Prevalence of burnout and its relation to the neuroendocrine system among pediatric residents during the early Covid-19 pandemic: A pilot feasibility study.

Background: Measuring burnout relies on infrequent and subjective surveys, which often do not reflect the underlying factors or biological mechanisms that promote or prevent it. Burnout correlates with cortisol levels and dysregulation of the hypothalamic-pituitary-adrenal axis, but the chronology and strength of this relationship are unknown. Objective: To determine the prevalence and feasibility of studying burnout in pediatric residents using hair cortisol and hair oxytocin concentrations. Design: /Methods: Longitudinal observational cohort study of pediatric residents. We assessed burnout using the Stanford Professional Fulfillment Index and hair cortisol (HCC), and hair oxytocin concentrations (HOC) at four 3-month intervals from January 2020-January 2021. We evaluated test-retest reliability, sensitivity to change using Pearson product-moment correlations, and relationships between burnout and hair biomarkers using hierarchical mixed-effects linear regression. Results: 17 Pediatrics residents provided 78 wellness surveys and 54 hair samples. Burnout symptoms were present in 39 (50%) of the surveys, with 14 (82%) residents reporting burnout in at least one time point. The lowest (41%) and highest (60%) burnout prevalence occurred in 04/2020 and 01/2021, respectively. No significant associations between burnout scores and HCC (ß -0.01, 95%CI: 0.14-0.13), HOC (ß 0.06, 95%CI: 0.06-0.19), or the HCC:HOC ratio (ß -0.04, 95%CI: 0.09-0.02) were noted in separate analyses. Intra-individual changes in hair cortisol concentration were not associated with changes in burnout score. Conclusions: Burnout was prevalent among Pediatrics residents, with highest prevalence noted in January 2021. This pilot longitudinal study demonstrates the feasibility of evaluating burnout with stress and resilience biomarkers in Pediatrics residents.

Drp1/p53 interaction mediates p53 mitochondrial localization and dysfunction in septic cardiomyopathy.

BACKGROUND: Previous studies have implicated p53-dependent mitochondrial dysfunction in sepsis induced end organ injury, including sepsis-induced myocardial dysfunction (SIMD). However, the mechanisms behind p53 localization to the mitochondria have not been well established. Dynamin-related protein 1 (Drp1), a mediator of mitochondrial fission, may play a role in p53 mitochondrial localization. Here we examined the role of Drp1/p53 interaction in SIMD using in vitro and murine models of sepsis. METHODS: H9c2 cardiomyoblasts and BALB/c mice were exposed to lipopolysaccharide (LPS) to model sepsis phenotype. Pharmacologic inhibitors of Drp1 activation (ψDrp1) and of p53 mitochondrial binding (pifithrin µ, PFTµ) were utilized to assess interaction between Drp1 and p53, and the subsequent downstream impact on mitochondrial morphology and function, cardiomyocyte function, and sepsis phenotype. RESULTS: Both in vitro and murine models demonstrated an increase in physical Drp1/p53 interaction following LPS treatment, which was associated with increased p53 mitochondrial localization, and mitochondrial dysfunction. This Drp1/p53 interaction was inhibited by ΨDrp1, suggesting that this interaction is dependent on Drp1 activation. Treatment of H9c2 cells with either ΨDrp1 or PFTµ inhibited the LPS mediated localization of Drp1/p53 to the mitochondria, decreased oxidative stress, improved cellular respiration and ATP production. Similarly, treatment of BALB/c mice with either ΨDrp1 or PFTµ decreased LPS-mediated mitochondrial localization of p53, mitochondrial ROS in cardiac tissue, and subsequently improved cardiomyocyte contractile function and survival. CONCLUSION: Drp1/p53 interaction and mitochondrial localization is a key prodrome to mitochondrial damage in SIMD and inhibiting this interaction may serve as a therapeutic target.

New and Progressive Medical Conditions After Pediatric Sepsis Hospitalization Requiring Critical Care.

Importance: Children commonly experience physical, cognitive, or emotional sequelae after sepsis. However, little is known about the development or progression of medical conditions after pediatric sepsis. Objective: To quantify the development and progression of 4 common conditions in the 6 months after sepsis and to assess whether they differed after hospitalization for sepsis vs nonsepsis among critically ill children. Design, Setting, and Participants: This cohort study of 101 511 children (<19 years) with sepsis or nonsepsis hospitalization used a national administrative claims database (January 1, 2010, to June 30, 2018). Data management and analysis were conducted from April 1, 2020, to July 7, 2022. Exposures: Intensive care unit hospitalization for sepsis vs all-cause intensive care unit hospitalizations, excluding sepsis. Main Outcomes and Measures: Primary outcomes were the development of 4 target conditions (chronic respiratory failure, seizure disorder, supplemental nutritional dependence, and chronic kidney disease) within 6 months of hospital discharge. Secondary outcomes were the progression of the 4 target conditions among children with the condition before hospitalization. Outcomes were identified via diagnostic and procedural codes, durable medical equipment codes, and prescription medications. Differences in the development and the progression of conditions between pediatric patients with sepsis and pediatric patients with nonsepsis who survived intensive care unit hospitalization were assessed using logistic regression with matching weights. Results: A total of 5150 survivors of pediatric sepsis and 96 361 survivors of nonsepsis intensive care unit hospitalizations were identified; 2593 (50.3%) were female. The median age was 9.5 years (IQR, 3-15 years) in the sepsis cohort and 7 years (IQR, 2-13 years) in the nonsepsis cohort. Of the 5150 sepsis survivors, 670 (13.0%) developed a new target condition, and 385 of 1834 (21.0%) with a preexisting target condition had disease progression. A total of 998 of the 5150 survivors (19.4%) had development and/or progression of at least 1 condition. New conditions were more common among sepsis vs nonsepsis hospitalizations (new chronic respiratory failure: 4.6% vs 1.9%; odds ratio [OR], 2.54 [95% CI, 2.19-2.94]; new supplemental nutritional dependence: 7.9% vs 2.7%; OR, 3.17 [95% CI, 2.80-3.59]; and new chronic kidney disease: 1.1% vs 0.6%; OR, 1.65 [95% CI, 1.25-2.19]). New seizure disorder was less common (4.6% vs 6.0%; OR, 0.77 [95% CI, 0.66-0.89]). Progressive supplemental nutritional dependence was more common (1.5% vs 0.5%; OR, 2.95 [95% CI, 1.60-5.42]), progressive epilepsy was less common (33.7% vs 40.6%; OR, 0.74 [95% CI, 0.65-0.86]), and progressive respiratory failure (4.4% vs 3.3%; OR, 1.35 [95% CI, 0.89-2.04]) and progressive chronic kidney disease (7.9% vs 9.2%; OR, 0.84 [95% CI, 0.18-3.91]) were similar among survivors of sepsis vs nonsepsis admitted to an intensive care unit. Conclusions and Relevance: In this national cohort of critically ill children who survived sepsis, 1 in 5 developed or had progression of a condition of interest after sepsis hospitalization, suggesting survivors of pediatric sepsis may benefit from structured follow-up to identify and treat new or worsening medical comorbid conditions.

GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas.

Diffuse intrinsic pontine glioma (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMGs) are universally lethal paediatric tumours of the central nervous system1. We have previously shown that the disialoganglioside GD2 is highly expressed on H3K27M-mutated glioma cells and have demonstrated promising preclinical efficacy of GD2-directed chimeric antigen receptor (CAR) T cells2, providing the rationale for a first-in-human phase I clinical trial (NCT04196413). Because CAR T cell-induced brainstem inflammation can result in obstructive hydrocephalus, increased intracranial pressure and dangerous tissue shifts, neurocritical care precautions were incorporated. Here we present the clinical experience from the first four patients with H3K27M-mutated DIPG or spinal cord DMG treated with GD2-CAR T cells at dose level 1 (1 × 106 GD2-CAR T cells per kg administered intravenously). Patients who exhibited clinical benefit were eligible for subsequent GD2-CAR T cell infusions administered intracerebroventricularly3. Toxicity was largely related to the location of the tumour and was reversible with intensive supportive care. On-target, off-tumour toxicity was not observed. Three of four patients exhibited clinical and radiographic improvement. Pro-inflammatory cytokine levels were increased in the plasma and cerebrospinal fluid. Transcriptomic analyses of 65,598 single cells from CAR T cell products and cerebrospinal fluid elucidate heterogeneity in response between participants and administration routes. These early results underscore the promise of this therapeutic approach for patients with H3K27M-mutated DIPG or spinal cord DMG.

Drp1/Fis1-Dependent Pathologic Fission and Associated Damaged Extracellular Mitochondria Contribute to Macrophage Dysfunction in Endotoxin Tolerance.

OBJECTIVES: Recent publications have shown that mitochondrial dynamics can govern the quality and quantity of extracellular mitochondria subsequently impacting immune phenotypes. This study aims to determine if pathologic mitochondrial fission mediated by Drp1/Fis1 interaction impacts extracellular mitochondrial content and macrophage function in sepsis-induced immunoparalysis. DESIGN: Laboratory investigation. SETTING: University laboratory. SUBJECTS: C57BL/6 and BALB/C mice. INTERVENTIONS: Using in vitro and murine models of endotoxin tolerance (ET), we evaluated changes in Drp1/Fis1-dependent pathologic fission and simultaneously measured the quantity and quality of extracellular mitochondria. Next, by priming mouse macrophages with isolated healthy mitochondria (MC) and damaged mitochondria, we determined if damaged extracellular mitochondria are capable of inducing tolerance to subsequent endotoxin challenge. Finally, we determined if inhibition of Drp1/Fis1-mediated pathologic fission abrogates release of damaged extracellular mitochondria and improves macrophage response to subsequent endotoxin challenge. MEASUREMENTS AND MAIN RESULTS: When compared with naïve macrophages (NMs), endotoxin-tolerant macrophages (ETM) demonstrated Drp1/Fis1-dependent mitochondrial dysfunction and higher levels of damaged extracellular mitochondria (Mitotracker-Green + events/50 µL: ETM = 2.42 × 106 ± 4,391 vs NM = 5.69 × 105 ± 2,478; p < 0.001). Exposure of NMs to damaged extracellular mitochondria (MH) induced cross-tolerance to subsequent endotoxin challenge, whereas MC had minimal effect (tumor necrosis factor [TNF]-α [pg/mL]: NM = 668 ± 3, NM + MH = 221 ± 15, and NM + Mc = 881 ± 15; p < 0.0001). Inhibiting Drp1/Fis1-dependent mitochondrial fission using heptapeptide (P110), a selective inhibitor of Drp1/Fis1 interaction, improved extracellular mitochondrial function (extracellular mitochondrial membrane potential, JC-1 [R/G] ETM = 7 ± 0.5 vs ETM + P110 = 19 ± 2.0; p < 0.001) and subsequently improved immune response in ETMs (TNF-α [pg/mL]; ETM = 149 ± 1 vs ETM + P110 = 1,150 ± 4; p < 0.0001). Similarly, P110-treated endotoxin tolerant mice had lower amounts of damaged extracellular mitochondria in plasma (represented by higher extracellular mitochondrial membrane potential, TMRM/MT-G: endotoxin tolerant [ET] = 0.04 ± 0.02 vs ET + P110 = 0.21 ± 0.02; p = 0.03) and improved immune response to subsequent endotoxin treatment as well as cecal ligation and puncture. CONCLUSIONS: Inhibition of Drp1/Fis1-dependent mitochondrial fragmentation improved macrophage function and immune response in both in vitro and in vivo models of ET. This benefit is mediated, at least in part, by decreasing the release of damaged extracellular mitochondria, which contributes to endotoxin cross-tolerance. Altogether, these data suggest that alterations in mitochondrial dynamics may play an important role in sepsis-induced immunoparalysis.

Outcomes Associated With Early RBC Transfusion in Pediatric Severe Sepsis: A Propensity-Adjusted Multicenter Cohort Study.

BACKGROUND: Little is known about the epidemiology of and outcomes related to red blood cell (RBC) transfusion in septic children across multiple centers. We performed propensity-adjusted secondary analyses of the Biomarker Phenotyping of Pediatric Sepsis and Multiple Organ Failure (PHENOMS) study to test the hypothesis that early RBC transfusion is associated with fewer organ failure-free days in pediatric severe sepsis. METHODS: Four hundred one children were enrolled in the parent study. Children were excluded from these analyses if they received extracorporeal membrane oxygenation (n = 22) or died (n = 1) before sepsis day 2. Propensity-adjusted analyses compared children who received RBC transfusion on or before sepsis day 2 (early RBC transfusion) with those who did not. Logistic regression was used to model the propensity to receive early RBC transfusion. A weighted cohort was constructed using stabilized inverse probability of treatment weights. Variables in the weighted cohort with absolute standardized differences >0.15 were added to final multivariable models. RESULTS: Fifty percent of children received at least one RBC transfusion. The majority (68%) of first transfusions were on or before sepsis day 2. Early RBC transfusion was not independently associated with organ failure-free (-0.34 [95%CI: -2, 1.3] days) or PICU-free days (-0.63 [-2.3, 1.1]), but was associated with the secondary outcome of higher mortality (aOR 2.9 [1.1, 7.9]). CONCLUSIONS: RBC transfusion is common in pediatric severe sepsis and may be associated with adverse outcomes. Future studies are needed to clarify these associations, to understand patient-specific transfusion risks, and to develop more precise transfusion strategies.

Quantifying paediatric intensive care unit staffing levels at a paediatric academic medical centre: A mixed-methods approach.

AIM: To identify, simulate and evaluate the formal and informal patient-level and unit-level factors that nurse managers use to determine the number of nurses for each shift. BACKGROUND: Nurse staffing schedules are commonly set based on metrics such as midnight census that do not account for seasonality or midday turnover, resulting in last-minute adjustments or inappropriate staffing levels. METHODS: Staffing schedules at a paediatric intensive care unit (PICU) were simulated based on nurse-to-patient assignment rules from interviews with nursing management. Multivariate regression modelled the discrepancies between scheduled and historical staffing levels and constructed rules to reduce these discrepancies. The primary outcome was the median difference between simulated and historical staffing levels. RESULTS: Nurse-to-patient ratios underestimated staffing by a median of 1.5 nurses per shift. Multivariate regression identified patient turnover as the primary factor accounting for this difference and subgroup analysis revealed that patient age and weight were also important. New rules reduced the difference to a median of 0.07 nurses per shift. CONCLUSION: Measurable, predictable indicators of patient acuity and historical trends may allow for schedules that better match demand. IMPLICATIONS FOR NURSING MANAGEMENT: Data-driven methods can quantify what drives unit demand and generate nurse schedules that require fewer last-minute adjustments.

Acute kidney injury after in-hospital cardiac arrest.

AIM: Determine 1) frequency and risk factors for acute kidney injury (AKI) after in-hospital cardiac arrest (IHCA) in the Therapeutic Hypothermia after Pediatric Cardiac Arrest In-Hospital (THAPCA-IH) trial and associated outcomes; 2) impact of temperature management on post-IHCA AKI. METHODS: Secondary analysis of THAPCA-IH; a randomized controlled multi-national trial at 37 children's hospitals. ELIGIBILITY: Serum creatinine (Cr) within 24 h of randomization. OUTCOMES: Prevalence of severe AKI defined by Stage 2 or 3 Kidney Disease Improving Global Outcomes Cr criteria. 12-month survival with favorable neurobehavioral outcome. Analyses stratified by entire cohort and cardiac subgroup. Risk factors and outcomes compared among cohorts with and without severe AKI. RESULTS: Subject randomization: 159 to hypothermia, 154 to normothermia. Overall, 80% (249) developed AKI (any stage), and 66% (207) developed severe AKI. Cardiac patients (204, 65%) were more likely to develop severe AKI (72% vs 56%,p = 0.006). Preexisting cardiac or renal conditions, baseline lactate, vasoactive support, and systolic blood pressure were associated with severe AKI. Comparing hypothermia versus normothermia, there were no differences in severe AKI rate (63% vs 70%,p = 0.23), peak Cr, time to peak Cr, or freedom from mortality or severe AKI (p = 0.14). Severe AKI was associated with decreased hospital survival (48% vs 65%,p = 0.006) and decreased 12-month survival with favorable neurobehavioral outcome (30% vs 53%,p < 0.001). CONCLUSION: Severe post-IHCA AKI occurred frequently especially in those with preexisting cardiac or renal conditions and peri-arrest hemodynamic instability. Severe AKI was associated with decreased survival with favorable neurobehavioral outcome. Hypothermia did not decrease incidence of severe AKI post-IHCA.

Clinician Accuracy in Identifying and Predicting Organ Dysfunction in Critically Ill Children.

OBJECTIVES: To determine clinician accuracy in the identification and prediction of multiple organ dysfunction syndrome. DESIGN: Prospective cohort study. SETTING: University of Michigan's C.S. Mott Children's Hospital PICU. PATIENTS: Patients admitted to the PICU with an anticipated PICU length of stay greater than 48 hours. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: For each patient, the clinical team (attending, fellow, resident/nurse practitioner) was surveyed regarding existing and anticipated organ dysfunction. The primary outcomes were clinicians' accuracy at identifying multiple organ dysfunction syndrome and predicting new or progressive multiple organ dysfunction syndrome, compared to the objective assessment of multiple organ dysfunction syndrome using Proulx criteria. We also measured sensitivity, specificity, negative and positive predictive values, and negative and positive likelihood ratios of clinician assessments. We tested for differences in accuracy by clinician type using chi-square tests. Clinicians rated their confidence in prediction on a 5-point Likert scale. There were 476 eligible PICU admissions, for whom 1,218 surveys were completed. Multiple organ dysfunction syndrome was present in 89 patients (18.7%) at enrollment, and new or progressive multiple organ dysfunction syndrome occurred in 39 (8.2%). Clinicians correctly identified multiple organ dysfunction syndrome with 79.9% accuracy and predicted additional organ dysfunction with 82.6% accuracy. However, the positive and negative likelihood ratios for new or progressive multiple organ dysfunction syndrome prediction were 3.0 and 0.7, respectively, indicating a weak relationship between the clinician prediction and development of new or progressive multiple organ dysfunction syndrome. The positive predictive value of new or progressive multiple organ dysfunction syndrome prediction was just 22.1%. We found no differences in accuracy by clinician type for either identification of multiple organ dysfunction syndrome (80.2% vs 78.2% vs 81.0%; p = 0.57) or prediction of new or progressive multiple organ dysfunction syndrome (84.8% vs 82.8% vs 80.3%; p = 0.26) for attendings, fellows, and residents/nurse practitioners, respectively. There was a weak correlation between the confidence and accuracy of prediction (pairwise correlation coefficient, 0.26; p < 0.001). CONCLUSIONS: PICU clinicians correctly identified multiple organ dysfunction syndrome and predicted new or progressive multiple organ dysfunction syndrome with 80% accuracy. However, only 8% of patients developed new or progressive multiple organ dysfunction syndrome, so accuracy was largely due to true negative predictions. The positive predictive value for new or progressive multiple organ dysfunction syndrome prediction was just 22%. Accuracy did not differ by clinician type, but was correlated with self-rated confidence and was higher for negative predictions.

New Medical Device Acquisition During Pediatric Severe Sepsis Hospitalizations.

OBJECTIVES: Severe sepsis is a significant cause of healthcare utilization and morbidity among pediatric patients. However, little is known about how commonly survivors acquire new medical devices during pediatric severe sepsis hospitalization. We sought to determine the rate of new device acquisition (specifically, tracheostomy placement, gastrostomy tube placement, vascular access devices, ostomy procedures, and amputation) among children surviving hospitalizations with severe sepsis. For contextualization, we compare this to rates of new device acquisition among three comparison cohorts: 1) survivors of all-cause pediatric hospitalizations; 2) matched survivors of nonsepsis infection hospitalizations; and 3) matched survivors of all-cause nonsepsis hospitalization with similar organ dysfunction. DESIGN: Observational cohort study. SETTING: Nationwide Readmission Database (2016), including all-payer hospitalizations from 27 states. PATIENTS: Eighteen-thousand two-hundred ten pediatric severe sepsis hospitalizations; 532,738 all-cause pediatric hospitalizations; 16,173 age- and sex-matched nonsepsis infection hospitalizations; 15,025 organ dysfunction matched all-cause nonsepsis hospitalizations; and all with live discharge. MEASUREMENTS AND MAIN RESULTS: Among 18,210 pediatric severe sepsis hospitalizations, 1,024 (5.6%) underwent device placement. Specifically, 3.5% had new gastrostomy, 3.1% new tracheostomy, 0.6% new vascular access devices, 0.4% new ostomy procedures, and 0.1% amputations. One-hundred forty hospitalizations (0.8%) included two or more new devices. After applying the Nationwide Readmissions Database sampling weights, there were 55,624 pediatric severe sepsis hospitalizations and 1,585,194 all-cause nonsepsis hospitalizations with live discharge in 2016. Compared to all-cause pediatric hospitalizations, severe sepsis hospitalizations were eight-fold more likely to involve new device acquisition (6.4% vs 0.8%; p < 0.001). New device acquisition was also higher in severe sepsis hospitalizations compared with matched nonsepsis infection hospitalizations (5.1% vs 1.2%; p < 0.01) and matched all-cause hospitalizations with similar organ dysfunction (4.7% vs 2.8%; p < 0.001). CONCLUSIONS: In this nationwide, all-payer cohort of U.S. pediatric severe sepsis hospitalizations, one in 20 children surviving severe sepsis experienced new device acquisition. The procedure rate was nearly eight-fold higher than all-cause, nonsepsis pediatric hospitalizations, and four-fold higher than matched nonsepsis infection hospitalizations.

Cardiac Dysfunction Identified by Strain Echocardiography Is Associated With Illness Severity in Pediatric Sepsis.

OBJECTIVES: Sepsis-induced myocardial dysfunction has been associated with illness severity and mortality in pediatrics. Although early sepsis-induced myocardial dysfunction diagnosis could aid in hemodynamic management, current echocardiographic metrics for assessing biventricular function are limited in detecting early impairment. Strain echocardiography is a validated quantitative measure that can detect subtle perturbations in left ventricular and right ventricular function. This investigation evaluates the utility of strain echocardiography in pediatric sepsis and compares with to conventional methods. DESIGN: Retrospective, observational study comparing left ventricular and right ventricular strain. Strain was compared with ejection fraction and fractional shortening and established sepsis severity of illness markers. SETTING: Tertiary care medical-surgical PICU from July 2013 to January 2018. PATIENTS: Seventy-nine septic children and 28 healthy controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Compared with healthy controls, patients with severe sepsis demonstrated abnormal left ventricular strain (left ventricular longitudinal strain: -13.0% ± 0.72; p = 0.04 and left ventricular circumferential strain: -16.5% ± 0.99; p = 0.046) and right ventricular (right ventricular longitudinal strain = -14.3% ± 6.3; p < 0.01) despite normal fractional shortening (36.0% ± 1.6 vs 38.1% ± 1.1; p = 0.5129) and ejection fraction (60.7% ± 2.2 vs 65.3% ± 1.5; p = 0.33). There was significant association between depressed left ventricular longitudinal strain and increased Vasotrope-Inotrope Score (r = 0.52; p = 0.034). Worsening left ventricular circumferential strain was correlated with higher lactate (r = 0.31; p = 0.03) and higher Pediatric Risk of Mortality-III score (r = 0.39; p < 0.01). Depressed right ventricular longitudinal strain was associated with elevated pediatric multiple organ dysfunction score (r = 0.44; p < 0.01) CONCLUSIONS:: Compared with healthy children, pediatric septic patients demonstrated abnormal left ventricular and right ventricular strain concerning for early signs of cardiac dysfunction. This was despite having normal ejection fraction and fractional shortening. Abnormal strain was associated with abnormal severity of illness markers. Strain echocardiography may have utility as an early indicator of sepsis-induced myocardial dysfunction in pediatric sepsis.

Loss of myeloid-specific protein phosphatase 2A enhances lung injury and fibrosis and results in IL-10-dependent sensitization of epithelial cell apoptosis.

Protein phosphatase 2A (PP2A), a ubiquitously expressed Ser/Thr phosphatase is an important regulator of cytokine signaling and cell function. We previously showed that myeloid-specific deletion of PP2A (LysMcrePP2A-/-) increased mortality in a murine peritoneal sepsis model. In the current study, we assessed the role of myeloid PP2A in regulation of lung injury induced by lipopolysaccharide (LPS) or bleomycin delivered intratracheally. LysMcrePP2A-/- mice experienced increased lung injury in response to both LPS and bleomycin. LysMcrePP2A-/- mice developed more exuberant fibrosis in response to bleomycin, elevated cytokine responses, and chronic myeloid inflammation. Bone marrow-derived macrophages (BMDMs) from LysMcrePP2A-/- mice showed exaggerated inflammatory cytokine release under conditions of both M1 and M2 activation. Notably, secretion of IL-10 was elevated under all stimulation conditions, including activation of BMDMs by multiple Toll-like receptor ligands. Supernatants collected from LPS-stimulated LysMcrePP2A-/- BMDMs induced epithelial cell apoptosis in vitro but this effect was mitigated when IL-10 was also depleted from the BMDMs by crossing LysMcrePP2A-/- mice with systemic IL-10-/- mice (LysMcrePP2A-/- × IL-10-/-) or when IL-10 was neutralized. Despite these findings, IL-10 did not directly induce epithelial cell apoptosis but sensitized epithelial cells to other mediators from the BMDMs. Taken together our results demonstrate that myeloid PP2A regulates production of multiple cytokines but that its effect is most pronounced on IL-10 production. Furthermore, IL-10 sensitizes epithelial cells to apoptosis in response to myeloid-derived mediators, which likely contributes to the pathogenesis of lung injury and fibrosis in this model.

Analysis of Inflammatory Cytokines in Postoperative Fontan Pleural Drainage.

Prolonged pleural drainage is a common complication in patients after Fontan palliation and is associated with short- and long- term morbidities. Among many potential etiologies, prolonged drainage has an inflammatory component, but there are no descriptions of cytokines in Fontan pleural drainage to date. This study aimed to examine the inflammatory make-up of Fontan pleural drainage. This prospective age-range-matched cohort study recruited 25 patients undergoing Fontan procedure and 15 bi-ventricular patients undergoing cardiopulmonary bypass (CPB). Chest tube samples were taken on postoperative day (POD) 1-4, 7, and 10. Cytokines were measured using Bio-Plex Assays. Univariate comparisons were made in patient characteristics and cytokine levels. Median age was 3.7 y (IQR 2.8-3.9) for controls and 2.5 y (IQR 2.1-2.9) in Fontan patients (p = 0.02). Median drainage duration and daily volume was higher in Fontan patients (both p < 0.001). Inflammatory cytokines (IL-17A, IFN-y, MIP-1ß, and TNF-α) were higher in Fontan patients than controls (all p < 0.02). There was an increase in pro-inflammatory cytokines (IL-8, MIP-1ß, and TNF-α) from POD1 to the last chest tube day (LCD) in Fontan patients (all p < 0.0001) and a decrease in the anti-inflammatory cytokine IL-10 (p = 0.001). There was no difference in cytokine concentration from POD1 to LCD among controls. There was a significant association with the cytokine concentration of TNF-α on POD1 and duration of chest tube drainage (p < 0.05). Inflammatory cytokine levels in the pleural fluid of Fontan patients are higher compared to bi-ventricular controls and rise over time where controls do not. This suggests ongoing localized inflammation that is not a result of CPB alone and may be an important contributor to pleural drainage in patients after the Fontan procedure.

The Functional Immune Response of Patients on Extracorporeal Life Support.

Extracorporeal life support (ECLS) is a widely used lifesaving technology. Whether ECLS results in immune dysregulation is unclear. This study's aim was to examine whether ECLS affected innate immune response. All patients placed on ECLS were eligible. Blood was obtained before, during, and after ECLS. Function of the innate immune system was measured by ex vivo lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) and plasma cytokine levels (interleukin [IL]-6, IL-8, IL-10, and TNF-α). Immunoparalysis was defined as ex vivo TNF-α levels less than 200 pg/ml. Nineteen patients were enrolled with twelve <18 years old. Median ECLS duration was 10 days (range: 3-108); nine patients died. After stratifying the cohort by the presence of immunoparalysis before ECLS, those immunoparalyzed showed increased response to LPS on days 1 and 3 (p = 0.016). Those without pre-ECLS immunoparalysis showed a transient decrease in response on day 3 (p = 0.008). Plasma IL-10 levels were elevated in those with pre-ECLS immunoparalysis and dropped significantly by day 1 (p = 0.031). The number treated with steroids was similar in the two groups. In conclusion, patients with immunoparalysis before ECLS showed a gradual increase in immune function during ECLS, whereas those without immunoparalysis had a transient decrease in responsiveness on day 3.

Acute kidney injury after out of hospital pediatric cardiac arrest.

IMPORTANCE: Many children with return of spontaneous circulation (ROSC) following cardiac arrest (CA) experience acute kidney injury (AKI). The impact of therapeutic hypothermia on the epidemiology of post-CA AKI in children has not been fully investigated. OBJECTIVE: The study aims were to: 1) describe the prevalence of severe AKI in comatose children following out-of-hospital CA (OHCA), 2) identify risk factors for severe AKI, 3) evaluate the impact of therapeutic hypothermia on the prevalence of severe AKI, and 4) evaluate the association of severe AKI with survival and functional outcomes. DESIGN: A post hoc secondary analysis of data from the Therapeutic Hypothermia after Pediatric Cardiac Arrest Out-of-Hospital (THAPCA-OH) trial. SETTING: Thirty-six pediatric intensive care units in the United States and Canada. PARTICIPANTS: Of 282 eligible subjects with an initial creatinine obtained within 24 h of randomization, 148 were randomized to therapeutic hypothermia and 134 were randomized to therapeutic normothermia. MAIN OUTCOMES AND MEASURES: Primary outcome was prevalence of severe AKI, as defined by stage 2 and 3 Kidney Disease Improving Global Outcomes (KDIGO) consensus definition; secondary outcome was survival with a favorable neurobehavioral outcome. For this study, risk factors and outcomes were compared between those with/without severe AKI. RESULTS: Of the 282 subjects enrolled, 180 (64%) developed AKI of which 117 (41% of all enrolled) developed severe AKI. Multivariable modeling found younger age, longer duration of chest compressions, higher lactate level at time of temperature intervention and higher number of vasoactive agents through day 1 of intervention associated with severe AKI. There was no difference in severe AKI between therapeutic hypothermia (39.9%) and therapeutic normothermia (43.3%) groups (p = 0.629). Survival was lower in those with severe AKI at 28 days (21% vs no severe AKI 49%, p < 0.001) and 12 months (21% vs no severe AKI 42%, p < 0.001). One year survival with favorable functional outcome was lower in those with severe AKI. CONCLUSIONS AND RELEVANCE: Severe AKI occurs frequently in children with ROSC after OHCA, especially in younger children and those with higher initial lactates and hemodynamic support. Severe AKI was associated with worse survival and functional outcome. Therapeutic hypothermia did not reduce the incidence of severe AKI.

Pathophysiology of Pediatric Multiple Organ Dysfunction Syndrome.

OBJECTIVE: To describe the pathophysiology associated with multiple organ dysfunction syndrome in children. DATA SOURCES: Literature review, research data, and expert opinion. STUDY SELECTION: Not applicable. DATA EXTRACTION: Moderated by an experienced expert from the field, pathophysiologic processes associated with multiple organ dysfunction syndrome in children were described, discussed, and debated with a focus on identifying knowledge gaps and research priorities. DATA SYNTHESIS: Summary of presentations and discussion supported and supplemented by relevant literature. CONCLUSIONS: Experiment modeling suggests that persistent macrophage activation may be a pathophysiologic basis for multiple organ dysfunction syndrome. Children with multiple organ dysfunction syndrome have 1) reduced cytochrome P450 metabolism inversely proportional to inflammation; 2) increased circulating damage-associated molecular pattern molecules from injured tissues; 3) increased circulating pathogen-associated molecular pattern molecules from infection or endogenous microbiome; and 4) cytokine-driven epithelial, endothelial, mitochondrial, and immune cell dysfunction. Cytochrome P450s metabolize endogenous compounds and xenobiotics, many of which ameliorate inflammation, whereas damage-associated molecular pattern molecules and pathogen-associated molecular pattern molecules alone and together amplify the cytokine production leading to the inflammatory multiple organ dysfunction syndrome response. Genetic and environmental factors can impede inflammation resolution in children with a spectrum of multiple organ dysfunction syndrome pathobiology phenotypes. Thrombocytopenia-associated multiple organ dysfunction syndrome patients have extensive endothelial activation and thrombotic microangiopathy with associated oligogenic deficiencies in inhibitory complement and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13. Sequential multiple organ dysfunction syndrome patients have soluble Fas ligand-Fas-mediated hepatic failure with associated oligogenic deficiencies in perforin and granzyme signaling. Immunoparalysis-associated multiple organ dysfunction syndrome patients have impaired ability to resolve infection and have associated environmental causes of lymphocyte apoptosis. These inflammation phenotypes can lead to macrophage activation syndrome. Resolution of multiple organ dysfunction syndrome requires elimination of the source of inflammation. Full recovery of organ functions is noted 6-18 weeks later when epithelial, endothelial, mitochondrial, and immune cell regeneration and reprogramming is completed.

AC Electroosmosis-Enhanced Nanoplasmofluidic Detection of Ultralow-Concentration Cytokine.

Label-free, nanoparticle-based plasmonic optical biosensing, combined with device miniaturization and microarray integration, has emerged as a promising approach for rapid, multiplexed biomolecular analysis. However, limited sensitivity prevents the wide use of such integrated label-free nanoplasmonic biosensors in clinical and life science applications where low-abundance biomolecule detection is needed. Here, we present a nanoplasmofluidic device integrated with microelectrodes for rapid, label-free analysis of a low-abundance cell signaling protein, detected by AC electroosmosis-enhanced localized surface plasmon resonance (ACE-LSPR) biofunctional nanoparticle imaging. The ACE-LSPR device is constructed using both bottom-up and top-down sensor fabrication methods, allowing the seamless integration of antibody-conjugated gold nanorod (AuNR) biosensor arrays with microelectrodes on the same microfluidic platform. Applying an AC voltage to microelectrodes while scanning the scattering light intensity variation of the AuNR biosensors results in significantly enhanced biosensing performance. The AC electroosmosis (ACEO) based enhancement of the biosensor performance enables rapid (5-15 min) quantification of IL-1ß, a pro-inflammatory cytokine biomarker, with a sensitivity down to 158.5 fg/mL (9.1 fM) for spiked samples in PBS and 1 pg/mL (58 fM) for diluted human serum. Together with the optimized detection sensitivity and speed, our study presents the first critical step toward the application of nanoplasmonic biosensing technology to immune status monitoring guided by low-abundance cytokine measurement.

Myeloid-Specific Gene Deletion of Protein Phosphatase 2A Magnifies MyD88- and TRIF-Dependent Inflammation following Endotoxin Challenge.

Protein phosphatase 2A (PP2A) is a member of the intracellular serine/threonine phosphatases. Innate immune cell activation triggered by pathogen-associated molecular patterns is mediated by various protein kinases, and PP2A plays a counter-regulatory role by deactivating these kinases. In this study, we generated a conditional knockout of the α isoform of the catalytic subunit of PP2A (PP2ACα). After crossing with myeloid-specific cre-expressing mice, effective gene knockout was achieved in various myeloid cells. The myeloid-specific knockout mice (lyM-PP2Afl/fl) showed higher mortality in response to endotoxin challenge and bacterial infection. Upon LPS challenge, serum levels of TNF-α, KC, IL-6, and IL-10 were significantly increased in lyM-PP2Afl/fl mice, and increased phosphorylation was observed in MAPK pathways (p38, ERK, JNK) and the NF-κB pathway (IKKα/ß, NF-κB p65) in bone marrow-derived macrophages (BMDMs) from knockout mice. Heightened NF-κB activation was not associated with degradation of IκBα; instead, enhanced phosphorylation of the NF-κB p65 subunit and p38 phosphorylation-mediated TNF-α mRNA stabilization appear to contribute to the increased TNF-α expression. In addition, increased IL-10 expression appears to be due to PP2ACα-knockout-induced IKKα/ß hyperactivation. Microarray experiments indicated that the Toll/IL-1R domain-containing adaptor inducing IFN-ß/ TNFR-associated factor 3 pathway was highly upregulated in LPS-treated PP2ACα-knockout BMDMs, and knockout BMDMs had elevated IFN-α/ß production compared with control BMDMs. Serum IFN-ß levels from PP2ACα-knockout mice treated with LPS were also greater than those in controls. Thus, we demonstrate that PP2A plays an important role in regulating inflammation and survival in the setting of septic insult by targeting MyD88- and Toll/IL-1R domain-containing adaptor inducing IFN-ß-dependent pathways.