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Extracellular vesicles (EVs) participate in cell-to-cell communication and contribute toward homeostasis under physiological conditions. But EVs can also contribute toward a wide array of pathophysiology like cancer, sepsis, sickle cell disease, and thrombotic disorders. COVID-19 infected patients are at an increased risk of aberrant coagulation, consistent with elevated circulating levels of ultra-high molecular weight VWF multimers, D-dimer and procoagulant EVs. The role of EVs in COVID-19 related hemostasis may depend on cells of origin, vesicular cargo and size, however this is not well defined. We hypothesized that the procoagulant potential of EV isolates from COVID-19 (+) patient plasmas could be defined by thrombin generation assays. Here we isolated small EVs (SEVs) and large EVs (LEVs) from hospitalized COVID-19 (+) patient (n = 21) and healthy donor (n = 20) plasmas. EVs were characterized by flow cytometry, Transmission electron microscopy, nanoparticle tracking analysis, plasma thrombin generation and a multi-omics approach to define coagulation potential. These data were consistent with differences in EV metabolite, lipid, and protein content when compared to healthy donor plasma isolated SEVs and LEVs. Taken together, the effect of EVs on plasma procoagulant potential as defined by thrombin generation and supported by multi-omics is enhanced in COVID-19. Further, we observe that this effect is driven both by EV size and phosphatidyl serine.

Background Thrombotic microangiopathy-induced thrombocytopenia-associated multiple organ failure and hyperinflammatory macrophage activation syndrome are important causes of late pediatric sepsis mortality that are often missed or have delayed diagnosis. The National Institutes of General Medical Science sepsis research working group recommendations call for application of new research approaches in extant clinical data sets to improve efficiency of early trials of new sepsis therapies. Our objective is to apply machine learning approaches to derive computable 24-h sepsis phenotypes to facilitate personalized enrollment in early anti-inflammatory trials targeting these conditions. Methods We applied consensus, k -means clustering analysis to our extant PHENOtyping sepsis-induced Multiple organ failure Study (PHENOMS) dataset of 404 children. 24-hour computable phenotypes are derived using 25 available bedside variables including C-reactive protein and ferritin. Results Four computable phenotypes (PedSep-A, B, C, and D) are derived. Compared to all other phenotypes, PedSep-A patients ( n  = 135; 2% mortality) were younger and previously healthy, with the lowest C-reactive protein and ferritin levels, the highest lymphocyte and platelet counts, highest heart rate, and lowest creatinine ( p  < 0.05); PedSep-B patients ( n  = 102; 12% mortality) were most likely to be intubated and had the lowest Glasgow Coma Scale Score ( p  < 0.05); PedSep-C patients ( n  = 110; mortality 10%) had the highest temperature and Glasgow Coma Scale Score, least pulmonary failure, and lowest lymphocyte counts ( p  < 0.05); and PedSep-D patients ( n  = 56, 34% mortality) had the highest creatinine and number of organ failures, including renal, hepatic, and hematologic organ failure, with the lowest platelet counts ( p  < 0.05). PedSep-D had the highest likelihood of developing thrombocytopenia-associated multiple organ failure (Adj OR 47.51 95% CI [18.83–136.83], p  < 0.0001) and macrophage activation syndrome (Adj OR 38.63 95% CI [13.26–137.75], p  < 0.0001). Conclusions Four computable phenotypes are derived, with PedSep-D being optimal for enrollment in early personalized anti-inflammatory trials targeting thrombocytopenia-associated multiple organ failure and macrophage activation syndrome in pediatric sepsis. A computer tool for identification of individual patient membership ( www.pedsepsis.pitt.edu ) is provided. Reproducibility will be assessed at completion of two ongoing pediatric sepsis studies.

Recent studies have underscored questions about the balance of risk and benefit of RBC transfusion. A better understanding of the nature and timing of molecular and functional changes in stored RBCs may provide strategies to improve the balance of benefit and risk of RBC transfusion. We analyzed changes occurring during RBC storage focusing on RBC deformability, RBC-dependent vasoregulatory function, and S-nitrosohemoglobin (SNO-Hb), through which hemoglobin (Hb) O₂ desaturation is coupled to regional increases in blood flow in vivo (hypoxic vasodilation). Five hundred ml of blood from each of 15 healthy volunteers was processed into leukofiltered, additive solution 3-exposed RBCs and stored at 1-6°C according to AABB standards. Blood was subjected to 26 assays at 0, 3, 8, 24 and 96 h, and at 1, 2, 3, 4, and 6 weeks. RBC SNO-Hb decreased rapidly (1.2 x 10⁻⁴ at 3 h vs. 6.5 x 10⁻⁴ (fresh) mol S-nitrosothiol (SNO)/mol Hb tetramer (P = 0.032, mercuric-displaced photolysis-chemiluminescence assay), and remained low over the 42-day period. The decline was corroborated by using the carbon monoxide-saturated copper-cysteine assay [3.0 x 10⁻⁵ at 3 h vs. 9.0 x 10⁻⁵ (fresh) mol SNO/mol Hb]. In parallel, vasodilation by stored RBCs was significantly depressed. RBC deformability assayed at a physiological shear stress decreased gradually over the 42-day period (P < 0.001). Time courses vary for several storage-induced defects that might account for recent observations linking blood transfusion with adverse outcomes. Of clinical concern is that SNO levels, and their physiological correlate, RBC-dependent vasodilation, become depressed soon after collection, suggesting that even \"fresh\" blood may have developed adverse biological characteristics.

Background Group model building (GMB) is a method to facilitate shared understanding of structures and relationships that determine system behaviors. This project aimed to determine the feasibility of GMB in a resource-limited setting and to use GMB to describe key barriers and facilitators to effective acute care delivery at a tertiary care hospital in Malawi. Methods Over 1 week, trained facilitators led three GMB sessions with two groups of healthcare providers to facilitate shared understanding of structures and relationships that determine system behaviors. One group aimed to identify factors that impact patient flow in the paediatric special care ward. The other aimed to identify factors impacting delivery of high-quality care in the paediatric accident and emergency room. Synthesized causal maps of factors influencing patient care were generated, revised, and qualitatively analyzed. Results Causal maps identified patient condition as the central modifier of acute care delivery. Severe illness and high volume of patients were identified as creating system strain in several domains: (1) physical space, (2) resource needs and utilization, (3) staff capabilities and (4) quality improvement. Stress in these domains results in worsening patient condition and perpetuating negative reinforcing feedback loops. Balancing factors inherent to the current system included (1) parental engagement, (2) provider resilience, (3) ease of communication and (4) patient death. Perceived strengths of the GMB process were representation of diverse stakeholder viewpoints and complex system synthesis in a visual causal pathway, the process inclusivity, development of shared understanding, new idea generation and momentum building. Challenges identified included time required for completion and potential for participant selection bias. Conclusions GMB facilitated creation of a shared mental model, as a first step in optimizing acute care delivery in a paediatric facility in this resource-limited setting.

Background One of five global deaths are attributable to sepsis. Hyperferritinemic sepsis (> 500 ng/mL) is associated with increased mortality in single-center studies. Our pediatric research network’s objective was to obtain rationale for designing anti-inflammatory clinical trials targeting hyperferritinemic sepsis. Methods We assessed differences in 32 cytokines, immune depression (low whole blood ex vivo TNF response to endotoxin) and thrombotic microangiopathy (low ADAMTS13 activity) biomarkers, seven viral DNAemias, and macrophage activation syndrome (MAS) defined by combined hepatobiliary dysfunction and disseminated intravascular coagulation, and mortality in 117 children with hyperferritinemic sepsis (ferritin level > 500 ng/mL) compared to 280 children with sepsis without hyperferritinemia. Causal inference analysis of these 41 variables, MAS, and mortality was performed. Results Mortality was increased in children with hyperferritinemic sepsis (27/117, 23% vs 16/280, 5.7%; Odds Ratio = 4.85, 95% CI [2.55–9.60]; z = 4.728; P -value < 0.0001). Hyperferritinemic sepsis had higher C-reactive protein, sCD163, IL-22, IL-18, IL-18 binding protein, MIG/CXCL9, IL-1β, IL-6, IL-8, IL-10, IL-17a, IFN-γ, IP10/CXCL10, MCP-1/CCL2, MIP-1α, MIP-1β, TNF, MCP-3, IL-2RA (sCD25), IL-16, M-CSF, and SCF levels; lower ADAMTS13 activity, sFasL, whole blood ex vivo TNF response to endotoxin, and TRAIL levels; more Adenovirus, BK virus, and multiple virus DNAemias; and more MAS ( P -value < 0.05). Among these variables, only MCP-1/CCL2 (the monocyte chemoattractant protein), MAS, and ferritin levels were directly causally associated with mortality. MCP-1/CCL2 and hyperferritinemia showed direct causal association with depressed ex vivo whole blood TNF response to endotoxin. MCP-1/CCL2 was a mediator of MAS. MCP-1/CCL2 and MAS were mediators of hyperferritinemia. Conclusions These findings establish hyperferritinemic sepsis as a high-risk condition characterized by increased cytokinemia, viral DNAemia, thrombotic microangiopathy, immune depression, macrophage activation syndrome, and death. The causal analysis provides rationale for designing anti-inflammatory trials that reduce macrophage activation to improve survival and enhance infection clearance in pediatric hyperferritinemic sepsis.

Intact red blood cells (RBCs) are required for phenotypic analyses. In order to allow separation (time and location) between subject encounter and sample analysis, we developed a research-specific RBC cryopreservation protocol and assessed its impact on data fidelity for key biochemical and physiological assays. RBCs drawn from healthy volunteers were aliquotted for immediate analysis or following glycerol-based cryopreservation, thawing, and deglycerolization. RBC phenotype was assessed by (1) scanning electron microscopy (SEM) imaging and standard morphometric RBC indices, (2) osmotic fragility, (3) deformability, (4) endothelial adhesion, (5) oxygen (O2) affinity, (6) ability to regulate hypoxic vasodilation, (7) nitric oxide (NO) content, (8) metabolomic phenotyping (at steady state, tracing with [1,2,3-13C3]glucose ± oxidative challenge with superoxide thermal source; SOTS-1), as well as in vivo quantification (following human to mouse RBC xenotransfusion) of (9) blood oxygenation content mapping and flow dynamics (velocity and adhesion). Our revised glycerolization protocol (40% v/v final) resulted in >98.5% RBC recovery following freezing (-80°C) and thawing (37°C), with no difference compared to the standard reported method (40% w/v final). Full deglycerolization (>99.9% glycerol removal) of 40% v/v final samples resulted in total cumulative lysis of ~8%, compared to ~12-15% with the standard method. The post cryopreservation/deglycerolization RBC phenotype was indistinguishable from that for fresh RBCs with regard to physical RBC parameters (morphology, volume, and density), osmotic fragility, deformability, endothelial adhesivity, O2 affinity, vasoregulation, metabolomics, and flow dynamics. These results indicate that RBC cryopreservation/deglycerolization in 40% v/v glycerol final does not significantly impact RBC phenotype (compared to fresh cells).

Outcomes after traumatic brain injury are worsened by secondary insults; modern intensive-care units address such challenges through use of best-practice pathways. Organisation of intensive-care units has an important role in pathway effectiveness. We aimed to assess the effect of a paediatric neurocritical care programme (PNCP) on outcomes for children with severe traumatic brain injury. We undertook a retrospective cohort study of 123 paediatric patients with severe traumatic brain injury (Glasgow coma scale scores ≤8, without gunshot or abusive head trauma, cardiac arrest, or Glasgow coma scale scores of 3 with fixed and dilated pupils) admitted to the paediatric intensive-care unit of the St Louis Children's Hospital (St Louis, MO, USA) between July 15, 1999, and Jan 15, 2012. The primary outcome was rate of categorised hospital discharge disposition before and after implementation of a PNCP on Sept 17, 2005. We developed an ordered probit statistical model to assess adjusted outcome as a function of initial injury severity. We assessed care-team behaviour by comparing timing of invasive neuromonitoring and scored intensity of therapies targeting intracranial hypertension. Characteristics of treated patients (aged 3–219 months) were much the same between treatment periods. Before PNCP implementation, 33 (52%) of 63 patients had unfavourable disposition at hospital discharge (death or admission to an inpatient facility) and 30 (48%) had a favourable disposition (home with or without treatment); after PNCP implementation, 20 (33%) of 60 patients had unfavourable disposition and 40 (67%) had favourable disposition (p=0·01). Seven (11%) patients died before PNCP implementation compared with two (3%) deaths after implementation. The probit model indicated that outcome improved across the spectrum of Glasgow coma scale scores after resuscitation (p=0·02); this improvement progressed with increasing injury severity. Kaplan-Meier analysis suggested that neuromonitoring was started earlier and maintained longer after implementation of the PNCP (p=0·03). Therapeutic intensity scores were increased for the first 3 days of treatment after PNCP implementation (p=0·0298 for day 1, p=0·0292 for day 2, and p=0·0471 for day 3). The probit model suggested that increasing age (p=0·03), paediatric risk of mortality III scores (p=0·0003), and injury severity scores (p=0·02) were reliably associated with increased probability of unfavourable outcomes whereas white race (p=0·01), use of intracranial pressure monitoring (p=0·001), and increasing Glasgow coma scale scores after resuscitation (p=0·04) were associated with increased probability of favourable outcomes. Outcomes for children with traumatic brain injury can be improved by altering the care system in a way that stably implements a cooperative programme of accepted best practice. St Louis Children's Hospital and the Sean Glanvill Foundations.

Inhaled nitric oxide (NO) exerts a variety of effects through metabolites and these play an important role in regulation of hemodynamics in the body. A detailed investigation into the generation of these metabolites has been overlooked. We investigated the kinetics of nitrite and S-nitrosothiol-hemoglobin (SNO-Hb) in plasma derived from inhaled NO subjects and how this modifies the cutaneous microvascular response. We enrolled 15 healthy volunteers. Plasma nitrite levels at baseline and during NO inhalation (15 minutes at 40 ppm) were 102 (86-118) and 114 (87-129) nM, respectively. The nitrite peak occurred at 5 minutes of discontinuing NO (131 (104-170) nM). Plasma nitrate levels were not significantly different during the study. SNO-Hb molar ratio levels at baseline and during NO inhalation were 4.7E-3 (2.5E-3-5.8E-3) and 7.8E-3 (4.1E-3-13.0E-3), respectively. Levels of SNO-Hb continued to climb up to the last study time point (30 min: 10.6E-3 (5.3E-3-15.5E-3)). The response to acetylcholine iontophoresis both before and during NO inhalation was inversely associated with the SNO-Hb level (r: -0.57, p = 0.03, and r: -0.54, p = 0.04, respectively). Both nitrite and SNO-Hb increase during NO inhalation. Nitrite increases first, followed by a more sustained increase in Hb-SNO. Nitrite and Hb-SNO could be a mobile reservoir of NO with potential implications on the systemic microvasculature.

It is proposed that the bond between nitric oxide (NO) and the Hb thiol Cys-β 93 (SNOHb) is favored when hemoglobin (Hb) is in the relaxed (R, oxygenated) conformation, and that deoxygenation to tense (T) state destabilizes the SNOHb bond, allowing transfer of NO from Hb to form other (vasoactive) S -nitrosothiols (SNOs). However, it has not previously been possible to measure SNOHb without extensive Hb preparation, altering its allostery and SNO distribution. Here, we have validated an assay for SNOHb that uses carbon monoxide (CO) and cuprous chloride (CuCl)-saturated Cys. This assay is specific for SNOs and sensitive to 2–5 pmol. Uniquely, it measures the total SNO content of unmodified erythrocytes (RBCs) (SNO RBC ), preserving Hb allostery. In room air, the ratio of SNO RBC to Hb in intact RBCs is stable over time, but there is a logarithmic loss of SNO RBC with oxyHb desaturation (slope, 0.043). This decay is accelerated by extraerythrocytic thiol (slope, 0.089; P < 0.001). SNO RBC stability is uncoupled from O 2 tension when Hb is locked in the R state by CO pretreatment. Also, SNO RBC is increased ≈20-fold in human septic shock ( P = 0.002) and the O 2 -dependent vasoactivity of RBCs is affected profoundly by SNO content in a murine lung bioassay. These data demonstrate that SNO content and O 2 saturation are tightly coupled in intact RBCs and that this coupling is likely to be of pathophysiological significance. sepsis nitric oxide vascular physiology

Nitric oxide (NO) has long been recognized to affect muscle contraction, both through activation of guanylyl cyclase and through modification of cysteines in proteins to yield S-nitrosothiols. While NO affects the contractile apparatus directly, the identities of the target myofibrillar proteins remain unknown. Here we report that nitrogen oxides directly regulate striated muscle myosins. Exposure of skeletal and cardiac myosins to physiological concentrations of nitrogen oxides, including the endogenous nitrosothiol S-nitroso-L-cysteine, reduced the velocity of actin filaments over myosin in a dose-dependent and oxygen-dependent manner, caused a doubling of force as measured in a laser trap transducer, and caused S-nitrosylation of cysteines in the myosin heavy chain. These biomechanical effects were not observed in response to S-nitroso-D-cysteine, demonstrating specificity for the naturally occurring isomer. Both myosin heavy chain isoforms in rats and cardiac myosin heavy chain from human were S-nitrosylated in vivo. These data show that nitrosylation signaling acts as a molecular \"gear shift\" for myosin--an altogether novel mechanism by which striated muscle and cellular biomechanics may be regulated.