RESUMEN
Inhibition of P300 acetyltransferase activity by specific inhibitor C646 has been shown to improve insulin signaling. However, the underlying molecular mechanism of this improvement remains unclear. In this study, we analyzed P300 levels of obese patients and found that they were significantly increased in liver hepatocytes. In addition, large amounts of P300 appeared in the cytoplasm. Inhibition of P300 acetyltransferase activity by C646 drastically increased tyrosine phosphorylation of the insulin receptor protein substrates (IRS1/2) without affecting the tyrosine phosphorylation of the beta subunit of the insulin receptor (IRß) in hepatocytes in the absence of insulin. Since IRS1/2 requires membrane translocation and binding to inositol compounds for normal functions, we also examined the role of acetylation on binding to phosphatidylinositol(4,5)P2 and found that IRS1/2 acetylation by P300 reduced this binding. In contrast, we show that inhibition of IRS1/2 acetylation by C646 facilitates IRS1/2 membrane translocation. Intriguingly, we demonstrate that C646 activates IRß's tyrosine kinase activity and directly promotes IRß interaction with IRS1/2, leading to the tyrosine phosphorylation of IRS1/2 and subsequent activation of insulin signaling even in the absence of insulin. In conclusion, these data reveal the unique effects of C646 in activating insulin signaling in patients with obesity and diabetes.
Asunto(s)
Benzoatos , Inhibidores Enzimáticos , Proteínas Sustrato del Receptor de Insulina , Nitrobencenos , Pirazolonas , Receptor de Insulina , Factores de Transcripción p300-CBP , Benzoatos/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Nitrobencenos/farmacología , Fosforilación , Pirazolonas/farmacología , Receptor de Insulina/metabolismo , Tirosina/metabolismo , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants and an orphan disease with no specific treatment. Most patients with confirmed NEC develop moderate-severe thrombocytopenia requiring one or more platelet transfusions. Here we used our neonatal murine model of NEC-related thrombocytopenia to investigate mechanisms of platelet depletion associated with this disease [K. Namachivayam, K. MohanKumar, L. Garg, B. A. Torres, A. Maheshwari, Pediatr. Res. 81, 817-824 (2017)]. In this model, enteral administration of immunogen trinitrobenzene sulfonate (TNBS) in 10-d-old mouse pups produces an acute necrotizing ileocolitis resembling human NEC within 24 h, and these mice developed thrombocytopenia at 12 to 15 h. We hypothesized that platelet activation and depletion occur during intestinal injury following exposure to bacterial products translocated across the damaged mucosa. Surprisingly, platelet activation began in our model 3 h after TNBS administration, antedating mucosal injury or endotoxinemia. Platelet activation was triggered by thrombin, which, in turn, was activated by tissue factor released from intestinal macrophages. Compared to adults, neonatal platelets showed enhanced sensitivity to thrombin due to higher expression of several downstream signaling mediators and the deficiency of endogenous thrombin antagonists. The expression of tissue factor in intestinal macrophages was also unique to the neonate. Targeted inhibition of thrombin by a nanomedicine-based approach was protective without increasing interstitial hemorrhages in the inflamed bowel or other organs. In support of these data, we detected increased circulating tissue factor and thrombin-antithrombin complexes in patients with NEC. Our findings show that platelet activation is an important pathophysiological event and a potential therapeutic target in NEC.
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Enterocolitis Necrotizante/metabolismo , Enterocolitis Necrotizante/patología , Enfermedades del Recién Nacido/metabolismo , Trombina/metabolismo , Animales , Animales Recién Nacidos , Plaquetas/metabolismo , Modelos Animales de Enfermedad , Humanos , Recién Nacido , Inflamación/metabolismo , Enfermedades Intestinales/patología , Intestinos/lesiones , Intestinos/patología , Macrófagos/metabolismo , Ratones , Trombocitopenia/metabolismoRESUMEN
OBJECTIVE: The study aimed to determine the association of surgical necrotizing enterocolitis (NEC) and its timing, with the development and timing of retinopathy of prematurity (ROP). STUDY DESIGN: This was a secondary data analysis of 7,483 preterm infants from the Postnatal Growth and Retinopathy of Prematurity Study. Associations between infants with surgical NEC, early-onset surgical NEC (8-28 days), and late-onset surgical NEC (over 28 days) with ROP were evaluated by using multivariable logistic regression models, controlling for birth weight, gestational age, small for gestational age status, chronic lung disease, intraventricular hemorrhage, hydrocephalus, patent ductus arteriosus, and periventricular leukomalacia. RESULTS: Three hundred fifty-six (4.8%) infants had surgical NEC, with 56% having early surgical NEC. Infants with surgical NEC had a higher risk of any ROP and severe ROP (adjusted odds ratio [OR]: 2.7; 95% CI: 1.9-3.7) and 2.5 (95% CI: 1.9-3.3), respectively; p < 0.001) compared with infants without surgical NEC. Infants with early surgical NEC were at the highest risk of developing ROP and severe ROP (adjusted OR: 3.1 [95% CI: 2.1-4.8], and 3.3 [95% CI: 2.3-4.7] respectively, p < 0.001). Infants with late surgical NEC were also at increased risk of developing ROP and severe ROP (adjusted OR: 2.1 [95% CI: 1.3-3.4], and 1.9 [95% CI: 1.3-2.8] respectively, p < 0.001) compared with infants without surgical NEC. CONCLUSION: Infants with surgical NEC, especially early surgical NEC, are at higher risk of ROP and severe ROP. KEY POINTS: · Infants with surgical NEC are at higher risk of ROP and severe ROP than those without surgical NEC.. · Increased ROP risk is seen in infants with both early- or later onset surgical NEC.. · Early-onset surgical NEC is associated with a higher ROP risk compared with later onset surgical NEC..
Asunto(s)
Enterocolitis Necrotizante , Retinopatía de la Prematuridad , Lactante , Femenino , Recién Nacido , Humanos , Recien Nacido Prematuro , Recién Nacido de Bajo Peso , Enterocolitis Necrotizante/epidemiología , Enterocolitis Necrotizante/cirugía , Retinopatía de la Prematuridad/epidemiología , Retinopatía de la Prematuridad/cirugía , Edad GestacionalRESUMEN
Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants and is a leading cause of morbidity and mortality in infants born between 23 and 28 weeks of gestation. Fifty to 95% of all infants with NEC develop thrombocytopenia (platelet counts <150 × 109/L) within 24-72 h of receiving this diagnosis. In many patients, thrombocytopenia is severe and is treated with one or more platelet transfusions. However, the underlying mechanism(s) and biological implications of NEC-related thrombocytopenia remain unclear. This review presents current evidence from human and animal studies on the clinical features and mechanisms of platelet depletion in NEC. Anecdotal clinical experience is combined with evidence from laboratory studies and from an extensive literature search in databases PubMed, EMBASE, and Scopus and the electronic archives of abstracts presented at the annual meetings of the Pediatric Academic Societies. To avoid bias in identification of existing studies, key words were short-listed prior to the actual search both from anecdotal experience and from PubMed's Medical Subject Heading (MeSH) thesaurus. IMPACT: Fifty to 95% of infants with necrotizing enterocolitis (NEC) develop idiopathic thrombocytopenia (platelet counts <150 × 109/L) within 24-72 h of disease onset. Early clinical trials suggest that moderate thrombocytopenia may be protective in human NEC, although further work is needed to fully understand this relationship. We have developed a neonatal murine model of NEC-related thrombocytopenia, where enteral administration of an immunological stimulant, trinitrobenzene sulfonate, on postnatal day 10 induces an acute necrotizing ileocolitis resembling human NEC. In this murine model, thrombocytopenia is seen at 15-18 h due to platelet consumption and mild-moderate thrombocytopenia is protective.
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Plaquetas/fisiología , Enterocolitis Necrotizante/sangre , Trombocitopenia/etiología , Animales , Plaquetas/efectos de los fármacos , Enterocolitis Necrotizante/complicaciones , Humanos , Recién Nacido , Enfermedades del Recién Nacido/sangre , Ratones , Activación Plaquetaria , Transfusión de Plaquetas , Trombina/farmacología , Trombocitopenia/terapiaRESUMEN
In the fetus and the neonate, altered macrophage function has been implicated not only in inflammatory disorders but also in developmental abnormalities marked by altered onset, interruption, or imbalance of key structural changes. The developmental role of macrophages were first noted nearly a century ago, at about the same time when these cells were being identified as central effectors in phagocytosis and elimination of microbes. Since that time, we have made considerable progress in understanding the diverse roles that these cells play in both physiology and disease. Here, we review the role of fetal and neonatal macrophages in immune surveillance, innate immunity, homeostasis, tissue remodeling, angiogenesis, and repair of damaged tissues. We also discuss the possibility of therapeutic manipulation of the relative abundance and activation status of macrophage subsets in various diseases. This article combines peer-reviewed evidence from our own studies with results of an extensive literature search in the databases PubMed, EMBASE, and Scopus. IMPACT: We have reviewed the structure, differentiation, and classification of macrophages in the neonatal period. Neonatal macrophages are derived from embryonic, hepatic, and bone marrow precursors. Macrophages play major roles in tissue homeostasis, innate immunity, inflammation, tissue repair, angiogenesis, and apoptosis of various cellular lineages in various infectious and inflammatory disorders. Macrophages and related inflammatory mediators could be important therapeutic targets in several neonatal diseases.
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Desarrollo Fetal , Recién Nacido/crecimiento & desarrollo , Macrófagos/citología , Diferenciación Celular , HumanosRESUMEN
Integrins are heterodimeric transmembrane cell adhesion molecules made up of alpha (α) and beta (ß) subunits arranged in numerous dimeric pairings. These complexes have varying affinities to extracellular ligands. Integrins regulate cellular growth, proliferation, migration, signaling, and cytokine activation and release and thereby play important roles in cell proliferation and migration, apoptosis, tissue repair, as well as in all processes critical to inflammation, infection, and angiogenesis. This review presents current evidence from human and animal studies on integrin structure and molecular signaling, with particular emphasis on signal transduction in infants. We have included evidence from our own laboratory studies and from an extensive literature search in databases PubMed, EMBASE, Scopus, and the electronic archives of abstracts presented at the annual meetings of the Pediatric Academic Societies. To avoid bias in identification of existing studies, key words were short-listed prior to the actual search both from anecdotal experience and from PubMed's Medical Subject Heading (MeSH) thesaurus. IMPACT: Integrins are a family of ubiquitous αß heterodimeric receptors that interact with numerous ligands in physiology and disease. Integrins play a key role in cell proliferation, tissue repair, inflammation, infection, and angiogenesis. This review summarizes current evidence from human and animal studies on integrin structure and molecular signaling and promising role in diseases of inflammation, infection, and angiogenesis in infants. This review shows that integrin receptors and ligands are novel therapeutic targets of clinical interest and hold promise as novel therapeutic targets in the management of several neonatal diseases.
Asunto(s)
Inflamación/fisiopatología , Integrinas/fisiología , Neovascularización Patológica , Animales , Niño , Humanos , Recién Nacido , Recien Nacido Prematuro , Ligandos , Unión ProteicaRESUMEN
BACKGROUND: Infants with advanced necrotizing enterocolitis (NEC) often need surgical resection of necrotic bowel. We hypothesized that incomplete resection of NEC lesions, signified by the detection of necrotic patches in margins of resected bowel loops, results in inferior clinical outcomes. METHODS: We reviewed the medical records of infants with surgical NEC in the past 15 years for demographic, clinical, and histopathological data. We also developed statistical models to predict mortality and hospital stay. RESULTS: Ninety infants with surgical NEC had a mean (±standard error) gestational age of 27.3 ± 0.4 weeks, birth weight 1008 ± 48 g, NEC onset at 25.2 ± 2.4 days, and resected bowel length of 29.2 ± 3.2 cm. Seventeen (18.9%) infants who had complete resection of the necrosed bowel had fewer (4; 23.5%) deaths and shorter lengths of hospital stay. In contrast, a group of 73 infants with some necrosis within the margins of resected bowel had significantly more (34; 46.6%) deaths and longer hospital stay. The combination of clinical and histopathological data gave better regression models for mortality and hospital stay. CONCLUSION: In surgical NEC, incomplete resection of necrotic bowel increased mortality and the duration of hospitalization. Regression models combining clinical and histopathological data were more accurate for mortality and the length of hospital stay. IMPACT: In infants with surgical NEC, complete resection of necrotic bowel reduced mortality and hospital stay. Regression models combining clinical and histopathological information were superior at predicting mortality and hospital stay than simpler models focusing on either of these two sets of data alone. Prediction of mortality improved with the combination of antenatal steroids, chorioamnionitis, and duration of post-operative ileus, with severity of inflammation and hemorrhages in resected intestine. Length of hospital stay was shorter in infants with higher gestational ages, but longer in those with greater depth of necrosis or needing prolonged parenteral nutrition or supervised feedings.
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Procedimientos Quirúrgicos del Sistema Digestivo/mortalidad , Enterocolitis Necrotizante/mortalidad , Enterocolitis Necrotizante/cirugía , Intestinos/cirugía , Procedimientos Quirúrgicos del Sistema Digestivo/efectos adversos , Enterocolitis Necrotizante/patología , Femenino , Edad Gestacional , Mortalidad Hospitalaria , Humanos , Recién Nacido , Recien Nacido Prematuro , Intestinos/patología , Tiempo de Internación , Masculino , Márgenes de Escisión , Necrosis , Nutrición Parenteral , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Factores de Tiempo , Resultado del TratamientoRESUMEN
Anemia is a frequent diagnosis in critically ill infants, but the clinical implications of severe anemia in these patients remain unclear. In this study, we examined preweaned mice to investigate the effects of severe anemia during early infancy on gut mucosal permeability. C57BL/6 mice were subjected to timed phlebotomy between postnatal days (P) 2-10 to induce severe anemia (hematocrits 20%-24%), and intestinal permeability was tracked longitudinally between P10 and P20 as intestine-to-plasma translocation of enteral macromolecules and bacterial translocation. Epithelial junctions were evaluated by electron microscopy, polymerase chain reactions, immunohistochemistry, and/or enzyme immunoassays on intestinal tissues, Caco-2 intestinal epithelial-like cells, and colonic organoids. Preweaned mouse pups showed an age-related susceptibility to severe anemia, with increased intestinal permeability to enteral macromolecules (dextran, ovalbumin, ß-lactoglobulin) and luminal bacteria. Electron micrographs showed increased paracellular permeability and ultrastructural abnormalities of the adherens junctions. These findings were explained by the loss of E-cadherin in epithelial cells, which was caused by destabilization of the E-cadherin (Cdh1) mRNA because of microRNA let-7e-5p binding to the 3'-untranslated region. Severe anemia resulted in a disproportionate and persistent increase in intestinal permeability in preweaned mice because of the disruption of epithelial adherens junctions. These changes are mediated via microRNA let-7e-mediated depletion of Cdh1 mRNA.NEW & NOTEWORTHY This research article shows that newborn infants with severe anemia show an age-related susceptibility to developing increased intestinal permeability to ingested macromolecules. This abnormal permeability develops because of abnormalities in intestinal epithelial junctions caused by a deficiency of the molecule E-cadherin in epithelial cells. The deficiency of E-cadherin is caused by destabilization of its mRNA precursor because of increased expression and binding of another molecule, the microRNA let-7e-5p, to the E-cadherin mRNA.
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Uniones Adherentes/patología , Anemia Neonatal/patología , Mucosa Intestinal/patología , Intestinos/patología , Uniones Adherentes/ultraestructura , Animales , Animales Recién Nacidos , Células CACO-2 , Cadherinas/genética , Cadherinas/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , MicroARNs/metabolismo , Permeabilidad , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
In recent years, several studies have shown that premature infants who develop NEC frequently display enteric dysbiosis with increased Gram-negative bacteria for several days to weeks prior to NEC onset. The importance of these findings, for the possibility of a causal role of these bacteria in NEC pathogenesis, and for potential value of gut dysbiosis as a biomarker of NEC, is well-recognized. In this review, we present current evidence supporting the association between NEC in premature infants and enteric dysbiosis, and its evaluation using the Bradford Hill criteria for causality. To provide an objective appraisal, we developed a novel scoring system for causal inference. Despite important methodological and statistical limitations, there is support for the association from several large studies and a meta-analysis. The association draws strength from strong biological plausibility of a role of Gram-negative bacteria in NEC and from evidence for temporality, that dysbiosis may antedate NEC onset. The weakness of the association is in the low level of consistency across studies, and the lack of specificity of effect. There is a need for an improved definition of dysbiosis, either based on a critical threshold of relative abundances or at higher levels of taxonomic resolution.
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Disbiosis , Enterocolitis Necrotizante/microbiología , Microbioma Gastrointestinal , Recien Nacido Prematuro , Intestinos/microbiología , Enterocolitis Necrotizante/diagnóstico , Edad Gestacional , Humanos , Recién Nacido , Medición de Riesgo , Factores de RiesgoRESUMEN
BACKGROUND: Prior studies have suggested an association between platelet transfusions (PTXs) and worse outcomes among infants with necrotizing enterocolitis (NEC), potentially mediated by proinflammatory factors released by platelets. However, the effects of storage on platelet proinflammatory factor release and the confounding role of illness severity on NEC outcomes have not been determined. STUDY DESIGN AND METHODS: First, neuropeptide Y (a potent splanchnic vasoconstrictor released by platelets) was measured by enzyme-linked immunosorbent assay in fresh frozen plasma and in the supernatant of leukoreduced apheresis-derived platelets at different times during storage. Next, we evaluated the relationship between PTX rates and death in a multicenter cohort of very-low-birth-weight infants with NEC, adjusting for illness severity. RESULTS: Neuropeptide Y levels increased over time in the supernatant of leukoreduced apheresis-derived platelets and were 4.4-fold and 8.9-fold higher than in fresh frozen plasma on Days 2 and 3 of storage, respectively (p < 0.001). Among 598 very-low-birth-weight infants, 44 developed NEC. In unadjusted analysis, PTX rate was 30.3 (95% confidence interval [CI], 11.5-80.1) per 100 infant-days among infants who died, compared to 6.0 (95% CI, 3.2-11.2) among survivors (incidence rate ratio, 5.1; 95% CI, 1.6-16.2; p = 0.006). In multivariable analysis, there was no association between PTX rate and mortality (incidence rate ratio, 3.0; 95% CI, 0.6-15.0; p = 0.18), although estimation was imprecise. CONCLUSION: Proinflammatory mediators accumulate in platelet suspensions during storage. Although PTX rates were not associated with increased mortality among infants with NEC in our study, our estimates suggest the potential for such an association that needs evaluation in larger studies.
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Enterocolitis Necrotizante/mortalidad , Enterocolitis Necrotizante/terapia , Transfusión de Plaquetas , Peso al Nacer , Enterocolitis Necrotizante/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Edad Gestacional , Humanos , Lactante , Recién Nacido , Recien Nacido Prematuro , Recién Nacido de muy Bajo Peso , Masculino , Neuropéptido Y/metabolismo , Factores de RiesgoRESUMEN
BACKGROUND: Premature infants often develop enteric dysbiosis with a preponderance of Gammaproteobacteria, which has been related to adverse clinical outcomes. We investigated the relationship between increasing fecal Gammaproteobacteria and mucosal inflammation, measured by fecal calprotectin (FC). METHODS: Stool samples were collected from very-low-birth weight (VLBW) infants at ≤2, 3, and 4 weeks' postnatal age. Fecal microbiome was surveyed using polymerase chain reaction amplification of the V4 region of 16S ribosomal RNA, and FC was measured by enzyme immunoassay. RESULTS: We enrolled 45 VLBW infants (gestation 27.9 ± 2.2 weeks, birth weight 1126 ± 208 g) and obtained stool samples at 9.9 ± 3, 20.7 ± 4.1, and 29.4 ± 4.9 days. FC was positively correlated with the genus Klebsiella (r = 0.207, p = 0.034) and its dominant amplicon sequence variant (r = 0.290, p = 0.003), but not with the relative abundance of total Gammaproteobacteria. Klebsiella colonized the gut in two distinct patterns: some infants started with low Klebsiella abundance and gained these bacteria over time, whereas others began with very high Klebsiella abundance. CONCLUSION: In premature infants, FC correlated with relative abundance of a specific pathobiont, Klebsiella, and not with that of the class Gammaproteobacteria. These findings indicate a need to define dysbiosis at genera or higher levels of resolution.
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Disbiosis/diagnóstico , Complejo de Antígeno L1 de Leucocito/análisis , Peso al Nacer , Disbiosis/microbiología , Enterocolitis Necrotizante/microbiología , Heces/química , Femenino , Gammaproteobacteria/aislamiento & purificación , Microbioma Gastrointestinal , Edad Gestacional , Humanos , Lactante , Recien Nacido Extremadamente Prematuro , Recién Nacido , Recien Nacido Prematuro , Enfermedades del Prematuro , Recién Nacido de muy Bajo Peso , Inflamación , Unidades de Cuidado Intensivo Neonatal , Infecciones por Klebsiella/diagnóstico , Masculino , Estudios Prospectivos , ARN Ribosómico 16S , Curva ROC , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Hyperglycemia is common in extremely low gestational age newborns (ELGAN) and is associated with increased mortality and morbidity, including abnormal neurodevelopment. Hippocampus-mediated cognitive deficits are common in this population, but the specific effects of hyperglycemia on the developing hippocampus are not known. METHODS: The objective of this study was to determine the acute and long-term effects of hyperglycemia on the developing hippocampus in neonatal rats using a streptozotocin (STZ)-induced model of hyperglycemia. STZ was injected on postnatal day (P) 2, and littermates in the control group were injected with an equivalent volume of citrate buffer. The acute effects of hyperglycemia on markers of oxidative stress, inflammatory cytokines, microglial activation, and reactive astrocytosis in the hippocampus were determined in the brain tissue collected on P6. The long-term effects on hippocampus-mediated behavior and hippocampal dendrite structure were determined on P90. RESULTS: On P6, the transcript and protein expression of markers of oxidative stress and inflammatory cytokines, including the CXCL10/CXCR3 pathway, were upregulated in the hyperglycemia group. Histological evaluation revealed microglial activation and astrocytosis. The long-term assessment on P90 demonstrated abnormal performance in Barnes maze neurobehavioral testing and altered dendrite structure in the hippocampus of formerly hyperglycemic rats. CONCLUSIONS: Neonatal hyperglycemia induces CXCL10/CXCR3 signaling, microglial activation, and astrocytosis in the rat hippocampus and alters long-term synaptogenesis and behavior. These results may explain the hippocampus-specific cognitive deficits common in ELGAN who experience neonatal hyperglycemia.
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Quimiocina CXCL10/metabolismo , Hipocampo/patología , Hiperglucemia/fisiopatología , Microglía/patología , Receptores CXCR3/metabolismo , Transducción de Señal/fisiología , Sinapsis/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Antibióticos Antineoplásicos/toxicidad , Antígenos CD11/metabolismo , Discapacidades del Desarrollo/etiología , Homólogo 4 de la Proteína Discs Large/metabolismo , Encefalitis/etiología , Femenino , Hiperglucemia/inducido químicamente , Hiperglucemia/complicaciones , Hiperglucemia/metabolismo , Masculino , Aprendizaje por Laberinto , Microglía/efectos de los fármacos , Microglía/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Estreptozocina/toxicidad , Sinapsis/efectos de los fármacosRESUMEN
BackgroundTwin studies suggest that genetic factors may account for up to 50% increased risk for necrotizing enterocolitis (NEC), but genome-wide association studies for NEC are lacking.MethodsGenotyping was done on Illumina BeadChip, followed by analysis using PLINK with logistic regression under an additive model.ResultsAmong 751 extremely-low-birth-weight (<1,000 g, >401 g) neonates, 30 had surgical NEC. Two hundred and sixty-one single-nucleotide polymorphisms (SNPs) showed association with NEC at P<0.05, of which 35 were significant at P<10-7. Minor allele(s) in a cluster of SNPs spanning a 43-kb region of chromosome 8 (8q23.3) conferred an odds ratio of 4.72 (95% confidence interval (CI): 2.51-8.88) for elevated risk of NEC. Two smaller clusters on chromosome 14 and chromosome 11 exhibited P values of 10-7-10-8. The chromosome 8 cluster is in an intergenic region between CUB and Sushi multiple domains 3 (-1.43 Mb) and trichorhinophalangeal syndrome I (+542 kb). RNA sequencing in this region identified a potential novel open-reading frame corresponding to a long interspersed element-1 retrotransposable element.ConclusionGenetic variation in an intergenic region of chromosome 8 is associated with increased risk for NEC with a mechanism that is yet to be identified.
Asunto(s)
Cromosomas Humanos Par 8 , ADN Intergénico , Enterocolitis Necrotizante/diagnóstico , Enterocolitis Necrotizante/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Estudios de Cohortes , Enterocolitis Necrotizante/cirugía , Femenino , Frecuencia de los Genes , Variación Genética , Genotipo , Humanos , Lactante , Recien Nacido con Peso al Nacer Extremadamente Bajo , Recién Nacido , Enfermedades del Recién Nacido , Recien Nacido Prematuro , Elementos de Nucleótido Esparcido Largo , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Polimorfismo de Nucleótido Simple , Respiración Artificial , Análisis de Secuencia de ARN , Transducción de SeñalRESUMEN
BACKGROUND: Thrombocytopenia is frequently encountered in infants with necrotizing enterocolitis (NEC). To develop a preclinical model of NEC-related thrombocytopenia, we measured serial platelet counts in 10-d-old (P10) mouse pups with trinitrobenzene sulfonic acid (TNBS)-induced NEC-like injury. We also measured platelet volume indices, immature platelet fraction (IPF), and megakaryocyte number/ploidy in these animals. METHODS: Platelet counts, platelet volume indices, and IPF were measured in control (N = 65) and TNBS-treated pups (N = 104) using an automated hematology analyzer. Bone marrow megakaryocyte number, ploidy and CD41 expression were measured by flow cytometry. These findings were confirmed in a small cohort of P3 mice with NEC-like injury. RESULTS: Murine pups with TNBS-mediated NEC-like injury developed thrombocytopenia at 15-24 h after exposure to TNBS. Intestinal injury was associated with increased platelet volume indices (mean platelet volume, platelet-to-large cell ratio, and platelet distribution width), and IPF, indicating increased thrombopoiesis. These mice also showed increased megakaryocyte number, ploidy, and CD41 expression, indicating increased megakaryocyte differentiation. CONCLUSION: Similar to human NEC, murine NEC-like injury was also associated with decreased platelet counts. There was evidence of increased megakaryocyte differentiation and thrombopoiesis, which favors peripheral consumption of platelets as the likely mechanism of thrombocytopenia in these animals, over decreased platelet production.
Asunto(s)
Plaquetas , Enterocolitis Necrotizante/sangre , Megacariocitos , Trombocitopenia/sangre , Trombopoyesis , Animales , Animales Recién Nacidos , Biomarcadores/sangre , Plaquetas/metabolismo , Plaquetas/patología , Modelos Animales de Enfermedad , Enterocolitis Necrotizante/inducido químicamente , Enterocolitis Necrotizante/patología , Intestinos/patología , Volúmen Plaquetario Medio , Megacariocitos/metabolismo , Megacariocitos/patología , Ratones Endogámicos C57BL , Recuento de Plaquetas , Glicoproteína IIb de Membrana Plaquetaria/sangre , Ploidias , Trombocitopenia/etiología , Trombocitopenia/patología , Factores de Tiempo , Ácido TrinitrobencenosulfónicoRESUMEN
BACKGROUND: We have shown previously that enteral administration of 2, 4, 6-trinitrobenzene sulfonic acid in 10-d-old C57BL/6 pups produces an acute necrotizing enterocolitis with histopathological and inflammatory changes similar to human necrotizing enterocolitis (NEC). To determine whether murine neonatal 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-mediated intestinal injury could be used as a NEC model, we compared gene expression profiles of TNBS-mediated intestinal injury and NEC. METHODS: Whole-genome microarray analysis was performed on proximal colon from control and TNBS-treated pups (n = 8/group). For comparison, we downloaded human microarray data of NEC (n = 5) and surgical control (n = 4) from a public database. Data were analyzed using the software programs Partek Genomics Suite and Ingenuity Pathway Analysis. RESULTS: We detected extensive changes in gene expression in murine TNBS-mediated intestinal injury and human NEC. Using fold-change cut-offs of ±1.5, we identified 4,440 differentially-expressed genes (DEGs) in murine TNBS-mediated injury and 1,377 in NEC. Murine TNBS-mediated injury and NEC produced similar changes in expression of orthologous genes (r = 0.611, P < 0.001), and also activated nearly-identical biological processes and pathways. Lipopolysaccharide was top predicted upstream regulator in both the murine and human datasets. CONCLUSION: Murine neonatal TNBS-mediated enterocolitis and human NEC activate nearly-identical biological processes, signaling pathways, and transcriptional networks.
Asunto(s)
Enterocolitis Necrotizante/genética , Redes Reguladoras de Genes/efectos de los fármacos , Mucosa Intestinal/metabolismo , Intestinos/lesiones , Ácido Trinitrobencenosulfónico/toxicidad , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Enterocolitis Necrotizante/etiología , Perfilación de la Expresión Génica , Humanos , Intestinos/efectos de los fármacos , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos C57BL , Especificidad de la EspecieRESUMEN
BACKGROUND: Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants. Based on our recent findings of increased Smad7 expression in surgically resected bowel affected by NEC, we hypothesized that NEC macrophages undergo inflammatory activation because increased Smad7 expression renders these cells resistant to normal, gut-specific, transforming growth factor (TGF)-ß-mediated suppression of inflammatory pathways. METHODS: We used surgically resected human NEC tissue, murine models of NEC-like injury, bone marrow-derived and intestinal macrophages, and RAW264.7 cells. Smad7 and IκB kinase-beta (IKK-ß) were measured by quantitative PCR, western blots, and immunohistochemistry. Promoter activation was confirmed in luciferase reporter and chromatin immunoprecipitation assays. RESULTS: NEC macrophages showed increased Smad7 expression, particularly in areas with severe tissue damage and high bacterial load. Lipopolysaccharide-induced Smad7 expression suppressed TGF-ß signaling and augmented nuclear factor-kappa B (NF-κB) activation and cytokine production in macrophages. Smad7-mediated NF-κB activation was likely mediated via increased expression of IKK-ß, which, further increased Smad7 expression in a feed-forward loop. We show that Smad7 induced IKK-ß expression through direct binding to the IKK-ß promoter and its transcriptional activation. CONCLUSION: Smad7 expression in NEC macrophages interrupts TGF-ß signaling and promotes NF-κB-mediated inflammatory signaling in these cells through increased expression of IKK-ß.
Asunto(s)
Enterocolitis Necrotizante/metabolismo , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Proteína smad7/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Humanos , Quinasa I-kappa B/metabolismo , Inflamación , Lipopolisacáridos/metabolismo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Células RAW 264.7 , Transducción de SeñalRESUMEN
Understanding mechanisms of childhood disease and development of rational therapeutics are fundamental to progress in pediatric intensive care specialties. However, Division Chiefs and Department Chairs face unique challenges when building effective laboratory-based research programs in Neonatal and Pediatric Intensive Care, owing to high clinical demands necessary to maintain competence as well as financial pressures arising from fund flow models and the current extramural funding climate. Given these factors, the role of institutional support that could facilitate successful transition of promising junior faculty to independent research careers is ever more important. Would standardized guidelines of such support provide greater consistency among institutions? We addressed preliminary questions during a national focus group, a workshop and a survey of junior and senior academicians to solicit recommendations for optimal levels of protected time and resources when starting an independent laboratory. The consensus was that junior faculty should be assigned no more than 8 wk clinical service and should obtain start-up funds of $500K-1M exclusive of a 5-y committed salary support. Senior respondents placed a higher premium on protected time than junior faculty.
Asunto(s)
Cuidados Críticos , Neonatología , Pediatría , Médicos , Investigación Biomédica Traslacional , Centros Médicos Académicos/organización & administración , Selección de Profesión , Cuidados Críticos/organización & administración , Grupos Focales , Guías como Asunto , Hospitales Pediátricos/organización & administración , Humanos , Satisfacción en el Trabajo , Cuerpo Médico de Hospitales , Mentores , Neonatología/organización & administración , Pediatría/organización & administración , Desarrollo de Programa , Encuestas y Cuestionarios , Investigación Biomédica Traslacional/organización & administración , Recursos HumanosRESUMEN
Human milk contains biologically important amounts of transforming growth factor-ß2 isoform (TGF-ß2), which is presumed to protect against inflammatory gut mucosal injury in the neonate. In preclinical models, enterally administered TGF-ß2 can protect against experimental necrotizing enterocolitis, an inflammatory bowel necrosis of premature infants. In this study, we investigated whether TGF-ß bioactivity in human preterm milk could be enhanced for therapeutic purposes by adding recombinant TGF-ß2 (rTGF-ß2) to milk prior to feeding. Milk-borne TGF-ß bioactivity was measured by established luciferase reporter assays. Molecular interactions of TGF-ß2 were investigated by nondenaturing gel electrophoresis and immunoblots, computational molecular modeling, and affinity capillary electrophoresis. Addition of rTGF-ß2 (20-40 nM) to human preterm milk samples failed to increase TGF-ß bioactivity in milk. Milk-borne TGF-ß2 was bound to chondroitin sulfate (CS) containing proteoglycan(s) such as biglycan, which are expressed in high concentrations in milk. Chondroitinase treatment of milk increased the bioactivity of both endogenous and rTGF-ß2, and consequently, enhanced the ability of preterm milk to suppress LPS-induced NF-κB activation in macrophages. These findings provide a mechanism for the normally low bioavailability of milk-borne TGF-ß2 and identify chondroitinase digestion of milk as a potential therapeutic strategy to enhance the anti-inflammatory effects of preterm milk.
Asunto(s)
Condroitinasas y Condroitín Liasas/metabolismo , Enterocolitis Necrotizante , Leche Humana , Factor de Crecimiento Transformador beta2/metabolismo , Animales , Antiinflamatorios/metabolismo , Disponibilidad Biológica , Línea Celular , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Enterocolitis Necrotizante/metabolismo , Enterocolitis Necrotizante/prevención & control , Humanos , Recién Nacido , Recien Nacido Prematuro , Inflamación/metabolismo , Mucosa Intestinal/metabolismo , Activación de Macrófagos/fisiología , Ratones , Leche Humana/enzimología , Leche Humana/metabolismo , FN-kappa B/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Vascular endothelial growth factor (VEGF), a well-characterized regulator of angiogenesis, has been mechanistically implicated in retinal neovascularization and in the pathogenesis of retinopathy of prematurity. However, the ontogeny of VEGF expression in the human fetal retina is not well known. Because retinal vasculature grows with gestational maturation, we hypothesized that VEGF expression also increases in the midgestation human fetal eye as a function of gestational age. METHODS: To identify changes in VEGF gene expression during normal human development, we measured VEGF mRNA by quantitative PCR and measured VEGF protein by enzyme-linked immunosorbent assay and western blots in 10-24 wk gestation fetal vitreous, retina, and serum. RESULTS: VEGF mRNA expression in the retina increased with gestational age. VEGF isoform A, particularly its VEGF121 splice variant, contributed to this positive correlation. Consistent with these findings, we detected increasing VEGF121 protein concentrations in vitreous humor from fetuses of 10-24 wk gestation, while VEGF concentrations decreased in fetal serum. CONCLUSION: VEGF121 mRNA and protein concentrations increase with increasing gestational age in the developing human retina. We speculate that VEGF plays an important role in normal retinal vascular development, and that preterm delivery affects production of this vascular growth factor.
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Regulación del Desarrollo de la Expresión Génica , ARN Mensajero/metabolismo , Retina/embriología , Neovascularización Retiniana , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cuerpo Vítreo/embriología , Actinas/metabolismo , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Edad Gestacional , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Recién Nacido , Recien Nacido Prematuro , ARN Ribosómico 18S/metabolismo , Retinopatía de la Prematuridad/metabolismoRESUMEN
PURPOSE OF REVIEW: The aim is to review normal blood neutrophil concentrations and the clinical approach to neutropenia in the neonatal period. A literature search on neonatal neutropenia was performed using the databases PubMed, EMBASE, and Scopus, and the electronic archive of abstracts presented at the annual meetings of the Pediatric Academic Societies. RECENT FINDINGS: The review summarizes current knowledge on the causes of neutropenia in premature and critically ill neonates, focusing on common causes such as maternal hypertension, neonatal sepsis, twin-twin transfusion, alloimmunization, and hemolytic disease. The article provides a rational approach to diagnosis and treatment of neonatal neutropenia, including current evidence on the role of recombinant hematopoietic growth factors. SUMMARY: Neutrophil counts should be carefully evaluated in premature and critically ill neonates. Although neutropenia is usually benign and runs a self-limited course in most neonates, it can be prolonged, and it constitutes a serious deficiency in antimicrobial defense in some infants.