Analyzing efficiency of a lentiviral shRNA knockdown system in human enteroids using western blot and flow cytometry.

Enteroids are in vitro models to study gastrointestinal pathologies and test personalized therapeutics; however, the inherent complexity of enteroids often renders standard gene editing approaches ineffective. Here, we introduce a refined lentiviral transfection protocol, ensuring sufficient lentiviral engagement with enteroids while considering spatiotemporal growth variability throughout the extracellular matrix. Additionally, we highlight a selection process for transduced cells, introduce a protocol to accurately measure transduction efficiency, and explore methodologies to gauge effects of gene knockdown on biological processes.

Impact of neonatal nutrition on necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the leading cause of morbidity and mortality in preterm infants. NEC is multifactorial and the result of a complex interaction of feeding, dysbiosis, and exaggerated inflammatory response. Feeding practices in the neonatal intensive care units (NICUs) can vary among institutions and have significant impact on the vulnerable gastointestinal tract of preterm infants. . These practices encompass factors such as the type of feeding and fortification, duration of feeding, and rate of advancement, among others. The purpose of this article is to review the data on some of the most common feeding practices in the NICU and their impact on the development of NEC in preterm infants. Data on the human milk bioactive component glycosaminoglycans, specifically hyaluronan, will also be discussed in the context of postnatal intestinal development and NEC prevention.

Clinical Characteristics and Potential Pathogenesis of Cardiac Necrotizing Enterocolitis in Neonates with Congenital Heart Disease: A Narrative Review.

Neonates with congenital heart disease (CHD) are at an increased risk of developing necrotizing enterocolitis (NEC), an acute inflammatory intestinal injury most commonly associated with preterm infants. The rarity of this complex disease, termed cardiac NEC, has resulted in a dearth of information on its pathophysiology. However, a higher incidence in term infants, effects on more distal regions of the intestine, and potentially a differential immune response may distinguish cardiac NEC as a distinct condition from the more common preterm, classical NEC. In this review, risk factors, differentiated from those of classical NEC, are discussed according to their potential contribution to the disease process, and a general pathogenesis is postulated for cardiac NEC. Additionally, biomarkers specific to cardiac NEC, clinical outcomes, and strategies for achieving enteral feeds are discussed. Working towards an understanding of the mechanisms underlying cardiac NEC may aid in future diagnosis of the condition and provide potential therapeutic targets.

Neonatal Microbiome, Intestinal Inflammation, and Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC), the most common gastrointestinal emergency in the neonatal intensive care unit (NICU), is a leading cause of preterm infant morbidity and mortality [...].

Placental Neutrophil Infiltration Associated with Tobacco Exposure but Not Development of Bronchopulmonary Dysplasia.

Objective: In utero inflammation is associated with bronchopulmonary dysplasia (BPD) in preterm infants. We hypothesized that maternal tobacco exposure (TE) might induce placental neutrophil infiltration, increasing the risk for BPD. Study design: We compared the composite outcome of BPD and death in a prospective pilot study of TE and no-TE mothers and their infants born <32 weeks. Placental neutrophil infiltration was approximated by neutrophil gelatinase-associated lipocalin (NGAL) ELISA, and total RNA expression was analyzed via NanoString© (Seattle, WA, USA). Result: Of 39 enrolled patients, 44% were classified as tobacco exposure. No significant difference was noted in the infant's composite outcome of BPD or death based on maternal tobacco exposure. NGAL was higher in placentas of TE vs. non-TE mothers (p < 0.05). Placental RNA analysis identified the upregulation of key inflammatory genes associated with maternal tobacco exposure. Conclusion: Tobacco exposure during pregnancy was associated with increased placental neutrophil markers and upregulated inflammatory gene expression. These findings were not associated with BPD.

Clinical and Laboratory Predictors for the Development of Low Cardiac Output Syndrome in Infants Undergoing Cardiopulmonary Bypass: A Pilot Study.

Cardiac surgery employing cardiopulmonary bypass exposes infants to a high risk of morbidity and mortality. The objective of this study was to assess the utility of clinical and laboratory variables to predict the development of low cardiac output syndrome, a frequent complication following cardiac surgery in infants. We performed a prospective observational study in the pediatric cardiovascular ICU in an academic children's hospital. Thirty-one patients with congenital heart disease were included. Serum levels of nucleosomes and a panel of 20 cytokines were measured at six time points in the perioperative period. Cardiopulmonary bypass patients were characterized by increased levels of interleukin-10, -6, and -1α upon admission to the ICU compared to non-bypass cardiac patients. Patients developing low cardiac output syndrome endured longer aortic cross-clamp time and required greater inotropic support at 12 h postoperatively compared to bypass patients not developing the condition. Higher preoperative interleukin-10 levels and 24 h postoperative interleukin-8 levels were associated with low cardiac output syndrome. Receiver operating characteristic curve analysis demonstrated a moderate capability of aortic cross-clamp duration to predict low cardiac output syndrome but not IL-8. In conclusion, low cardiac output syndrome was best predicted in our patient population by the surgical metric of aortic cross-clamp duration.

The Protective Influence of Chondroitin Sulfate, a Component of Human Milk, on Intestinal Bacterial Invasion and Translocation.

BACKGROUND: Human milk is known to be protective against necrotizing enterocolitis, a devastating intestinal inflammatory disease affecting the preterm population. Although the pathogenesis of necrotizing enterocolitis is yet to be solidified, intestinal integrity dysfunction, bacterial invasion and/or translocation, and inflammation may play important roles. Glycosaminoglycans, compounds naturally prevalent in both human milk and the intestine, are thought to be anti-inflammatory and capable of altering bacterial interactions within the gut. RESEARCH AIM: In this study, we aimed to evaluate the potential of chondroitin sulfate, the most prominent class of glycosaminoglycans in human milk, to protect against bacterial infection in an intestinal in vitro model. METHODS: T84 cell monolayers were treated with chondroitin sulfate and cell viability was assessed across a number of doses. Monolayers were then pretreated with chondroitin sulfate and subsequently challenged with E. coli invasion and translocation to evaluate any protective role of the compound against infection. Tight junction barrier function was assessed by transepithelial electrical resistance, and cytokine levels were evaluated. RESULTS: Chondroitin sulfate at any dose up to 750 µg/ml was not associated with any statistically significant decrease in cell viability. Additionally, chondroitin sulfate at 750 µg/ml was associated with a 75% decrease in both bacterial invasion and translocation compared to control. CONCLUSIONS: These data suggest chondroitin sulfate may protect against bacterial infection through a reduction in both invasion and translocation, importantly without attendant reduction in cell viability.