A Comparison of Racial and Ethnic Disparities in Complications Following Burn Injury in Adult Patients.

Burn injury contributes to significant morbidity and mortality in the United States. Despite an increased focus on racial and ethnic disparities in healthcare, there remains a critical knowledge gap in our understanding of the effect of these disparities on complications experienced by burn patients. The American Burn Association's National Burn Repository data were reviewed from 2010-2018. Information regarding demographics, burn mechanism and severity, complications, and clinical outcomes were recorded. Data analysis was performed using 1:1 propensity-score-matching and logistic regression modeling. A separate analysis of Hispanic and non-Hispanic patients was performed using Chi squared tests. Among 215,071 patients, racial distribution was 65.16% white, 19.13% black, 2.18% Asian, 0.74% American Indian/Alaskan Native, and 12.78% other. Flame injuries were the most common cause (35.2%), followed by scald burns (23.3%). All comparisons were made in reference to the white population. Black patients were more likely to die (OR: 1.28; 95%CI: 1.17-1.40), experience all (OR: 1.08; 95%CI: 1.03-1.14), cardiovascular (OR: 1.24; 95%CI: 1.08-1.43), or infectious (OR: 1.64; 95%CI: 1.40-1.91) complications, and less likely to experience airway complications (OR: 0.83; 95%CI: 0.74-0.94). American Indian/Alaskan Native patients were more likely to experience any complication (OR: 1.33; 95%CI: 1.05-1.70). All minority groups had increased length of hospital stay. Black, Asian, and other patients had longer length of ICU stay. Black patients had longer ventilator duration. Among 82,775 patients, 24,075 patients were identified as Hispanic and 58,700 as non-Hispanic. Statistically significant differences were noted between groups in age, TBSA, proportion of 2nd degree burn, and proportion of 3rd degree burn (p<0.01). These findings highlight the need for further work to determine the etiology of these disparities to improve burn care for all patients.

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.

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.

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.

A single institution case series of ReCell® use in treating pediatric burns.

BACKGROUND: Thermal injury has a significant impact on disability and morbidity in pediatric patients. Challenges in caring for pediatric burn patients include limited donor sites for large total body surface area (TBSA) burn as well as optimization of wound management for long term growth and cosmesis. ReCell® technology produces autologous skin cell suspensions from minimal donor split-thickness skin samples, allowing for expanded coverage using minimal donor skin. Most literature on outcomes reports on adult patients. OBJECTIVE: We present the largest to-date retrospective review of ReCell® technology use in pediatric patients at a single pediatric burn center. METHOD: Patients were treated at a quaternary care, free-standing, American Burn Association verified Pediatric Burn Center. A retrospective chart review was performed from September 2019 to March 2022, during which time twenty-one pediatric burn patients had been treated with ReCell® technology. Patient information was collected, including demographics, hospital course, burn wound characteristics, number of ReCell® applications, adjunct procedures, complications, healing time, Vancouver scar scale measurements, and follow-up. A descriptive analysis was performed, and medians were reported. RESULTS: Median TBSA burn on initial presentation was 31% (ranging 4%-86%). The majority of patients (95.2%) had placement of a dermal substrate prior to ReCell® application. Four patients did not receive split thickness skin grafting with their ReCell® treatment. The median time between date of burn injury and first ReCell® application was 18 days (ranging 5-43 days). The number of ReCell® applications ranged from 1-4 per patient. Median time until wound was classified as healed was 81 days (ranging 39-573 days). The median maximum Vancouver scar scale measurement per patient at time healed was 8, ranging from 3-14. Five patients who received skin grafts had graft loss and three of these patients had graft loss from areas with ReCell®. CONCLUSION: ReCell® technology provides an additional method for wound coverage, either on its own or in conjunction with split thickness skin grafting, and is safe and effective in pediatric patients.

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.

DEMOGRAPHICS TO DEFINE PEDIATRIC BURN PATIENTS AT RISK OF ADVERSE OUTCOMES.

ABSTRACT: Background: There is currently no standard definition of a severe burn in the pediatric patient population to identify those at higher risk of infectious complications. Our aim was to correlate total burn surface area (TBSA), burn depth, and type of burn injury to nosocomial infection rates and systemic immune system responses to better define risk factors associated with adverse outcomes. Methods: A prospective observational study at a single-center, quaternary-care, American Burn Association-verified pediatric burn center was conducted from 2016 to 2021. Blood was collected within 72 h of injury from 103 pediatric patients. Whole blood was incubated with lipopolysaccharide or phytohemagglutinin stimulation reagent to measure innate and adaptive immune response, respectively. Flow cytometry was performed on whole blood samples to measure both innate and adaptive immune cells. Unstimulated plasma was also extracted, and IL-6 and IL-10 as well as soluble proteins B- and T-lymphocyte attenuator, CD27, and T-cell immunoglobulin mucin 3 were quantified. Results: There was a significant increased risk for nosocomial infection in pediatric patients with TBSA burns of ≥20%, full-thickness burn injuries ≥5%, or flame burn injuries. There was an overall decrease in both innate and adaptive immune function in patients with TBSA burns ≥20% or full-thickness burn injuries ≥5%. Both burn injury characteristics were also associated with a significant increase in unstimulated IL-6 and IL-10 and soluble immunoregulatory checkpoint proteins. We observed a significant decrease in soluble B- and T-lymphocyte attenuator for those with a flame injury, but there were no other differences between flame injury and scald/contact burns in terms of innate and adaptive immune function. Conclusion: Burns with ≥20% TBSA or ≥5% full thickness in pediatric patients are associated with systemic immune dysfunction and increased risk of nosocomial infections.

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.

A miRNA-based classification of renal cell carcinoma subtypes by PCR and in situ hybridization.

Renal cell carcinoma (RCC) constitutes an array of morphologically and genetically distinct tumors the most prevalent of which are clear cell, papillary, and chromophobe RCC. Accurate distinction between the typically benign-behaving renal oncocytoma and RCC subtypes is a frequent challenge for pathologists. This is critical for clinical decision making. Subtypes also have different survival outcomes and responses to therapy. We extracted RNA from ninety formalin-fixed paraffin-embedded (FFPE) tissues (27 clear cell, 29 papillary, 19 chromophobe, 4 unclassified RCC and 11 oncocytomas). We quantified the expression of six miRNAs (miR-221, miR-222, miR-126, miR-182, miR-200b and miR-200c) by qRT-PCR, and by in situ hybridization in an independent set of tumors. We developed a two-step classifier. In the first step, it uses expression of either miR-221 or miR-222 to distinguish the clear cell and papillary subtypes from chromophobe RCC and oncocytoma (miR-221 AUC: 0.96, 95% CI: 0.9132-1.014, p < 0.0001 and miR-222 AUC: 0.91, 95% CI: 0.8478-0.9772, p < 0.0001). In the second step, it uses miR-126 to discriminate clear cell from papillary RCC (AUC: 1, p < 0.0001) and miR-200b to discriminate chromophobe RCC from oncocytoma (AUC: 0.95, 95% CI: 0.8933-1.021, p < 0.0001). In situ hybridization showed a nuclear staining pattern. miR-126, miR-222 and miR-200b were significantly differentially expressed between the subtypes by in situ hybridization. miRNA expression could distinguish RCC subtypes and oncocytoma. miRNA expression assessed by either PCR or in situ hybridization can be a clinically useful diagnostic tool to complement morphologic renal tumor classification, improving diagnosis and patient management.

Integrated Phenotypic/Genotypic Analysis of Papillary Renal Cell Carcinoma Subtypes: Identification of Prognostic Markers, Cancer-related Pathways, and Implications for Therapy.

BACKGROUND: Two histologic subtypes are recognized for papillary renal cell carcinoma (PRCC). Studies have shown that the subtypes differ in characteristic genetic alterations and clinical behavior. Clinically, the subtypes are managed similarly. OBJECTIVES: To analyze the biological differences between the two PRCC histological subtypes, in order to further guide their clinical management. DESIGN, SETTING, AND PARTICIPANTS: PRCC cohort consisting of 317 patients from the Cancer Genome Atlas database and our institution. Patients were stratified according to histologic criteria as type 1, type 2, or not otherwise specified (NOS). Gene and miRNA expression data for the cohort were examined via unsupervised and supervised clustering. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Significant molecular signatures for each subtype were used to unravel the implicated molecular pathways via bioinformatics analysis. Survival was compared between the subtypes. Newly discovered biomarkers were used to further stratify survival of patients in the NOS category. RESULTS AND LIMITATIONS: Tumor genotyping revealed two distinct PRCC subtypes. The top molecular pathways enriched in PRCC1 were WNT, Hedgehog, and Notch signaling (p=0.001-0.01); highlighting an embryonic developmental theme to the pathogenesis of this subtype. PRCC2 showed enrichment in the mTOR, VEGF (p=7.49E-09) and HIF (p=7.63E-05) signaling pathways. Overall survival and disease-free survival significantly differed between the types. ABCC2 expression was identified as a significant prognostic biomarker for the NOS group in univariate (log rank p<0.0001; hazard ratio [HR] >11.63) and multivariate analysis (p=0.003; HR >2.12). ABCC2 expression and its effect on survival should be further validated at the protein level. CONCLUSIONS: The classical PRCC types 1 and 2 have two distinct genotypes. We unraveled pathways that indicate that the two types could potentially respond differently to current therapies. We also identified biomarkers that stratify tumors within the PRCC NOS category into prognostic subgroups. Our findings highlight the need for molecular markers to accurately subtype PRCC and guide clinical management. PATIENT SUMMARY: The two types of papillary renal cancer are treated similarly. We show that the two types have a different genetic makeup, and hence they should be considered two different tumors. There is a different biology underlying each tumor type that can potentially affect the way they respond to treatment. We uncovered genes that can be tested for to guide therapy in some problematic cases for which it hard to define the tumor type.

Toward Biological Subtyping of Papillary Renal Cell Carcinoma With Clinical Implications Through Histologic, Immunohistochemical, and Molecular Analysis.

Papillary renal cell carcinoma (PRCC) has 2 histologic subtypes. Almost half of the cases fail to meet all morphologic criteria for either type, hence are characterized as PRCC not otherwise specified (NOS). There are yet no markers to resolve the PRCC NOS category. Accurate classification can better guide the management of these patients. In our previous PRCC study we identified markers that can distinguish between the subtypes. A PRCC patient cohort of 108 cases was selected for the current study. A panel of potentially distinguishing markers was chosen from our previous genomic analysis, and assessed by immunohistochemistry. The panel exhibited distinct staining patterns between the 2 classic PRCC subtypes; and successfully reclassified the NOS (45%) cases. Moreover, these immunomarkers revealed a third subtype, PRCC3 (35% of the cohort). Molecular testing using miRNA expression and copy number variation analysis confirmed the presence of 3 distinct molecular signatures corresponding to the 3 subtypes. Disease-free survival was significantly enhanced in PRCC1 versus 2 and 3 (P=0.047) on univariate analysis. The subtypes stratification was also significant on multivariate analysis (P=0.025; hazard ratio, 6; 95% confidence interval, 1.25-32.2). We propose a new classification system of PRCC integrating morphologic, immunophenotypical, and molecular analysis. The newly described PRCC3 has overlapping morphology between PRCC1 and PRCC2, hence would be subtyped as NOS in the current classification. Molecularly PRCC3 has a distinct signature and clinically it behaves similar to PRCC2. The new classification stratifies PRCC patients into clinically relevant subgroups and has significant implications on the management of PRCC.

S100A11 is a potential prognostic marker for clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is one of few cancers with rising incidence in North America. The prognosis of ccRCC is variable and difficult to predict. Stratification of patients according to disease aggressiveness can significantly improve patient management. We investigated the expression of the S100A11 protein in 385 patients with primary ccRCC using immunohistochemistry on tissue microarrays. We compared its expression with clinicopathologic parameters and patients' survival. We also validated our results at the mRNA level on an independent set from The Cancer Genome Atlas. As a dichotomous variable (low vs. high expression), there was a significant association between S100A11 expression and tumor grade, with higher expression associated with higher tumor grades (p < 0.001). High expression was also significantly more frequently seen in higher versus lower stages (56 vs. 28 %). In the univariate analysis, high S100A11 expression was associated with significantly shorter disease-free survival (DFS) (HR = 2.28; p = 0.001). This was maintained in the multivariate analysis (HR = 1.69; p = 0.042). Expression was not associated with overall survival (OS) (p = 0.10). Comparable results were obtained when S100A11 expression was analyzed as a trichotomous variable (low, moderate, or high expression). The Kaplan­Meier survival analyses showed that higher S100A11 expression was associated with statistically significant decrease in DFS (p < 0.001), but not OS (p = 0.1).

An integrated genomic analysis of papillary renal cell carcinoma type 1 uncovers the role of focal adhesion and extracellular matrix pathways.

Papillary renal cell carcinoma (pRCC) is the second most common RCC subtype and can be further classified as type 1 (pRCC1) or 2 (pRCC2). There is currently minimal understanding of pRCC1 pathogenesis, and treatment decisions are mostly empirical. The aim of this study was to identify biological pathways that are involved in pRCC1 pathogenesis using an integrated genomic approach. By microarray analysis, we identified a number of significantly dysregulated genes and microRNAs (miRNAs) that were unique to pRCC1. Integrated bioinformatics analyses showed enrichment of the focal adhesion and extracellular matrix (ECM) pathways. We experimentally validated that many members of these pathways are dysregulated in pRCC1. We identified and experimentally validated the downregulation of miR-199a-3p in pRCC1. Using cell line models, we showed that miR-199a-3p plays an important role in pRCC1 pathogenesis. Gain of function experiments showed that miR-199a-3p overexpression significantly decreased cell proliferation (p = 0.013). We also provide evidence that miR-199a-3p regulates the expression of genes linked to the focal adhesion and ECM pathways, such as caveolin 2 (CAV2), integrin beta 8 (ITGB8), MET proto-oncogene and mammalian target of rapamycin (MTOR). Using a luciferase reporter assay, we further provide evidence that miR-199a-3p overexpression decreases the expression of MET and MTOR. Using an integrated gene/miRNA approach, we provide evidence linking miRNAs to the focal adhesion and ECM pathways in pRCC1 pathogenesis. This novel information can contribute to the development of effective targeted therapies for pRCC1, for which there is none currently available in the clinic.