RNA Sequencing Analysis of CD4+ T Cells Exposed to Airway Fluid From Children With Pediatric Acute Respiratory Distress Syndrome.

CD4+ T cells contribute to lung inflammation in acute respiratory distress syndrome. The CD4+ T-cell response in pediatric acute respiratory distress syndrome (PARDS) is unknown. OBJECTIVES: To identify differentially expressed genes and networks using a novel transcriptomic reporter assay with donor CD4+ T cells exposed to the airway fluid of intubated children with mild versus severe PARDS. DESIGN: In vitro pilot study. SETTING: Laboratory-based study using human airway fluid samples admitted to a 36-bed university-affiliated pediatric intensive care unit. PATIENTS/SUBJECTS: Seven children with severe PARDS, nine children with mild PARDS, and four intubated children without lung injury as controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We performed bulk RNA sequencing using a transcriptomic reporter assay of CD4+ T cells exposed to airway fluid from intubated children to discover gene networks differentiating severe from mild PARDS. We found that innate immunity pathways, type I (α and ß), and type II (γ) interferon response and cytokine/chemokine signaling are downregulated in CD4+ T cells exposed to airway fluid from intubated children with severe PARDS compared with those with mild PARDS. CONCLUSIONS: We identified gene networks important to the PARDS airway immune response using bulk RNA sequencing from a novel CD4+ T-cell reporter assay that exposed CD4+ T cells to airway fluid from intubated children with severe and mild PARDS. These pathways will help drive mechanistic investigations into PARDS. Validation of our findings using this transcriptomic reporter assay strategy is needed.

Identification of a pediatric acute hypoxemic respiratory failure signature in peripheral blood leukocytes at 24 hours post-ICU admission with machine learning.

Background: There is no generalizable transcriptomics signature of pediatric acute respiratory distress syndrome. Our goal was to identify a whole blood differential gene expression signature for pediatric acute hypoxemic respiratory failure (AHRF) using transcriptomic microarrays within twenty-four hours of diagnosis. We used publicly available human whole-blood gene expression arrays of a Berlin-defined pediatric acute respiratory distress syndrome (GSE147902) cohort and a sepsis-triggered AHRF (GSE66099) cohort within twenty-four hours of diagnosis and compared those children with a PaO2/FiO2 < 200 to those with a PaO2/FiO2 ≥ 200. Results: We used stability selection, a bootstrapping method of 100 simulations using logistic regression as a classifier, to select differentially expressed genes associated with a PaO2/FiO2 < 200 vs. PaO2/FiO2 ≥ 200. The top-ranked genes that contributed to the AHRF signature were selected in each dataset. Genes common to both of the top 1,500 ranked gene lists were selected for pathway analysis. Pathway and network analysis was performed using the Pathway Network Analysis Visualizer (PANEV) and Reactome was used to perform an over-representation gene network analysis of the top-ranked genes common to both cohorts. Changes in metabolic pathways involved in energy balance, fundamental cellular processes such as protein translation, mitochondrial function, oxidative stress, immune signaling, and inflammation are differentially regulated early in pediatric ARDS and sepsis-induced AHRF compared to both healthy controls and to milder acute hypoxemia. Specifically, fundamental pathways related to the severity of hypoxemia emerged and included (1) ribosomal and eukaryotic initiation of factor 2 (eIF2) regulation of protein translation and (2) the nutrient, oxygen, and energy sensing pathway, mTOR, activated via PI3K/AKT signaling. Conclusions: Cellular energetics and metabolic pathways are important mechanisms to consider to further our understanding of the heterogeneity and underlying pathobiology of moderate and severe pediatric acute respiratory distress syndrome. Our findings are hypothesis generating and support the study of metabolic pathways and cellular energetics to understand heterogeneity and underlying pathobiology of moderate and severe acute hypoxemic respiratory failure in children.

Cluster analysis of plasma cytokines identifies two unique endotypes of children with asthma in the pediatric intensive care unit.

Children with life-threatening asthma exacerbations who are admitted to a pediatric intensive care unit (PICU) are a heterogeneous group with poorly studied inflammatory features. We hypothesized that distinct clusters of children with asthma in a PICU would be identified based on differences in plasma cytokine levels and that these clusters would have differing underlying inflammation and asthma outcomes within 1 year. Plasma cytokines and differential gene expression were measured in neutrophils isolated from children admitted to a PICU for asthma. Participants were clustered by differential plasma cytokine abundance. Gene expression differences were compared by cluster and pathway over-representation analysis was performed. We identified two clusters in 69 children with no clinical differences. Cluster 1 (n = 41) had higher cytokines compared to Cluster 2 (n = 28). Cluster 2 had a hazard ratio of 2.71 (95% CI 1.11-6.64) compared to Cluster 1 for time to subsequent exacerbation. Gene expression pathways that differed by cluster included interleukin-10 signaling; nucleotide-binding domain, leucine rich repeat containing receptor (NLR signaling); and toll-like receptor (TLR) signaling. These observations suggest that a subset of children may have a unique pattern of inflammation during PICU hospitalization that might require alternative treatment approaches.

Expression Patterns of Airway Fluid Cytokines From Intubated Children With Pediatric Acute Respiratory Distress Syndrome.

Pediatric acute respiratory distress syndrome (PARDS) is a heterogeneous illness affecting 6% of mechanically ventilated children and with an overall mortality of 17%. Studies in PARDS have mainly focused on plasma biomarkers which may not reflect airway biomarkers. We lack adequate understanding of the inflammatory mediators and underlying immune responses in the airways of PARDS patients. Our objective was to compare the levels of cytokines in the airway fluid of intubated children with severe versus nonsevere acute respiratory distress syndrome. DESIGN: Prospective observational cohort study. SETTING: Single 36-bed quaternary care academic safety-net hospital PICU. PATIENTS: Children intubated for acute respiratory failure between January 2018 and November 2021 stratified by Pediatric Acute Lung Injury Consensus Conference-1 criteria for PARDS. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We measured levels of 23 cytokines, chemokines, and protein biomarkers in the tracheal aspirate from 82 intubated children, between 14 days and 17 years old, at risk for or with PARDS. Levels of interleukin-4, -5, -7, -8, -12(p-70), -17a, -21, and fractalkine were higher in patients with severe versus nonsevere PARDS. There were no associations between airway and plasma cytokines. CONCLUSIONS: Proinflammatory cytokines are elevated in the airway fluid from intubated children with severe PARDS and reflect diverse patterns of airway inflammation.

Text message interventions for follow up of infants born to mothers positive for Chagas disease in Tucumán, Argentina: a feasibility study.

BACKGROUND: Diagnosis of congenital Chagas disease occurs at 9 months of age, making effective treatment challenging due to loss to follow-up. Mobile health (mHealth) has been utilized to improve communication and treatment adherence in many chronic diseases, although no studies of mHealth in Trypanosoma cruzi-infected individuals have been conducted. Text message interventions, a subset of mHealth, has shown to improve appointment attendance and is relatively simple to set up, thus making it an ideal mechanism to facilitate communication with individuals in low-resource settings. OBJECTIVE: The aim of this study is to understand the acceptability, utilization, and barriers of an SMS-based appointment reminder to confirm a post-partum home visit to women in Tucumán, Argentina and whether these factors differ in urban and rural populations. METHODS: Women that tested positive for Chagas disease were invited to receive SMS reminders of their follow-up 4-week postpartum home visit. Demographic information and SMS contact preferences were collected at hospital discharge, and variables on mHealth utilization and barriers were recorded at follow-up. RESULTS: 77 (70.6%) of women possessed a cell phone for personal use. All eligible women owned phones compatible with SMS messages. The appointment reminder SMS was widely accepted with 64/72 (88.9%) enrolled women receiving the SMS message and 58/64 (90.6%) replying. Ninety-two percent of women stated that the text message was a useful reminder for the follow-up home visit. Women living in rural areas were less likely to own a cell phone for personal use and were significantly less likely to have internet access on their phone than women living in urban areas (RR 0.30, 95% CI 0.10-0.89). Furthermore, women from rural areas faced barriers to mHealth uptake such as change of phone number and response to messages from the hospital team at higher rates than women from urban areas, although these differences were not statistically significant. CONCLUSIONS: There is generally widespread acceptance and utilization of mHealth among this group of women with access to cell phones. However, there are still many barriers to overcome before mHealth interventions attain complete penetration in a population, most notably the issue of cell phone for personal use.

Activation of c-Myc and Cyclin D1 by JCV T-Antigen and ß-catenin in colon cancer.

During the last decade, mounting evidence has implicated the human neurotropic virus JC virus in the pathology of colon cancer. However, the mechanisms of JC virus-mediated oncogenesis are still not fully determined. One candidate to mediate these effects is the viral early transcriptional product T-Antigen, which has the ability to inactivate cell cycle regulatory proteins such as p53. In medulloblastomas, T-Antigen has been shown to bind the Wnt signaling pathway protein ß-catenin; however, the effects of this interaction on downstream cell cycle regulatory proteins remain unknown. In light of these observations, we investigated the association of T-Antigen and nuclear ß-catenin in colon cancer cases and the effects of this complex in the activation of the transcription and cell cycle regulators c-Myc and Cyclin D1 in vitro. Gene amplification demonstrated the presence of viral sequences in 82.4% of cases and we detected expression of T-Antigen in 64.6% of cases by immunohistochemistry. Further, we found that T-Antigen and ß-catenin co-localized in the nuclei of tumor cells and we confirmed the physical binding between these two proteins in vitro. The nuclear presence of T-Antigen and ß-catenin resulted in the significant enhancement of TCF-dependent promoter activity and activation of the ß-catenin downstream targets, c-Myc and Cyclin D1. These observations provide further evidence for a role of JCV T-Antigen in the dysregulation of the Wnt signaling pathway and in the pathogenesis of colon cancer.

Immunomodulation with IL-4R alpha antisense oligonucleotide prevents respiratory syncytial virus-mediated pulmonary disease.

Respiratory syncytial virus (RSV) causes significant morbidity and mortality in infants worldwide. Severe RSV infections in infants cause bronchiolitis, wheeze, and/or cough and significantly increase the risk for developing asthma. RSV pathogenesis is thought to be due to a Th2-type immune response initiated in response to RSV infection, specifically in the infant. Using a neonatal mouse system as an appropriate model for human infants, we sought to determine whether local inhibition of IL-4Rα expression during primary RSV infection in the neonate would prevent Th2-skewed responses to secondary RSV infection and improve long-term pulmonary function. To reduce IL-4Rα expression, antisense oligonucleotides (ASOs) specific for IL-4Rα were administered intranasally to neonatal mice at the time of primary infection. Mice were initially infected with RSV at 1 wk of age and were reinfected at 6 wk of age. Administration of IL-4Rα ASOs during primary RSV infection in neonatal mice abolished the pulmonary dysfunction normally observed following reinfection in the adult. This ablation of pulmonary dysfunction correlated with a persistent rebalancing of the Th cell compartment with decreased Th2 responses (i.e., reduced goblet cell hyperplasia, Th2 cells, and cytokine secretion) and increased Th1 responses (i.e., elevated Th1 cell numbers and type I Abs and cytokines). Our data support our hypothesis that a reduction in the Th2 immune response during primary infection in neonates prevents Th2-mediated pulmonary pathology initially and upon reinfection and further suggest that vaccine strategies incorporating IL-4Rα ASOs may be of significant benefit to infants.