Emanuela Ferretti

Mesenchymal regulation of differentiation of intestinal epithelial cells.

Intestinal epithelial differentiation requires the presence of mesenchyme, but the exact nature of this epithelial-mesenchymal interaction is unknown. The role of humoral and extracellular matrix factors in determining intestinal epithelial differentiation was examined by exposing a nontransformed, immature, rat small-intestinal cell line (IEC-6) to intestinal fibroblast-conditioned media or by growing the cells on matrix deposited by intestinal fibroblasts. Differentiation was assessed morphologically and by induction of the disaccharidase sucrase, which is a marker of intestinal epithelial maturation. When exposed to fetal intestinal fibroblast-conditioned medium or grown on extracellular matrix prepared from fetal intestinal fibroblasts, IEC-6 cells showed a significant increase in sucrase activity. The addition of cortisol to IEC-6 cells cultured on intestinal matrix also led to increased sucrase expression, as measured by enzyme activity and protein and messenger RNA levels. Ultrastructurally, cells cultured on intestinal fibroblast matrix in the presence of cortisol displayed a more differentiated phenotype. In contrast, there was no induction of sucrase activity in cells exposed to lung-conditioned media or grown on lung fibroblast matrix. These studies suggest that both humoral and matrix factors from intestinal mesenchyme are involved in intestinal epithelial differentiation and that these factors appear to be organ specific.

How we teach ethics and communication during a Canadian neonatal perinatal medicine residency: An interactive experience

Background: Ethically challenging clinical situations frequently confront health care professionals in neonatology. These situations require neonatologists to exercise professionalism by communicating effectively throughout evolving physician–parent relationships in order to arrive at shared decisions for care that are in the best interest of the neonate and grounded solidly in ethical precepts. Aim: This article describes the process by which a well-delineated, interactive program to teach ethical reasoning and skillful communication with parents was implemented at the University of Ottawa, Canada. Methods: A revised ethics program implemented in 2009 identified competencies that should be demonstrated at the end of the Neonatal-Perinatal Medicine (NPM) residency. Several seminars were refined while new workshops, problem-based learning in ethics, and a personal portfolio were added. Results: All teaching strategies were well received based on the average level of satisfaction (5.8 out of 7, SD 0.4). We are now moving forward by formally assessing our program including the impact on knowledge acquisition and behavior. Conclusion: A dedicated, interactive competency-based neonatal ethics teaching program is vital to support NPM trainees in learning how to integrate ethical thinking with competencies in communication.

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

BackgroundNecrotizing 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.MethodsDeep 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.ResultsData 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.ConclusionsGene 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.

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.

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.

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

BACKGROUND AND AIMnNO 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.nnnMETHODSnGene expression profiles of cultured mid-gestation human intestinal explants were investigated in the absence or presence of a physiological concentration of EGF (50xa0ng/ml) or 1xa0μM INDO for 48xa0h using Illumina whole genome microarrays, Ingenuity Pathway Analysis software and quantitative PCR to investigate the expression of NOS2 and NOS2-pathway related genes.nnnRESULTSnIn 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.nnnCONCLUSIONnThese 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.

Early Onset Allergic Proctitis in a Preterm Neonate—A Case Report and Review of the Literature

Cow’s milk protein allergy/intolerance (CMPA/CMPI) is a common entity in the pediatric population with a nonspecific presentation ranging from gastrointestinal symptoms to systemic manifestations. Most infants with CMPI are term, and symptoms often appear in the week following the introduction of cow’s milk-based formula. There is typically a significant delay in the onset of milk allergy in premature infants compared to full term. We report a rare case of a premature neonate who presented with symptoms of CMPA within the first 2 days of life.

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

BackgroundThe 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.MethodsThe 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.ResultsWe 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.ConclusionsOur 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.

Disseminated Neonatal Herpes Simplex Virus Type 1 After a Water Birth

Neonatal herpes simplex virus (NHSV) infections are associated with significant morbidity and mortality. Numerous factors influence the transmission of HSV infection to newborns; however, immersion in water during labor has received very little attention as a possible risk factor despite the increasing popularity of water births. We report a case of disseminated NHSV type 1 infection, possibly acquired during a water birth. The purpose of this report is to alert healthcare providers to this potential route of transmission and to highlight the importance of screening guidelines for HSV before a water birth. Furthermore, it is essential to consider NHSV infection in any febrile infant who is not responding to standard empirical antibiotic management, even in the absence of herpetic lesions.