PEGylated Polyester Nanoparticles Trigger Adverse Events in a Large Animal Model of Trauma and in Naïve Animals: Understanding Cytokine and Cellular Correlations with These Events.

Intravenously infusible nanoparticles to control bleeding have shown promise in rodents, but translation into preclinical models has been challenging as many of these nanoparticle approaches have resulted in infusion responses and adverse outcomes in large animal trauma models. We developed a hemostatic nanoparticle technology that was screened to avoid one component of the infusion response: complement activation. We administered these hemostatic nanoparticles, control nanoparticles, or saline volume controls in a porcine polytrauma model. While the hemostatic nanoparticles promoted clotting as marked by a decrease in prothrombin time and both the hemostatic nanoparticles and controls did not active complement, in a subset of the animals, hard thrombi were found in uninjured tissues in both the hemostatic and control nanoparticle groups. Using data science methods that allow one to work across heterogeneous data sets, we found that the presence of these thrombi correlated with changes in IL-6, INF-alpha, lymphocytes, and neutrophils. While these findings might suggest that this formulation would not be a safe one for translation for trauma, they provide guidance for developing screening tools to make nanoparticle formulations in the complex milieux of trauma as well as for therapeutic interventions more broadly. This is important as we look to translate intravenously administered nanoparticle formulations for therapies, particularly considering the vascular changes seen in a subset of patients following COVID-19. We need to understand adverse events like thrombi more completely and screen for these events early to make nanomaterials as safe and effective as possible.

Evaluation of prolonged 'Permissive Hypotension': results from a 6-hour hemorrhage protocol in swine.

BACKGROUND: Tactical Combat Casualty Care guidelines for hemorrhage recommend resuscitation to systolic blood pressure (SBP) of 85±5 mm Hg during prehospital care. Success depends on transport to definitive care within the 'golden hour'. As future conflicts may demand longer prehospital/transport times, we sought to determine safety of prolonged permissive hypotension (PH). METHODS: Adult male swine were randomized into three experimental groups. Non-shock (NS)/normotensive underwent anesthesia only. NS/PH was bled to SBP of 85±5 mm Hg for 6 hours of prolonged field care (PFC) with SBP maintained via crystalloid, then recovered. Experimental group underwent controlled hemorrhage to mean arterial pressure 30 mm Hg until decompensation (Decomp/PH), followed by 6 hours of PFC. Hemorrhaged animals were then resuscitated with whole blood and observed for 24 hours. Physiologic variables, blood, tissue samples, and neurologic scores were collected. RESULTS: Survival of all groups was 100%. Fluid volumes to maintain targeted SBP in PFC were significantly higher in the hemorrhage group than sham groups. After 24 hours' recovery, no significant differences were observed in neurologic scores or cerebrospinal fluid markers of brain injury. No significant changes in organ function related to treatment were observed during PFC through recovery, as assessed by serum chemistry and histological analysis. CONCLUSIONS: After 6 hours, a prolonged PH strategy showed no detrimental effect on survival or neurologic outcome despite the increased ischemic burden of hemorrhage. Significant fluid volume was required to maintain SBP-a potential logistic burden for prehospital care. Further work to define maximum allowable time of PH is needed. STUDY TYPE: Translational animal model. LEVEL OF EVIDENCE: N/A.

Prehospital whole blood resuscitation prevents coagulopathy and improves acid-base status at hospital arrival in a nonhuman primate hemorrhagic shock model.

BACKGROUND: Hemorrhage remains the primary cause of preventable death in civilian and military trauma. The Committee on Tactical Combat Casualty Care recommends prehospital (PH) resuscitation with whole blood (WB). However, 6% hetastarch in lactated electrolyte (HEX) and crystalloids are more commonly available and used for PH resuscitation in military and civilian environments, respectively. The mechanistic benefits of PH WB resuscitation have not been well studied and remain to be elucidated. STUDY DESIGN AND METHODS: The aim of this study was to evaluate the differences in simulated PH WB and HEX resuscitation, specifically with regards to coagulation, physiologic, and metabolic outcomes to better elucidate the mechanistic benefits of WB. In a randomized study, the physiologic, coagulation, and metabolic responses to simulated PH WB (n = 12) or HEX (n = 12) were evaluated in a nonhuman primate model of severe polytraumatic hemorrhagic shock. RESULTS: Notable findings included 1) equivalence of shock reversal between simulated PH WB and HEX treatment groups as determined by hemodynamics and base deficit and 2) prevention of coagulopathy at simulated hospital arrival with initial WB resuscitation as determined by viscoelastic and plasmatic coagulation assays. CONCLUSION: The major benefit of WB, as compared to HEX, in simulated PH resuscitation appears to be prevention of coagulopathy at hospital arrival. Both fluids effectively reversed shock in this model, implying that efficacious provision preload (cardiac output support and hence oxygen delivery) and coagulation proteins (prevention of coagulopathy) are mechanisms underlying WB's effectiveness in early resuscitation of hemorrhagic shock.

Prehospital adenosine, lidocaine, and magnesium has inferior survival compared with tactical combat casualty care resuscitation in a porcine model of prolonged hemorrhagic shock.

BACKGROUND: Adenosine, lidocaine, and magnesium (ALM) is a cardioplegic agent shown to improve survival by improving cardiac function, tissue perfusion, and coagulopathy in animal models of shock. We hypothesized prehospital ALM treatment in hemorrhagic shock would improve survival compared to current Tactical Combat Casualty Care (TCCC) resuscitation beyond the golden hour. METHODS: Swine were randomized to: (1) TCCC, (2) 2 mL·kg vehicle control (VC), (3) 2 mL·kg ALM + drip, (4) 4 mL·kg ALM + drip, 5) 4 mL·kg ALM + delayed drip at 0.5 mL·kg·h, 6) 4 mL/kg VC, 7) 4 mL·kg ALM for 15 minutes + delayed drip at 3 mL·kg·h. Animals underwent pressure controlled hemorrhage to mean arterial pressure (MAP) of 30 mm Hg (S = 0). Treatment was administered at T = 0. After 120 minutes of simulated prehospital care (T = 120) blood product resuscitation commenced. Physiologic variables were recorded and laboratories were drawn at specified time points. RESULTS: Tactical Combat Casualty Care demonstrated superior survival to all other agents. The VC and ALM groups had lower MAPs and systolic blood pressures compared with TCCC. Except for the VC groups, lactate levels remained similar with correction of base deficit after prehospital resuscitation in all groups. Kidney function and liver function remained comparable across all groups. Compared with baseline values, TCCC demonstrated significant hypocoagulability. CONCLUSION: Adenosine, lidocaine, and magnesium, as administered in this study, are inferior to current Hextend-based resuscitation for survival from prolonged hemorrhagic shock in this model. In survivors, ALM groups had lower systolic blood pressures and MAPs, but provided a protective effect on coagulopathy as compared to TCCC. Adenosine, lidocaine, and magnesium do not appear to be a suitable low volume replacement to current TCCC resuscitation. The reduced coagulopathy compared to TCCC warrants future studies of ALM, perhaps as a therapeutic adjunct.

Whole blood mitigates the acute coagulopathy of trauma and avoids the coagulopathy of crystalloid resuscitation.

INTRODUCTION: The contributions of type and timing of fluid resuscitation to coagulopathy in trauma remain controversial. As part of a multifunctional resuscitation fluid research effort, we sought to further characterize the coagulation responses to resuscitation, specifically as compared to whole blood. We hypothesized that early whole blood administration mitigates the acute coagulopathy of trauma by avoiding the coagulopathy of CR resuscitation. METHODS: Anesthetized rhesus macaques underwent polytraumatic, hemorrhagic shock, then a crossover study design resuscitation (n = 6 each) with either whole blood first (WB-1st) followed by crystalloid (CR); or CR-1st followed by WB. Resuscitation strategies were the following: WB-1st received 50% shed blood in 30minutes, followed by twice the shed blood volume (SBV) of CR over 30minutes and one times the SBV CR over 60minutes, where CR-1st received twice the SBV of CR over 30minutes, followed by 50% of shed blood in 30minutes, and one times the SBV CR over 60minutes. Blood samples were collected at baseline, end-of-shock, end-of-first and end-of-second resuscitation stages, and end-of-resuscitation for assessment (thromboelastometry, platelet aggregation, and plasmatic coagulation factors). Statistical analyses were conducted using two-way analysis of variance ANOVA with Bonferroni correction and t-tests; significance was at p < 0.05. RESULTS: Survival, blood loss, hemodynamics, and shock duration were equivalent between the groups. Compared to baseline, parameters measured at first and second resuscitation stage time points directly following CR infusion revealed abnormalities in thromboelastometry (clot formation time, α angle, and maximum clot firmness), platelet aggregation response (to collagen, arachidonic acid, and adenosine diphosphate), and plasmatic coagulation (prothrombin time, anti-thrombin 3, and fibrinogen), while whole blood infusion resulted in stabilization or correction of these parameters following its administration. CONCLUSIONS: These data suggest that in the setting of trauma and hemorrhagic shock, the coagulation alterations begin before intervention/resuscitation; however, these are significantly aggravated by CR resuscitation and could perhaps be best termed acute coagulopathy of resuscitation. STUDY TYPE: Translational animal model.

Whole blood and Hextend: Bookends of modern tactical combat casualty care field resuscitation and starting point for multifunctional resuscitation fluid development.

BACKGROUND: Hemorrhage is the leading cause of preventable death in traumatically injured civilian and military populations. Prehospital resuscitation largely relies on crystalloid and colloid intravascular expansion, as whole blood and component blood therapy are logistically arduous. In this experiment, we evaluated the bookends of Tactical Combat Casualty Care Guidelines recommendations of prehospital resuscitation with Hextend and whole blood in a controlled hemorrhagic shock model within non-human primates, as means of a multifunctional resuscitative fluid development. METHODS: In the nonhuman primate, a multiple injuries model was used, consisting of a musculoskeletal injury (femur fracture), soft tissue injury (15-cm laparotomy), and controlled hemorrhage to a mean arterial pressure of 20 mm Hg, demarcating the beginning of the shock period. Animals were randomized to prehospital interventions of whole blood or Hextend at T = 0 minutes, and at T = 90 minutes definitive surgical interventions and balanced sanguineous damage control resuscitation could be implemented. All animals were euthanized at T = 480 minutes. Data are expressed as mean ± SEM; significance, p < 0.05. RESULTS: No significant differences in survival (83% vs. 100%; p = 0.3), tissue perfusion (EtCO2 and StO2) or endpoints of resuscitation (base deficit, lactate, pH) between Hextend and whole blood were identified. Second, whole blood compared with Hextend demonstrated significantly earlier normalization of clot formation time, maximal clot firmness, and α angle. CONCLUSION: A future multifunctional resuscitative fluid including an asanguineous, oncotic, non-oxygen-carrying component to facilitate intravascular volume expansion, and a component with synthetic coagulation factors and fibrinogen to deter coagulopathy may show equivalence to whole blood. LEVEL OF EVIDENCE: N/A: Study type: translational animal model.

All animals are equal but some animals are more equal than others: Plasma lactate and succinate in hemorrhagic shock-A comparison in rodents, swine, nonhuman primates, and injured patients.

BACKGROUND: Plasma levels of lactate and succinate are predictors of mortality in critically injured patients in military and civilian settings. In relative terms, these metabolic derangements have been recapitulated in rodent, swine, and nonhuman primate models of severe hemorrhage. However, no direct absolute quantitative comparison has been evaluated across these species. METHODS: Ultra-high pressure liquid chromatography-mass spectrometry with stable isotope standards was used to determine absolute concentrations of baseline and postshock levels of lactate and succinate in rats, pigs, macaques, and injured patients. RESULTS: Baseline levels of lactate and succinate were most comparable to humans in macaques, followed by pigs and rats. Baseline levels of lactate in pigs and baseline and postshock levels of lactate and succinate in rats were significantly higher than those measured in macaques and humans. Postshock levels of lactate and succinate in pigs and macaques, respectively, were directly comparable to measurements in critically injured patients. CONCLUSION: Acknowledging the caveats associated with the variable degrees of shock in the clinical cohort, our data indicate that larger mammals represent a better model than rodents when investigating metabolic derangements secondary to severe hemorrhage.

Nonhuman primate model of polytraumatic hemorrhagic shock recapitulates early platelet dysfunction observed following severe injury in humans.

BACKGROUND: Platelet dysfunction has been described as an early component of trauma-induced coagulopathy. The platelet component of trauma-induced coagulopathy remains to be fully elucidated and translatable animal models are required to facilitate mechanistic investigations. We sought to determine if the early platelet dysfunction described in trauma patients could be recapitulated in a nonhuman primate model of polytraumatic hemorrhagic shock. METHODS: Twenty-four male rhesus macaques weighting 7 to 14 kg were subjected to 60 minutes (min) of severe pressure-targeted controlled hemorrhagic shock (HS) with and without other injuries. After 60 min, resuscitation with 0.9% NaCl and whole blood was initiated. Platelet counts and platelet aggregation assays were performed at baseline (BSLN), end of shock (EOS; T = 60 min), end of resuscitation (EOR; T = 180 min), and T = 360 min on overall cohort. Results are reported as mean ± standard deviation (SD) or median (interquartile range). Statistical analysis was conducted using Spearmen correlation, one-way analysis of variance, two-way repeated-measures analysis of variance, paired t-test or Wilcoxon nonparametric test, with p < 0.05 considered significant. RESULTS: Platelet count in all injury cohorts decreased over time, but no animals developed thrombocytopenia. Correlations were observed between platelet aggregation and platelet count for all agonists: adenosine diphosphate, thrombin recognition-activating peptide-6, collagen, and arachidonic acid. Overall, compared to BSLN, platelet aggregation decreased for all agonist at EOS, EOR, and T = 360 min. When normalized to platelet count, platelet aggregation in response to agonist thrombin recognition-activating peptide-6 demonstrated no change from BSLN at subsequent time points. Aggregation to adenosine diphosphate was significantly less at EOR but not EOS or T = 360 min compared to BSLN. Platelet aggregation to collagen and arachidonic acid was not significantly different at EOS compared to BSLN but was significantly less at EOR and T = 360 min. CONCLUSION: Nonhuman primates manifest early platelet dysfunction in response to polytraumatic hemorrhagic shock, consistent with that reported in severely injured human patients. Nonhuman primate models potentially are translationally valuable for understanding the mechanisms and pathophysiology of trauma-induced platelet dysfunction.

Severe Hemorrhagic Shock Induces Acute Activation and Expansion of IL-8+/IL-10+ Neutrophils with Enhanced Oxidative Reactivity in Non-Human Primates.

BACKGROUND: Neutrophilic inflammation is a mediator of morbidity and mortality in response to hemorrhagic shock. Although injury-induced neutrophil margination has long been observed, the nature of neutrophils' role in the "second hit" paradigm remains to be fully elucidated. We sought to extensively characterize neutrophil phenotype and functionality in response to severe hemorrhage in non-human primates (NHPs). METHODS: NHPs (n = 8) were subjected to severe hemorrhagic shock and resuscitation. Blood was obtained at baseline (T = 0 min), end of shock (T = 60 min), end of resuscitation (T = 180 min), T = 360 min, and 24 h (T = 1440 min). Neutrophils were quantified by complete blood count and flow cytometry. IL-8 and IL-10 production was determined by intracellular flow cytometry. Oxidation of dihydrorhodamine-123 (DHR-123) was used to determine neutrophil oxidative bursts (untreated), priming (+fMLP), and burst capacity (+PMA/ionomycin) via microplate reader ex vivo. Data are reported as mean ±â€ŠSEM; statistical significance was measured using repeated measures ANOVA with Bonferroni adjustment. P < 0.05 is considered significant. RESULTS: CD45CD11bCD16 neutrophils doubled postinjury (P < 0.0001); this was due to activated IL-8/IL-10 neutrophils that increased in frequency in relation to resting IL-8IL-10 cells. At 24 h, the proportions of activated to resting neutrophils returned to baseline levels. Resuscitative measures initially decreased neutrophil oxidative output; however, oxidative bursts, priming, and burst capacity were significantly increased at 24 h (P < 0.0025, 0.0124, and 0.0118, respectively). CONCLUSION: These results demonstrate an acute expansion and phenotypic activation of circulating neutrophils postinjury followed by a return to homeostatic proportions within 24 h; paradoxically, phenotypically "resting" neutrophils at 24 h have significantly higher oxidative potential, predisposing for exaggerated inflammatory responses. These data are consistent with clinical literature and provide important functional insight into neutrophil-mediated shock pathology.

Rapid Detection of Neutrophil Oxidative Burst Capacity is Predictive of Whole Blood Cytokine Responses.

BACKGROUND: Maladaptive immune responses, particularly cytokine and chemokine-driven, are a significant contributor to the deleterious inflammation present in many types of injury and infection. Widely available applications to rapidly assess individual inflammatory capacity could permit identification of patients at risk for exacerbated immune responses and guide therapy. Here we evaluate neutrophil oxidative burst (NOX) capacity measured by plate reader to immuno-type Rhesus Macaques as an acute strategy to rapidly detect inflammatory capacity and predict maladaptive immune responses as assayed by cytokine array. METHODS: Whole blood was collected from anesthetized Rhesus Macaques (n = 25) and analyzed for plasma cytokine secretion (23-plex Luminex assay) and NOX capacity. For cytokine secretion, paired samples were either unstimulated or ex-vivo lipopolysaccharide (LPS)-stimulated (100µg/mL/24h). NOX capacity was measured in dihydrorhodamine-123 loaded samples following phorbol 12-myristate 13-acetate (PMA)/ionomycin treatment. Pearson's test was utilized to correlate NOX capacity with cytokine secretion, p<0.05 considered significant. RESULTS: LPS stimulation induced secretion of the inflammatory molecules G-CSF, IL-1ß, IL-1RA, IL-6, IL-10, IL-12/23(p40), IL-18, MIP-1α, MIP-1ß, and TNFα. Although values were variable, several cytokines correlated with NOX capacity, p-values≤0.0001. Specifically, IL-1ß (r = 0.66), IL-6 (r = 0.74), the Th1-polarizing cytokine IL-12/23(p40) (r = 0.78), and TNFα (r = 0.76) were strongly associated with NOX. CONCLUSION: NOX capacity correlated with Th1-polarizing cytokine secretion, indicating its ability to rapidly predict inflammatory responses. These data suggest that NOX capacity may quickly identify patients at risk for maladaptive immune responses and who may benefit from immuno-modulatory therapies. Future studies will assess the in-vivo predictive value of NOX in animal models of immune-mediated pathologies.

Highly active combination of BRD4 antagonist and histone deacetylase inhibitor against human acute myelogenous leukemia cells.

The bromodomain and extra-terminal (BET) protein family members, including BRD4, bind to acetylated lysines on histones and regulate the expression of important oncogenes, for example, c-MYC and BCL2. Here, we demonstrate the sensitizing effects of the histone hyperacetylation-inducing pan-histone deacetylase (HDAC) inhibitor panobinostat on human acute myelogenous leukemia (AML) blast progenitor cells (BPC) to the BET protein antagonist JQ1. Treatment with JQ1, but not its inactive enantiomer (R-JQ1), was highly lethal against AML BPCs expressing mutant NPM1c+ with or without coexpression of FLT3-ITD or AML expressing mixed lineage leukemia fusion oncoprotein. JQ1 treatment reduced binding of BRD4 and RNA polymerase II to the DNA of c-MYC and BCL2 and reduced their levels in the AML cells. Cotreatment with JQ1 and the HDAC inhibitor panobinostat synergistically induced apoptosis of the AML BPCs, but not of normal CD34(+) hematopoietic progenitor cells. This was associated with greater attenuation of c-MYC and BCL2, while increasing p21, BIM, and cleaved PARP levels in the AML BPCs. Cotreatment with JQ1 and panobinostat significantly improved the survival of the NOD/SCID mice engrafted with OCI-AML3 or MOLM13 cells (P < 0.01). These findings highlight cotreatment with a BRD4 antagonist and an HDAC inhibitor as a potentially efficacious therapy of AML.

Thermal unfolding of the N-terminal region of p53 monitored by circular dichroism spectroscopy.

It has been estimated that 30% of eukaryotic protein and 70% of transcription factors are intrinsically disordered (ID). The biochemical significance of proteins that lack stable tertiary structure, however, is not clearly understood, largely owing to an inability to assign well-defined structures to specific biological tasks. In an attempt to investigate the structural character of ID protein, we have measured the circular dichroism spectrum of the N-terminal region of p53 over a range of temperatures and solution conditions. p53 is a well-studied transcription factor that has a proline-rich N-terminal ID region containing two activation domains. High proline content is a property commonly associated with ID, and thus p53 may be a good model system for investigating the biochemical importance of ID. The spectra presented here suggest that the N-terminal region of p53 may adopt an ordered structure under physiological conditions and that this structure can be thermally unfolded in an apparent two-state manner. The midpoint temperature for this thermal unfolding of the N-terminal region of p53 was at the near-physiological temperature of 39°C, suggesting the possibility of a physiological role for the observed structural equilibrium.