucosa of group 2 was significantly (58.57%, p<0.001) lower compared to group 1 (75.12%). The mRNA expression of the inflammatory cytokines IL-6, IL-8 and COX-2 was significantly 2-, 5- and 10-fold increased in group 2 compared to group 1.. Formula fed newborn rats displayed an inflammatory enterocolitis similar to human NEC. Our study demonstrates a significant loss of mucosa in animals with NEC having increased expression levels of IL-6, IL-8 and COX-2. Mucosal loss appears to be a distinct feature of experimental NEC and has to be correlated with the human disease.

Topics: Animals; Animals, Newborn; Body Weight; Cyclooxygenase 2; Disease Models, Animal; Enterocolitis, Necrotizing; Humans; Ileum; Infant; Infant Formula; Inflammation; Interleukin-6; Interleukin-8; Intestinal Mucosa; Milk; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; RNA, Messenger; Time Factors

Necrotizing enterocolitis (NEC), an extensive intestinal inflammatory disease of premature infants, is caused, in part, by an excessive inflammatory response to initial bacterial colonization due to the immature expression of innate immune response genes. In a randomized placebo-controlled clinical trial, supplementation of very low birth weight infants with probiotics significantly reduced the incidence of NEC. The primary goal of this study was to determine whether secreted products of these two clinically effective probiotic strains, Bifidobacterium infantis and Lactobacillus acidophilus, prevented NEC by accelerating the maturation of intestinal innate immune response genes and whether both strains are required for this effect. After exposure to probiotic conditioned media (PCM), immature human enterocytes, immature human intestinal xenografts, and primary enterocyte cultures of NEC tissue (NEC-IEC) were assayed for an IL-8 and IL-6 response to inflammatory stimuli. The latter two models were also assayed for innate immune response gene expression. In the immature xenograft, PCM exposure significantly attenuated LPS and IL-1β-induced IL-8 and IL-6 expression, decreased TLR2 mRNA and TLR4 mRNA, and increased mRNA levels of specific negative regulators of inflammation, SIGIRR and Tollip. In NEC-IEC, PCM decreased TLR2-dependent IL-8 and IL-6 induction and increased SIGIRR and Tollip expression. The attenuated inflammatory response with PCM was reversed with Tollip siRNA-mediated knockdown. The anti-inflammatory secreted factor is a 5- to 10-kDa molecule resistant to DNase, RNase, protease, heat stress, and acid exposure. B. infantis-conditioned media showed superior anti-inflammatory properties to that of L. acidophilus in immature human enterocytes, suggesting a strain specificity to this effect. We conclude that PCM promotes maturation of innate immune response gene expression, potentially explaining the protective effects of probiotics in clinical NEC.

Topics: Animals; Bifidobacterium; Cells, Cultured; Culture Media, Conditioned; Enterocolitis, Necrotizing; Enterocytes; Gene Expression Regulation; Humans; Immunity, Innate; Inflammation Mediators; Interleukin-6; Interleukin-8; Intestine, Small; Intracellular Signaling Peptides and Proteins; Lactobacillus acidophilus; Mice; Mice, SCID; Organ Culture Techniques; Primary Cell Culture; Probiotics; Receptors, Interleukin-1; RNA Interference; Toll-Like Receptor 2; Toll-Like Receptor 4

The aim of this study was to test the predictive value of interleukin (IL) 8 in the assessment of intestinal involvement in necrotizing enterocolitis (NEC).. Forty infants with surgically treated NEC were classified into 3 groups based on intestinal involvement during laparotomy: focal (n = 11), multifocal (n = 16), and panintestinal (n = 13). Preoperatively obtained serum levels of IL-8, C-reactive protein, white blood cell count, and platelet count were correlated with intestinal involvement using logistic regression models.. Interleukin 8 correlated significantly with intestinal involvement in infants with surgically treated NEC (odds ratio, 1.74; confidence interval, 1.27-2.39; P < .001). An exploratory IL-8 cutoff value of 449 pg/mL provided a specificity of 81.8% and sensitivity of 82.8% to discriminate focal from multifocal and panintestinal disease. An IL-8 cutoff value of 1388 pg/mL provided a specificity of 77.8% and a sensitivity of 76.9% to discriminate panintestinal disease from focal and multifocal disease.. To our knowledge, this is the first study to demonstrate a significant correlation of IL-8 with intestinal involvement in advanced NEC in a large patient population. Our results indicate that IL-8 may be a promising biomarker for assessing intestinal involvement in infants with advanced NEC.

Topics: Bacteremia; Biomarkers; C-Reactive Protein; Enterocolitis, Necrotizing; Female; Gestational Age; Humans; Infant; Infant, Newborn; Infant, Premature; Infant, Premature, Diseases; Interleukin-8; Intestines; Laparotomy; Leukocyte Count; Male; Pneumatosis Cystoides Intestinalis; Predictive Value of Tests; Preoperative Care; ROC Curve; Sensitivity and Specificity; Severity of Illness Index

The aim of this study was to analyze whether the mucosal innate immune response of extremely-low-birth-weight (ELBW) infants might play a role in the development of necrotizing enterocolitis (NEC).. Between April 2008 and December 2009 antimicrobial peptides were prospectively measured in fecal samples of ELBW infants. In cases requiring abdominal surgery, full-thickness gut biopsies were analyzed for expression of human β-defensin 2 (hBD2), interleukin-8 (IL-8), villin, MD2, and Toll-like receptor 4 (TLR4).. Fecal hBD1 concentrations were consistently low in all patients, whereas hBD2 concentrations were high in meconium, particularly in clinical chorioamnionitis, and then dropped, followed by a steady increase after day 14. Infants with moderate NEC showed significantly increased fecal hBD2 concentrations before clinical symptoms, in contrast to infants developing severe NEC. Analysis of intestinal resection material obtained from patients with severe NEC revealed low hBD2 mRNA and protein levels, and increased expression of the inflammatory cytokine IL-8.. High hBD2 concentrations, reflecting strong intestinal immune responses, were associated with moderate courses of the disease. In severe NEC, low hBD2 expression was accompanied by low TLR4/MD2 expression, suggesting an inadequate response to luminal bacteria, possibly predisposing those infants to the development of NEC.

Topics: Analysis of Variance; beta-Defensins; Biomarkers; Biopsy; Enterocolitis, Necrotizing; Feces; Female; Gene Expression Regulation; Germany; Gestational Age; Humans; Immunity, Mucosal; Infant, Extremely Low Birth Weight; Infant, Newborn; Interleukin-8; Intestinal Mucosa; Lymphocyte Antigen 96; Male; Meconium; Microfilament Proteins; Prevalence; Prognosis; Prospective Studies; RNA, Messenger; Severity of Illness Index; Time Factors; Toll-Like Receptor 4

Necrotizing enterocolitis (NEC) is a devastating neonatal intestinal inflammatory disease, occurring primarily in premature infants, causing significant morbidity and mortality. The pathogenesis of NEC is associated with an excessive inflammatory IL-8 response. In this study, we hypothesized that this excessive inflammatory response is related to an immature expression of innate immune response genes. To address this hypothesis, intestinal RNA expression analysis of innate immune response genes was performed after laser capture microdissection of resected ileal epithelium from fetuses, NEC patients and children and confirmed in ex vivo human intestinal xenografts. Changes in mRNA levels of toll-like receptors (TLR)-2 and -4, their signaling molecules and transcription factors (MyD88, TRAF-6 and NFκB1) and negative regulators (SIGIRR, IRAK-M, A-20 and TOLLIP) and the effector IL-8 were characterized by qRT-PCR. The expression of TLR2, TLR4, MyD88, TRAF-6, NFκB1 and IL-8 mRNA was increased while SIGIRR, IRAK-M, A-20 and TOLLIP mRNA were decreased in fetal vs. mature human enterocytes and further altered in NEC enterocytes. Similar changes in mRNA expression were observed in immature, but not mature, human intestinal xenografts. Confirmation of gene expression was also validated with selective protein measurements and with suggested evidence that immature TRL4 enterocyte surface expression was internalized in mature enterocytes. Cortisone, an intestinal maturation factor, treatment corrected the mRNA differences only in the immature intestinal xenograft. Using specific siRNA to attenuate expression of primary fetal enterocyte cultures, both TOLLIP and A-20 were confirmed to be important when knocked down by exhibiting the same excessive inflammatory response seen in the NEC intestine. We conclude that the excessive inflammatory response of the immature intestine, a hallmark of NEC, is due to a developmental immaturity in innate immune response genes.

Topics: Animals; Blotting, Western; Electrophoresis, Polyacrylamide Gel; Enterocolitis, Necrotizing; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gene Knockdown Techniques; Humans; Immunity, Innate; Infant, Newborn; Interleukin-8; Mice; RNA, Messenger; Toll-Like Receptor 2; Toll-Like Receptor 4

This pilot study in parenteral nutrition-dependent infants with short bowel syndrome (SBS) evaluated the impact of feeding route and intestinal permeability on bloodstream infection (BSI), small bowel bacterial overgrowth (SBBO), and systemic immune responses, as well as fecal calprotectin as a biomarker for SBBO.. Ten infants (ages 4.2-15.4 months) with SBS caused by necrotizing enterocolitis were evaluated. Nutritional assessment, breath hydrogen testing, intestinal permeability, fecal calprotectin, serum flagellin- and lipopolysaccharide-specific antibody titers, and proinflammatory cytokine concentrations (tumor necrosis factor-alpha [TNF-alpha], interleukin-1 beta, -6, and -8) were performed at baseline and at 60 and 120 days. Healthy, age-matched control subjects (n = 5) were recruited.. BSI incidence was high (80%), and SBBO was common (50%). SBBO increased the odds for BSI (>7-fold; P = .009). Calprotectin levels were higher in children with SBS and SBBO versus those without SBBO and healthy control subjects (P < .05). Serum TNF-alpha, was elevated at baseline versus controls. Serum TNF-alpha and interleukin-1 beta, -6, and -8 levels diminished with increased enteral nutrition. Anti-flagellin and anti-lipopolysaccharide immunoglobulin G levels in children with SBS were lower versus control subjects and rose over time.. In children with SBS, SBBO increases the risk for BSI, and systemic proinflammatory response decreases with increasing enteral feeding and weaning parenteral nutrition.

Topics: Enteral Nutrition; Enterocolitis, Necrotizing; Feces; Female; Flagellin; Humans; Incidence; Infant; Interleukin-1beta; Interleukin-6; Interleukin-8; Intestine, Small; Leukocyte L1 Antigen Complex; Male; Pilot Projects; Sepsis; Short Bowel Syndrome; Tumor Necrosis Factor-alpha

The purpose of this study was to determine cord blood cytokine levels and their relationship with morbidity and mortality in neonates with prolonged, premature rupture of membranes (PPROM). Forty two premature neonates of 29-35 weeks gestational age with PPROM exceeding 24 hours were considered as the PPROM group and simultaneously, 41 premature neonates without PPROM were considered as the control group. All the neonates were admitted to the Neonatology Unit for further evaluation of subsequent complications such as early neonatal sepsis, pneumonia, intraventicular haemorrhage (IVH), respiratory distress syndrome (RDS), necrotizing enterocolitis (NEC) and chronic lung disease (CLD). Cord blood and mothers' blood samples were obtained during delivery in both groups and tested for IL-6, IL-8 and TNF-alpha levels. Twenty one percent of patients with PPROM had histological chorioamnionitis. The risk for developing early neonatal sepsis increased significantly in neonates whose mothers had histological chorioamnionitis (p < 0.05). There was a statistically significant relationship between PPROM and risk of developing NEC (p < 0.05); no significant increase was seen as regards early neonatal sepsis, IVH, RDS, pneumonia, or BPD. The mean IL-8 levels in cord blood and mothers' serum were significantly higher in the PPROM group (p < 0.001, p< 0.005). In addition, IL-6 levels found in mothers' serum were significantly higher than those found in the control group (p < 0.01). However, levels in cord blood were similar (p > 0.05). TNF-alpha levels were similar in both groups (p > 0.05). Neonates who developed NEC had higher IL-8 levels in their cord blood when compared to those without NEC (p < 0.05). In conclusion, the presence of PPROM increases the risk of chorioamnionitis. In addition, PPROM increases the risk of NEC, and patients who developed NEC had significantly higher cord blood IL-8 values. We may conclude that patients with PPROM and higher IL-8 levels in cord blood might be considered as at possible risk of NEC.

Topics: Adult; Chorioamnionitis; Cytokines; Enterocolitis, Necrotizing; Female; Fetal Blood; Fetal Membranes, Premature Rupture; Humans; Infant, Newborn; Infant, Premature; Interleukin-6; Interleukin-8; Neonatology; Pregnancy; Risk; Sepsis

Necrotizing enterocolitis (NEC) is a major inflammatory disease of the premature human intestine that can be prevented by glucocorticoids if given prenatally before the 34th wk of gestation. This observation suggests that a finite period of steroid responsiveness exists as has been demonstrated in animal models. Human intestinal xenografts were used to determine whether a glucocorticoid responsive period exists in the developing human intestine. Developmental responsiveness was measured by lactase activity and inflammatory responsiveness by IL-8, IL-6, and monocyte chemotactic protein-1 (MCP-1) induction after an endogenous (IL-1 beta) or exogenous (LPS) proinflammatory stimulus, respectively. Functional development of ileal xenografts were monitored for 30 wk posttransplantation, and the lactase activity recapitulated that predicted by in utero development. Cortisone acetate accelerated the ontogeny of lactase at 20 wk (immature) but the effect was lost by 30 wk (mature) posttransplant. Concomitant with accelerated maturation, the IL-8 response to both IL-1 beta and LPS was significantly dampened (from 6- to 3-fold) by glucocorticoid pretreatment in the immature but not mature xenografts. The induction of IL-8 was reflected at the level of IL-8 mRNA, suggesting transcriptional regulation. The excessive activation of IL-8 in the immature gut was mediated by a prolonged activation of ERK and p38 kinases and nuclear translocation of NF-kappa B due to low levels of I kappa B. Steroid pretreatment in immature intestine dampens activation of all three signaling pathways in response to proinflammatory stimuli. Therefore, accelerating intestinal maturation by glucocorticoids within the responsive period by accelerating functional and inflammatory maturation may provide an effective preventive therapy for NEC.

Topics: Animals; Child; Child, Preschool; Cortisone; Enterocolitis, Necrotizing; Glucocorticoids; Humans; Ileum; Infant; Infant, Newborn; Inflammation; Interleukin-8; Mice; Mice, SCID; Signal Transduction; Transcription, Genetic; Transplantation, Heterologous

To investigate the role of macrophage migration inhibitory factor (MIF) and its downstream cytokine cascade in necrotizing enterocolitis (NEC).. The expression of MIF mRNA and protein in NEC guts was assayed by in situ hybridization and immunohistochemistry, respectively. Concentrations of MIF, interleukin (IL)-6 and IL-8 in the serum and in the supernatant of macrophage cultures were examined by ELISA. Increased expression of MIF mRNA and protein was observed in the NEC guts, mainly in the infiltrating macrophages in the mucosa and submucosal layers. Up-regulation of MIF was associated with the accumulation of macrophages and T cells. In addition, serum levels of MIF, IL-6 and IL-8 in NEC patients during the acute stage of the disease were significantly increased. The expression of MIF decreased both locally and systemically after the disease was resolved. MIF was also found to increase the secretion of IL-6 and IL-8 by macrophages isolated from healthy individuals in vitro in NEC.. MIF acts by stimulating macrophage production of IL-6 and IL-8. This further aggravates the inflammatory process by increasing the infiltration of neutrophils and activating inflammatory cells. The results of this study suggest that MIF plays an important role in the pathogenesis of NEC and may serve as a target for therapeutic intervention in NEC.

Topics: Acute Disease; Cells, Cultured; Culture Media, Conditioned; Enterocolitis, Necrotizing; Enzyme-Linked Immunosorbent Assay; Humans; Immunohistochemistry; In Situ Hybridization; Infant, Newborn; Interleukin-6; Interleukin-8; Macrophage Migration-Inhibitory Factors; Macrophages; RNA, Messenger; Up-Regulation

Neonatal necrotizing enterocolitis (NEC), which is a disease with a poor prognosis, is considered to be caused by the coincidence of intestinal ischemia-reperfusion injury and systemic inflammation due to the colonization of pathogenic bacteria. Interleukin (IL)-8, a proinflammatory cytokine, plays an important role in the pathophysiology of NEC. It was recently reported that IL-1beta activates the IL-8 gene by regulating the transcriptional nuclear factor kappaB (NF-kappaB) signaling pathways in intestinal cells. The protective role of maternal milk in NEC pathogenesis has been reported in both human and animal studies. In this study, we show that human breast milk dramatically suppressed the IL-1beta-induced activation of the IL-8 gene promoter by inhibiting the activation pathway of NF-kappaB. Moreover, we also show that human breast milk induced the production of IkappaBalpha. These results suggest that human breast milk could be protective and therapeutic in neonates with NEC by inhibiting the activation pathway of NF-kappaB.

Topics: Blotting, Western; Caco-2 Cells; Electrophoresis, Polyacrylamide Gel; Enterocolitis, Necrotizing; Enzyme-Linked Immunosorbent Assay; Gene Expression; Humans; Interleukin-1; Interleukin-8; Intestinal Mucosa; Milk, Human; NF-kappa B; Signal Transduction; Transcription, Genetic

Necrotizing enterocolitis (NEC) seems to result from the inflammatory response of an immature intestine. Human milk is protective against NEC via an unknown mechanism. We hypothesized that specific factors found in human milk would decrease stimulated IL-8 secretion in intestinal epithelial cells. HT29-cl19A and Caco2 cells were compared with the fetal human primary intestinal epithelial cell line H4 and temperature-sensitive conditionally immortalized fetal human intestinal (tsFHI) cells. Cells were pretreated with transforming growth factor-beta (TGF-beta), erythropoietin (Epo), IL-10, or epidermal growth factor (EGF) at physiologic concentrations before stimulation with tumor necrosis factor-alpha (TNF-alpha) or IL-1beta, and then IL-8 was measured by ELISA. The fetal cells produced significantly more IL-8 when stimulated by TNF-alpha or IL-1beta. There were also differences in the pattern of alteration of IL-8 secretion by human milk factors. In HT29-cl19A cells, IL-10 inhibited TNF-alpha-stimulated IL-8 secretion by 52%, and EGF increased secretion by 144%. In H4 cells, TGF-beta1 and Epo inhibited TNF-alpha-stimulated IL-8 secretion to control levels, and EGF increased secretion by 29%. IL-1beta-stimulated IL-8 secretion was inhibited 25% by TGF-beta1 in Caco2 cells and in H4 cells was inhibited by TGF-beta1, Epo, and TGF-beta2. TsFHI cells confirmed H4 cell results. Fetal human enterocytes have an exaggerated IL-8 secretion in response to TNF-alpha and IL-1beta. TGF-beta and Epo decrease this stimulated IL-8 secretion, which may partially explain the protective effect of human milk in NEC.

Topics: Age Factors; Caco-2 Cells; Enterocolitis, Necrotizing; Epidermal Growth Factor; Epithelial Cells; Erythropoietin; Fetus; HT29 Cells; Humans; In Vitro Techniques; Interleukin-1; Interleukin-10; Interleukin-8; Intestinal Mucosa; Milk, Human; Transforming Growth Factor beta

The authors have shown previously that surgical specimens from infants with acute necrotizing enterocolitis (NEC) show upregulation of inducible nitric oxide (NO) synthase (iNOS) and interferon-gamma mRNA. However, the contribution of other inflammatory cytokines such as interleukin-8 (IL-8), IL-11, and IL-12 has not been defined. Likewise, the role of GTP-cyclohydrolase, the rate-limiting enzyme in tetrahydrobiopterin synthesis, and thus NO production by iNOS is unclear. In this study, the authors sought to further define the pattern of cytokine expression seen in infants with acute NEC.. The authors measured intestinal cytokine mRNA expression by semiquantitative reverse transcriptase polymerase chain reaction in 21 infants with histologically confirmed NEC, 18 with other inflammatory conditions, and in 9 patients without intestinal inflammation. Guanosine triphosphate-cyclohydrolase (GTP-CH) activity was measured by specific enzyme assay. Univariate exact logistic regression analysis was performed to identify predictors of outcome.. IL-8 and IL-11 mRNA were upregulated in patients with acute NEC compared with those with other inflammatory conditions or those without disease; these levels returned to baseline at the time of stoma closure. Increased IL-11 mRNA decreased the likelihood of pan-necrosis (odds ratio, 0.93; P =.002). Increased IL-12 levels (but not IL-8) seemed to protect against pan-necrosis (odds ratio, 0.70; P =.06).. Local upregulation of IL-11 may represent an adaptive response designed to limit the extent of intestinal damage in NEC. Decreased IL-12 levels may contribute to the pathogenesis of NEC by allowing bacteria to escape host defenses.

Topics: Acute Disease; Analysis of Variance; Culture Techniques; Cytokines; Enterocolitis, Necrotizing; Female; Gene Expression Regulation; Genetic Markers; Guanosine Triphosphate; Humans; Immunohistochemistry; Infant; Infant, Newborn; Interleukin-11; Interleukin-12; Interleukin-8; Logistic Models; Male; Prognosis; Reference Values; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Severity of Illness Index

Necrotizing enterocolitis (NEC), a major cause of morbidity and mortality in premature infants, occurs after the introduction of oral feedings in conjunction with initial bacterial colonization of the gut and is hypothesized to be due to an immature (inappropriate) enterocyte response to bacterial stimuli. To test this hypothesis, we compared the enterocyte IL-8 response to inflammatory stimuli [lipopolysaccharide (LPS) and IL-1beta] in immature vs. mature human small intestine. Initial in vitro studies comparing confluent Caco-2 cells, a model for mature human enterocytes, with a primary human fetal intestinal cell line (H4 cells) demonstrated that after inflammatory stimulation fetal cells secreted more IL-8 (LPS, 8-fold; IL-1beta, 20-fold) than Caco-2 cells. IL-8 mRNA activity in fetal compared to Caco-2 cells was proportionately increased by the same magnitude with both stimuli. To validate the in vitro observations, small intestinal organ cultures from fetuses vs. older children were exposed to LPS and IL-1beta. Again in human organ cultures from fetuses compared to older children, IL-8 secretion was greater (LPS, 2.5-fold; IL-1beta, 200-fold) and mRNA activity after stimulation was comparably higher, suggesting that increased transcription of the IL-8 gene may account for the excessive response. Using immunohistochemical staining to identify the cellular source of IL-8, activity was noted predominantly in villous and crypt epithelium but also in a few immunoresponsive lymphoid cells. The observation that immature human enterocytes react with excessive pro-inflammatory cytokine production after inflammatory stimulation may help in part explain why prematures exposed to initial colonizing bacteria develop necrotizing enterocolitis.

Topics: Adenocarcinoma; Age Factors; Cell Line; Child; Colonic Neoplasms; Duodenum; Enterocolitis, Necrotizing; Gestational Age; Gram-Negative Bacteria; Humans; Inflammation; Interleukin-1; Interleukin-8; Intestinal Mucosa; Lipopolysaccharides; Organ Culture Techniques; RNA, Messenger; Tumor Cells, Cultured

To evaluate the relationship between the severity of necrotizing enterocolitis (NEC) and circulating concentrations of proinflammatory cytokines interleukin (IL)-1beta and IL-8 and counterinflammatory cytokines IL-1 receptor antagonist (IL-1ra) and IL-10. These cytokines have been associated with bowel injury or inflammation and may be released more slowly or later than previously examined cytokines. Also, to determine if any one of these cytokines will predict the eventual severity of NEC when measured at symptom onset.. Serial blood samples at onset, 8, 24, 48, and 72 hours were obtained from newborn infants with predefined signs and symptoms of NEC. Normal levels were defined from weight-, gestation-, and age-matched controls. Concentrations of the four cytokines were determined by enzyme-linked immunosorbent assay and compared throughout the time period by stage of NEC, using sepsis as a co-factor. Mean concentrations of each cytokine at onset were compared with the controls. Threshold values were obtained with the best combination of high sensitivity and high specificity for defining stage 1 NEC or for diagnosing stage 3 NEC at onset.. There were 12 cases of stage 1, 18 cases of stage 2, and 6 cases of stage 3 NEC included in the study, as well as 20 control infants. Concentrations of IL-8 and IL-10 were significantly higher in infants with stage 3 NEC from onset through 24 hours compared with infants with less severe NEC. At onset, concentrations of all four cytokines were significantly higher in stage 3 NEC. To identify, at onset, the infants with a final diagnosis of stage 3 NEC, an IL-1ra concentration of >130 000 pg/mL had a sensitivity of 100% and a specificity of 92%. At 8 hours, an IL-10 concentration of >250 pg/mL had a sensitivity of 100% and a specificity of 90% in identifying stage 3 NEC in infants with symptoms suggestive of NEC at onset.. The severity of NEC and its systemic signs and symptoms are not due to a deficiency of counterregulatory cytokines. In fact, mean concentrations of IL-1ra in NEC are higher than what has been reported in other populations. The cytokines IL-8, IL-1ra, and IL-10 are released later or more slowly after a stimulus and may be more useful in identifying, within hours of symptom onset, which infant will develop significant NEC.

Topics: Enterocolitis, Necrotizing; Humans; Infant, Newborn; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Interleukin-10; Interleukin-8; Interleukins; Prognosis; Receptors, Interleukin-1; Severity of Illness Index; Sialoglycoproteins