Exposure to chlorine (Cl2) gas can occur during accidents and intentional release scenarios. However, biomarkers that specifically indicate Cl2 exposure and Cl2-derived products that mediate ...postexposure toxicity remain unclear. We hypothesized that chlorinated lipids (Cl-lipids) formed by direct reactions between Cl2 gas and plasmalogens serve as both biomarkers and mediators of post-Cl2 gas exposure toxicities. The 2-chloropalmitaldehyde (2-Cl-Pald), 2-chlorostearaldehyde (2-Cl-Sald), and their oxidized products, free- and esterified 2-chloropalmitic acid (2-Cl-PA) and 2-chlorostearic acid were detected in the lungs and plasma of mouse and rat models of Cl2 gas exposure. Levels of Cl-lipids were highest immediately post-Cl2 gas exposure, and then declined over 72 h with levels remaining 20- to 30-fold higher at 24 h compared with baseline. Glutathione adducts of 2-Cl-Pald and 2-Cl-Sald also increased with levels peaking at 4 h in plasma. Notably, 3-chlorotyrosine also increased after Cl2 gas exposure, but returned to baseline within 24 h. Intranasal administration of 2-Cl-PA or 2-Cl-Pald at doses similar to those formed in the lung after Cl2 gas exposure led to increased distal lung permeability and inflammation and systemic endothelial dysfunction characterized by loss of eNOS-dependent vasodilation. These data suggest that Cl-lipids could serve as biomarkers and mediators for Cl2 gas exposure and toxicity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Trauma is the leading cause of death and disability in patients aged 1-46 y. Severely injured patients experience considerable blood loss and hemorrhagic shock requiring treatment with massive ...transfusion of red blood cells (RBCs). Preclinical and retrospective human studies in trauma patients have suggested that poorer therapeutic efficacy, increased severity of organ injury, and increased bacterial infection are associated with transfusion of large volumes of stored RBCs, although the mechanisms are not fully understood.
We developed a murine model of trauma hemorrhage (TH) followed by resuscitation with plasma and leukoreduced RBCs (in a 1:1 ratio) that were banked for 0 (fresh) or 14 (stored) days. Two days later, lungs were infected with Pseudomonas aeruginosa K-strain (PAK). Resuscitation with stored RBCs significantly increased the severity of lung injury caused by P. aeruginosa, as demonstrated by higher mortality (median survival 35 h for fresh RBC group and 8 h for stored RBC group; p < 0.001), increased pulmonary edema (mean 95% CI 106.4 μl 88.5-124.3 for fresh RBCs and 192.5 μl 140.9-244.0 for stored RBCs; p = 0.003), and higher bacterial numbers in the lung (mean 95% CI 1.2 × 10(7) -1.0 × 10(7) to 2.5 × 10(7) for fresh RBCs and 3.6 × 10(7) 2.5 × 10(7) to 4.7 × 10(7) for stored RBCs; p = 0.014). The mechanism underlying this increased infection susceptibility and severity was free-heme-dependent, as recombinant hemopexin or pharmacological inhibition or genetic deletion of toll-like receptor 4 (TLR4) during TH and resuscitation completely prevented P. aeruginosa-induced mortality after stored RBC transfusion (p < 0.001 for all groups relative to stored RBC group). Evidence from studies transfusing fresh and stored RBCs mixed with stored and fresh RBC supernatants, respectively, indicated that heme arising both during storage and from RBC hemolysis post-resuscitation plays a role in increased mortality after PAK (p < 0.001). Heme also increased endothelial permeability and inhibited macrophage-dependent phagocytosis in cultured cells. Stored RBCs also increased circulating high mobility group box 1 (HMGB1; mean 95% CI 15.4 ng/ml 6.7-24.0 for fresh RBCs and 50.3 ng/ml 12.3-88.2 for stored RBCs), and anti-HMGB1 blocking antibody protected against PAK-induced mortality in vivo (p = 0.001) and restored macrophage-dependent phagocytosis of P. aeruginosa in vitro. Finally, we showed that TH patients, admitted to the University of Alabama at Birmingham ER between 1 January 2015 and 30 April 2016 (n = 50), received high micromolar-millimolar levels of heme proportional to the number of units transfused, sufficient to overwhelm endogenous hemopexin levels early after TH and resuscitation. Limitations of the study include lack of assessment of temporal changes in different products of hemolysis after resuscitation and the small sample size precluding testing of associations between heme levels and adverse outcomes in resuscitated TH patients.
We provide evidence that large volume resuscitation with stored blood, compared to fresh blood, in mice increases mortality from subsequent pneumonia, which occurs via mechanisms sensitive to hemopexin and TLR4 and HMGB1 inhibition.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Transfusion of stored red blood cells (RBCs) is associated with increased morbidity and mortality in trauma patients. Pro-oxidant, pro-inflammatory, and nitric oxide (NO) scavenging properties of ...stored RBCs are thought to underlie this association. In this study we determined the effects of RBC washing and nitrite and antiheme therapy on stored RBC-dependent toxicity in the setting of trauma-induced hemorrhage. A murine (C57BL/6) model of trauma–hemorrhage and resuscitation with 1 or 3 units of RBCs stored for 0–10 days was used. Tested variables included washing RBCs to remove lower MW components that scavenge NO, NO-repletion therapy using nitrite, or mitigation of free heme toxicity by heme scavenging or preventing TLR4 activation. Stored RBC toxicity was determined by assessment of acute lung injury indices (airway edema and inflammation) and survival. Transfusion with 5 day RBCs increased acute lung injury indexed by BAL protein and neutrophil accumulation. Washing 5 day RBCs prior to transfusion did not decrease this injury, whereas nitrite therapy did. Transfusion with 10 day RBCs elicited a more severe injury resulting in ~90% lethality, compared to <15% with 5 day RBCs. Both washing and nitrite therapy significantly protected against 10 day RBC-induced lethality, suggesting that washing may be protective when the injury stimulus is more severe. Finally, a spectral deconvolution assay was developed to simultaneously measure free heme and hemoglobin in stored RBC supernatants, which demonstrated significant increases of both in stored human and mouse RBCs. Transfusion with free heme partially recapitulated the toxicity mediated by stored RBCs. Furthermore, inhibition of TLR4 signaling, which is stimulated by heme, using TAK-242, or hemopexin-dependent sequestration of free heme significantly protected against both 5 day and 10 day mouse RBC-dependent toxicity. These data suggest that RBC washing, nitrite therapy, and/or antiheme and TLR4 strategies may prevent stored RBC toxicities.
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•RBC storage results in increased free hemoglobin and free heme.•Transfusion with stored but not fresh RBC causes acute lung injury (ALI) in trauma–hemorrhage.•Stored RBC toxicity is prevented by pretransfusion washing, nitrite therapy, heme sequestration, or blocking TLR4 signaling.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Recent studies have suggested that in addition to oxygen transport, red blood cells (RBC) are key regulators of vascular function by both inhibiting and promoting nitric oxide (NO)-mediated ...vasodilation. Most studies assume that RBC are homogenous, but, in fact, they comprise cells of differing morphology and biochemical composition which are dependent on their age, parameters that control NO reactions. We tested the hypothesis that distinct RBC populations will have differential effects on NO signaling.
Young and old RBC were separated by density gradient centrifugation. Consistent with previous reports, old RBC had decreased levels of surface N-acetyl neuraminic acid and increased oxygen binding affinities. Competition kinetic experiments showed that older RBCs scavenged NO∼2-fold faster compared with younger RBC, which translated to a more potent inhibition of both acetylcholine and NO-donor dependent vasodilation of isolated aortic rings. Moreover, nitrite oxidation kinetics was faster with older RBC compared with younger RBC; whereas no differences in nitrite-reduction kinetics were observed. This translated to increased inhibitory effect of older RBC to nitrite-dependent vasodilation under oxygenated and deoxygenated conditions. Finally, leukodepleted RBC storage also resulted in more dense RBC, which may contribute to the greater NO-inhibitory potential of stored RBC.
These results suggest that a key element in vascular NO-homeostasis mechanisms is the distribution of RBC ages across the physiological spectrum (0-120 days) and suggest a novel mechanism for inhibited NO bioavailability in diseases which are characterized by a shift to an older RBC phenotype.
Older RBC inhibit NO bioavailability by increasing NO- and nitrite scavenging.
Purpose
Hemodilutional anemia is associated with acute kidney injury (AKI) and mortality in patients undergoing cardiac surgery by mechanisms that may include tissue hypoxia. Our hypothesis was to ...assess if changes in the potential hypoxic biomarkers, including methemoglobin and erythropoietin, correlated with a decrease in hemoglobin (Hb) concentration following hemodilution on cardiopulmonary bypass (CPB).
Methods
Arterial blood samples were taken from patients (
n
= 64) undergoing heart surgery and CPB at baseline, during CPB, following CPB, and in the intensive care unit (ICU). Potential hypoxic biomarkers were measured, including methemoglobin, plasma Hb, and erythropoietin. Data were analyzed by repeated measures one-way analysis of variance on ranks and linear regression.
Results
Hemoglobin levels decreased following CPB and methemoglobin increased in the ICU (
P
< 0.001 for both). No correlation was observed between the change in Hb and methemoglobin (
P
= 0.23). By contrast, reduced Hb on CPB correlated with increased lactate, reduced pH, and increased erythropoietin levels following CPB (
P
≤ 0.004 for all). Increased plasma Hb (
P
< 0.001) also correlated with plasma erythropoietin levels (
P
< 0.001).
Conclusion
These data support the hypothesis that erythropoietin rather than methemoglobin is a potential biomarker of anemia-induced tissue hypoxia. The observed relationships between decreased Hb during CPB and the increase in lactate, reduced pH, and increase in erythropoietin levels suggest that early changes in plasma erythropoietin may be a pragmatic early biomarker of anemia-induced renal hypoxia. Further study is required to determine if anemia-induced increases in erythropoietin may predict AKI in patients undergoing cardiac surgery.
Trial registration
www.clinicaltrials.gov
(NCT01883713). Registered 21 June 2013.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Acute respiratory distress syndrome (ARDS) due to sepsis has a high mortality rate with limited treatment options. High density lipoprotein (HDL) exerts innate protective effects in systemic ...inflammation. However, its role in ARDS has not been well studied. Peptides such as L-4F mimic the secondary structural features and functions of apolipoprotein (apo)A-I, the major protein component of HDL. We set out to measure changes in HDL in sepsis-mediated ARDS patients, and to study the potential of L-4F to prevent sepsis-mediated ARDS in a rodent model of lipopolysaccharide (LPS)-mediated acute lung injury, and a combination of primary human leukocytes and human ARDS serum. We also analyzed serum from non-lung disease intubated patients (controls) and sepsis-mediated ARDS patients. Compared to controls, ARDS demonstrates increased serum endotoxin and IL-6 levels, and decreased HDL, apoA-I and activity of anti-oxidant HDL-associated paraoxanase-1. L-4F inhibits the activation of isolated human leukocytes and neutrophils by ARDS serum and LPS in vitro. Further, L-4F decreased endotoxin activity and preserved anti-oxidant properties of HDL both in vitro and in vivo. In a rat model of severe endotoxemia, L-4F significantly decreased mortality and reduces lung and liver injury, even when administered 1 hour post LPS. Our study suggests the protective role of the apoA-I mimetic peptide L-4F in ARDS and gram-negative endotoxemia and warrant further clinical evaluation. The main protective mechanisms of L-4F are due to direct inhibition of endotoxin activity and preservation of HDL anti-oxidant activity.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Slope was significantly non-zero for number of units (r2 = 0.91, p<0.001). https://doi.org/10.1371/journal.pmed.1002991.g001 In the Results, in the subsection “Heme levels in patients after TH and ...resuscitation”, there is an error in the fifth sentence of the first paragraph. While the absolute concentration of these mediators is important, the relative concentrations of hemoglobin and free heme, compared to Hp and Hpx respectively is likely of greater clinical significance; Hp and Hpx are the endogenous primary defense mechanisms protecting against hemolysis-dependent injury. (2018) Role of heme in lung bacterial infection after trauma hemorrhage and stored red blood cell transfusion: A preclinical experimental study.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
High density lipoprotein (HDL) and apolipoprotein A-I (apoA-I) reduce inflammatory responses to lipopolysaccharide (LPS). We tested the hypothesis that the apoA-I mimetic peptide 4F prevents ...LPS-induced defects in blood pressure and vascular reactivity. Systolic blood pressure (SBP) was measured in rats at baseline and 6 h after injection of LPS (10 mg/kg) or saline vehicle. Subgroups of LPS-treated rats also received 4F (10 mg/kg) or scrambled 4F (Sc-4F). LPS administration reduced SBP by 35% compared with baseline. 4F attenuated the reduction in SBP in LPS-treated rats (17% reduction), while Sc-4F was without effect. Ex vivo studies showed a reduced contractile response to phenylephrine (PE) in aortae of LPS-treated rats (ED50= 459 ± 83 nM) compared with controls (ED50= 57 ± 6 nM). This was associated with nitric oxide synthase 2 (NOS2) upregulation. 4F administration improved vascular contractility (ED50= 60 ± 9 nM), reduced aortic NOS2 protein, normalized plasma levels of NO metabolites, and reduced mortality in LPS-treated rats. These changes were associated with a reduction in plasma endotoxin activity. In vivo administration of 14C-4F and Bodipy-LPS resulted in their colocalization and retention in the HDL fraction. It is proposed that 4F promotes the localization of LPS to the HDL fraction, resulting in endotoxin neutralization. 4F may thus prevent LPS-induced hemodynamic changes associated with NOS2 induction.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•Chlorine gas induces acute lung injury and death in rabbits.•Post-chlorine exposure administration of IM nitrite improves 18h survival.•Post-chlorine exposure administration of IM nitrite prevents ...lung leak and airway accumulation of neutrophils.
Chlorine (Cl2) gas exposure and toxicity remains a concern in military and industrial sectors. While post-Cl2 exposure damage to the lungs and other tissues has been documented and major underlying mechanisms elucidated, no targeted therapeutics that are effective when administered post-exposure, and which are amenable to mass-casualty scenarios have been developed. Our recent studies show nitrite administered by intramuscular (IM) injection post-Cl2 exposure is effective in preventing acute lung injury and improving survival in rodent models. Our goal in this study was to develop a rabbit model of Cl2 toxicity and test whether nitrite affords protection in a non-rodent model. Exposure of New Zealand White rabbits to Cl2 gas (600ppm, 45min) caused significant increases in protein and neutrophil accumulation in the airways and ∼35% mortality over 18h. Nitrite administered 30min post Cl2 exposure by a single IM injection, at 1mg/kg or 10mg/kg, prevented indices of acute lung injury at 6h by up to 50%. Moreover, all rabbits that received nitrite survived over the study period. These data provide further rationale for developing nitrite as post-exposure therapeutic to mitigate against Cl2 gas exposure injury.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Traumatic brain injury (TBI) is the leading cause of injury-related death and disability in patients under the age of 46 years. Survivors of the initial injury often endure systemic complications ...such as pulmonary infection, and
is one of the most common causes of nosocomial pneumonia in intensive care units. Female patients are less likely to develop secondary pneumonia after TBI, and pre-clinical studies have revealed a salutary role for estrogen after trauma. Therefore, we hypothesized that female mice would experience less mortality after post-TBI pneumonia with
. We employed a mouse model of TBI followed by
pneumonia. Male mice had greater mortality and impaired lung bacterial clearance after post-TBI pneumonia compared with female mice. This was confirmed as a difference in sex hormones, as oophorectomized wild-type mice had mortality and lung bacterial clearance similar to male mice. There were differences in tumor necrosis factor-α secretion in male and female alveolar macrophages after
infection. Finally, injection of male or oophorectomized wild-type female mice with estrogen restored lung bacterial clearance and prevented mortality. Our model of TBI followed by
pneumonia is among the first to reveal sex dimorphism in secondary, long-term TBI complications.
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