The Polyanionic Drug Suramin Neutralizes Histones and Prevents Endotheliopathy.

Drugs are needed to protect against the neutrophil-derived histones responsible for endothelial injury in acute inflammatory conditions such as trauma and sepsis. Heparin and other polyanions can neutralize histones but challenges with dosing or side effects such as bleeding limit clinical application. In this study, we demonstrate that suramin, a widely available polyanionic drug, completely neutralizes the toxic effects of individual histones, but not citrullinated histones from neutrophil extracellular traps. The sulfate groups on suramin form stable electrostatic interactions with hydrogen bonds in the histone octamer with a dissociation constant of 250 nM. In cultured endothelial cells (Ea.Hy926), histone-induced thrombin generation was significantly decreased by suramin. In isolated murine blood vessels, suramin abolished aberrant endothelial cell calcium signals and rescued impaired endothelial-dependent vasodilation caused by histones. Suramin significantly decreased pulmonary endothelial cell ICAM-1 expression and neutrophil recruitment caused by infusion of sublethal doses of histones in vivo. Suramin also prevented histone-induced lung endothelial cell cytotoxicity in vitro and lung edema, intra-alveolar hemorrhage, and mortality in mice receiving a lethal dose of histones. Protection of vascular endothelial function from histone-induced damage is a novel mechanism of action for suramin with therapeutic implications for conditions characterized by elevated histone levels.

Lung infection by Pseudomonas aeruginosa induces neuroinflammation and blood-brain barrier dysfunction in mice.

BACKGROUND: Severe lung infection can lead to brain dysfunction and neurobehavioral disorders. The mechanisms that regulate the lung-brain axis of inflammatory response to respiratory infection are incompletely understood. This study examined the effects of lung infection causing systemic and neuroinflammation as a potential mechanism contributing to blood-brain barrier (BBB) leakage and behavioral impairment. METHODS: Lung infection in mice was induced by instilling Pseudomonas aeruginosa (PA) intratracheally. We determined bacterial colonization in tissue, microvascular leakage, expression of cytokines and leukocyte infiltration into the brain. RESULTS: Lung infection caused alveolar-capillary barrier injury as indicated by leakage of plasma proteins across pulmonary microvessels and histopathological characteristics of pulmonary edema (alveolar wall thickening, microvessel congestion, and neutrophil infiltration). PA also caused significant BBB dysfunction characterized by leakage of different sized molecules across cerebral microvessels and a decreased expression of cell-cell junctions (VE-cadherin, claudin-5) in the brain. BBB leakage peaked at 24 h and lasted for 7 days post-inoculation. Additionally, mice with lung infection displayed hyperlocomotion and anxiety-like behaviors. To test whether cerebral dysfunction was caused by PA directly or indirectly, we measured bacterial load in multiple organs. While PA loads were detected in the lungs up to 7 days post-inoculation, bacteria were not detected in the brain as evidenced by negative cerebral spinal fluid (CSF) cultures and lack of distribution in different brain regions or isolated cerebral microvessels. However, mice with PA lung infection demonstrated increased mRNA expression in the brain of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α), chemokines (CXCL-1, CXCL-2) and adhesion molecules (VCAM-1 and ICAM-1) along with CD11b + CD45+ cell recruitment, corresponding to their increased blood levels of white cells (polymorphonuclear cells) and cytokines. To confirm the direct effect of cytokines on endothelial permeability, we measured cell-cell adhesive barrier resistance and junction morphology in mouse brain microvascular endothelial cell monolayers, where administration of IL-1ß induced a significant reduction of barrier function coupled with tight junction (TJ) and adherens junction (AJ) diffusion and disorganization. Combined treatment with IL-1ß and TNFα augmented the barrier injury. CONCLUSIONS: Lung bacterial infection is associated with BBB disruption and behavioral changes, which are mediated by systemic cytokine release.

Lung infection by P. aeruginosa induces neuroinflammation and blood-brain barrier dysfunction in mice.

Background: Severe lung infection can lead to brain dysfunction and neurobehavioral disorders. The mechanisms that regulate the lung-brain axis of inflammatory response to respiratory infection are incompletely understood. This study examined the effects of lung infection causing systemic and neuroinflammation as a potential mechanism contributing to blood-brain barrier (BBB) leakage and behavioral impairment. Methods: Pneumonia was induced in adult C57BL/6 mice by intratracheal inoculation of Pseudomonas aeruginosa (PA). Solute extravasation, histology, immunofluorescence, RT-PCR, multiphoton imaging and neurological testing were performed in this study. Results: Lung infection caused alveolar-capillary barrier injury as indicated by leakage of plasma proteins across pulmonary microvessels and histopathological characteristics of pulmonary edema (alveolar wall thickening, microvessel congestion, and neutrophil infiltration). PA also caused significant BBB dysfunction characterized by leakage of different sized molecules across cerebral microvessels and a decreased expression of cell-cell junctions (VE-cadherin, claudin-5) in the brain. BBB leakage peaked at 24 hours and lasted for 7 days post-inoculation. Additionally, mice with lung infection displayed hyperlocomotion and anxiety-like behaviors. To test whether cerebral dysfunction was caused by PA directly or indirectly, we measured bacterial load in multiple organs. While PA loads were detected in the lungs up to 7 days post-inoculation, bacteria were not detected in the brain as evidenced by negative cerebral spinal fluid (CSF) cultures and lack of distribution in different brain regions or isolated cerebral microvessels. However, mice with PA lung infection demonstrated increased mRNA expression in the brain of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α), chemokines (CXCL-1, CXCL-2) and adhesion molecules (VCAM-1 and ICAM-1) along with CD11b+ cell recruitment, corresponding to their increased blood levels of white cells (polymorphonuclear cells) and cytokines. To confirm the direct effect of cytokines on endothelial permeability, we measured cell-cell adhesive barrier resistance and junction morphology in mouse brain microvascular endothelial cell monolayers, where administration of IL-1ß induced a significant reduction of barrier function coupled with tight junction (TJ) diffusion and disorganization. Combined treatment with IL-1ß and TNFα augmented the barrier injury. Conclusions: These results suggest that lung bacterial infection causes cerebral microvascular leakage and neuroinflammation via a mechanism involving cytokine-induced BBB injury.

Lung infection by P. aeruginosa induces neuroinflammation and blood-brain barrier dysfunction in mice.

Background Severe lung infection can lead to brain dysfunction and neurobehavioral disorders. The mechanisms that regulate the lung-brain axis of inflammatory response to respiratory infection are incompletely understood. This study examined the effects of lung infection causing systemic and neuroinflammation as a potential mechanism contributing to blood-brain barrier (BBB) leakage and behavioral impairment. Methods Pneumonia was induced in adult C57BL/6 mice by intratracheal inoculation of Pseudomonas aeruginosa (PA). Solute extravasation, histology, immunofluorescence, RT-PCR, multiphoton imaging and neurological testing were performed in this study. Results Lung infection caused alveolar-capillary barrier injury as indicated by leakage of plasma proteins across pulmonary microvessels and histopathological characteristics of pulmonary edema (alveolar wall thickening, microvessel congestion, and neutrophil infiltration). PA also caused significant BBB dysfunction characterized by leakage of different sized molecules across cerebral microvessels and a decreased expression of cell-cell junctions (VE-cadherin, claudin-5) in the brain. BBB leakage peaked at 24 hours and lasted for 7 days post-inoculation. Additionally, mice with lung infection displayed hyperlocomotion and anxiety-like behaviors. To test whether cerebral dysfunction was caused by PA directly or indirectly, we measured bacterial load in multiple organs. While PA loads were detected in the lungs up to 7 days post-inoculation, bacteria were not detected in the brain as evidenced by negative cerebral spinal fluid (CSF) cultures and lack of distribution in different brain regions or isolated cerebral microvessels. However, mice with PA lung infection demonstrated increased mRNA expression in the brain of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α), chemokines (CXCL-1, CXCL-2) and adhesion molecules (VCAM-1 and ICAM-1) along with CD11b + cell recruitment, corresponding to their increased blood levels of white cells (polymorphonuclear cells) and cytokines. To confirm the direct effect of cytokines on endothelial permeability, we measured cell-cell adhesive barrier resistance and junction morphology in mouse brain microvascular endothelial cell monolayers, where administration of IL-1ß induced a significant reduction of barrier function coupled with tight junction (TJ) diffusion and disorganization. Combined treatment with IL-1ß and TNFα augmented the barrier injury. Conclusions These results suggest that lung bacterial infection causes cerebral microvascular leakage and neuroinflammation via a mechanism involving cytokine-induced BBB injury.

Endothelial glycocalyx-associated molecules as potential serological markers for sepsis-associated encephalopathy: A systematic review and meta-analysis.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is characterized by a diffuse cerebral dysfunction that accompanies sepsis in the absence of direct central nervous system infection. The endothelial glycocalyx is a dynamic mesh containing heparan sulfate linked to proteoglycans and glycoproteins, including selectins and vascular/intercellular adhesion molecules (V/I-CAMs), which protects the endothelium while mediating mechano-signal transduction between the blood and vascular wall. During severe inflammatory states, components of the glycocalyx are shed into the circulation and can be detected in soluble forms. Currently, SAE remains a diagnosis of exclusion and limited information is available on the utility of glycocalyx-associated molecules as biomarkers for SAE. We set out to synthesize all available evidence on the association between circulating molecules released from the endothelial glycocalyx surface during sepsis and sepsis-associated encephalopathy. METHODS: MEDLINE (PubMed) and EMBASE were searched since inception until May 2, 2022 to identify eligible studies. Any comparative observational study: i) evaluating the association between sepsis and cognitive decline and ii) providing information on level of circulating glycocalyx-associated molecules was eligible for inclusion. RESULTS: Four case-control studies with 160 patients met the inclusion criteria. Meta-analysis of biomarkers ICAM-1 (SMD 0.41; 95% CI 0.05-0.76; p = 0.03; I2 = 50%) and VCAM-1 (SMD 0.55; 95% CI 0.12-0.98; p = 0.01; I2 = 82%) revealed higher pooled mean concentration in patients with SAE compared to the patients with sepsis alone. Single studies reported elevated levels of P-selectin (MD 0.80; 95% CI -17.77-19.37), E-selectin (MD 96.40; 95% Cl 37.90-154.90), heparan sulfate NS2S (MD 19.41; 95% CI 13.37-25.46), and heparan sulfate NS+NS2S+NS6S (MD 67.00; 95% CI 31.00-103.00) in patients with SAE compared to the patients with sepsis alone. CONCLUSION: Plasma glycocalyx-associated molecules are elevated in SAE and may be useful for early identification of cognitive decline in sepsis patients.

Burn Injury-Induced Extracellular Vesicle Production and Characteristics.

ABSTRACT: Extracellular vesicles (EVs) are nano-sized membrane-bound particles containing biologically active cargo molecules. The production and molecular composition of EVs reflect the physiological state of parent cells, and once released into the circulation, they exert pleiotropic functions via transferring cargo contents. Thus, circulating EVs not only serve as biomarkers, but also mediators in disease processes or injury responses. In the present study, we performed a comprehensive analysis of plasma EVs from burn patients and healthy subjects, characterizing their size distribution, concentration, temporal changes, cell origins, and cargo protein contents. Our results indicated that burn injury induced a significant increase in circulating EVs, the response peaked at the time of admission and declined over the course of recovery. Importantly, EV production correlated with injury severity, as indicated by the total body surface area and depth of burn, requirement for critical care/ICU stay, hospitalization length, wound infection, and concurrence of sepsis. Burn patients with inhalation injury showed a higher level of EVs than those without inhalation injury. We also evaluated patient demographics (age and sex) and pre-existing conditions (hypertension, obesity, and smoking) and found no significant correlation between these conditions and overall EV production. At the molecular level, flow cytometric analysis showed that the burn-induced EVs were largely derived from leukocytes and endothelial cells (ECs), which are known to be activated postburn. Additionally, a high level of zona-occludens-1 (ZO-1), a major constituent of tight junctions, was identified in burn EV cargos, indicative of injury in tissues that form barriers via tight junctions. Moreover, when applied to endothelial cell monolayers, burn EVs caused significant barrier dysfunction, characterized by decreased transcellular barrier resistance and disrupted cell-cell junction continuity. Taken together, these data suggest that burn injury promotes the production of EVs containing unique cargo proteins in a time-dependent manner; the response correlates with injury severity and worsened clinical outcomes. Functionally, burn EVs serve as a potent mediator capable of reducing endothelial barrier resistance and impairing junction integrity, a pathophysiological process underlying burn-associated tissue dysfunction. Thus, further in-depth characterization of circulating EVs will contribute to the development of new prognostic tools or therapeutic targets for advanced burn care.

Circulating lymphocyte trafficking to the bone marrow contributes to lymphopenia in myocardial infarction.

Some patients with myocardial infarction (MI) exhibit lymphopenia, a reduction in blood lymphocyte count. Moreover, lymphopenia inversely correlates with patient prognosis. The objective of this study was to elucidate the underlying mechanisms that cause lymphopenia after MI. Multiparameter flow cytometric analysis demonstrated that MI induced profound B and T lymphopenia in a mouse model, peaking at day 1 post-MI. The finding that non-MI control and MI mice exhibited similar apoptotic rate for blood B and T lymphocytes argues against apoptosis being essential for MI-induced lymphopenia. Interestingly, the bone marrow in day 1 post-MI mice contained more B and T cells but showed less B- and T-cell proliferation compared with day 0 controls. This suggests that blood lymphocytes may travel to the bone marrow after MI. This was confirmed by adoptive transfer experiments demonstrating that MI caused the loss of transferred lymphocytes in the blood, but the accumulation of transferred lymphocytes in the bone marrow. To elucidate the underlying signaling pathways, ß2-adrenergic receptor or sphingosine-1-phosphate receptor type 1 (S1PR1) was pharmacologically blocked, respectively. ß2-receptor inhibition had no significant effect on blood lymphocyte count, whereas S1PR1 blockade aggravated lymphopenia in MI mice. Furthermore, we discovered that MI-induced glucocorticoid release triggered lymphopenia. This was supported by the findings that adrenalectomy (ADX) completely prevented mice from MI-induced lymphopenia, and supplementation with corticosterone in adrenalectomized MI mice reinduced lymphopenia. In conclusion, our study demonstrates that MI-associated lymphopenia involves lymphocyte redistribution from peripheral blood to the bone marrow, which is mediated by glucocorticoids.NEW & NOTEWORTHY Lymphopenia, a reduction in blood lymphocyte count, is known to inversely correlate with the prognosis for patients with myocardial infarction (MI). However, the underlying mechanisms by which cardiac ischemia induces lymphopenia remain elusive. This study provides the first evidence that MI activates the hypothalamic-pituitary-adrenal (HPA) axis to increase glucocorticoid secretion, and elevated circulating glucocorticoids induce blood lymphocytes trafficking to the bone marrow, leading to lymphopenia.

Protein palmitoylation regulates extracellular vesicle production and function in sepsis.

Extracellular vesicles (EVs) are bioactive membrane-encapsulated particles generated by a series of events involving membrane budding, fission and fusion. Palmitoylation, mediated by DHHC palmitoyl acyltransferases, is a lipidation reaction that increases protein lipophilicity and membrane localization. Here, we report palmitoylation as a novel regulator of EV formation and function during sepsis. Our results showed significantly decreased circulating EVs in mice with DHHC21 functional deficiency (Zdhhc21dep/dep), compared to wild-type (WT) mice 24 h after septic injury. Furthermore, WT and Zdhhc21dep/dep EVs displayed distinct palmitoyl-proteomic profiles. Ingenuity pathway analysis indicated that sepsis altered several inflammation related pathways expressed in EVs, among which the most significantly activated was the complement pathway; however, this sepsis-induced complement enrichment in EVs was greatly blunted in Zdhhc21dep/dep EVs. Functionally, EVs isolated from WT mice with sepsis promoted neutrophil adhesion, transmigration, and neutrophil extracellular trap production; these effects were significantly attenuated by DHHC21 loss-of-function. Furthermore, Zdhhc21dep/dep mice displayed reduced neutrophil infiltration in lungs and improved survival after CLP challenges. These findings indicate that blocking palmitoylation via DHHC21 functional deficiency can reduce sepsis-stimulated production of complement-enriched EVs and attenuates their effects on neutrophil activity.

Traumatic Brain Injury Impairs Systemic Vascular Function Through Disruption of Inward-Rectifier Potassium Channels.

Trauma can lead to widespread vascular dysfunction, but the underlying mechanisms remain largely unknown. Inward-rectifier potassium channels (Kir2.1) play a critical role in the dynamic regulation of regional perfusion and blood flow. Kir2.1 channel activity requires phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane phospholipid that is degraded by phospholipase A2 (PLA2) in conditions of oxidative stress or inflammation. We hypothesized that PLA2-induced depletion of PIP2 after trauma impairs Kir2.1 channel function. A fluid percussion injury model of traumatic brain injury (TBI) in rats was used to study mesenteric resistance arteries 24 hours after injury. The functional responses of intact arteries were assessed using pressure myography. We analyzed circulating PLA2, hydrogen peroxide (H2O2), and metabolites to identify alterations in signaling pathways associated with PIP2 in TBI. Electrophysiology analysis of freshly-isolated endothelial and smooth muscle cells revealed a significant reduction of Ba2+-sensitive Kir2.1 currents after TBI. Additionally, dilations to elevated extracellular potassium and BaCl2- or ML 133-induced constrictions in pressurized arteries were significantly decreased following TBI, consistent with an impairment of Kir2.1 channel function. The addition of a PIP2 analog to the patch pipette successfully rescued endothelial Kir2.1 currents after TBI. Both H2O2 and PLA2 activity were increased after injury. Metabolomics analysis demonstrated altered lipid metabolism signaling pathways, including increased arachidonic acid, and fatty acid mobilization after TBI. Our findings support a model in which increased H2O2-induced PLA2 activity after trauma hydrolyzes endothelial PIP2, resulting in impaired Kir2.1 channel function.

The Endothelial Glycocalyx as a Double-Edged Sword in Microvascular Homeostasis and Pathogenesis.

Expressed on the endothelial cell (EC) surface of blood vessels, the glycocalyx (GCX), a mixture of carbohydrates attached to proteins, regulates the access of cells and molecules in the blood to the endothelium. Besides protecting endothelial barrier integrity, the dynamic microstructure of the GCX confers remarkable functions including mechanotransduction and control of vascular tone. Recently, a novel perspective has emerged supporting the pleiotropic roles of the endothelial GCX (eGCX) in cardiovascular health and disease. Because eGCX degradation occurs in certain pathological states, the circulating levels of eGCX degradation products have been recognized to have diagnostic or prognostic values. Beyond their biomarker roles, certain eGCX fragments serve as pathogenic factors in disease progression. Pharmacological interventions that attenuate eGCX degradation or restore its integrity have been sought. This review provides our current understanding of eGCX structure and function across the microvasculature in different organs. We also discuss disease or injury states, such as infection, sepsis and trauma, where eGCX dysfunction contributes to severe inflammatory vasculopathy.

DHHC21 deficiency attenuates renal dysfunction during septic injury.

Renal dysfunction is one of the most common complications of septic injury. One critical contributor to septic injury-induced renal dysfunction is renal vascular dysfunction. Protein palmitoylation serves as a novel regulator of vascular function. Here, we examined whether palmitoyl acyltransferase (PAT)-DHHC21 contributes to septic injury-induced renal dysfunction through regulating renal hemodynamics. Multispectral optoacoustic imaging showed that cecal ligation and puncture (CLP)-induced septic injury caused impaired renal excretion, which was improved in DHHC21 functional deficient (Zdhhc21dep/dep) mice. DHHC21 deficiency attenuated CLP-induced renal pathology, characterized by tissue structural damage and circulating injury markers. Importantly, DHHC21 loss-of-function led to better-preserved renal perfusion and oxygen saturation after CLP. The CLP-caused reduction in renal blood flow was also ameliorated in Zdhhc21dep/dep mice. Next, CLP promoted the palmitoylation of vascular α1-adrenergic receptor (α1AR) and the activation of its downstream effector ERK, which were blunted in Zdhhc21dep/dep mice. Vasoreactivity analysis revealed that renal arteries from Zdhhc21dep/dep mice displayed reduced constriction response to α1AR agonist phenylephrine compared to those from wild-type mice. Consistently, inhibiting PATs with 2-bromopalmitate caused a blunted vasoconstriction response to phenylephrine in small arteries isolated from human kidneys. Therefore, DHHC21 contributes to impaired renal perfusion and function during septic injury via promoting α1AR palmitoylation-associated vasoconstriction.

Gut Ischemia Reperfusion Injury Induces Lung Inflammation via Mesenteric Lymph-Mediated Neutrophil Activation.

Gut ischemia/reperfusion (I/R) injury is a common clinical problem associated with significant mortality and morbidities that result from systemic inflammation and remote organ dysfunction, typically acute lung injury. The mechanisms underlying the dissemination of gut-derived harmful mediators into the circulation are poorly understood. The objective of our study was to determine the role of mesenteric lymphatic circulation in the systemic and pulmonary inflammatory response to gut I/R. Using a murine intestinal I/R model, we evaluated whether and how blocking mesenteric lymph flow affects the inflammatory response in local tissues (gut) and remote organs (lungs). We further explored the mechanisms of post-I/R lymph-induced systemic inflammation by examining neutrophil activity and interaction with endothelial cells in vitro. Mice subjected to intestinal I/R displayed a significant inflammatory response in local tissues, evidenced by neutrophil infiltration into mucosal areas, as well as lung inflammation, evidenced by increased myeloperoxidase levels, neutrophil infiltration, and elevated microvascular permeability in the lungs. Mesenteric lymph duct ligation (MLDL) had no effect on gut injury per se, but effectively attenuated lung injury following gut I/R. Cell experiments showed that lymph fluid from post-I/R animals, but not pre-I/R, increased neutrophil surface CD11b expression and their ability to migrate across vascular endothelial monolayers. Moreover, post-I/R lymph upregulated neutrophil expression of pro-inflammatory cytokines and chemokines, which was mediated by a mechanism involving nuclear factor (NF)-κB signaling. Consistently, gut I/R activated NF-κB in lung neutrophils, which was alleviated by MLDL. In conclusion, all these data indicate that mesenteric lymph circulation contributes to neutrophil activation and lung inflammation following gut I/R injury partly through activating NF-κB.

Site-specific opening of the blood-brain barrier by extracellular histones.

BACKGROUND: Increased extracellular histones in the bloodstream are known as a biomarker for vascular dysfunction associated with severe trauma or sepsis. There is limited information regarding the pathogenic role of circulating histones in neuroinflammation and cerebrovascular endothelial injury. Particularly, it remains unclear whether histones affect the blood-brain barrier (BBB) permeability function. METHODS: The direct effects of unfractionated histones on endothelial barrier properties were first assessed in brain microvascular endothelial cell monolayers by measuring transendothelial electrical resistance and solute flux. This was followed by in vivo mouse experiments, where BBB function was assessed by quantifying brain tissue accumulation of intravenously injected tracers of different molecular sizes, and comparison was made in mice receiving a sublethal dose of histones versus sterile saline. In parallel, the endothelial barrier ultrastructure was examined in histone- and saline-injected animals under transmission electron microscopy, corresponding to the expression of tight junction and adherens junction proteins. RESULTS: Histones increased paracellular permeability to sodium fluorescein and reduced barrier resistance at 100 µg/mL; these responses were accompanied by discontinuous staining of the tight junction proteins claudin-5 and zona ocludens-1. Interestingly, the effects of histones did not seem to result from cytotoxicity, as evidenced by negative propidium iodide staining. In vivo, histones increased the paracellular permeability of the BBB to small tracers of < 1-kDa, whereas tracers larger than 3-kDa remained impermeable across brain microvessels. Further analysis of different brain regions showed that histone-induced tracer leakage and loss of tight junction protein expression mainly occurred in the hippocampus, but not in the cerebral cortex. Consistently, opening of tight junctions was found in hippocampal capillaries from histone-injected animals. Protein expression levels of GFAP and iBA1 remained unchanged in histone-injected mice indicating that histones did not affect reactive gliosis. Moreover, cell membrane surface charge alterations are involved in histone-induced barrier dysfunction and tight junction disruption. CONCLUSIONS: Extracellular histones cause a reversible, region-specific increase in BBB permeability to small molecules by disrupting tight junctions in the hippocampus. We suggest that circulating histones may contribute to cerebrovascular injury or brain dysfunction by altering BBB structure and function.

Diagnostic Performance of Carbon Monoxide Testing by Pulse Oximetry in the Emergency Department.

BACKGROUND: Carbon monoxide (CO) exposure causes roughly 40,000 emergency department (ED) visits annually and is commonly misdiagnosed. Whereas the standard method of carboxyhemoglobin (HbCO) measurement utilizes blood gas analysis, a noninvasive, FDA-cleared alternative exists. We evaluated the performance of pulse oximetry (SpCO) for identification of CO exposure in ED patients. METHODS: We compared pulse oximetry to blood HbCO levels in a prospective observational study of adult and pediatric subjects recruited from the ED. Nurses screened a convenience sample of patients and referred those with SpCO ≥ 10% to research staff. Researchers also approached individuals who presented with signs and symptoms of CO toxicity. We determined diagnostic performance with a Bland-Altman analysis and calculated sensitivity and specificity for detection of elevated HbCO at thresholds of ≥ 10% and ≥ 15%. To optimize the potential sensitivity of SpCO for detection of CO toxicity, research technicians performed 3 SpCO readings within 5 min of the blood draw for laboratory measurement. A positive SpCO test was defined as any SpCO ≥ 10%. RESULTS: 42,000 patients were screened, 212 were evaluated, and 126 subjects were enrolled. Median HbCO level was 6% (range 1.6-21.9%). Limits of agreement were -10.3% and 8.1%. Of 23 individuals with elevated HbCO ≥ 10%, 13 were not suspected based on clinical assessment. Critically elevated HbCO was present in 6 individuals. Based on our a priori threshold of 10% for a positive test, pulse oximetry identified 14 of 23 subjects with HbCO ≥ 10%, with a sensitivity of 61% (95% CI 39-80%) and a specificity of 86% (95% CI 78-92%), and 5 of 6 subjects with HbCO ≥ 15%, with a sensitivity of 83% (95% CI 36-100%) and a specificity of 81% (95% CI 73-87%). CONCLUSIONS: Pulse oximetry underestimated HbCO and produced false negative results (ie, SpCO < 10% for all three measurements) in 17% of ED subjects with elevated HbCO ≥ 15%. Triage screening with pulse oximetry detected cases of elevated HbCO that were not suspected by the clinical provider.

Myogenic tone contributes to the regulation of permeability in mesenteric microvessels.

The microvascular endothelium plays a key role in regulating solute permeability in the gut, but the contribution of vascular smooth muscle to barrier function is unknown. We sought to determine the role of vascular smooth muscle and its myogenic tone in the vascular barrier to solutes in mesenteric microvessels. We determined vascular permeability to 4.4 kDa and 70 kDa dextrans in isolated mouse mesenteric arteries at increasing pressure increments. The myogenic response was simultaneously monitored using video edge-detection of vessel diameter and wall thickness. We expressed permeability as the apparent permeability coefficient, or the solute flux per second normalized to surface area and concentration gradient. We compared the effects of myogenic tone, L-type calcium channel blockade, calcium elimination, and endothelial removal on the permeability of each dextran. We found arteries resisted changes in 4.4 kDa and 70 kDa dextran permeability coefficients at intravascular pressures associated with myogenic tone. Manipulations that reduced or eliminated myogenic tone (L-type calcium channel blockade or calcium elimination) caused vasodilation and increased permeability coefficients. Thus, the maintenance of a reactive mesenteric vascular smooth muscle layer and its myogenic tone prevents increases in vascular permeability that would otherwise occur with increasing pressure. Conditions that impact vascular tone, such as trauma, stroke, or major surgery could diminish the gut-vascular barrier against dissemination of the microbiome.

Extracellular histones induce calcium signals in the endothelium of resistance-sized mesenteric arteries and cause loss of endothelium-dependent dilation.

Histone proteins are elevated in the circulation after traumatic injury owing to cellular lysis and release from neutrophils. Elevated circulating histones in trauma contribute to coagulopathy and mortality through a mechanism suspected to involve endothelial cell (EC) dysfunction. However, the functional consequences of histone exposure on intact blood vessels are unknown. Here, we sought to understand the effects of clinically relevant concentrations of histones on the endothelium in intact, resistance-sized, mesenteric arteries (MAs). EC Ca2+ was measured with high spatial and temporal resolution in MAs from mice selectively expressing the EC-specific, genetically encoded ratiometric Ca2+ indicator, Cx40-GCaMP-GR, and vessel diameter was measured by edge detection. Application of purified histone protein directly to the endothelium of en face mouse and human MA preparations produced large Ca2+ signals that spread within and between ECs. Surprisingly, luminal application of histones had no effect on the diameter of pressurized arteries. Instead, after prolonged exposure (30 min), it reduced dilations to endothelium-dependent vasodilators and ultimately caused death of ~25% of ECs, as evidenced by markedly elevated cytosolic Ca2+ levels (793 ± 75 nM) and uptake of propidium iodide. Removal of extracellular Ca2+ but not depletion of intracellular Ca2+ stores prevented histone-induced Ca2+ signals. Histone-induced signals were not suppressed by transient receptor potential vanilloid 4 (TRPV4) channel inhibition (100 nM GSK2193874) or genetic ablation of TRPV4 channels or Toll-like receptor receptors. These data demonstrate that histones are robust activators of noncanonical EC Ca2+ signaling, which cause vascular dysfunction through loss of endothelium-dependent dilation in resistance-sized MAs. NEW & NOTEWORTHY We describe the first use of the endothelial cell (EC)-specific, ratiometric, genetically encoded Ca2+ indicator, Cx40-GCaMP-GR, to study the effect of histone proteins on EC Ca2+ signaling. We found that histones induce an influx of Ca2+ in ECs that does not cause vasodilation but instead causes Ca2+ overload, EC death, and vascular dysfunction in the form of lost endothelium-dependent dilation.

Accuracy of Point-of-Care Testing for Anemia in the Emergency Department.

BACKGROUND: Pulse oximetry has become the standard of care in emergency medicine, operating rooms, and medical wards for the monitoring of oxygenation, but the use of pulse oximetry for assessment of hemoglobin (Hb) is controversial. The purpose of this study was to compare the accuracy and precision of 2 point-of-care Hb measurement devices, the Pronto-7 and the HemoCue 201+, to laboratory testing. METHODS: We studied a convenience sample of patients in the emergency department who required a complete blood count. We excluded patients in critical condition or those with elevated methemoglobin, impaired perfusion, or finger deformities. Each subject provided 2 capillary samples for measurement with the HemoCue 201+ and 2 consecutive readings with the Pronto-7. We used Bland-Altman analysis to compare the performance of the point-of-care devices to laboratory measurements. We also determined the diagnostic performance for the detection of anemia by sex (Hb < 11.6 g/dL for females, Hb < 13.8 g/dL for males). RESULTS: 201 of the 350 subjects enrolled (57%) were female. Mean (SD) age was 50.9 (19.0) y. Complete data were available for 297 (84.9%) of the Pronto-7 readings and 323 (92.3%) of the HemoCue 201+ readings. Mean (SD) laboratory Hb was 13.1 g/dL (2.3). Mean bias (Bland-Altman limits of agreement) for the Pronto-7 was -0.52 g/dL (-3.29 to 2.25), and for the HemoCue 201+ the mean bias was -0.98 g/dL (-3.57 to 1.61). Sensitivity and specificity for diagnosis of anemia were 81.6% (95% CI 72.5-88.7) and 75.4% (95% CI 68.8-81.1) for the Pronto-7 and 99.1% (95% CI 94.8-100.0) and 71.0% (95% CI 64.4-76.9) for HemoCue 201+. CONCLUSION: Both devices provided clinically useful methods to screen for anemia.

Optimization of a diagnostic platform for oxidation-reduction potential (ORP) measurement in human plasma.

OBJECTIVES: Oxidation-reduction potential (ORP) measurement can demonstrate the extent of oxidative stress in patients with severe illness and/or injury. A novel ORP diagnostic platform using disposable sensors (RedoxSYS) has been validated by comparison to mass spectrometry, but the optimal methods of sample handling for best performance of the device have not been described. METHODS: We sought to optimize ORP measurement in human plasma under controlled conditions. We hypothesized that the anticoagulant, freeze-thawing, and storage duration would influence measured ORP levels. RESULTS: The platform was sensitive to exogenous oxidation with hydrogen peroxide and reduction with ascorbic acid. Plasma anticoagulated with heparin was more sensitive to differences in ORP than plasma prepared in citrate. ORP measurements decreased slightly after a freeze-thaw cycle, but once frozen, ORP was stable for up to one month. DISCUSSION: We confirm that ORP detects oxidative stress in plasma samples. Optimal measurement of plasma ORP requires blood collection in heparin anticoagulant tubes and immediate analysis without a freeze-thaw cycle.