Using redox potential as a feasible marker for banked blood quality and the state of oxidative stress in stored red blood cells.

BACKGROUND: Stored red blood cells (RBCs) may undergo oxidative stress over time, with functional changes affecting oxygen delivery. Central to these changes are oxidation-reduction (redox) reactions and redox potential (RP) that must be maintained for cell function. RP imbalance can lead to oxidative stress that may contribute to storage lesions. This study's purpose was to identify changes in RP over time in banked RBCs, and among RBCs of similar age. METHODS: Multiple random RBC segments from RBC units were tested (n = 32), ranging in age from 5 to 40 days, at 5-day intervals. RP was recorded by measuring open circuit potential of RBCs using nanoporous gold electrodes with Ag/AgCl reference. RP measures were also performed on peripheral venous blood from 10 healthy volunteers. RP measures were compared between RBC groups, and with volunteer blood. RESULTS: Stored RBCs show time-dependent RP increases. There were significant differences in Day 5 RP compared to all other groups (p ≤ 0.005), Day 10-15 vs. ages ≥ Day 20 (p ≤ 0.025), Day 20-25 vs. Day 40 (p = 0.039), and all groups compared to healthy volunteers. RP became more positive over time suggesting ongoing oxidation as RBCs age; however, storage time alone was not predictive of RP measured in a particular unit/segment. CONCLUSIONS: There are significant differences in RP between freshly stored RBCs and all others, with RP becoming more positive over time. However, storage time alone does not predict RP, indicating RP screening may be an important measure of RBC oxidative stress and serve as an RBC quality marker.

Monitoring Severity of Multiple Organ Dysfunction Syndrome: New Technologies.

OBJECTIVE: To describe new technologies (biomarkers and tests) used to assess and monitor the severity and progression of multiple organ dysfunction syndrome in children as discussed as part of the Eunice Kennedy Shriver National Institute of Child Health and Human Development MODS Workshop (March 26-27, 2015). DATA SOURCES: Literature review, research data, and expert opinion. STUDY SELECTION: Not applicable. DATA EXTRACTION: Moderated by an experienced expert from the field, investigators developing and assessing new technologies to improve the care and understanding of critical illness presented their research and the relevant literature. DATA SYNTHESIS: Summary of presentations and discussion supported and supplemented by relevant literature. CONCLUSIONS: There are many innovative tools and techniques with the potential application for the assessment and monitoring of severity of multiple organ dysfunction syndrome. If the reliability and added value of these candidate technologies can be established, they hold promise to enhance the understanding, monitoring, and perhaps, treatment of multiple organ dysfunction syndrome in children.

Electrochemical properties of nanostructured porous gold electrodes in biofouling solutions.

The effect of electrode porosity on the electrochemical response of redox active molecules (potassium ferricyanide, ruthenium(III) hexammine, and ferrocene methanol) in the presence of bovine serum albumin or fibrinogen was studied at macroporous (pore diameter: 1200 nm), hierarchical (1200/60 nm), and nanoporous (<50 nm) gold. These electrodes were prepared using standard templating or dealloying techniques, and cyclic voltammetry (CV) was utilized to evaluate the effect of protein adsorption on the electron transfer of the diffusing redox probes. Following exposure to albumin (or fibrinogen) under near neutral pH conditions, planar gold electrodes showed an immediate reduction in Faradaic peak current and increase in peak splitting for potassium ferricyanide. The rate at which the CV curves changed was highly dependent on the morphology of the electrode. For example, the time required for the Faradaic current to drop to one-half of its original value was 3, 12, and 38 min for planar gold, macroporous gold, and hierarchical gold, respectively. Remarkably, for nanoporous gold, only a few percent drop in the peak Faradaic current was observed after an hour in solution. A similar suppression in the voltammetry at planar gold was also noted for ruthenium hexammine at pH 3 after exposure to albumin for several hours. At nanoporous gold, no significant loss in response was observed. The order of performance of the electrodes as judged by their ability to efficiently transfer electrons in the presence of biofouling agents tracked porosity with the electrode having the smallest pore size and largest surface area, providing near ideal results. Nanoporous gold electrodes when immersed in serum or heparinized blood containing potassium ferricyanide showed ideal voltammetry while significant fouling was evident in the electrochemical response at planar gold. The small nanopores in this 3D open framework are believed to restrict the transport of large biomolecules, thus minimizing passivation of the inner surfaces while permitting access to small redox probes to efficiently exchange electrons.

Improved outcomes for stem cell transplant recipients requiring pediatric intensive care.

OBJECTIVES: Survival for hematopoietic stem cell transplant patients requiring pediatric intensive care unit admission may be improving. This study was conducted to review outcomes for patients undergoing hematopoietic stem cell transplantation requiring admission to our pediatric intensive care unit and to identify variables impacting survival. DESIGN: Retrospective database review. SETTING: Pediatric intensive care unit and bone marrow transplant service of a children's hospital. PATIENTS: Patients undergoing hematopoietic stem cell transplantation at our center from July 2004 through June 2010 requiring pediatric intensive care unit admission during the same period. MEASUREMENTS AND MAIN RESULTS: Thirty-five percent of patients (155 of 448) undergoing hematopoietic stem cell transplantation required 319 admissions over this period. Of these 155 patients, 63% (97 of 155) were discharged alive following their most recent admission with a 100-day survival of 51% (79 of 155). Forty-five percent (69 of 155) of patients were still alive on long-term follow-up. Intubation and mechanical ventilation were required for 57% (88 of 155) of patients, with 39% (34 of 88) of patients surviving their last pediatric intensive care unit admission. Renal support was utilized for 25% (38 of 155) of patients with 34% (13 of 38) survival to pediatric intensive care unit discharge. Admissions surviving to pediatric intensive care unit discharge had significantly lower Pediatric Risk of Mortality II scores, shorter pediatric intensive care unit length of stay, lower utilization of intubation and mechanical ventilation with fewer ventilator days, and lower use of renal support when compared to nonsurvivors. Of note, each prior pediatric intensive care unit admission significantly reduced the odds of pediatric intensive care unit survival. CONCLUSIONS: We report a 63% survival to pediatric intensive care unit discharge, with 45% surviving at a median follow-up of over 2 yrs for all hematopoietic stem cell transplantation patients admitted to our pediatric intensive care unit over a 6-yr period. Our data suggest improved survival outcomes for this high risk patient population.

Redox Potential Correlates with Changes in Metabolite Concentrations Attributable to Pathways Active in Oxidative Stress Response in Swine Traumatic Shock.

INTRODUCTION: Oxidation-reduction (redox) reactions, and the redox potential (RP) that must be maintained for proper cell function, lie at the heart of physiologic processes in critical illness. Imbalance in RP reflects systemic oxidative stress, and whole blood RP measures have been shown to correlate with oxygen debt level over time in swine traumatic shock. We hypothesize that RP measures reflect changing concentrations of metabolites involved in oxidative stress. To test this hypothesis, we compared blood and urine RP with concentrations of multiple metabolites in a swine traumatic shock model to identify meaningful RP-metabolite relationships. METHODS: Seven swine were subjected to traumatic shock. Mixed venous (MV) RP, urine RP, and concurrent MV and urine metabolite concentrations were assessed at baseline, max O 2 Debt (80 mL/kg), end resuscitation, and 2 h post-resuscitation. RP was measured at collection via open circuit potential using nanoporous gold electrodes with Ag/AgCl reference and a ParstatMC potentiostat. Metabolite concentrations were measured by quantitative 1 H-NMR spectroscopy. MV and urine RP were compared with time-matched metabolites across all swine. LASSO regression with leave-one-out cross validation was used to determine meaningful RP/metabolite relationships. Metabolites had to maintain magnitude and direction of coefficients across 6 or more swine to be considered as having a meaningful relationship. KEGG IDs of these metabolites were uploaded into Metscape for pathway identification and evaluation for physiologic function. RESULTS: Meaningful metabolite relationships (and mean coefficients across cross-validation folds) with MV RP included: choline (-6.27), ATP (-4.39), glycine (5.93), ADP (1.84), glucose (15.96), formate (-13.09), pyruvate (6.18), and taurine (-7.18). Relationships with urine RP were: betaine (4.81), urea (4.14), glycine (-2.97), taurine (10.32), 3-hydroxyisobutyrate (-7.67), N-phenylacetylglycine, PAG (-14.52), hippurate (12.89), and formate (-5.89). These meaningful metabolites were found to scavenge extracellular peroxide (pyruvate), inhibit ROS and activate cellular antioxidant defense (taurine), act as indicators of antioxidant mobilization against oxidative stress (glycine + PAG), and reflect renal hydroxyl radical trapping (hippurate), among other activities. CONCLUSIONS: Real-time RP measures demonstrate significant relationships with metabolites attributable to metabolic pathways involved in systemic responses to oxidative stress, as well as those involved in these processes. These data support RP measures as a feasible, biologically relevant marker of oxidative stress. As a direct measure of redox state, RP may be a useful biomarker and clinical tool in guiding diagnosis and therapy in states of increased oxidative stress and may offer value as a marker for organ injury in these states as well.

A novel swine model of the acute respiratory distress syndrome using clinically relevant injury exposures.

To date, existing animal models of the acute respiratory distress syndrome (ARDS) have failed to translate preclinical discoveries into effective pharmacotherapy or diagnostic biomarkers. To address this translational gap, we developed a high-fidelity swine model of ARDS utilizing clinically relevant lung injury exposures. Fourteen male swine were anesthetized, mechanically ventilated, and surgically instrumented for hemodynamic monitoring, blood, and tissue sampling. Animals were allocated to one of three groups: (1) Indirect lung injury only: animals were inoculated by direct injection of Escherichia coli into the kidney parenchyma, provoking systemic inflammation and distributive shock physiology; (2) Direct lung injury only: animals received volutrauma, hyperoxia, and bronchoscope-delivered gastric particles; (3) Combined indirect and direct lung injury: animals were administered both above-described indirect and direct lung injury exposures. Animals were monitored for up to 12 h, with serial collection of physiologic data, blood samples, and radiographic imaging. Lung tissue was acquired postmortem for pathological examination. In contrast to indirect lung injury only and direct lung injury only groups, animals in the combined indirect and direct lung injury group exhibited all of the physiological, radiographic, and histopathologic hallmarks of human ARDS: impaired gas exchange (mean PaO2 /FiO2 ratio 124.8 ± 63.8), diffuse bilateral opacities on chest radiographs, and extensive pathologic evidence of diffuse alveolar damage. Our novel porcine model of ARDS, built on clinically relevant lung injury exposures, faithfully recapitulates the physiologic, radiographic, and histopathologic features of human ARDS and fills a crucial gap in the translational study of human lung injury.

A comprehensive assessment of multi-system responses to a renal inoculation of uropathogenic E. coli in swine.

BACKGROUND: The systemic responses to infection and its progression to sepsis remains poorly understood. Progress in the field has been stifled by the shortcomings of experimental models which include poor replication of the human condition. To address these challenges, we developed and piloted a novel large animal model of severe infection that is capable of generating multi-system clinically relevant data. METHODS: Male swine (n = 5) were anesthetized, mechanically ventilated, and surgically instrumented for continuous hemodynamic monitoring and serial blood sampling. Animals were inoculated with uropathogenic E. coli by direct injection into the renal parenchyma and were maintained until a priori endpoints were met. The natural history of the infection was studied. Animals were not resuscitated. Multi-system data were collected hourly to 6 hours; all animals were euthanized at predetermined physiologic endpoints. RESULTS: Core body temperature progressively increased from mean (SD) 37.9(0.8)°C at baseline to 43.0(1.2)°C at experiment termination (p = 0.006). Mean arterial pressure did not begin to decline until 6h post inoculation, dropping from 86(9) mmHg at baseline to 28(5) mmHg (p = 0.005) at termination. Blood glucose progressively declined but lactate levels did not elevate until the last hours of the experiment. There were also temporal changes in whole blood concentrations of a number of metabolites including increases in the catecholamine precursors, tyrosine (p = 0.005) and phenylalanine (p = 0.005). Lung, liver, and kidney function parameters worsened as infection progressed and at study termination there was histopathological evidence of injury in these end-organs. CONCLUSION: We demonstrate a versatile, multi-system, longitudinal, swine model of infection that could be used to further our understanding of the mechanisms that underlie infection-induced multi-organ dysfunction and failure, optimize resuscitation protocols and test therapeutic interventions. Such a model could improve translation of findings from the bench to the bedside, circumventing a significant obstacle in sepsis research.

3D-printed, externally-implanted, bioresorbable airway splints for severe tracheobronchomalacia.

OBJECTIVES/HYPOTHESIS: To report the clinical safety and efficacy of three-dimensional (3D)-printed, patient-specific, bioresorbable airway splints in a cohort of critically ill children with severe tracheobronchomalacia. STUDY DESIGN: Case series. METHODS: From 2012 to 2018, 15 subjects received 29 splints on their trachea, right and/or left mainstem bronchi. The median age at implantation was 8 months (range, 3-25 months). Nine children were female. Five subjects had a history of extracorporeal membrane oxygenation (ECMO), and 11 required continuous sedation, six of whom required paralytics to maintain adequate ventilation. Thirteen were chronically hospitalized, unable to be discharged, and seven were hospitalized their entire lives. At the time of splint implantation, one subject required ECMO, one required positive airway pressure, and 13 subjects were tracheostomy and ventilator dependent, requiring a median positive end-expiratory pressure (PEEP) of 14 cm H2 O (range, 6-20 cm H2 0). Outcomes collected included level of respiratory support, disposition, and splint-related complications. RESULTS: At the time of discharge from our institution, at a median of 28 days postimplantation (range, 10-56 days), the subject on ECMO was weaned from extracorporeal support, and the subjects who were ventilated via tracheostomy had a median change in PEEP (discharge-baseline) of -2.5 cm H2 O (range, -15 to 2 cm H2 O, P = .022). At median follow-up of 8.5 months (range, 0.3-77 months), all but one of the 12 surviving subjects lives at home. Of the 11 survivors who were tracheostomy dependent preoperatively, one is decannulated, one uses a speaking valve, six use a ventilator exclusively at night, and three remain ventilator dependent. CONCLUSIONS: This case series demonstrates the initial clinical efficacy of the 3D-printed bioresorbable airway splint device in a cohort of critically ill children with severe tracheobronchomalacia. LEVEL OF EVIDENCE: 4 Laryngoscope, 129:1763-1771, 2019.

Whole Blood Redox Potential Correlates With Progressive Accumulation of Oxygen Debt and Acts as A Marker of Resuscitation in A Swine Hemorrhagic Shock Model.

INTRODUCTION: Oxidation-reduction reactions involve electron exchanges that require optimal balance for proper cell function. This balance is measured via redox potential and reflects oxidative stress. Despite the critical role of oxidative stress in critical illness and injury, little is known regarding redox potential. We hypothesize redox potential measurements will correlate with accumulation of O2 debt produced by hemorrhage over time. METHODS: Ten swine were studied using a polytrauma hemorrhagic shock model. Whole blood and plasma redox potential measures were obtained at defined stages of O2 debt (20 mL/kg, 40 mL/kg, 60 mL/kg, 80 mL/kg), and through resuscitation. Redox potential was determined by measuring open circuit potential using novel gold nanoporous electrodes with Ag/AgCl reference. RESULTS: Whole blood redox potential showed negative change as O2 debt accumulated, exhibiting positive response during resuscitation, and correlated with O2 debt across all animals (P < 0.001). Redox potential changes throughout O2 debt accrual were significant compared with baseline (P≤0.05), and at end resuscitation compared with O2 debt 60 mL/kg (P = 0.05) and 80 mL/kg (P = 0.02). Whole blood redox potential measures also correlated with oxygen extraction ratio, ScvO2, and lactic acid, appearing very sensitive to acute changes. Plasma redox potential showed no correlation with O2 debt. CONCLUSIONS: Whole blood redox potential demonstrates significant correlation to O2 debt at all stages in this model. These results set the stage for further study of redox potential as a direct measure of oxidative stress and potential clinical tool. Given redox potential plasma performance, these measures should be made in whole blood versus plasma.

Renal complications and therapy in the PICU: hypertension, CKD, AKI, and RRT.

This article provides the bedside clinician an overview of the unique renal complications that are seen commonly in the pediatric intensive care unit. These sections are purposely succinct to give a quick guide to the clinician for the care of these children. We have identified four major areas that should result in discussion and cooperative care between intensive care physicians and nephrologists for the care of these children: (1) hypertension, (2) chronic kidney failure, (3) acute kidney injury, and (4) renal replacement therapy.

Human T-cell leukemia virus-I tax oncoprotein functionally targets a subnuclear complex involved in cellular DNA damage-response.

The virally encoded oncoprotein Tax has been implicated in HTLV-1-mediated cellular transformation. The exact mechanism by which this protein contributes to the oncogenic process is not known. However, it has been hypothesized that Tax induces genomic instability via repression of cellular DNA repair. We examined the effect of de novo Tax expression upon the cell cycle, because appropriate activation of cell cycle checkpoints is essential to a robust damage-repair response. Upon induction of tax expression, Jurkat T-cells displayed a pronounced accumulation in G2/M that was reversible by caffeine. We examined the G2-specific checkpoint signaling response in these cells and found activation of the ATM/chk2-mediated pathway, whereas the ATR/chk1-mediated response was unaffected. Immunoprecipitation with anti-chk2 antibody results in co-precipitation of Tax demonstrating a direct interaction of Tax with a chk2-containing complex. We also show that Tax targets a discrete nuclear site and co-localizes with chk2 and not chk1. This nuclear site, previously identified as Tax Speckled Structures (TSS), also contains the early damage response factor 53BP1. The recruitment of 53BP1 to TSS is dependent upon ATM signaling and requires expression of Tax. Specifically, Tax expression induces redistribution of diffuse nuclear 53BP1 to the TSS foci. Taken together these data suggest that the TSS describe a unique nuclear site involved in DNA damage recognition, repair response, and cell cycle checkpoint activation. We suggest that association of Tax with this multifunctional subnuclear site results in disruption of a subset of the site-specific activities and contributes to cellular genomic instability.