ROCK1 inhibitor stabilizes E-cadherin and improves barrier function in experimental necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of newborns. Although incompletely understood, NEC is associated with intestinal barrier dysfunction. E-cadherin, an adherens junction, is a protein complex integral in maintaining normal barrier homeostasis. Rho-associated protein kinase-1 (ROCK1) is a kinase that regulates the E-cadherin complex, and p120-catenin is a subunit of the E-cadherin complex that has been implicated in stabilizing the cadherin complex at the plasma membrane. We hypothesized that E-cadherin is decreased in NEC and that inhibition of ROCK1 would protect against adherens junction disruption. To investigate this, a multimodal approach was used: In vitro Caco-2 model of NEC (LPS/TNFα), rap pup model (hypoxia + bacteria-containing formula), and human intestinal samples. E-cadherin was decreased in NEC compared with controls, with relocalization from the cell border to an intracellular location. ROCK1 exhibited a time-dependent response to disease, with increased early expression in NEC and decreased expression at later time points and disease severity. Administration of ROCK1 inhibitor (RI) resulted in preservation of E-cadherin expression at the cell border, preservation of intestinal villi on histological examination, and decreased apoptosis. ROCK1 upregulation in NEC led to decreased association of E-cadherin to p120 and increased intestinal permeability. RI helped maintain the stability of the E-cadherin-p120 complex, leading to improved barrier integrity and protection from experimental NEC.NEW & NOTEWORTHY This paper is the first to describe the effect of ROCK1 on E-cadherin expression in the intestinal epithelium and the protective effects of ROCK inhibitor on E-cadherin stability in necrotizing enterocolitis.

Identification of suitable reference microRNA for qPCR analysis in pediatric inflammatory bowel disease.

Pediatric inflammatory bowel disease (IBD) accounts for 10-15% of IBD and is associated with considerable morbidity for patients. Dysregulated microRNAs (miRNA, miR), small noncoding RNA molecules that modulate gene expression, have been the target of research in IBD diagnosis, surveillance, and therapy. Proper selection of reference genes, which are a prerequisite for accurate measurement of miRNA expression, is currently lacking. We hypothesize that appropriate normalization requires unique reference genes for different tissue and disease types. Through the study of 28 pediatric intestinal samples, we sought to create a protocol for selection of suitable endogenous reference genes. Candidate reference genes (miR-16, 193a, 27a, 103a, 191) were analyzed by RT-quantitative (q)PCR. Criteria used for designation of suitable reference genes were as follows: 1) ubiquitous: present in all tissue samples with quantification cycle value 15-35; 2) uniform expression: no differential expression between control and disease samples (P > 0.05); 3) stability: stability value <0.5 by NormFinder. Our results suggest the use of miR-27a/191 for Crohn's disease small bowel, none of the five candidate genes for Crohn's disease colon, and miR-16/27a for ulcerative colitis. Additionally, target miR-874 had differential expression when normalized with different reference genes. Our results demonstrate that reference gene choice for qPCR analysis has a significant effect on study results and that proper data normalization is imperative.

Impact of Decolonization Protocols and Recurrence in Pediatric MRSA Skin and Soft-Tissue Infections.

BACKGROUND: Methicillin-resistant staphylococcus aureus (MRSA) colonization is associated with the development of skin and soft-tissue infection in children. Although MRSA decolonization protocols are effective in eradicating MRSA colonization, they have not been shown to prevent recurrent MRSA infections. This study analyzed the prescription of decolonization protocols, rates of MRSA abscess recurrence, and factors associated with recurrence. MATERIALS AND METHODS: This study is a single-institution retrospective review of patients ≤18 y of age diagnosed with MRSA culture-positive abscesses who underwent incision and drainage (I&D) at a tertiary-care children's hospital. The prescription of an MRSA decolonization protocol was recorded. The primary outcome was MRSA abscess recurrence. RESULTS: Three hundred ninety-nine patients with MRSA culture-positive abscesses who underwent I&D were identified. Patients with previous history of abscesses, previous MRSA infection groin/genital region abscesses, higher number of family members with a history of abscess/cellulitis or MRSA infection, and I&D by a pediatric surgeon were more likely to be prescribed decolonization. Decolonized patients did not have lower rates of recurrence. Recurrence was more likely to occur in patients with previous abscesses, previous MRSA infection, family history of abscesses, family history of MRSA infection, Hispanic ethnicity, and those with fever on admission. CONCLUSIONS: MRSA decolonization did not decrease the rate of recurrence of MRSA abscesses in our patient cohort. Patients at high risk for MRSA recurrence such as personal or family history of abscess or MRSA infection, Hispanic ethnicity, or fever on admission did not benefit from decolonization.

Outcomes and associated ethical considerations of long-run pediatric ECMO at a single center institution.

PURPOSE: Survival of neonatal and pediatric patients undergoing extracorporeal membrane oxygenation (ECMO) ≥ 21 days has not been well described. We hypothesized that patients would have poor survival and increased long-term complications. METHODS: Retrospective, single center, review and case analysis. Tertiary-care university children's hospital including neonatal, pediatric and cardiac intensive care units. After institutional review board approval, the charts of all patients < 18 years of age undergoing ECMO for ≥ 21 continuous days were performed, and they were compared to comparative patients undergoing shorter runs. Overall survival, incidence of complications, and post-discharge recovery were recorded. RESULTS: Overall survival was 36% in patients undergoing ≥ 21 days of ECMO (N = 14). 5/8 patients with cardiopulmonary failure from acquired etiologies survived versus 0/6 patients with congenital anomalies. 1/5 survivors achieved complete recovery with no neurologic deficits. The remaining survivors suffer from multiple medical and neurodevelopmental morbidities. CONCLUSION: ECMO support for ≥ 21 days is associated with poor survival, particularly in neonates with congenital anomalies. Long-term outcomes for survivors ought to be carefully weighed and discussed with parents given the high incidence of neurologic morbidities in this population.

Rho kinase inhibition maintains intestinal and vascular barrier function by upregulation of occludin in experimental necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a deadly disease that occurs in 5-10% of neonates. Although NEC has been extensively studied, no single therapeutic target has been identified. Rho kinase (ROCK) is a serine/threonine kinase that affects multiple cellular processes, including tight junction (TJ) function, cellular permeability, and apoptosis. We hypothesized that ROCK inhibition would decrease cellular permeability, stabilize TJ proteins (occludin), and decrease the severity of NEC. To test this hypothesis, human colon epithelial cells (Caco-2) and human endothelial cells were studied. Cells were treated with lipopolysaccharide to simulate an in vitro model of NEC. The effect of ROCK inhibition was measured by transepithelial membrane resistance (TEER) and cellular permeability to FITC-dextran. The effects of ROCK inhibition in vivo were analyzed in the rat pup model of NEC. NEC was induced by feeding formula supplemented with Cronobacter sakazakii with or without gavaged ROCK inhibitor. Rat intestines were scored based on histological degree of injury. RNA and protein assays for occludin protein were performed for all models of NEC. Treatment with ROCK inhibitor significantly decreased cellular permeability in Caco-2 cells and increased TEER. Intestinal injury scoring revealed decreased scores in ROCK inhibitor-treated pups compared with NEC only. Both cell and rat pup models demonstrated an upregulation of occludin expression in the ROCK inhibitor-treated groups. Therefore, we conclude that ROCK inhibition protects against experimental NEC by strengthening barrier function via upregulation of occludin. These data suggest that ROCK may be a potential therapeutic target for patients with NEC. NEW & NOTEWORTHY These studies are the first to demonstrate an upregulation of occludin tight junction protein in response to Rho kinase (ROCK) inhibition. Furthermore, we have demonstrated that ROCK inhibition in experimental models of necrotizing enterocolitis (NEC) is protective against NEC in both in vitro and in vivo models of disease.

Analysis of factors predicting surgical intervention and associated costs in pediatric breast masses: a single center study.

PURPOSE: Finding a breast mass in a child provokes apprehension in parents, especially in those with a family history of breast cancer. Clinicians must decide between serial imaging or biopsy of the mass. Herein, we identify management differences in those with and without a positive family history, as well as identify cost differences. METHODS: An institutional retrospective review was performed of patients (2-18 years of age) with a diagnosis of breast mass. Patient demographics, presentation, medical and surgical history, physical exam, imaging, and pathologic diagnosis were collected. Cost data were acquired from the pediatric health information system (PHIS). Costs were compared between patients managed by biopsy versus serial ultrasounds. Bivariate analyses including Pearson's Chi-square, student's t tests, and logistic regression were performed. RESULTS: The probability of biopsy increases with age (p = 0.0001) and female gender (p = 0.006). Biopsy rate is higher for larger masses (p < 0.0001), growing size (p < 0.0001), and in patients with a positive family history of breast cancer (p < 0.0001). The average cost of care for management with initial excisional biopsy was $4491 versus those with serial ultrasounds ($986) (p < 0.0001). CONCLUSIONS: In patients with small lesions, even with a family history of breast cancer, non-operative monitoring is a safe and cost-effective alternative to invasive biopsy.

Central venous access in children: indications, devices, and risks.

PURPOSE OF REVIEW: Central venous catheters (CVCs) have a prominent role in the diagnostic and therapy of neonates and children. Herein, we describe the multiple indications for CVC use and the different devices available for central venous access. Given the prevalent use of CVCs, healthcare systems are focused on reducing complications from their use, particularly central line-associated bloodstream infections (CLABSIs). The most up-to-date information available sheds light on best practices and future areas of investigation. RECENT FINDINGS: Large systematic reviews of randomized trials suggest that ultrasound guidance for placement of CVCs in children is safer than using blind technique, at least for internal jugular vein access. Appropriate catheter tip placement is associated with decreased complications. Furthermore, the prophylactic use of ethanol lock between cycles of parenteral nutrition administration has reduced the rates of CLABSI. A recent randomized trial in pediatric CVCs showed a benefit with antibiotic-coated CVCs. SUMMARY: Based on the available evidence, multiple techniques for CVC placement are still valid, including the landmark technique based on practitioner experience, but ultrasound guidance has been shown to decrease complications from line placement. Adherence to CVC care protocols is essential in reducing infectious complications.

Experimental Modeling of Necrotizing Enterocolitis in Human Infant Intestinal Enteroids.

METHODS: There are several limitations when using a single cell culture to recapitulate the findings in a complex organism and results often vary between species, when proxy animal models are studied. RESULTS: Human enteroids have allowed for study of human disease in complex multicellular culture systems. Here we present the novel use of human infant enteroids generated from premature infant intestine to study necrotizing enterocolitis (NEC), which is a devastating intestinal disorder that affects our most vulnerable pediatric population. CONCLUSIONS: We demonstrate that NEC can be induced in premature human enteroids as supported by corresponding alterations in inflammation, apoptosis, tight junction expression, and permeability by treatment with lipopolysaccharide.

Surgical Residents in the Battle Against COVID-19.

PERSPECTIVES: In times of public health crises, medical residency program leaders are responsible to maintain the wellbeing of their residents and ensure uninterrupted training. COVID-19 caused significant impact on healthcare industry, depleting resources and manpower, which led to disruption to graduate medical education and residency training. Surgical residents were affected by the pandemic both by reduced operative opportunities in most training centers and inducing stress and concerns about safety and wellbeing among residents. Spread of the SARS-Cov-2 was naturally accompanied with a gradual decrease in numbers of healthcare personnel which consequently increased the burden on residents. During these times of crisis and uncertainty, it is crucial that residency programs find alternative learning opportunities and deploy pre-designed, dynamic operational strategies to ensure high quality surgical services while optimizing resident safety and wellbeing. The COVID-19 crisis was a natural call for the essential need to add another dimension to residency competencies, which is Crisis-based learning and practice. Times of public health crisis are opportunities to reflect on the medical practice from an interdisciplinary and interprofessional perspective and train the residents to function as part of a larger, globally responsible team. It also calls upon adopting innovative instructional and learning strategies such as utilizing digital and online learning tools to complement learning. A holistic approach to the crisis was taken by the surgical residency program at the University of Illinois in Chicago, which addressed the issue from a resident, hospital, and public health standpoints. An operational strategy was introduced to optimize resident safety, maximize learning opportunities, support other non-surgical services, and promote online teaching and learning. This strategy is meant to serve as a dynamic reference for surgical residency programs and as an infrastructure for dealing with this and any upcoming healthcare crises in an efficient and resident-centered way.

A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease of newborn infants. It is characterized by multiple pathophysiologic alterations in the human intestinal epithelium, leading to increased intestinal permeability, impaired restitution, and increased cell death. Although there are numerous animal models of NEC, response to injury and therapeutic interventions may be highly variable between species. Furthermore, it is ethically challenging to study disease pathophysiology or novel therapeutic agents directly in human subjects, especially children. Therefore, it is highly desirable to develop a novel model of NEC using human tissue. Enteroids are 3-dimensional organoids derived from intestinal epithelial cells. They are ideal for the study of complex physiologic interactions, cell signaling, and host-pathogen defense. In this manuscript we describe a protocol that cultures human enteroids after isolating intestinal stem cells from patients undergoing bowel resection. The crypt cells are cultured in media containing growth factors that encourage differentiation into the various cell types native of the human intestinal epithelium. These cells are grown in a synthetic, collagenous mix of proteins that serve as a scaffold, mimicking the extra-cellular basement membrane. As a result, enteroids develop apical-basolateral polarity. Co-administration of lipopolysaccharide (LPS) in media causes an inflammatory response in the enteroids, leading to histologic, genetic, and protein expression alterations similar to those seen in human NEC. An experimental model of NEC using human tissue may provide a more accurate platform for drug and treatment testing prior to human trials, as we strive to identify a cure for this disease.

Caveolin 1 is Associated with Upregulated Claudin 2 in Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency of neonates. Epithelial tight junction (TJ) proteins, such as claudins, are essential for regulation and function of the intestinal barrier. Rho kinase (ROCK) affects cellular permeability and TJ regulation. We hypothesized that TJ protein changes would correlate with increased permeability in experimental NEC, and ROCK inhibitors would be protective against NEC by regulation of key claudin proteins. We tested this hypothesis using an in vivo rat pup model, an in vitro model of experimental NEC, and human intestinal samples from patients with and without NEC. Experimental NEC was induced in rats via hypoxia and bacteria-containing formula, and in Caco-2 cells by media inoculated with LPS. The expression of claudins was measured by gene and protein analysis. Experimental NEC in rat pups and Caco-2 cells had increased permeability compared to controls. Gene and protein expression of claudin 2 was increased in experimental NEC. Sub-cellular fractionation localized increased claudin 2 protein to the cytoskeleton. ROCK inhibition was associated with normalization of these alterations and decreased severity of experimental NEC. Co-immunoprecipitation of caveolin-1 with claudin 2 suggests that caveolin-1 may act as a shuttle for the internalization of claudin 2 seen in experimental NEC. In conclusion, NEC is associated with intestinal permeability and increased expression of claudin 2, increased binding of caveolin-1 and claudin 2, and increased trafficking of claudin 2 to the cytoskeleton.

The science and necessity of using animal models in the study of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) remains one of the highest causes of mortality and of acute and long-term morbidity in premature infants. Multiple factors are involved in the pathophysiology of NEC including the immaturity of the immune system and the complex changing composition of the intestinal microbiome. This is compounded by the fact that the premature infant should ideally still be a developing fetus and has an immature intestinal tract. Because these complexities are beyond the scope of studies in single-cell cultures, animal models are absolutely essential to understand the mechanisms involved in the pathophysiology of NEC and the effects of inflammation on the immature intestinal tract. To this end, investigators have utilized many different species (e.g., rats, mice, rabbits, quails, piglets, and non-human primates) and conditions to develop models of NEC. Each animal has distinct advantages and drawbacks related to its preterm viability, body size, genetic variability, and cost. The choice of animal model is strongly influenced by the scientific question being addressed. While no model perfectly mimics human NEC, each has greatly improved our understanding of disease. Examples of recent discoveries in NEC pathogenesis and prevention underscore the importance of continued animal research in NEC.

Utility of liver biopsy in the evaluation of pediatric total parenteral nutrition cholestasis.

BACKGROUND: Cholestasis is a serious complication of total parenteral nutrition (TPN) in neonates. Liver biopsies may be requested to assess the severity of cholestasis and fibrosis. We hypothesized that liver biopsy would not lead to changes in management or improved patient outcomes. METHODS: A single institution retrospective review of infants with TPN cholestasis from January 2008 to January 2016. OUTCOMES: length of stay, complications, change in management and mortality. Statistical analysis was performed using Fisher's exact test. RESULTS: Twenty-seven out of 95 patients with TPN cholestasis underwent liver biopsy. Liver biopsy was associated with increased utilization or ursodeoxycholic acid (p = 0.001). There were no differences in length of stay (LOS) or mortality. One patient had a complication following anesthesia for liver biopsy, there were no bleeding complications recorded. CONCLUSIONS: Liver biopsy in patients with TPN cholestasis was associated with an increase in utilization of ursodeoxycholic acid. The effects of this are not fully understood; however, liver biopsy was not associated with improved patient outcomes such as LOS or mortality.

Effect of preadmission bowel preparation on outcomes of elective colorectal procedures in young children.

BACKGROUND: The utility of mechanical bowel preparation (MBP) to minimize infectious complications in elective colorectal surgery is contentious. Though data is scarce in children, adult studies suggest a benefit to MBP when administered with oral antibiotics (OAB). METHODS: After IRB approval, the Pediatric Health Information System (PHIS) was queried for young children undergoing elective colon surgery from 2011 to 2014. Patients were divided into: no bowel preparation (Group 1), MBP (Group 2), and MBP plus OAB (Group 3). Statistical significance was determined using univariate and multivariate analysis with GEE models accounting for clustering by hospital. RESULTS: One thousand five hundred eighty-one patients met study criteria: 63.7% in Group 1, 27.1% in Group 2, and 9.2% in Group 3. Surgical complication rate was higher in Group 1 (23.3%) compared to Groups 2 and 3 (14.2% and 15.5%; P<0.001). However, median length of stay was shorter in Group 1 (4, IQR 4days) compared to Group 2 (5, IQR 3) and Group 3 (6, IQR 3) (P<0.001). 30-day readmission rates were similar. In multivariate analysis compared to patients in Group 1, the odds of surgical complications were 0.72 (95% CI 0.40-1.29, P=0.28) with MBP alone (Group 2), 1.79 (95% CI 1.28-2.52, P=0.0008) with MBP+OAB (Group 3), and 1.13 (95% CI 0.81-1.58, P=0.46) for the aggregate Group 2 plus 3. CONCLUSION: Utilization of bowel preparation in children is variable across children's hospitals nationally, and the benefit is unclear. Given the discrepancy with adult literature, a three-armed pediatric-specific randomized controlled trial is warranted. LEVEL OF EVIDENCE: Level III treatment study - retrospective comparative study.