Contemporary use of prophylactic probiotics in NICUs in the United States: a survey update.

OBJECTIVE: In 2015, 14.0% of US NICUs administered probiotics to very low birth weight infants. Current probiotic use prior to and after the Fall of 2023 (when FDA warnings were issued) remains unknown. STUDY DESIGN: A survey was distributed to the American Academy of Pediatrics Section on Neonatal and Perinatal Medicine (August-November/2022) and Neonatology Solutions' Level III/IV NICUs (January-April/2023). Probiotic administration practices were investigated. RESULTS: In total, 289 unique NICUs and 406 providers responded to the survey. Of those, 29.1% of NICUs administered prophylactic probiotics to premature neonates, however, this decreased considerably after FDA warnings were issued. Additionally, 71.4% of providers stated willingness to administer probiotics to premature infants if there was an FDA-approved formulation. CONCLUSIONS: Probiotic use in US NICUs increased between 2015 and the Fall of 2023 and then dropped dramatically following warning letters from the FDA. The introduction of an FDA-approved probiotic may further expand administration.

An Electronic Health Record-integrated Web Application Augments a QI-directed Morbidity & Mortality Conference and Improves Quality of Care.

BACKGROUND: In 2014, we developed a QI-directed Morbidity and Mortality (M&M) Conference, prioritizing discussion of individual and system failures, as well as development of action items to prevent failure recurrence. However, due to a reliance on individual electronic documents to store M&M data, our ability to assess trends in failures and action item implementation was hindered. To address this issue, in 2019, we created a secure electronic health record (EHR)-integrated web application (web app) to store M&M data. STUDY DESIGN: In this study, we assessed the impact of our web app on efficient review and tracking of M&M data, including system failure occurrence and closure of action items. Additionally, in 2021, it was discovered that a backlog of action items existed. To address this issue, we implemented a QI initiative to reduce the backlog, and used the web app to compare action item closure over time. RESULTS: Use of the web app dramatically improved review of M&M data. During the study period, there was a 67.0% reduction in the occurrence of the most common system failures. Additionally, our QI initiative resulted in a 97.7% reduction in the duration of time to complete a single action item and a 61.1% increase in the on-time closure rate for action items. CONCLUSIONS: Integration of a web app into a QI-directed M&M Conference enhanced our ability to track system level failures and action item closure over time. Using this web app, we demonstrated that our M&M Conference achieved its intended goal of improving the quality of patient care. LEVEL OF EVIDENCE: IV.

Superior performance of biofilm versus planktonic Limosilactobacillus reuteri in protection of the intestines and brain in a piglet model of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal-related death in premature infants. Its etiology is multifactorial, with intestinal dysbiosis playing a major role. Probiotics are a logical preventative therapy for NEC, however their benefits have been inconsistent. We previously developed a novel probiotic delivery system in which planktonic (free-living) Limosilactobacillus reuteri (Lr) is incubated with biocompatible dextranomer microspheres (DM) loaded with maltose (Lr-DM-maltose) to induce biofilm formation. Here we have investigated the effects of Lr-DM-maltose in an enteral feed-only piglet model of NEC. We found a significant decrease in the incidence of Definitive NEC (D-NEC), death associated with D-NEC, and activated microglia in the brains of piglets treated with Lr-DM-maltose compared to non-treated piglets. Microbiome analyses using 16S rRNA sequencing of colonic contents revealed a significantly different microbial community composition between piglets treated with Lr-DM-maltose compared to non-treated piglets, with an increase in Lactobacillaceae and a decrease in Clostridiaceae in Lr-DM-maltose-treated piglets. Furthermore, there was a significant decrease in the incidence of D-NEC between piglets treated with Lr-DM-maltose compared to planktonic Lr. These findings validate our previous results in rodents, and support future clinical trials of Lr in its biofilm state for the prevention of NEC in premature neonates.

Inequities in the Diagnosis of Pediatric Appendicitis in Tertiary Children's Hospitals and the Consequences of Delayed Diagnosis.

INTRODUCTION: Missed diagnosis (MD) of acute appendicitis is associated with increased risk of appendiceal perforation. This study aimed to investigate whether racial/ethnic disparities exist in the diagnosis of pediatric appendicitis by comparing rates of MD versus single-encounter diagnosis (SED) between racial/ethnic groups. METHODS: Patients 0-18 y-old admitted for acute appendicitis from February 2017 to December 2021 were identified in the Pediatric Health Information System (PHIS). International Classification of Diseases, 10th Revision, Clinical Modification diagnosis codes for Emergency Department visits within 7 d prior to diagnosis were evaluated to determine whether the encounter represented MD. Generalized mixed models were used to assess the association between MD and patient characteristics. A similar model assessed independent predictors of perforation. RESULTS: 51,164 patients admitted for acute appendicitis were included; 50,239 (98.2%) had SED and 925 (1.8%) had MD. Compared to non-Hispanic White patients, patients of non-Hispanic Black (odds ratio 2.5, 95% confidence interval 2.0-3.1), Hispanic (2.1, 1.8-2.5), and other race/ethnicity (1.6, 1.2-2.1) had higher odds of MD. There was a significant interaction between race/ethnicity and imaging (P < 0.0001). Among patients with imaging, race/ethnicity was not significantly associated with MD. Among patients without imaging, there was an increase in strength of association between race/ethnicity and MD (non-Hispanic Black 3.6, 2.7-4.9; Hispanic 3.3, 2.6-4.1; other 2.0, 1.4-2.8). MD was associated with increased risk of perforation (2.5, 2.2-2.8). CONCLUSIONS: Minority children were more likely to have MD. Future efforts should aim to mitigate the risk of MD, including implementation of algorithms to standardize the workup of abdominal pain to reduce potential consequences of implicit bias.

Probiotics and novel probiotic delivery systems.

Necrotizing enterocolitis (NEC) is an infectious and inflammatory intestinal disease that is the most common surgical emergency in the premature patient population. Although the etiology of the disease is multifactorial, intestinal dysbiosis is a hallmark of this disease. Based on this, probiotics may play a therapeutic role in NEC by introducing beneficial bacteria with immunomodulating, antimicrobial, and anti-inflammatory functions into the gastrointestinal tract. Currently, there is no Food and Drug Administration (FDA)-approved probiotic for the prevention and treatment of NEC. All probiotic clinical studies to date have administered the bacteria in their planktonic (free-living) state. This review will discuss established probiotic delivery systems including planktonic probiotics, prebiotics, and synbiotics, as well as novel probiotic delivery systems such as biofilm-based and designer probiotics. We will also shed light on whether or not probiotic efficacy is influenced by administration with breast milk. Finally, we will consider the challenges associated with developing an FDA-approved probiotic for NEC.

Development of a novel definitive scoring system for an enteral feed-only model of necrotizing enterocolitis in piglets.

Introduction: Necrotizing enterocolitis (NEC) is a complex inflammatory disorder of the human intestine that most often occurs in premature newborns. Animal models of NEC typically use mice or rats; however, pigs have emerged as a viable alternative given their similar size, intestinal development, and physiology compared to humans. While most piglet NEC models initially administer total parenteral nutrition prior to enteral feeds, here we describe an enteral-feed only piglet model of NEC that recapitulates the microbiome abnormalities present in neonates that develop NEC and introduce a novel multifactorial definitive NEC (D-NEC) scoring system to assess disease severity. Methods: Premature piglets were delivered via Caesarean section. Piglets in the colostrum-fed group received bovine colostrum feeds only throughout the experiment. Piglets in the formula-fed group received colostrum for the first 24 h of life, followed by Neocate Junior to induce intestinal injury. The presence of at least 3 of the following 4 criteria were required to diagnose D-NEC: (1) gross injury score ≥4 of 6; (2) histologic injury score ≥3 of 5; (3) a newly developed clinical sickness score ≥5 of 8 within the last 12 h of life; and (4) bacterial translocation to ≥2 internal organs. Quantitative reverse transcription polymerase chain reaction was performed to confirm intestinal inflammation in the small intestine and colon. 16S rRNA sequencing was performed to evaluate the intestinal microbiome. Results: Compared to the colostrum-fed group, the formula-fed group had lower survival, higher clinical sickness scores, and more severe gross and histologic intestinal injury. There was significantly increased bacterial translocation, D-NEC, and expression of IL-1α and IL-10 in the colon of formula-fed compared to colostrum-fed piglets. Intestinal microbiome analysis of piglets with D-NEC demonstrated lower microbial diversity and increased Gammaproteobacteria and Enterobacteriaceae. Conclusions: We have developed a clinical sickness score and a new multifactorial D-NEC scoring system to accurately evaluate an enteral feed-only piglet model of NEC. Piglets with D-NEC had microbiome changes consistent with those seen in preterm infants with NEC. This model can be used to test future novel therapies to treat and prevent this devastating disease.

Current and future methods of probiotic therapy for necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a complex intestinal disease that primarily affects premature neonates. Given its significant mortality and morbidity, there is an urgent need to develop improved prophylactic measures against the disease. One potential preventative strategy for NEC is the use of probiotics. Although there has been significant interest for decades in probiotics in neonatal care, no clear guidelines exist regarding which probiotic to use or for which patients, and no FDA-approved products exist on the market for NEC. In addition, there is lack of agreement regarding the benefits of probiotics in neonates, as well as some concerns about the safety and efficacy of available products. We discuss currently available probiotics as well as next-generation probiotics and novel delivery strategies which may offer an avenue to capitalize on the benefits of probiotics, while minimizing the risks. Thus, probiotics may still prove to be an effective prevention strategy for NEC, although further product development and research is needed to support use in the preterm population.

Next-Generation Probiotic Therapy to Protect the Intestines From Injury.

Probiotics are live microorganisms that, when administered in adequate amounts, provide health benefits to the host. Some strains of the probiotic Lactobacillus reuteri (L. reuteri) have both antimicrobial and anti-inflammatory properties that may be exploited for the treatment and prevention of different gastrointestinal diseases, including necrotizing enterocolitis (NEC) and Clostridioides difficile (C. difficile) infection. Our laboratory has developed a new delivery system for L. reuteri in which the probiotic is incubated with biocompatible, semipermeable, porous dextranomer microspheres (DM) that can be loaded with beneficial and diffusible cargo. L. reuteri can be induced to form a biofilm by incubating the bacteria on the surface of these microspheres, which enhances the efficacy of the probiotic. Loading the DM with sucrose or maltose induces L. reuteri to produce more biofilm, further increasing the efficacy of the probiotic. Using a rat model of NEC, L. reuteri administered in its biofilm state significantly increases animal survival, reduces the incidence of NEC, preserves gut barrier function, and decreases intestinal inflammation. In a murine model of Clostridiodes difficile infection, L. reuteri administered in its biofilm state decreases colitis when administered either before or after C. difficile induction, demonstrating both prophylactic and therapeutic efficacy. There are currently no FDA-approved probiotic preparations for human use. An FDA-approved phase I clinical trial of L. reuteri in its biofilm state in healthy adults is currently underway. The results of this trial will be used to support a phase 1 clinical trial in neonates, with the goal of utilizing L. reuteri in its biofilm state to prevent NEC in premature neonates in the future.

Guidance for Structuring a Pediatric Intermediate Care Unit.

The purpose of this policy statement is to update the 2004 American Academy of Pediatrics clinical report and provide enhanced guidance for institutions, administrators, and providers in the development and operation of a pediatric intermediate care unit (IMCU). Since 2004, there have been significant advances in pediatric medical, surgical, and critical care that have resulted in an evolution in the acuity and complexity of children potentially requiring IMCU admission. A group of 9 clinical experts in pediatric critical care, hospital medicine, intermediate care, and surgery developed a consensus on priority topics requiring updates, reviewed the relevant evidence, and, through a series of virtual meetings, developed the document. The intended audience of this policy statement is broad and includes pediatric critical care professionals, pediatric hospitalists, pediatric surgeons, other pediatric medical and surgical subspecialists, general pediatricians, nurses, social workers, care coordinators, hospital administrators, health care funders, and policymakers, primarily in resource-rich settings. Key priority topics were delineation of core principles for an IMCU, clarification of target populations, staffing recommendations, and payment.

Development of an academic RVU (aRVU) system to promote pediatric surgical academic productivity.

BACKGROUND/PURPOSE: Improvements in patient care are directly affected by scientific discovery, and surgeons have historically played a vital role in this process. However, increasing clinical demands and incentivization for pure clinical productivity present challenges for promoting academic productivity. The objective of this work was to analyze the effects of adding an academic relative value unit (aRVU) scoring system to an existing work RVU (wRVU)-based incentivization plan on academic productivity in a Department of Pediatric Surgery. METHODS: Prior to 2012, incentive bonuses in our Department were mainly based on clinical wRVU activities. A weighted scoring system for 30 specific aRVUs was established in 2012. Incentivization for wRVUs vs. aRVUs was based on the clinical full-time equivalent (cFTE) of each faculty member. Academic activities incentivized included grant submissions/funding, peer reviewed publications, national presentations, Study Section participation, education and mentoring activities, receipt of research or teaching awards, initiation of Institutional Review Board (IRB) protocols, new academic society committee memberships/chairpersons, and patents. Academic progress was analyzed from 2012 to 2020. RESULTS: During the study period, annual external federal funding increased from $750,168 to $5,768,243 (7.7-fold increase); annual peer-reviewed publications increased from 24 to 140 (5.8-fold increase); annual national presentations accepted for oral/poster presentations nearly doubled; and faculty members and their trainees received 41 competitive research awards including 8 American Pediatric Surgical Association Awards, 9 American Academy of Pediatrics Section on Surgery Awards, and 3 American College of Surgeons Awards. During the same study period, wRVUs increased by 8%. CONCLUSIONS: Incentivization based on the addition of an aRVU system to a pre-existing wRVU system was associated with a significant increase in academic productivity, while still maintaining clinical productivity. Implementing an aRVU program is an important means of increasing academic productivity in Pediatric and other Surgery Departments.

Antibacterial and anti-inflammatory effects of Lactobacillus reuteri in its biofilm state contribute to its beneficial effects in a rat model of experimental necrotizing enterocolitis.

INTRODUCTION: Necrotizing enterocolitis (NEC) remains a significant surgical emergency in neonates. We have demonstrated the efficacy of Lactobacillus reuteri (Lr) in protecting against experimental NEC when administered as a biofilm by incubation with maltose loaded dextranomer microspheres. Lr possesses antimicrobial and anti-inflammatory properties. We developed mutant strains of Lr to examine the importance of its antimicrobial and anti-inflammatory properties in protecting the intestines from NEC. METHODS: Premature rat pups were exposed to hypoxia/hypothermia/hypertonic feeds to induce NEC. To examine the importance of antimicrobial reuterin and anti-inflammatory histamine, pups received either native or mutant forms of Lr, in either its planktonic or biofilm states, prior to induction of NEC. Intestinal histology was examined upon sacrifice. RESULTS: Compared to no treatment, administration of a single dose of Lr in its biofilm state significantly decreased the incidence of NEC (67% vs. 18%, p < 0.0001), whereas Lr in its planktonic state had no significant effect. Administration of reuterin-deficient or histamine-deficient forms of Lr, in either planktonic or biofilm states, resulted in significant loss of efficacy. CONCLUSION: Antimicrobial and anti-inflammatory effects of Lr contribute to its beneficial effects against NEC. This suggests that both infectious and inflammatory components contribute to the etiology of NEC.

Initial Laparotomy Versus Peritoneal Drainage in Extremely Low Birthweight Infants With Surgical Necrotizing Enterocolitis or Isolated Intestinal Perforation: A Multicenter Randomized Clinical Trial.

OBJECTIVE: The aim of this study was to determine which initial surgical treatment results in the lowest rate of death or neurodevelopmental impairment (NDI) in premature infants with necrotizing enterocolitis (NEC) or isolated intestinal perforation (IP). SUMMARY BACKGROUND DATA: The impact of initial laparotomy versus peritoneal drainage for NEC or IP on the rate of death or NDI in extremely low birth weight infants is unknown. METHODS: We conducted the largest feasible randomized trial in 20 US centers, comparing initial laparotomy versus peritoneal drainage. The primary outcome was a composite of death or NDI at 18 to 22 months corrected age, analyzed using prespecified frequentist and Bayesian approaches. RESULTS: Of 992 eligible infants, 310 were randomized and 96% had primary outcome assessed. Death or NDI occurred in 69% of infants in the laparotomy group versus 70% with drainage [adjusted relative risk (aRR) 1.0; 95% confidence interval (CI): 0.87-1.14]. A preplanned analysis identified an interaction between preoperative diagnosis and treatment group (P = 0.03). With a preoperative diagnosis of NEC, death or NDI occurred in 69% after laparotomy versus 85% with drainage (aRR 0.81; 95% CI: 0.64-1.04). The Bayesian posterior probability that laparotomy was beneficial (risk difference <0) for a preoperative diagnosis of NEC was 97%. For preoperative diagnosis of IP, death or NDI occurred in 69% after laparotomy versus 63% with drainage (aRR, 1.11; 95% CI: 0.95-1.31); Bayesian probability of benefit with laparotomy = 18%. CONCLUSIONS: There was no overall difference in death or NDI rates at 18 to 22 months corrected age between initial laparotomy versus drainage. However, the preoperative diagnosis of NEC or IP modified the impact of initial treatment.

Lactobacillus reuteri in its biofilm state promotes neurodevelopment after experimental necrotizing enterocolitis in rats.

Necrotizing enterocolitis (NEC) is a devastating disease affecting premature newborns with no known cure. Up to half of survivors subsequently exhibit cognitive impairment and neurodevelopmental defects. We created a novel probiotics delivery system in which the probiotic Lactobacillus reuteri (Lr) was induced to form a biofilm [Lr (biofilm)] by incubation with dextranomer microspheres loaded with maltose (Lr-DM-maltose). We have previously demonstrated that a single dose of the probiotic Lr administered in its biofilm state significantly reduces the incidence of NEC and decreases inflammatory cytokine production in an animal model of the disease. The aim of our current study was to determine whether a single dose of the probiotic Lr administered in its biofilm state protects the brain after experimental NEC. We found that rat pups exposed to NEC reached developmental milestones significantly slower than breast fed pups, with mild improvement with Lr (biofilm) treatment. Exposure to NEC had a negative effect on cognitive behavior, which was prevented by Lr (biofilm) treatment. Lr administration also reduced anxiety-like behavior in NEC-exposed rats. The behavioral effects of NEC were associated with increased numbers of activated microglia, decreased myelin basic protein (MBP), and decreased neurotrophic gene expression, which were prevented by administration of Lr (biofilm). Our data indicate early enteral treatment with Lr in its biofilm state prevented the deleterious effects of NEC on developmental impairments.

Lactobacillus reuteri in Its Biofilm State Improves Protection from Experimental Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease predominately found in premature infants that is associated with significant morbidity and mortality. Despite decades of research, medical management with broad spectrum antibiotics and bowel rest has remained relatively unchanged, with no significant improvement in patient outcomes. The etiology of NEC is multi-factorial; however, gastrointestinal dysbiosis plays a prominent role in a neonate's vulnerability to and development of NEC. Probiotics have recently emerged as a new avenue for NEC therapy. However, current delivery methods are associated with potential limitations, including the need for at least daily administration in order to obtain any improvement in outcomes. We present a novel formulation of enterally delivered probiotics that addresses the current limitations. A single enteral dose of Lactobacillus reuteri delivered in a biofilm formulation increases probiotic survival in acidic gastric conditions, increases probiotic adherence to gastrointestinal epithelial cells, and reduces the incidence, severity, and neurocognitive sequelae of NEC in experimental models.

Urine-derived extracellular vesicle miRNAs as possible biomarkers for and mediators of necrotizing enterocolitis: A proof of concept study.

BACKGROUND: Early-stage symptomology of necrotizing enterocolitis (NEC) is similar in presentation to non-NEC sepsis, though the treatment plans differ based on antibiotic administration and withholding of feeds. Improved diagnostics for NEC differentiation would allow clinicians to more rapidly set individual patients on a targeted treatment path. Extracellular vesicle-derived miRNAs, have previously demonstrated efficacy as disease biomarkers. To determine if these miRNAs are differentially-expressed in NEC infants, we performed transcriptomic analysis of urine-derived extracellular vesicle-derived miRNAs. METHODS: Urine was non-invasively obtained from infants in one of four groups (n ≥ 8) (Medical NEC, Surgical NEC, non-NEC sepsis, and healthy age-matched controls). EV-derived miRNAs were isolated and transcriptomic analysis was performed. RESULTS: Multiple miRNAs, including miR-376a, miR-518a-3p and miR-604, were significantly altered when comparing NEC to non-NEC sepsis and healthy controls, and could potentially be used as specific NEC biomarkers. Additionally, Ingenuity Pathway Analysis demonstrated that miRs differentially-expressed in NEC were associated with inflammatory disease and intestinal disease. Signal transduction molecules associated with NEC including TP53 and RPS15, which were also reduced transcriptionally in a rat model of NEC. CONCLUSION: These data indicate that there is a pool of potential urine EV-derived miRNAs that may be validated as NEC biomarkers in the differentiation of NEC from non-NEC sepsis and from age-matched controls. Additionally, signal transduction molecules associated with miRNAs differentially-expressed in human NEC are altered in a murine model of NEC, suggesting potential crossover between murine models of the disease and actual human presentation. LEVEL OF EVIDENCE: Level III Study of Diagnostic Test.

Improving care through standardized treatment of spontaneous pneumothorax.

PURPOSE: The objective of this quality improvement (QI) initiative was to implement a standardized clinical treatment protocol for patients presenting with primary spontaneous pneumothorax (PSP) in order to decrease hospital length of stay (LOS), diagnostic radiation exposure, and related cost. METHODS: Baseline data from patients admitted with PSP from January 1, 2016 to July 31, 2018 were compared to data from patients managed using a newly developed evidence-based treatment pathway from August 1, 2018 to December 31, 2019. Standard QI methodology was used to track results. RESULTS: Fifty-six episodes of PSP were observed during the baseline period and 40 episodes of PSP following initiation of the PSP protocol. The average LOS decreased from 4.5 days to 2.9 days. Patients underwent an average of 8.8 X-rays per admission preintervention versus 5.9 postintervention. The rate of CT scans decreased from 45% to 15% (p = 0.002). There was no significant difference in the rates of 30-day recurrence between the preintervention (13%) and postintervention (10%) groups (p = 0.7). Average admission costs per patient decreased by $1322 after adoption of the pathway. CONCLUSIONS: Adoption of a standardized treatment protocol for PSP led to a reduction in LOS, diagnostic imaging utilization, and cost without increasing clinical recurrence. TYPE OF STUDY: Quality improvement. LEVEL OF EVIDENCE: Level III.

A novel probiotic therapeutic in a murine model of Clostridioides difficile colitis.

For prophylactic therapy, mice received an oral antibiotic cocktail followed by clindamycin injection, followed by probiotic administration (planktonic vs. biofilm state), followed by C. difficile oral gavage. For treatment therapy, mice received antibiotics and C. difficile first, followed by probiotic administration. Clinical sickness scores (CSS) and intestinal histologic injury scores (HIS) were assigned.In the Prophylactic Therapy model, CSS: 67% of untreated mice exposed to C. difficile demonstrated CSS ≥ 6, which is consistent with C. difficile infection (p< .001 compared to unexposed mice). In mice treated with planktonic Lr, 55% had a CSS ≥ 6, but only 19% of mice treated with Lr in its biofilm state had CSS ≥ 6 (p< .001). Mice receiving Lr + DM-Maltose lost the least amount of weight compared to mice receiving saline (p = .004676) or to mice receiving Lr (p= .003185). HIS: 77% of untreated mice exposed to C. difficile had HIS scores ≥4, which is consistent with C. difficile infection. In mice treated with planktonic Lr, 62% had HIS ≥4, but only 19% of mice treated with Lr in its biofilm state had HIS ≥4. (p< .001). Additionally, mice treated with Lr in its biofilm state had better survival compared to untreated mice and to mice treated with planktonic Lr (p ≤ 0.05). Similar findings for weight loss, CSS, HIS and survival were obtained for Treatment Therapy.A single dose of Lactobacillus reuteri in its biofilm state reduces the severity and incidence of experimental C. difficile infection when administered as both prophylactic and treatment therapy.

Hope on the horizon: promising novel therapies for necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) remains among the most common and devastating diseases in neonates. Despite advances in neonatal clinical care, specific treatment strategies and diagnostic modalities remain lacking. As a result, morbidity and mortality remain high. Improved understanding of the pathogenesis of NEC has the potential for improved therapeutics. Some of the areas of research leading to promising discoveries include inhibition of Toll-like receptor signaling, modulation of vascular endothelial growth factor signal pathways, defining metabolomic alterations in NEC to discover potential biomarkers, probing for genetic predispositions to NEC susceptibility, determining mechanistic relations between anemia and NEC, and microflora modulation through the use of probiotics. All of these areas may represent novel promising approaches to the prevention and treatment of NEC. This review will focus on these current and possible therapeutic perspectives.

Intestinal proinflammatory macrophages induce a phenotypic switch in interstitial cells of Cajal.

Interstitial cells of Cajal (ICCs) are pacemaker cells in the intestine, and their function can be compromised by loss of C-KIT expression. Macrophage activation has been identified in intestine affected by Hirschsprung disease-associated enterocolitis (HAEC). In this study, we examined proinflammatory macrophage activation and explored the mechanisms by which it downregulates C-KIT expression in ICCs in colon affected by HAEC. We found that macrophage activation and TNF-α production were dramatically increased in the proximal dilated colon of HAEC patients and 3-week-old Ednrb-/- mice. Moreover, ICCs lost their C-KIT+ phenotype in the dilated colon, resulting in damaged pacemaker function and intestinal dysmotility. However, macrophage depletion or TNF-α neutralization led to recovery of ICC phenotype and restored their pacemaker function. In isolated ICCs, TNF-α-mediated phosphorylation of p65 induced overexpression of microRNA-221 (miR-221), resulting in suppression of C-KIT expression and pacemaker currents. We also identified a TNF-α/NF-κB/miR-221 pathway that downregulated C-KIT expression in ICCs in the colon affected by HAEC. These findings suggest the important roles of proinflammatory macrophage activation in a phenotypic switch of ICCs, representing a promising therapeutic target for HAEC.