Metformin alleviates lung-endothelial hyperpermeability by regulating cofilin-1/PP2AC pathway.

Background: Microvascular endothelial hyperpermeability is an earliest pathological hallmark in Acute Lung Injury (ALI), which progressively leads to Acute Respiratory Distress Syndrome (ARDS). Recently, vascular protective and anti-inflammatory effect of metformin, irrespective of glycemic control, has garnered significant interest. However, the underlying molecular mechanism(s) of metformin's barrier protective benefits in lung-endothelial cells (ECs) has not been clearly elucidated. Many vascular permeability-increasing agents weakened adherens junctions (AJ) integrity by inducing the reorganization of the actin cytoskeleton and stress fibers formation. Here, we hypothesized that metformin abrogated endothelial hyperpermeability and strengthen AJ integrity via inhibiting stress fibers formation through cofilin-1-PP2AC pathway. Methods: We pretreated human lung microvascular ECs (human-lung-ECs) with metformin and then challenged with thrombin. To investigate the vascular protective effects of metformin, we studied changes in ECs barrier function using electric cell-substrate impedance sensing, levels of actin stress fibers formation and inflammatory cytokines IL-1ß and IL-6 expression. To explore the downstream mechanism, we studied the Ser3-phosphorylation-cofilin-1 levels in scramble and PP2AC-siRNA depleted ECs in response to thrombin with and without metformin pretreatment. Results: In-vitro analyses showed that metformin pretreatment attenuated thrombin-induced hyperpermeability, stress fibers formation, and the levels of inflammatory cytokines IL-6 and IL-ß in human-lung-ECs. We found that metformin mitigated Ser3-phosphorylation mediated inhibition of cofilin-1 in response to thrombin. Furthermore, genetic deletion of PP2AC subunit significantly inhibited metformin efficacy to mitigate thrombin-induced Ser3-phosphorylation cofilin-1, AJ disruption and stress fibers formation. We further demonstrated that metformin increases PP2AC activity by upregulating PP2AC-Leu309 methylation in human-lung-ECs. We also found that the ectopic expression of PP2AC dampened thrombin-induced Ser3-phosphorylation-mediated inhibition of cofilin-1, stress fibers formation and endothelial hyperpermeability. Conclusion: Together, these data reveal the unprecedented endothelial cofilin-1/PP2AC signaling axis downstream of metformin in protecting against lung vascular endothelial injury and inflammation. Therefore, pharmacologically enhancing endothelial PP2AC activity may lead to the development of novel therapeutic approaches for prevention of deleterious effects of ALI on vascular ECs.

Robust genome editing in adult vascular endothelium by nanoparticle delivery of CRISPR-Cas9 plasmid DNA.

Vascular endothelium plays a crucial role in vascular homeostasis and tissue fluid balance. To target endothelium for robust genome editing, we developed poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PEG-b-PLGA) copolymer-based nanoparticle formulated with polyethyleneimine. A single i.v. administration of mixture of nanoparticles and plasmid DNA expressing Cas9 controlled by CDH5 promoter and guide RNA (U6 promoter) induced highly efficient genome editing in endothelial cells (ECs) of the vasculatures, including lung, heart, aorta, and peripheral vessels in adult mice. Western blotting and immunofluorescent staining demonstrated an ∼80% decrease of protein expression selectively in ECs, resulting in a phenotype similar to that of genetic knockout mice. Nanoparticle delivery of plasmid DNA could induce genome editing of two genes or genome editing and transgene expression in ECs simultaneously. Thus, nanoparticle delivery of plasmid DNA is a powerful tool to rapidly and efficiently alter expression of gene(s) in ECs for cardiovascular research and potential gene therapy.

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.

Risk Factors for Mortality in Refractory Pediatric Septic Shock Supported with Extracorporeal Life Support.

Risk factors for mortality in children with refractory pediatric septic shock who are supported with extracorporeal life support (ECLS) are largely unknown. Therefore, we performed univariable and multivariable analyses to determine risk factors for mortality among children (<19 years) who underwent an ECLS run between January 2012 and September 2014 at eight tertiary pediatric hospitals, and who had septic shock based on 2005 International Consensus Criteria. Of the 514 children treated with ECLS during the study period, 70 were identified with septic shock. The mortality rate was similar between those with (54.3%) and without septic shock (43.7%). Among those with septic shock, significant risk factors for mortality included cardiac failure or extracorporeal cardiopulmonary resuscitation (ECPR) as indication for ECLS cannulation compared with respiratory failure (P = 0.003), having a new neurologic event following cannulation (P = 0.032), acquiring a new infection following cannulation (P = 0.005), inability to normalize pH in the 48 hours following ECLS cannulation (P = 0.010), and requiring higher daily volume of platelet transfusions (P = 0.005). These findings can be used to help guide clinical decision making for children with septic shock that is refractory to medical management.

Platelet Transfusion Practice and Related Outcomes in Pediatric Extracorporeal Membrane Oxygenation.

OBJECTIVE: To describe factors associated with platelet transfusion during pediatric extracorporeal membrane oxygenation and the relationships among platelet transfusion, complications, and mortality. DESIGN: Secondary analysis of data collected prospectively by the Collaborative Pediatric Critical Care Research Network between December 2012 and September 2014. SETTING: Eight Collaborative Pediatric Critical Care Research Network-affiliated hospitals. PATIENTS: Age less than 19 years old and treated with extracorporeal membrane oxygenation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Of 511 children, 496 (97.1%) received at least one platelet transfusion during extracorporeal membrane oxygenation. Neonatal age, venoarterial extracorporeal membrane oxygenation, and various acute and chronic diagnoses were associated with increased average daily platelet transfusion volume (milliliters per kilogram body weight). On multivariable analysis, average daily platelet transfusion volume was independently associated with mortality (per 1 mL/kg; odds ratio, 1.05; CI, 1.03-1.08; p < 0.001), whereas average daily platelet count was not (per 1 × 10/L up to 115 × 10/L; odds ratio, 1.00; CI, 0.98-1.01; p = 0.49). Variables independently associated with increased daily bleeding risk included increased platelet transfusion volume on the previous extracorporeal membrane oxygenation day, a primary cardiac indication for extracorporeal membrane oxygenation, adolescent age, and an acute diagnosis of congenital cardiovascular disease. Variables independently associated with increased daily thrombotic risk included increased platelet transfusion volume on the previous extracorporeal membrane oxygenation day and venoarterial extracorporeal membrane oxygenation. Variables independently associated with decreased daily thrombotic risk included full-term neonatal age and an acute diagnosis of airway abnormality. CONCLUSIONS: Platelet transfusion was common in this multisite pediatric extracorporeal membrane oxygenation cohort. Platelet transfusion volume was associated with increased risk of mortality, bleeding, and thrombosis.

The association of immediate post cardiac arrest diastolic hypertension and survival following pediatric cardiac arrest.

AIM: In-hospital cardiac arrest occurs in >5000 children each year in the US and almost half will not survive to discharge. Animal data demonstrate that an immediate post-resuscitation burst of hypertension is associated with improved survival. We aimed to determine if systolic and diastolic invasive arterial blood pressures immediately (0-20 min) after return of spontaneous circulation (ROSC) are associated with survival and neurologic outcomes at hospital discharge. METHODS: This is a secondary analysis of the Pediatric Intensive Care Quality of CPR (PICqCPR) study of invasively measured blood pressures during intensive care unit CPR. Patients were eligible if they achieved ROSC and had at least one invasively measured blood pressure within the first 20 min following ROSC. Post-ROSC blood pressures were normalized for age, sex and height. "Immediate hypertension" was defined as at least one systolic or diastolic blood pressure >90th percentile. The primary outcome was survival to hospital discharge. RESULTS: Of 102 children, 70 (68.6%) had at least one episode of immediate post-CPR diastolic hypertension. After controlling for pre-existing hypotension, duration of CPR, calcium administration, and first documented rhythm, patients with immediate post-CPR diastolic hypertension were more likely to survive to hospital discharge (79.3% vs. 54.5%; adjusted OR = 2.93; 95%CI, 1.16-7.69). CONCLUSIONS: In this post hoc secondary analysis of the PICqCPR study, 68.6% of subjects had diastolic hypertension within 20 min of ROSC. Immediate post-ROSC hypertension was associated with increased odds of survival to discharge, even after adjusting for covariates of interest.

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.

miR-144-mediated Inhibition of ROCK1 Protects against LPS-induced Lung Endothelial Hyperpermeability.

Dysfunctional endothelial cell (EC) barrier and increased lung vascular permeability is a cardinal feature of acute lung injury and sepsis that may result in a pathophysiological condition characterized by alveolar flooding, pulmonary edema, and subsequent hypoxemia. In lung ECs, activation of Rho-associated kinase-1 (ROCK1) phosphorylates myosin light chain (MLC)-associated phosphatase at its inhibitory site, which favors phosphorylation of MLC, stress fiber formation, and hyperpermeability during acute lung injury. The role of microRNA-144 (miR-144) has been well investigated in many human diseases, including cardiac ischemia/reperfusion-induced injury, lung cancer, and lung viral infection; however, its role in pulmonary EC barrier regulation remains obscure. Here, we investigated the miR-144-mediated mechanism in the protection of endothelial barrier function in an LPS-induced lung injury model. By using transendothelial electrical resistance and transwell permeability assay to examine in vitro permeability and immunofluorescence microscopy to determine barrier integrity, we showed that ectopic expression of miR-144 effectively blocked lung EC barrier disruption and hyperpermeability in response to proinflammatory agents. Furthermore, using a gain-and-loss-of-function strategy, overexpression of miR-144 significantly decreased ROCK1 expression. Concomitantly, miR-144 inhibits ROCK1-mediated phosphorylation of MLC phosphataseThr853 and thus phosphorylation of MLCThr18/Ser19 to counteract stress fiber formation in LPS-activated EC. Finally, in LPS-challenged mice, intranasal delivery of miR-144 mimic via liposomes attenuated endotoxemia-induced increases in lung wet/dry ratio, vascular permeability, and inflammation. In conclusion, these data suggest that miR-144-attenuated activation of inflammatory ROCK1/MLC pathway in vascular ECs is a promising therapeutic strategy to counter inflammatory lung injury.

Hemolysis During Pediatric Extracorporeal Membrane Oxygenation: Associations With Circuitry, Complications, and Mortality.

OBJECTIVES: To describe factors associated with hemolysis during pediatric extracorporeal membrane oxygenation and the relationships between hemolysis, complications, and mortality. DESIGN: Secondary analysis of data collected prospectively by the Collaborative Pediatric Critical Care Research Network between December 2012 and September 2014. SETTING: Three Collaborative Pediatric Critical Care Research Network-affiliated hospitals. PATIENTS: Age less than 19 years and treated with extracorporeal membrane oxygenation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Hemolysis was defined based on peak plasma free hemoglobin levels during extracorporeal membrane oxygenation and categorized as none (< 0.001 g/L), mild (0.001 to < 0.5 g/L), moderate (0.5 to < 1.0 g/L), or severe (≥ 1.0 g/L). Of 216 patients, four (1.9%) had no hemolysis, 67 (31.0%) had mild, 51 (23.6%) had moderate, and 94 (43.5%) had severe. On multivariable analysis, variables independently associated with higher daily plasma free hemoglobin concentration included the use of in-line hemofiltration or other continuous renal replacement therapy, higher hemoglobin concentration, higher total bilirubin concentration, lower mean heparin infusion dose, lower body weight, and lower platelet count. Using multivariable Cox modeling, daily plasma free hemoglobin was independently associated with development of renal failure during extracorporeal membrane oxygenation (defined as creatinine > 2 mg/dL [> 176.8 µmol/L] or use of in-line hemofiltration or continuous renal replacement therapy) (hazard ratio, 1.04; 95% CI, 1.02-1.06; p < 0.001), but not mortality (hazard ratio, 1.01; 95% CI, 0.99-1.04; p = 0.389). CONCLUSIONS: Hemolysis is common during pediatric extracorporeal membrane oxygenation. Hemolysis may contribute to the development of renal failure, and therapies used to manage renal failure such as in-line hemofiltration and other forms of continuous renal replacement therapy may contribute to hemolysis. Hemolysis was not associated with mortality after controlling for other factors. Monitoring for hemolysis should be a routine part of extracorporeal membrane oxygenation practice, and efforts to reduce hemolysis may improve patient care.

Acquired infection during neonatal and pediatric extracorporeal membrane oxygenation.

INTRODUCTION: Our objectives are to (1) describe the pathogens, site, timing and risk factors for acquired infection during neonatal and pediatric ECMO and (2) explore the association between acquired infection and mortality. METHODS: Secondary analysis of prospective data collected by the Collaborative Pediatric Critical Care Research Network between December 2012 and September 2014. Clinical factors associated with acquired infection were assessed with multivariable Cox regression. Factors associated with mortality were assessed with logistic regression. RESULTS: Of 481 patients, 247 (51.3%) were neonates and 400 (83.2%) received venoarterial ECMO. Eighty (16.6%) patients acquired one or more infections during ECMO; 60 (12.5%) patients had bacterial, 21 (4.4%) had fungal and 11 (2.3%) had viral infections. The site of infection included respiratory for 53 (11.0%) patients, bloodstream for 21 (4.4%), urine for 20 (4.2%) and other for 7 (1.5%). Candida species were most common. Median time to infection was 5.2 days (IQR 2.3, 9.6). On multivariable analysis, a greater number of procedures for ECMO cannula placement was independently associated with increased risk of acquired infection during ECMO (Hazard Ratio 2.13 (95% CI 1.22, 3.72), p<0.01) and receiving ECMO in a neonatal ICU compared to a pediatric or cardiac ICU was associated with decreased risk (Hazard Ratio pediatric ICU 4.25 (95% CI 2.20, 8.20), cardiac ICU 2.91 (95% CI 1.48, 5.71), neonatal ICU as reference, p<0.001). Acquired infection was not independently associated with mortality. CONCLUSION: ECMO procedures and location may contribute to acquired infection risk; however, acquired infection did not predict mortality in this study.

RBC Transfusion Practice in Pediatric Extracorporeal Membrane Oxygenation Support.

OBJECTIVES: To determine RBC transfusion practice and relationships between RBC transfusion volume and mortality in infants and children treated with extracorporeal membrane oxygenation. DESIGN: Secondary analysis of a multicenter prospective observational study. SETTING: Eight pediatric institutions within the Eunice Kennedy Shriver National Institute of Child Health and Human Development's Collaborative Pediatric Critical Care Research Network. PATIENTS: Patients age less than 19 years old treated with extracorporeal membrane oxygenation at a participating center. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Clinical data and target hemoglobin or hematocrit values (if set) were recorded daily by trained bedside extracorporeal membrane oxygenation specialists and research coordinators. Laboratory values, including hemoglobin and hematocrit, were recorded daily using the value obtained closest to 8:00 AM. RBC transfusion was recorded as total daily volume in mL/kg. Multivariable logistic regression was used to determine the relationship between RBC transfusion volume and hospital mortality, accounting for potential confounders. Average goal hematocrits varied across the cohort with a range of 27.5-41.3%. Overall, actual average daily hematocrit was 36.8%, and average RBC transfusion volume was 29.4 mL/kg/d (17.4-49.7 mL/kg/d) on extracorporeal membrane oxygenation. On multivariable analysis, each additional 10 mL/kg/d of RBC transfusion volume was independently associated with a 9% increase in odds of hospital mortality (adjusted odds ratio, 1.09 [1.02-1.16]; p = 0.009). CONCLUSIONS: In this multicenter cohort of pediatric extracorporeal membrane oxygenation patients, daily hematocrit levels were maintained at normal or near-normal values and RBC transfusion burden was high. RBC transfusion volume was independently associated with odds of mortality. Future clinical studies to identify optimum RBC transfusion thresholds for pediatric extracorporeal membrane oxygenation are urgently needed.

Hyperoxia and Hypocapnia During Pediatric Extracorporeal Membrane Oxygenation: Associations With Complications, Mortality, and Functional Status Among Survivors.

OBJECTIVES: To determine the frequency of hyperoxia and hypocapnia during pediatric extracorporeal membrane oxygenation and their relationships to complications, mortality, and functional status among survivors. DESIGN: Secondary analysis of data collected prospectively by the Collaborative Pediatric Critical Care Research Network. SETTING: Eight Collaborative Pediatric Critical Care Research Network-affiliated hospitals. PATIENTS: Age less than 19 years and treated with extracorporeal membrane oxygenation. INTERVENTIONS: Hyperoxia was defined as highest PaO2 greater than 200 Torr (27 kPa) and hypocapnia as lowest PaCO2 less than 30 Torr (3.9 kPa) during the first 48 hours of extracorporeal membrane oxygenation. Functional status at hospital discharge was evaluated among survivors using the Functional Status Scale. MEASUREMENTS AND MAIN RESULTS: Of 484 patients, 420 (86.7%) had venoarterial extracorporeal membrane oxygenation and 64 (13.2%) venovenous; 69 (14.2%) had extracorporeal membrane oxygenation initiated during cardiopulmonary resuscitation. Hyperoxia occurred in 331 (68.4%) and hypocapnia in 98 (20.2%). Hyperoxic patients had higher mortality than patients without hyperoxia (167 [50.5%] vs 48 [31.4%]; p < 0.001), but no difference in functional status among survivors. Hypocapnic patients were more likely to have a neurologic event (49 [50.0%] vs 143 (37.0%]; p = 0.021) or hepatic dysfunction (49 [50.0%] vs 121 [31.3%]; p < 0.001) than patients without hypocapnia, but no difference in mortality or functional status among survivors. On multivariable analysis, factors independently associated with increased mortality included highest PaO2 and highest blood lactate concentration in the first 48 hours of extracorporeal membrane oxygenation, congenital diaphragmatic hernia, and being a preterm neonate. Factors independently associated with lower mortality included meconium aspiration syndrome. CONCLUSIONS: Hyperoxia is common during pediatric extracorporeal membrane oxygenation and associated with mortality. Hypocapnia appears to occur less often and although associated with complications, an association with mortality was not observed.

Cognitive Development One Year After Infantile Critical Pertussis.

OBJECTIVES: Pertussis can cause life-threatening illness in infants. Data regarding neurodevelopment after pertussis remain scant. The aim of this study was to assess cognitive development of infants with critical pertussis 1 year after PICU discharge. DESIGN: Prospective cohort study. SETTING: Eight hospitals comprising the Eunice Kennedy Shriver National Institute for Child Health and Human Development Collaborative Pediatric Critical Care Research Network and 18 additional sites across the United States. PATIENTS: Eligible patients had laboratory confirmation of pertussis infection, were less than 1 year old, and were admitted to the PICU for at least 24 hours. INTERVENTIONS: The Mullen Scales of Early Learning was administered at a 1-year follow-up visit. Functional status was determined by examination and parental interview. MEASUREMENTS AND MAIN RESULTS: Of 196 eligible patients, 111 (57%) completed the Mullen Scales of Early Learning. The mean scores for visual reception, receptive language, and expressive language domains were significantly lower than the norms (p < 0.001), but not fine and gross motor domains. Forty-one patients (37%) had abnormal scores in at least one domain and 10 (9%) had an Early Learning Composite score 2 or more SDs below the population norms. Older age (p < 0.003) and Hispanic ethnicity (p < 0.008) were associated with lower mean Early Learning Composite score, but presenting symptoms and PICU course were not. CONCLUSIONS: Infants who survive critical pertussis often have neurodevelopmental deficits. These infants may benefit from routine neurodevelopmental screening.

Variability in Usual Care Mechanical Ventilation for Pediatric Acute Respiratory Distress Syndrome: Time for a Decision Support Protocol?

OBJECTIVES: Although pediatric intensivists philosophically embrace lung protective ventilation for acute lung injury and acute respiratory distress syndrome, we hypothesized that ventilator management varies. We assessed ventilator management by evaluating changes to ventilator settings in response to blood gases, pulse oximetry, or end-tidal CO2. We also assessed the potential impact that a pediatric mechanical ventilation protocol adapted from National Heart Lung and Blood Institute acute respiratory distress syndrome network protocols could have on reducing variability by comparing actual changes in ventilator settings to those recommended by the protocol. DESIGN: Prospective observational study. SETTING: Eight tertiary care U.S. PICUs, October 2011 to April 2012. PATIENTS: One hundred twenty patients (age range 17 d to 18 yr) with acute lung injury/acute respiratory distress syndrome. MEASUREMENTS AND MAIN RESULTS: Two thousand hundred arterial and capillary blood gases, 3,964 oxygen saturation by pulse oximetry, and 2,757 end-tidal CO2 values were associated with 3,983 ventilator settings. Ventilation mode at study onset was pressure control 60%, volume control 19%, pressure-regulated volume control 18%, and high-frequency oscillatory ventilation 3%. Clinicians changed FIO2 by ±5 or ±10% increments every 8 hours. Positive end-expiratory pressure was limited at ~10 cm H2O as oxygenation worsened, lower than would have been recommended by the protocol. In the first 72 hours of mechanical ventilation, maximum tidal volume/kg using predicted versus actual body weight was 10.3 (8.5-12.9) (median [interquartile range]) versus 9.2 mL/kg (7.6-12.0) (p < 0.001). Intensivists made changes similar to protocol recommendations 29% of the time, opposite to the protocol's recommendation 12% of the time and no changes 56% of the time. CONCLUSIONS: Ventilator management varies substantially in children with acute respiratory distress syndrome. Opportunities exist to minimize variability and potentially injurious ventilator settings by using a pediatric mechanical ventilation protocol offering adequately explicit instructions for given clinical situations. An accepted protocol could also reduce confounding by mechanical ventilation management in a clinical trial.

Potential Acceptability of a Pediatric Ventilator Management Computer Protocol.

OBJECTIVES: To examine issues regarding the granularity (size/scale) and potential acceptability of recommendations in a ventilator management protocol for children with pediatric acute respiratory distress syndrome. DESIGN: Survey/questionnaire. SETTING: The eight PICUs in the Collaborative Pediatric Critical Care Research Network. PARTICIPANTS: One hundred twenty-two physicians (attendings and fellows). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We used an online questionnaire to examine attitudes and assessed recommendations with 50 clinical scenarios. Overall 80% of scenario recommendations were accepted. Acceptance did not vary by provider characteristics but did vary by ventilator mode (high-frequency oscillatory ventilation 83%, pressure-regulated volume control 82%, pressure control 75%; p = 0.002) and variable adjusted (ranging from 88% for peak inspiratory pressure and 86% for FIO2 changes to 69% for positive end-expiratory pressure changes). Acceptance did not vary based on child size/age. There was a preference for smaller positive end-expiratory pressure changes but no clear granularity preference for other variables. CONCLUSIONS: Although overall acceptance rate for scenarios was good, there was little consensus regarding the size/scale of ventilator setting changes for children with pediatric acute respiratory distress syndrome. An acceptable protocol could support robust evaluation of ventilator management strategies. Further studies are needed to determine if adherence to an explicit protocol leads to better outcomes.

Education and training of clinical and translational study investigators and research coordinators: A competency-based approach.

INTRODUCTION: Training for the clinical research workforce does not sufficiently prepare workers for today's scientific complexity; deficiencies may be ameliorated with training. The Enhancing Clinical Research Professionals' Training and Qualifications developed competency standards for principal investigators and clinical research coordinators. METHODS: Clinical and Translational Science Awards representatives refined competency statements. Working groups developed assessments, identified training, and highlighted gaps. RESULTS: Forty-eight competency statements in 8 domains were developed. CONCLUSIONS: Training is primarily investigator focused with few programs for clinical research coordinators. Lack of training is felt in new technologies and data management. There are no standardized assessments of competence.

Functional Status of Neonatal and Pediatric Patients After Extracorporeal Membrane Oxygenation.

OBJECTIVES: To describe functional status at hospital discharge for neonatal and pediatric patients treated with extracorporeal membrane oxygenation, and identify factors associated with functional status and mortality. DESIGN: Secondary analysis of observational data collected by the Collaborative Pediatric Critical Care Research Network between December 2012 and September 2014. SETTING: Eight hospitals affiliated with the Collaborative Pediatric Critical Care Research Network. PATIENTS: Patients were less than 19 years old and treated with extracorporeal membrane oxygenation. INTERVENTIONS: Functional status was evaluated among survivors using the Functional Status Scale. Total Functional Status Scale scores range from 6 to 30 and are categorized as 6-7 (good), 8-9 (mildly abnormal), 10-15 (moderately abnormal), 16-21 (severely abnormal), and greater than 21 (very severely abnormal). MEASUREMENTS AND MAIN RESULTS: Of 514 patients, 267 (52%) were neonates (≤ 30 d old). Indication for extracorporeal membrane oxygenation was respiratory for 237 (46%), cardiac for 207 (40%), and extracorporeal cardiopulmonary resuscitation for 70 (14%). Among 282 survivors, 89 (32%) had good, 112 (40%) mildly abnormal, 67 (24%) moderately abnormal, and 14 (5%) severely or very severely abnormal function at hospital discharge. Among neonates, development of renal failure and longer hospitalization were independently associated with worse Functional Status Scale. Chronic conditions, prematurity, venoarterial extracorporeal membrane oxygenation, increased red cell transfusion in the first 24 hours of extracorporeal membrane oxygenation, and longer extracorporeal membrane oxygenation duration were independently associated with mortality. Among pediatric patients, chronic neurologic conditions, tracheostomy or home ventilator, extracorporeal cardiopulmonary resuscitation, hepatic dysfunction, and longer ICU stay were independently associated with worse Functional Status Scale. Chronic cardiac conditions, hepatic dysfunction, and neurologic or thrombotic complications were independently associated with mortality. Achieving blood lactate concentration less than or equal to 2 mmol/L during extracorporeal membrane oxygenation was independently associated with survival in both neonatal and pediatric patients. CONCLUSIONS: In this study, about half of extracorporeal membrane oxygenation patients survived with good, mildly abnormal, or moderately abnormal function at hospital discharge. Patient and extracorporeal membrane oxygenation-related factors are associated with functional status and mortality.

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.

Enhancing Clinical Research Professionals' Training and Qualifications (ECRPTQ): Recommendations for Good Clinical Practice (GCP) training for investigators and study coordinators.

INTRODUCTION: The translation of discoveries to drugs, devices, and behavioral interventions requires well-prepared study teams. Execution of clinical trials remains suboptimal due to varied quality in design, execution, analysis, and reporting. A critical impediment is inconsistent, or even absent, competency-based training for clinical trial personnel. METHODS: In 2014, the National Center for Advancing Translational Science (NCATS) funded the project, Enhancing Clinical Research Professionals' Training and Qualifications (ECRPTQ), aimed at addressing this deficit. The goal was to ensure all personnel are competent to execute clinical trials. A phased structure was utilized. RESULTS: This paper focuses on training recommendations in Good Clinical Practice (GCP). Leveraging input from all Clinical and Translational Science Award hubs, the following was recommended to NCATS: all investigators and study coordinators executing a clinical trial should understand GCP principles and undergo training every 3 years, with the training method meeting the minimum criteria identified by the International Conference on Harmonisation GCP. CONCLUSIONS: We anticipate that industry sponsors will acknowledge such training, eliminating redundant training requests. We proposed metrics to be tracked that required further study. A separate task force was composed to define recommendations for metrics to be reported to NCATS.

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.