Sepsis is a major cause of death in hospitalized patients. Approximately 50% of patients in intensive care units develop sepsis, and the overall mortality rate is 29% ( 1 ). Mortality is due, in large part, to the cytotoxic actions of lipopolysaccharide (LPS), an endotoxic component of the outer membrane of Gram-negative bacteria. LPS is released from bacterial membranes and activates Toll-like receptors (TLR) on monocytes, neutrophils, and other target cells ( 2-5 ). TLRs transduce LPS action by activating nuclear factor (NF)-B-dependent signaling ( 4-6 ). By this mechanism, LPS stimulates the synthesis/release of infl ammatory cytokines, which play an important role in the innate immune response ( 7,8 ). Dysregulation of this infl ammatory response can lead to intravascular coagulation and multiple organ failure. LPS-induced cytokine production also increases expression of nitric oxide synthase 2 (NOS2) and cyclooxygenase 2 (COX-2), thus enhancing the synthesis of nitric oxide (NO) and arachidonic acidderived vasoactive prostanoids. These products have been implicated in hemodynamic disturbances in sepsis by reducing peripheral vascular resistance ( 9-14 ). Cardiovascular (CV) failure, characterized by severe hypotension and cardiac dysfunction, is linked to increased mortality in patients with severe sepsis ( 15-17 ).Lipoproteins are thought to play a role in the neutralization/detoxifi cation of endotoxin (18)(19)(20). Low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), and high-density lipoprotein (HDL) bind to LPS and direct it to the liver for metabolism and excretion (18)(19)(20). HDL is most effective in clearing endotoxin, a property attributed to its relatively high phospholipid content compared with LDL and VLDL ( 18,21 ). With increasing Abstract High density lipoprotein (HDL) and apolipoprotein A-I (apoA-I) reduce infl ammatory 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 LPStreated 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 LPStreated 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 (ED 50 = 459 ± 83 nM) compared with controls (ED 50 = 57 ± 6 nM). This was associated with nitric oxide synthase 2 (NOS2) upregulation. 4F administration improved vascular contractility (ED 50 = 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 , , GM-082952 (C.R.W. and G.D.) and HL-85282 (H.G.) Abbreviations: apoA-I, apolipoprotein A-I; COX-2,...

BackgroundTrauma 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.Methods and findingsWe 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 × 107 [−1.0 × 107 to 2.5 × 107] for fresh RBCs and 3.6 × 107 [2.5 × 107 to 4.7 × 107] 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.ConclusionsWe provide evidence that ...

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 RBC are thought to underlie this association. In this study we determined the effects of RBC washing, nitrite and anti-heme 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 RBC stored for 0–10d was used. Tested variables included whether washing RBC to remove lower MWt 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 5d RBC increased acute lung injury indexed by BAL protein and neutrophil accumulation. Washing 5d RBC prior to transfusion did not decrease this injury, whereas nitrite therapy did. Transfusion with 10d RBC elicited a more severe injury resulting in ~90% lethality, compared to <15% with 5d RBC. Both washing and nitrite therapy significantly protected against 10d 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 RBC. Transfusion with free heme partially recapitulated the toxicity mediated by stored RBC. Furthermore, inhibition of TLR4 signaling, which is stimulated by heme, using TAK-242, or hemopexin-dependent sequestration of free heme significantly protected against both 5d and 10d mouse RBC-dependent toxicity. These data suggest that RBC washing, nitrite therapy and / or anti-heme and TLR4 strategies may prevent stored RBC toxicities.

Chlorine gas (Cl 2 ) exposure during accidents or in the military setting results primarily in injury to the lungs. However, the potential for Cl 2 exposure to promote injury to the systemic vasculature leading to compromised vascular function has not been studied. We hypothesized that Cl 2 promotes extrapulmonary endothelial dysfunction characterized by a loss of endothelial nitric oxide synthase (eNOS)-derived signaling. Male Sprague Dawley rats were exposed to Cl 2 for 30 minutes, and eNOS-dependent vasodilation of aorta as a function of Cl 2 dose (0-400 ppm) and time after exposure (0-48 h) were determined. Exposure to Cl 2 (250-400 ppm) significantly inhibited eNOS-dependent vasodilation (stimulated by acetycholine) at 24 to 48 hours after exposure without affecting constriction responses to phenylephrine or vasodilation responses to an NO donor, suggesting decreased NO formation. Consistent with this hypothesis, eNOS protein expression was significantly decreased (z 60%) in aorta isolated from Cl 2 -exposed versus airexposed rats. Moreover, inducible nitric oxide synthase (iNOS) mRNA was up-regulated in circulating leukocytes and aorta isolated 24 hours after Cl 2 exposure, suggesting stimulation of inflammation in the systemic vasculature. Despite decreased eNOS expression and activity, no changes in mean arterial blood pressure were observed. However, injection of 1400W, a selective inhibitor of iNOS, increased mean arterial blood pressure only in Cl 2 -exposed animals, suggesting that iNOS-derived NO compensates for decreased eNOS-derived NO. These results highlight the potential for Cl 2 exposure to promote postexposure systemic endothelial dysfunction via disruption of vascular NO homeostasis mechanisms.

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.