Lipocalin-2 and calprotectin as stool biomarkers for predicting necrotizing enterocolitis in premature neonates.

BACKGROUND: Necrotizing enterocolitis (NEC) is a major challenge for premature infants in neonatal intensive care units and efforts toward the search for indicators that could be used to predict the development of the disease have given limited results until now. METHODS: In this study, stools from 132 very low birth weight infants were collected daily in the context of a multi-center prospective study aimed at investigating the potential of fecal biomarkers for NEC prediction. Eight infants (~6%) received a stage 3 NEC diagnosis. Their stools collected up to 10 days before diagnosis were included and matched with 14 non-NEC controls and tested by ELISA for the quantitation of eight biomarkers. RESULTS: Biomarkers were evaluated in all available stool samples leading to the identification of lipocalin-2 and calprotectin as the two most reliable predicting markers over the 10-day period prior to NEC development. Pooling the data for each infant confirmed the significance of lipocalin-2 and calprotectin, individually and in combination 1 week in advance of the NEC clinical diagnosis. CONCLUSIONS: The lipocalin-2 and calprotectin tandem represents a significant biomarker signature for predicting NEC development. Although not yet fulfilling the "perfect biomarker" criteria, it represents a first step toward it. IMPACT: Stool biomarkers can be used to predict NEC development in very low birth weight infants more than a week before the diagnosis. LCN2 was identified as a new robust biomarker for predicting NEC development, which used in conjunction with CALPRO, allows the identification of more than half of the cases that will develop NEC in very low birth weight infants. Combining more stool markers with the LCN2/CALPRO tandem such as PGE2 can further improve the algorithm for the prediction of NEC development.

Proteomics Profiling of Stool Samples from Preterm Neonates with SWATH/DIA Mass Spectrometry for Predicting Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a life-threatening condition for premature infants in neonatal intensive care units. Finding indicators that can predict NEC development before symptoms appear would provide more time to apply targeted interventions. In this study, stools from 132 very-low-birth-weight (VLBW) infants were collected daily in the context of a multi-center prospective study aimed at investigating the potential of fecal biomarkers for NEC prediction using proteomics technology. Eight of the VLBW infants received a stage-3 NEC diagnosis. Stools collected from the NEC infants up to 10 days before their diagnosis were available for seven of them. Their samples were matched with those from seven pairs of non-NEC controls. The samples were processed for liquid chromatography-tandem mass spectrometry analysis using SWATH/DIA acquisition and cross-compatible proteomic software to perform label-free quantification. ROC curve and principal component analyses were used to explore discriminating information and to evaluate candidate protein markers. A series of 36 proteins showed the most efficient capacity with a signature that predicted all seven NEC infants at least a week in advance. Overall, our study demonstrates that multiplexed proteomic signature detection constitutes a promising approach for the early detection of NEC development in premature infants.

Impaired antimicrobial response and mucosal protection induced by ibuprofen in the immature human intestine.

BACKGROUND: The use of nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin (INDO) and ibuprofen (IBU) has been shown to be an effective therapy for the closure of patent ductus arteriosus (PDA). However, this treatment has been associated with an increased risk of developing enteropathies in neonates. Whether the use of IBU is safer than INDO for the immature intestine remains to be elucidated. METHODS: The direct impact of IBU on the human immature intestinal transcriptome was investigated using serum-free organ culture. Differentially expressed genes were analyzed with Ingenuity Pathway Analysis software and compared with those previously reported with INDO. Validation of differentially expressed genes was confirmed by qPCR. RESULTS: We identified several biological processes that were significantly modulated by IBU at similar levels to what had previously been observed with INDO, while the expression of genes involved in "antimicrobial response" and "mucus production" was significantly decreased exclusively by IBU in the immature intestine. CONCLUSIONS: Our findings indicate that IBU has a harmful influence on the immature intestine. In addition to exerting many of the INDO observed deleterious effects, IBU alters pathways regulating microbial colonization and intestinal epithelial defense.

The nitric oxide synthase 2 pathway is targeted by both pro- and anti-inflammatory treatments in the immature human intestine.

BACKGROUND AND AIM: NO synthase 2 (NOS2) was recently identified as one the most overexpressed genes in intestinal samples of premature infants with necrotizing enterocolitis (NEC). NOS2 is widely implicated in the processes of epithelial cell injury/apoptosis and host immune defense but its specific role in inflammation of the immature human intestinal mucosa remains unclear. Interestingly, factors that prevent NEC such as epidermal growth factor (EGF) attenuate the inflammatory response in the mid-gestation human small intestine using serum-free organ culture while drugs that are associated with NEC occurrence such as the non-steroidal anti-inflammatory drug, indomethacin (INDO), exert multiple detrimental effects on the immature human intestine. In this study we investigate the potential role of NOS2 in modulating the gut inflammatory response under protective and stressful conditions by determining the expression profile of NOS2 and its downstream pathways in the immature intestine. METHODS: Gene expression profiles of cultured mid-gestation human intestinal explants were investigated in the absence or presence of a physiological concentration of EGF (50 ng/ml) or 1 µM INDO for 48 h using Illumina whole genome microarrays, Ingenuity Pathway Analysis software and quantitative PCR to investigate the expression of NOS2 and NOS2-pathway related genes. RESULTS: In the immature intestine, NOS2 expression was found to be increased by EGF and repressed by INDO. Bioinformatic analysis identified differentially regulated pathways where NOS2 is known to play an important role including citrulline/arginine metabolism, epithelial cell junctions and oxidative stress. At the individual gene level, we identified many differentially expressed genes of the citrulline/arginine metabolism pathway such as ARG1, ARG2, GLS, OAT and OTC in response to EGF and INDO. Gene expression of tight junction components such as CLDN1, CLDN2, CLDN7 and OCN and of antioxidant markers such as DUOX2, GPX2, SOD2 were also found to be differentially modulated by EGF and INDO. CONCLUSION: These results suggest that the protective effect of EGF and the deleterious influence of INDO on the immature intestine could be mediated via regulation of NOS2. Pathways downstream of NOS2 involved with these effects include metabolism linked to NO production, epithelial barrier permeability and antioxidant expression. These results suggest that NOS2 is a likely regulator of the inflammatory response in the immature human gut and may provide a mechanistic basis for the protective effect of EGF and the deleterious effects of INDO.

Deleterious effects of indomethacin in the mid-gestation human intestine.

The use of the anti-inflammatory drug indomethacin (INDO) in preterm infants has been associated with an increased risk of developing enteropathies. In this study, we have investigated the direct impact of INDO on the human mid-gestation intestinal transcriptome using serum-free organ culture. After determining the optimal dose of 1 µM of INDO (90% inhibition of intestinal prostaglandin E2 production and range of circulating levels in treated preterm babies), global gene expression profiles were determined using Illumina bead chip microarrays in both small and large intestines after 48 h of INDO treatment. Using Ingenuity Pathway Analysis software, we identified critical metabolic pathways that were significantly altered by INDO in both intestinal segments including inflammation and also glycolysis, oxidative phosphorylation and free radical scavenging/oxidoreductase activity, which were confirmed by qPCR at the level of individual genes. Taken together, these data revealed that INDO directly exerts multiple detrimental effects on the immature human intestine.

Malnutrition in hospitalized children: prevalence, impact, and management.

PURPOSE: Malnutrition in hospitalized children has been reported since the late 1970s. The prevalence of acute and chronic malnutrition was examined in hospitalized patients in a general pediatric unit, and the impact and management of malnutrition were assessed. METHODS: The nutritional risk score (NRS) and nutritional status (NS) (weight, height, body mass index, and skinfold thickness) of children aged zero to 18 years were assessed upon hospital admission. Growth and energy intake were monitored every three days until discharge. RESULTS: A total of 173 children (median age three years, 88 girls) participated; 79.8% had a moderate to severe NRS and 13.3% were acutely and/or chronically malnourished. A high NRS was associated with a longer hospital stay in children older than three years (P<0.05), while a poor NS (weight for height percentile) was correlated with prolonged hospitalization in children aged three years or younger (P<0.05). Although weight did not change during hospitalization, a decrease in skinfolds was documented (n=43, P<0.05). Patients with a high NRS had lower energy intake than those not at risk. However, children with abnormal NS received 92.5% of recommended energy intake. CONCLUSIONS: This study suggests that all children admitted to hospital should have an evaluation of their NRS and NS, so that they can receive appropriate nutrition interventions provided by a multidisciplinary nutrition team.

Burden of rotavirus disease: A population-based study in Eastern Townships, Quebec.

BACKGROUND: Since July 2010, the National Advisory Committee on Immunization of Canada has recommended rotavirus vaccination for all healthy infants. However, before implementing this vaccine in routine health programs, Canadian provinces need to establish current epidemiological data on rotavirus-associated acute gastroenteritis (AGE). METHODS: A retrospective cohort study of children <5 years of age with AGE from 2002 to 2008 was performed in Eastern Townships, Quebec (population in 2006: 298,780). Data were collected on visits to outpatient clinics, emergency department (ED) visits, hospitalizations (standard and short-stay units) and nosocomial AGE. The winter residual estimation and Brandt methods were used to estimate the proportion of AGE attributable to rotaviruses. RESULTS: During the six-year study period, a total of 1435 hospitalizations, 3631 ED visits and 6220 ambulatory care visits were attributed to AGE. The specific rotavirus burden was estimated to be 449 to 666 for hospitalizations, 1050 to 1361 for ED visits and 1633 to 1687 for outpatient visits. The epidemic curve showed a periodicity with higher incidence in March and April. Short-stay unit hospitalizations represented 58% of all hospitalizations. The annual incidence rate of rotaviruses was estimated to be 50 to 74 per 10,000 children for hospitalizations, 117 to 152 per 10,000 children for ED visits and 182 to 188 per 10,000 children for outpatient visits. CONCLUSION: Most available retrospective studies probably underestimate rotavirus-associated hospitalizations because they do not take into account short-stay unit hospitalizations. Furthermore, these data on emergency and outpatient visits provide an exhaustive appraisal of the rotavirus burden, which serves as crucial information for the evaluation of immunization programs.

HISTORIQUE: Depuis juillet 2010, le Comité consultatif national de l'immunisation recommande de vacciner tous les nourrissons en santé contre le rotavirus. Cependant, avant d'ajouter ce vaccin aux programmes de vaccination systématique, les provinces canadiennes doivent obtenir des données épidémiologiques à jour sur la gastro-entérite aiguë (GEA) associée au rotavirus. MÉTHODOLOGIE: Les chercheurs ont effectué une étude de cohorte rétrospective auprès d'enfants de moins de cinq ans ayant eu une GEA entre 2002 et 2008 en Estrie, au Québec (population de 298 780 habitants en 2006). Ils ont colligé les données aux consultations externes, au département d'urgence (DU), chez les patients hospitalisés (unités standard et de courte durée) et relatives aux cas de GEA nosocomiales. Ils ont utilisé l'estimation hivernale résiduelle et les méthodologies de Brandt pour évaluer la proportion de GEA attribuables aux rotavirus. RÉSULTATS: Pendant la période d'étude de six ans, 1 435 hospitalisations, 3 631 consultations au DU et 6 220 consultations externes étaient attribuables à la GEA. On estimait que le fardeau propre à la GEA se situait entre 449 et 666 pour les hospitalisations, entre 1 050 et 1 361 pour les consultations au DU et entre 1 633 et 1 687 pour les consultations externes. La courbe épidémique a révélé une périodicité à l'incidence plus élevée en mars et avril. Les hospitalisations de courte durée représentaient 58 % de toutes les hospitalisations. On estime que le taux d'incidence annuel des rotavirus oscille entre 50 et 74 cas sur 10 000 enfants sur le plan des hospitalisations, entre 117 et 152 cas sur 10 000 enfants sur le plan des consultations au DU et entre 182 et 188 cas sur 10 000 enfants sur le plan des consultations externes. CONCLUSION: Selon toute probabilité, la plupart des études rétrospectives disponibles sous-estiment le nombre d'hospitalisations associées au rotavirus parce qu'elles ne tiennent pas compte des hospitalisations dans les unités de courte durée. De plus, ces données sur les consultations externes et à l'urgence fournissent une évaluation détaillée du fardeau du rotavirus, qui contient de l'information capitale pour évaluer les programmes de vaccination.

Anti-inflammatory effects of epidermal growth factor on the immature human intestine.

The inflammatory response of the preterm infants' intestine underlines its inability to respond to hemodynamic stress, microbes, and nutrients. Recent evidence suggests that exogenous epidermal growth factor (EGF) exerts a therapeutic influence on neonatal enteropathies. However, the molecular mechanisms underlying the beneficial effects of EGF remain to be clarified. The purpose of this study was to evaluate the impact of EGF on the gene expression profiles of the developing human small and large intestine at midgestation in serum-free organ cultures using microarrays. The gene expression profiles of cultured human fetal ileal and colonic explants were investigated in the absence or presence of a physiological concentration of 50 ng/ml EGF for 48 h. Data were analyzed with the Ingenuity Pathway Analysis (IPA) software and confirmed by qPCR. We found a total of 6,474 differentially expressed genes in the two segments in response to EGF. IPA functional analysis revealed that in addition to differentially modulating distinct cellular, molecular, and physiological functions in the small and large intestine, EGF regulated the inflammatory response in both intestinal segments in a distinct manner. For instance, several intestinal-derived chemokines such as CCL2, CCL25, CXCL5, and CXCL10 were found to be differentially regulated by EGF in the immature ileum and colon. The findings showing the anti-inflammatory influence of exogenous EGF suggests a mechanistic basis for the beneficial effects of EGF on neonatal enteropathies. These results reinforce growing evidence that by midgestation, the human small intestine and colon rely on specific and distinct regulatory pathways.

Gene-expression profile analysis in the mid-gestation human intestine discloses greater functional immaturity of the colon as compared with the ileum.

BACKGROUND AND OBJECTIVES: The occurrence of many neonatal inflammatory intestinal diseases in preterm infants highlights the susceptibility of the immature intestine to responding inadequately to nutrients and microbes. A better understanding of functional intestinal development is essential for the design of optimal treatments ensuring survival and growth of premature infants. The purpose of this study was to evaluate the gene expression profiles of the human ileum and colon at mid-gestation because these 2 segments are considered to be similar at this stage and are the sites of the most frequent pathologies in preterm infants. SUBJECTS AND METHODS: We compared the gene-expression profiles of human fetal small and large intestines using a cDNA microarray and analyzed the data with Ingenuity Pathway Analysis software. RESULTS: We found that a significant proportion of the genes was differentially expressed in the 2 segments. Gene cluster analysis revealed an even higher level of transcriptional dissimilarity at the functional level. For instance, segment-specific/overexpressed gene clusters in the ileum included genes involved with amino acid, vitamin, and mineral metabolism, reflecting the higher level of maturity of the small intestine as compared with the colon in which genes involved with cell cycle, cell death, and cell signaling were the predominant clusters of genes expressed. CONCLUSIONS: Functional clustering analysis of the differentially expressed genes revealed important functional differences between the 2 segments and a relative immaturity of the colon, suggesting that already at mid-gestation, the 2 intestinal segments should be considered as 2 distinct organs.

IL-17-related signature genes linked to human necrotizing enterocolitis.

OBJECTIVE: Necrotizing enterocolitis (NEC) is the most frequent life-threatening gastrointestinal disease experienced by premature infants in neonatal intensive care units all over the world. The objective of the present study was to take advantage of RNA-Seq data from the analysis of intestinal specimens of preterm infants diagnosed with NEC. Function enrichments with Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes were used to analyse previous data in order to identify biological and functional processes, which could provide more insight into the pathogenesis of NEC in infants. RESULTS: Gene set enrichment analysis indicated that the most significant biological pathways over-represented in NEC neonates were closely associated with innate immune functions. One of the striking observations was the highly modulated expression of inflammatory genes related to the IL-17 pathway including such as pro-inflammatory cytokines (CXCL8), chemokines (CXCL5 and CXCL10) and antimicrobials (DEF5A, DEF6A, LCN2, NOS2) in the intestine of neonates diagnosed with NEC. Interestingly, the increase in IL-17 expression appeared to be under the IL-17F form, as reported in Crohn's disease, another inflammatory bowel disease. Further investigation is thus still needed to determine the precise role of IL-17F and its downstream targets in NEC.

Gene expression profiling in necrotizing enterocolitis reveals pathways common to those reported in Crohn's disease.

BACKGROUND: Necrotizing enterocolitis (NEC) is the most frequent life-threatening gastrointestinal disease experienced by premature infants in neonatal intensive care units. The challenge for neonatologists is to detect early clinical manifestations of NEC. One strategy would be to identify specific markers that could be used as early diagnostic tools to identify preterm infants most at risk of developing NEC or in the event of a diagnostic dilemma of suspected disease. As a first step in this direction, we sought to determine the specific gene expression profile of NEC. METHODS: Deep sequencing (RNA-Seq) was used to establish the gene expression profiles in ileal samples obtained from preterm infants diagnosed with NEC and non-NEC conditions. Data were analyzed with Ingenuity Pathway Analysis and ToppCluster softwares. RESULTS: Data analysis indicated that the most significant functional pathways over-represented in NEC neonates were associated with immune functions, such as altered T and B cell signaling, B cell development, and the role of pattern recognition receptors for bacteria and viruses. Among the genes that were strongly modulated in neonates with NEC, we observed a significant degree of similarity when compared with those reported in Crohn's disease, a chronic inflammatory bowel disease. CONCLUSIONS: Gene expression profile analysis revealed a predominantly altered immune response in the intestine of NEC neonates. Moreover, comparative analysis between NEC and Crohn's disease gene expression repertoires revealed a surprisingly high degree of similarity between these two conditions suggesting a new avenue for identifying NEC biomarkers.

Hypersensitivity reaction to parenteral nutrition in an intrauterine growth-restricted newborn: a case report.

Hypersensitivity reaction to parenteral nutrition (PN) in infants is a rare condition, especially in the neonatal period. The authors report the case of a neonate with intrauterine growth restriction (IUGR) who presented symptoms of anaphylaxis while receiving standard PN. Given the very common practice of neonatal PN use, especially in newborns with IUGR, clinicians should be alerted about possible acute reactions to this useful therapy.

Luminal fermentation and colonocyte metabolism in a rat model of enteral nutrition.

Large intestinal fermentation and nutrient metabolism in colonocytes were investigated in a rat model of enteral feeding. Male Wistar rats (240-280 g) were submitted to 7 or 14 days of treatment: intragastric feeding (elemental formula) versus oral feeding (isocaloric and isonitrogenous diet, containing 5% purified cellulose) in the control group. Fermentation products and bacterial populations were analyzed in cecal contents. Colonic cells were isolated and tested for their capacities to metabolize [1-(14)C] butyrate and [U-(14)C]glutamine. After 7 days of enteral nutrition, short-chain fatty acid concentrations represented 52% of those measured in the control group, but colonocyte metabolism remained unchanged. After 14 days of enteral nutrition, short-chain fatty acid concentrations were still decreasing, although bacterial counts remained unchanged. In parallel, ammonia and lactate concentrations were significantly increased. The capacities to utilize butyrate and glutamine in colonocytes were only slightly affected. However, there was a dramatic increase in the ratio of beta-OH-butyrate to acetoacetate fluxes, suggesting a more reduced redox mitochondrial state associated with enteral feeding.