Modulation of angiopoietin-2 and Tie2: Organ specific effects of microvascular leakage and edema in mice.

INTRODUCTION: Critical illness is associated with organ failure, in which endothelial hyperpermeability and tissue edema play a major role. The endothelial angiopoietin/Tie2 system, a regulator of endothelial permeability, is dysbalanced during critical illness. Elevated circulating angiopoietin-2 and decreased Tie2 receptor levels are reported, but it remains unclear whether they cause edema independent of other critical illness-associated alterations. Therefore, we have studied the effect of angiopoietin-2 administration and/or reduced Tie2 expression on microvascular leakage and edema under normal conditions. METHODS: Transgenic male mice with partial deletion of Tie2 (heterozygous exon 9 deletion, Tie2+/-) and wild-type controls (Tie2+/+) received 24 or 72 pg/g angiopoietin-2 or PBS as control (n = 12 per group) intravenously. Microvascular leakage and edema were determined by Evans blue dye (EBD) extravasation and wet-to-dry weight ratio, respectively, in lungs and kidneys. Expression of molecules related to endothelial angiopoietin/Tie2 signaling were determined by ELISA and RT-qPCR. RESULTS: In Tie2+/+ mice, angiopoietin-2 administration increased EBD extravasation (154 %, p < 0.05) and wet-to-dry weight ratio (133 %, p < 0.01) in lungs, but not in the kidney compared to PBS. Tie2+/- mice had higher pulmonary (143 %, p < 0.001), but not renal EBD extravasation, compared to wild-type control mice, whereas a more pronounced wet-to-dry weight ratio was observed in lungs (155 %, p < 0.0001), in contrast to a minor higher wet-to-dry weight ratio in kidneys (106 %, p < 0.05). Angiopoietin-2 administration to Tie2+/- mice did not further increase pulmonary EBD extravasation, pulmonary wet-to-dry weight ratio, or renal wet-to-dry weight ratio. Interestingly, angiopoietin-2 administration resulted in an increased renal EBD extravasation in Tie2+/- mice compared to Tie2+/- mice receiving PBS. Both angiopoietin-2 administration and partial deletion of Tie2 did not affect circulating angiopoietin-1, soluble Tie2, VEGF and NGAL as well as gene expression of angiopoietin-1, -2, Tie1, VE-PTP, ELF-1, Ets-1, KLF2, GATA3, MMP14, Runx1, VE-cadherin, VEGFα and NGAL, except for gene and protein expression of Tie2, which was decreased in Tie2+/- mice compared to Tie2+/+ mice. CONCLUSIONS: In mice, the microvasculature of the lungs is more vulnerable to angiopoietin-2 and partial deletion of Tie2 compared to those in the kidneys with respect to microvascular leakage and edema.

Effect of antibody-mediated connective tissue growth factor neutralization on lung edema in ventilator-induced lung injury in rats.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is characterized by alveolar edema that can progress to septal fibrosis. Mechanical ventilation can augment lung injury, termed ventilator-induced lung injury (VILI). Connective tissue growth factor (CTGF), a mediator of fibrosis, is increased in ARDS patients. Blocking CTGF inhibits fibrosis and possibly vascular leakage. This study investigated whether neutralizing CTGF reduces pulmonary edema in VILI. METHODS: Following LPS administration, rats were mechanically ventilated for 6 h with low (6 mL/kg; low VT) or moderate (10 mL/kg; mod VT) tidal volume and treated with a neutralizing CTGF antibody (FG-3154) or placebo lgG (vehicle). Control rats without LPS were ventilated for 6 h with low VT. Lung wet-to-dry weight ratio, FITC-labeled dextran permeability, histopathology, and soluble RAGE were determined. RESULTS: VILI was characterized by reduced PaO2/FiO2 ratio (low VT: 540 [381-661] vs. control: 693 [620-754], p < 0.05), increased wet-to-dry weight ratio (low VT: 4.8 [4.6-4.9] vs. control: 4.5 [4.4-4.6], p < 0.05), pneumonia (low VT: 30 [0-58] vs. control: 0 [0-0]%, p < 0.05) and interstitial inflammation (low VT: 2 [1-3] vs. control: 1 [0-1], p < 0.05). FG-3154 did not affect wet-to-dry weight ratio (mod VT + FG-3154: 4.8 [4.7-5.0] vs. mod VT + vehicle: 4.8 [4.8-5.0], p > 0.99), extravasated dextrans (mod VT + FG-3154: 0.06 [0.04-0.09] vs. mod VT + vehicle: 0.04 [0.03-0.09] µg/mg tissue, p > 0.99), sRAGE (mod VT + FG-3154: 1865 [1628-2252] vs. mod VT + vehicle: 1885 [1695-2159] pg/mL, p > 0.99) or histopathology. CONCLUSIONS: 'Double hit' VILI was characterized by inflammation, impaired oxygenation, pulmonary edema and histopathological lung injury. Blocking CTGF does not improve oxygenation nor reduce pulmonary edema in rats with VILI.

Transfusion of female blood in a rat model is associated with red blood cells entrapment in organs.

Transfusion of red blood cells (RBCs) has been associated with adverse outcomes. Mechanisms may be related to donor sex and biological age of RBC. This study hypothesized that receipt of female blood is associated with decreased post-transfusion recovery (PTR) and a concomitant increased organ entrapment in rats, related to young age of donor RBCs. Donor rats underwent bloodletting to stimulate production of new, young RBCs, followed by Percoll fractionation for further enrichment of young RBCs based on their low density. Control donors did not undergo these procedures. Male rats received either a (biotinylated) standard RBC product or a product enriched for young RBCs, derived from either male or female donors. Controls received saline. Organs and blood samples were harvested after 24 hours. This study found no difference in PTR between groups, although only the group receiving young RBCs from females failed to reach a PTR of 75%. Receipt of both standard RBCs and young RBCs from females was associated with increased entrapment of donor RBCs in the lung, liver, and spleen compared to receiving blood from male donors. Soluble ICAM-1 and markers of hemolysis were higher in recipients of female blood compared to control. In conclusion, transfusing RBCs from female donors, but not from male donors, is associated with trapping of donor RBCs in organs, accompanied by endothelial activation and hemolysis.

Influence of bacterial and alveolar cell co-culture on microbial VOC production using HS-GC/MS.

Volatile organic compounds (VOCs) found in exhaled breath continue to garner interest as an alternative diagnostic tool in pulmonary infections yet, their clinical integration remains a challenge with difficulties in translating identified biomarkers. Alterations in bacterial metabolism secondary to host nutritional availability may explain this but is often inadequately modelled in vitro. The influence of more clinically relevant nutrients on VOC production for two common respiratory pathogens was investigated. VOCs from Staphylococcus aureus (S.aureus) and Pseudomonas aeruginosa (P.aeruginosa) cultured with and without human alveolar A549 epithelial cells were analyzed using headspace extraction coupled with gas chromatography-mass spectrometry. Untargeted and targeted analyses were performed, volatile molecules identified from published data, and the differences in VOC production evaluated. Principal component analysis (PCA) could differentiate alveolar cells from either S. aureus or P. aeruginosa when cultured in isolation based on PC1 (p = 0.0017 and 0.0498, respectively). However, this separation was lost for S. aureus (p = 0.31) but not for P. aeruginosa (p = 0.028) when they were cultured with alveolar cells. S. aureus cultured with alveolar cells led to higher concentrations of two candidate biomarkers, 3-methyl-1-butanol (p = 0.001) and 3-methylbutanal (p = 0.002) when compared to S. aureus, alone. P. aeruginosa metabolism resulted in less generation of pathogen-associated VOCs when co-cultured with alveolar cells compared to culturing in isolation. VOC biomarkers previously considered indicative of bacterial presence are influenced by the local nutritional environment and this should be considered when evaluating their biochemical origin.

The impact of biological age of red blood cell on in vitro endothelial activation markers.

Introduction: Blood donor characteristics influence red blood cell transfusion outcomes. As donor sex affects the distribution of young to old RBCs in the circulation, we hypothesized that the amount of circulating young RBCs in the blood product are associated with immune suppression. Materials and Methods: Blood samples were collected from healthy volunteers and density fractionated into young and old subpopulations. In an activated endothelial cell model, RBC adhesion to endothelium and secretion of endothelial activation markers were assessed. The impact of RBC biological age was also assessed in a T cell proliferation assay and in a whole blood stimulation assay. Results: After Percoll fractionation, young RBCs contained more reticulocytes compared to old RBCs. Young RBCs associated with lower levels of E-selectin, ICAM-1, and vWF from activated endothelial cells compared to old RBCs. RBC subpopulations did not affect T cell proliferation or cytokine responses following whole blood stimulation. Conclusion: Young RBCs contain more reticulocytes which are associated with lower levels of endothelial activation markers compared to old RBCs.

Validation of volatile metabolites of pulmonary oxidative injury: a bench to bedside study.

Background: Changes in exhaled volatile organic compounds (VOCs) can be used to discriminate between respiratory diseases, and increased concentrations of hydrocarbons are commonly linked to oxidative stress. However, the VOCs identified are inconsistent between studies, and translational studies are lacking. Methods: In this bench to bedside study, we captured VOCs in the headspace of A549 epithelial cells after exposure to hydrogen peroxide (H2O2), to induce oxidative stress, using high-capacity polydimethylsiloxane sorbent fibres. Exposed and unexposed cells were compared using targeted and untargeted analysis. Breath samples of invasively ventilated intensive care unit patients (n=489) were collected on sorbent tubes and associated with the inspiratory oxygen fraction (F IO2 ) to reflect pulmonary oxidative stress. Headspace samples and breath samples were analysed using gas chromatography and mass spectrometry. Results: In the cell, headspace octane concentration was decreased after oxidative stress (p=0.0013), while the other VOCs were not affected. 2-ethyl-1-hexanol showed an increased concentration in the headspace of cells undergoing oxidative stress in untargeted analysis (p=0.00014). None of the VOCs that were linked to oxidative stress showed a significant correlation with F IO2 (Rs range: -0.015 to -0.065) or discriminated between patients with F IO2 ≥0.6 or below (area under the curve range: 0.48 to 0.55). Conclusion: Despite a comprehensive translational approach, validation of known and novel volatile biomarkers of oxidative stress was not possible in patients at risk of pulmonary oxidative injury. The inconsistencies observed highlight the difficulties faced in VOC biomarker validation, and that caution is warranted in the interpretation of the pathophysiological origin of discovered exhaled breath biomarkers.

Shock-Driven Endotheliopathy in Trauma Patients Is Associated with Leucocyte Derived Extracellular Vesicles.

Endotheliopathy following trauma is associated with poor outcome, but the underlying mechanisms are unknown. This study hypothesized that an increased extracellular vesicle (EV) concentration is associated with endotheliopathy after trauma and that red blood cell (RBC) transfusion could further enhance endotheliopathy. In this post hoc sub study of a multicentre observational trial, 75 trauma patients were stratified into three groups based on injury severity score or shock. In patient plasma obtained at hospital admission and after transfusion of four RBC transfusions, markers for endotheliopathy were measured and EVs were labelled with anti CD41 (platelet EVs), anti CD235a (red blood cell EVs), anti CD45 (leucocyte EVs), anti CD144 (endothelial EVs) or anti CD62e (activated endothelial EVs) and EV concentrations were measured with flow cytometry. Statistical analysis was performed by a Kruskall Wallis test with Bonferroni correction or Wilcoxon rank test for paired data. In patients with shock, syndecan-1 and von Willebrand Factor (vWF) were increased compared to patients without shock. Additionally, patients with shock had increased red blood cell EV and leucocyte EV concentrations compared to patients without shock. Endotheliopathy markers correlated with leucocyte EVs (ρ = 0.263, p = 0.023), but not with EVs derived from other cells. Injury severity score had no relation with EV release. RBC transfusion increased circulating red blood cell EVs but did not impact endotheliopathy. In conclusion, shock is (weakly) associated with EVs from leucocytes, suggesting an immune driven pathway mediated (at least in part) by shock.

Aged versus fresh autologous platelet transfusion in a two-hit healthy volunteer model of transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a severe complication of blood transfusion that is thought of as a two-hit event: first the underlying patient condition (e.g., sepsis), and then the transfusion. Transfusion factors include human leukocyte antigen antibodies or biologic response modifiers (BRMs) accumulating during storage. Preclinical studies show an increased TRALI risk with longer stored platelets, clinical studies are conflicting. We aim to discover whether longer platelet concentrate (PC) storage time increases TRALI risk in a controlled human experiment. STUDY DESIGN AND METHODS: In a randomized controlled trial, 18 healthy male volunteers received a first hit of experimental endotoxemia (2 ng/kg lipopolysaccharide), and a second hit of fresh (2-day old) or aged (7-day old) autologous PC, or physiological saline. After 6 h, changes in TRALI pathways were determined using spirometry, chest X-ray, and bronchoalveolar lavage (BAL). RESULTS: All subjects reacted adequately to lipopolysaccharide infusion and satisfied SIRS criteria (increased pulse [>90/min] and temperature [>38°C]). There were no differences between the saline, fresh, and aged PC groups in BAL-fluid protein (95 ± 33 µg/ml; 83 ± 21 µg/ml and 104 ± 29 µg/ml, respectively) and relative neutrophil count (1.5 ± 0.5%; 1.9 ± 0.8% and 1.3 ± 0.8%, respectively), nor in inflammatory BAL-fluid BRMs (Interleukin-6, CXCL8, TNFα , and myeloperoxidase), clinical respiratory parameters, and spirometry results. All chest X-rays were normal. CONCLUSIONS: In a human endotoxemia model of autologous platelet transfusion, with an adequate first hit and platelet storage lesion, transfusion of 7-day-old PC does not increase pulmonary inflammation compared with 2-day-old PC.

Clinical features and prognostic factors in Covid-19: A prospective cohort study.

BACKGROUND: Mortality rates are high among hospitalized patients with COVID-19, especially in those intubated on the ICU. Insight in pathways associated with unfavourable outcome may lead to new treatment strategies. METHODS: We performed a prospective cohort study of patients with COVID-19 admitted to general ward or ICU who underwent serial blood sampling. To provide insight in the pathways involved in disease progression, associations were estimated between outcome risk and serial measurements of 64 biomarkers in potential important pathways of COVID-19 infection (inflammation, tissue damage, complement system, coagulation and fibrinolysis) using joint models combining Cox regression and linear mixed-effects models. For patients admitted to the general ward, the primary outcome was admission to the ICU or mortality (unfavourable outcome). For patients admitted to the ICU, the primary outcome was 12-week mortality. FINDINGS: A total of 219 patients were included: 136 (62%) on the ward and 119 patients (54%) on the ICU; 36 patients (26%) were included in both cohorts because they were transferred from general ward to ICU. On the general ward, 54 of 136 patients (40%) had an unfavourable outcome and 31 (23%) patients died. On the ICU, 54 out of 119 patients (45%) died. Unfavourable outcome on the general ward was associated with changes in concentrations of IL-6, IL-8, IL-10, soluble Receptor for Advanced Glycation End Products (sRAGE), vascular cell adhesion molecule 1 (VCAM-1) and Pentraxin-3. Death on the ICU was associated with changes in IL-6, IL-8, IL-10, sRAGE, VCAM-1, Pentraxin-3, urokinase-type plasminogen activator receptor, IL-1-receptor antagonist, CD14, procalcitonin, tumor necrosis factor alfa, tissue factor, complement component 5a, Growth arrest-specific 6, angiopoietin 2, and lactoferrin. Pathway analysis showed that unfavourable outcome on the ward was mainly driven by chemotaxis and interleukin production, whereas death on ICU was associated with a variety of pathways including chemotaxis, cell-cell adhesion, innate host response mechanisms, including the complement system, viral life cycle regulation, angiogenesis, wound healing and response to corticosteroids. INTERPRETATION: Clinical deterioration in patients with severe COVID-19 involves multiple pathways, including chemotaxis and interleukin production, but also endothelial dysfunction, the complement system, and immunothrombosis. Prognostic markers showed considerable overlap between general ward and ICU patients, but we identified distinct differences between groups that should be considered in the development and timing of interventional therapies in COVID-19. FUNDING: Amsterdam UMC, Amsterdam UMC Corona Fund, and Dr. C.J. Vaillant Fonds.

Induced normothermia ameliorates the procoagulant host response in human endotoxaemia.

BACKGROUND: Dysregulation of coagulation occurs commonly in sepsis, ranging from mild coagulopathy with decreased platelets to disseminated intravascular coagulation (DIC). We investigated the effect of induced normothermia on coagulation during lipopolysaccharide (LPS)-induced endotoxaemia in healthy volunteers. METHODS: Twelve volunteers received an infusion of bacterial lipopolysaccharide (Escherichia coli; 2 ng kg-1) and were assigned to either induced normothermia or control. Induced normothermia to maintain core temperature at 37°C consisted of external surface cooling, cold i.v. fluids, and medication to reduce shivering (buspirone, clonidine, and magnesium sulphate). The primary outcome was the DIC score (International Society on Thrombosis and Haemostasis guideline). Prothrombin time (PT), activated partial thromboplastin time (aPTT), D-dimer, plasma von Willebrand factor (vWf), and rotational thromboelastometry (ROTEM) were measured before and 1, 3, 6, and 8 h after LPS infusion. Differences between groups were tested with a mixed effects model. RESULTS: In control subjects, lipopolysaccharide caused a fever, transiently decreased platelet levels and lowered activated partial thromboplastin time, while prolonging prothrombin time and increasing D-Dimer and vWf levels. Normothermia prevented the DIC-score exceeding 4, which occurred in 50% of control subjects. Normothermia also reduced the fall in platelet count by 67x109 L-1([95%CI:27-107]; p=0.002), aPTT (mean difference:3s [95%CI:1-5]; p=0.005) and lowered vWf levels by 89% ([95%CI:6-172]; p=0.03), compared to the fever group. ROTEM measurements were unaffected by lipopolysaccharide. CONCLUSION: In human endotoxaemia, induced normothermia decreases markers of endothelial activation and DIC. Maintaining normothermia may reduce coagulopathy in hyperinflammatory states.

Early increase in anti-inflammatory biomarkers is associated with the development of multiple organ dysfunction syndrome in severely injured trauma patients.

BACKGROUND: As a result of improvements in the early resuscitation phase of trauma, mortality is largely driven by later mortality due to multiple organ dysfunction syndrome (MODS), which may be mediated by an early overdrive in the host immune response. If patients at risk for MODS could be identified early, preventive treatment measures could be taken. The aim of this study is to investigate whether specific biomarkers are associated with MODS. METHODS: Multiple trauma patients presenting to the Amsterdam University Medical Centers, location Academic Medical Center, between 2012 and 2018 with an Injury Severity Score of 16 or higher were sampled on arrival at the emergency department. A wide variety of inflammatory cytokines, endothelial and lung-specific markers were determined. Comparisons were made between patients with and without MODS. Univariate and multivariate logistic regression was used to determine associations between specific biomarkers and MODS. A p value of 0.05 was considered to be statistically significant. RESULTS: In total, 147 multiple trauma patients were included. Of these, 32 patients developed MODS (21.7%). Patients who developed MODS were more severely injured, had more traumatic brain injury and showed more deranged markers of coagulation when compared with patients without MODS. Overall, both proinflammatory and anti-inflammatory cytokines were higher in patients with MODS, indicative of a host immune reaction. In the multivariate analysis, the combination of anti-inflammatory proteins interleukin 1 receptor antagonist (IL-1RA) (OR 1.27 (1.07-1.51), p=0.002) and Clara cell protein 16 (CC-16) (1.06 (1.01-1.05), p=0.031) was most strongly associated with the development MODS. CONCLUSIONS: In trauma, anti-inflammatory proteins IL-1RA and CC-16 have the potential to early identify patients at risk for development of MODS. Further research is warranted to prospectively validate these results. LEVEL OF EVIDENCE: Prognostic study, level III.

Association between pre-operative biological phenotypes and postoperative pulmonary complications: An unbiased cluster analysis.

BACKGROUND: Biological phenotypes have been identified within several heterogeneous pulmonary diseases, with potential therapeutic consequences. OBJECTIVE: To assess whether distinct biological phenotypes exist within surgical patients, and whether development of postoperative pulmonary complications (PPCs) and subsequent dependence of intra-operative positive end-expiratory pressure (PEEP) differ between such phenotypes. SETTING: Operating rooms of six hospitals in Europe and USA. DESIGN: Secondary analysis of the 'PROtective Ventilation with HIgh or LOw PEEP' trial. PATIENTS: Adult patients scheduled for abdominal surgery who are at risk of PPCs. INTERVENTIONS: Measurement of pre-operative concentrations of seven plasma biomarkers associated with inflammation and lung injury. MAIN OUTCOME MEASURES: We applied unbiased cluster analysis to identify biological phenotypes. We then compared the proportion of patients developing PPCs within each phenotype, and associations between intra-operative PEEP levels and development of PPCs among phenotypes. RESULTS: In total, 242 patients were included. Unbiased cluster analysis clustered the patients within two biological phenotypes. Patients with phenotype 1 had lower plasma concentrations of TNF-α (3.8 [2.4 to 5.9] vs. 10.2 [8.0 to 12.1] pg ml; P < 0.001), IL-6 (2.3 [1.5 to 4.0] vs. 4.0 [2.9 to 6.5] pg ml; P < 0.001) and IL-8 (4.7 [3.1 to 8.1] vs. 8.1 [6.0 to 13.9] pg ml; P < 0.001). Phenotype 2 patients had the highest incidence of PPC (69.8 vs. 34.2% in type 1; P < 0.001). There was no interaction between phenotype and PEEP level for the development of PPCs (43.2% in high PEEP vs. 25.6% in low PEEP in phenotype 1, and 73.6% in high PEEP and 65.7% in low PEEP in phenotype 2; P for interaction = 0.503). CONCLUSION: Patients at risk of PPCs and undergoing open abdominal surgery can be clustered based on pre-operative plasma biomarker concentrations. The two identified phenotypes have different incidences of PPCs. Biologic phenotyping could be useful in future randomised controlled trials of intra-operative ventilation. TRIAL REGISTRATION: The PROtective Ventilation with HIgh or LOw PEEP trial, including the substudy from which data were used for the present analysis, was registered at ClinicalTrials.gov (NCT01441791).

Transfusion of 35-day-stored red blood cells does not alter lipopolysaccharide tolerance during human endotoxemia.

BACKGROUND: Transfusion-related immunomodulation (TRIM) encompasses immunosuppressive and proinflammatory effects induced by red blood cell (RBC) transfusion. Changes that occur during storage in the RBC product have been hypothesized to underlie TRIM, mediated by tolerance of toll-like receptors (TLR). We investigated whether transfusion of 35-day-stored autologous RBCs alters cytokine production in response to stimulation with lipopolysaccharide (LPS) or lipotheic acid (LTA), in a clinically relevant model of endotoxemia. STUDY DESIGN AND METHODS: Eighteen volunteers received 2 ng/kg LPS intravenously, followed by normal saline or 2- or 35-day-stored autologous RBC transfusion. Before LPS, before transfusion, and 6 hours after transfusion blood was collected to measure cytokine gene expression. Whole blood was used for ex vivo stimulation with LPS and LTA, after which cytokine levels were measured with enzyme-linked immunosorbent assay. RESULTS: In vivo LPS induced a biphasic response in cytokine mRNA with peak values 2 hours after LPS infusion. Storage time of RBC transfusion did not influence cytokine mRNA levels. In vivo infusion of LPS resulted in tolerance for ex vivo stimulation with LPS and LTA. However, transfusion of either fresh or stored RBCs did not further affect the capacity to produce cytokines after ex vivo stimulation. CONCLUSION: In a clinically relevant model of human endotoxemia, autologous transfusion of 35-day-stored RBCs does not influence cytokine mRNA levels nor does it change the capacity of white blood cells in whole blood to produce cytokines after ex vivo stimulation with LPS or LTA.

Transfusion of 35-Day Stored RBCs in the Presence of Endotoxemia Does Not Result in Lung Injury in Humans.

OBJECTIVE: Transfusion-related acute lung injury is the leading cause of transfusion-related mortality. Preclinical studies have shown that aged RBCs can induce transfusion-related acute lung injury in the presence of a "first hit" (e.g., sepsis). Clinical studies, however, show conflicting results on this matter. We tested whether maximally stored RBCs are able to induce lung injury in the presence of a "first hit" in humans (Dutch Trial Register: NTR4455). DESIGN: Open-label, randomized controlled trial. PATIENTS: Healthy male volunteers. INTERVENTIONS: Eighteen healthy male volunteers donated one unit of autologous RBCs 2 or 35 days before the experiment. The experiment was started by infusion of 2 ng/kg lipopolysaccharide ("first hit"). After 2 hours, volunteers received normal saline (n = 6), 2-day stored transfusion (n = 6), or 35-day stored transfusion (n = 6) ("second hit"). Blood was sampled hourly. Six hours after transfusion, the diffusion capacity of the lungs for carbon monoxide was tested and volunteers underwent spirometry, chest x-ray study, and a bronchoalveolar lavage. MEASUREMENTS AND MAIN RESULTS: All volunteers fulfilled sepsis criteria after lipopolysaccharide injection. The stored blood transfusion did not result in significant changes in either hemodynamic or respiratory variables compared with the control groups. Furthermore, chest x-rays, lung function, and PaO2/FIO2 ratios did not differ between groups. Transfusion of stored autologous RBCs did not result in an increased level of protein in the lungs or neutrophil influx. CONCLUSIONS: Transfusion of 35-day stored autologous RBCs in the presence of endotoxemia does not result in lung injury in humans.

Plasma suPAR as a prognostic biological marker for ICU mortality in ARDS patients.

PURPOSE: We investigated the prognostic value of plasma soluble urokinase plasminogen activator receptor (suPAR) on day 1 in patients with the acute respiratory distress syndrome (ARDS) for intensive care unit (ICU) mortality and compared it with established disease severity scores on day 1. METHODS: suPAR was determined batchwise in plasma obtained within 24 h after admission. RESULTS: 632 ARDS patients were included. Significantly (P = 0.02) higher median levels of suPAR were found with increasing severity of ARDS: 5.9 ng/ml [IQR 3.1-12.8] in mild ARDS (n = 82), 8.4 ng/ml [IQR 4.1-15.0] in moderate ARDS (n = 333), and 9.0 ng/ml [IQR 4.5-16.0] in severe ARDS (n = 217). Non-survivors had higher median levels of suPAR [12.5 ng/ml (IQR 5.1-19.5) vs. 7.4 ng/ml (3.9-13.6), P < 0.001]. The area under the receiver operator characteristic curve (ROC-AUC) for mortality of suPAR (0.62) was lower than the ROC-AUC of the APACHE IV score (0.72, P = 0.007), higher than that of the ARDS definition classification (0.53, P = 0.005), and did not differ from that of the SOFA score (0.68, P = 0.07) and the oxygenation index (OI) (0.58, P = 0.29). Plasma suPAR did not improve the discrimination of the established disease severity scores, but did improve net reclassification of the APACHE score (29%), SOFA score (23%), OI (38%), and Berlin definition classification (39%). CONCLUSION: As a single biological marker, the prognostic value for death of plasma suPAR in ARDS patients is low. Plasma suPAR, however, improves the net reclassification, suggesting a potential role for suPAR in ICU mortality prediction models.

Cardiac arrest patients have an impaired immune response, which is not influenced by induced hypothermia.

INTRODUCTION: Induced hypothermia is increasingly applied as a therapeutic intervention in ICUs. One of the underlying mechanisms of the beneficial effects of hypothermia is proposed to be reduction of the inflammatory response. However, a fear of reducing the inflammatory response is an increased infection risk. Therefore, we studied the effect of induced hypothermia on immune response after cardiac arrest. METHODS: A prospective observational cohort study in a mixed surgical-medical ICU. Patients admitted at the ICU after surviving cardiac arrest were included and during 24 hours body temperature was strictly regulated at 33°C or 36°C. Blood was drawn at three time points: after reaching target temperature, at the end of the target temperature protocol and after rewarming to 37°C. Plasma cytokine levels and response of blood leucocytes to stimulation with toll-like receptor (TLR) ligands lipopolysaccharide (LPS) from Gram-negative bacteria and lipoteicoic acid (LTA) from Gram-positive bacteria were measured. Also, monocyte HLA-DR expression was determined. RESULTS: In total, 20 patients were enrolled in the study. Compared to healthy controls, cardiac arrest patients kept at 36°C (n = 9) had increased plasma cytokines levels, which was not apparent in patients kept at 33°C (n = 11). Immune response to TLR ligands in patients after cardiac arrest was generally reduced and associated with lower HLA-DR expression. Patients kept at 33°C had preserved ability of immune cells to respond to LPS and LTA compared to patients kept at 36°C. These differences disappeared over time. HLA-DR expression did not differ between 33°C and 36°C. CONCLUSIONS: Patients after cardiac arrest have a modest systemic inflammatory response compared to healthy controls, associated with lower HLA-DR expression and attenuated immune response to Gram-negative and Gram-positive antigens, the latter indicative of an impaired immune response to bacteria. Patients with a body temperature of 33°C did not differ from patients with a body temperature of 36°C, suggesting induced hypothermia does not affect immune response in patients with cardiac arrest. TRIAL REGISTRATION: ClinicalTrials.gov NCT01020916, registered 25 November 2009.

TLR2 deficiency aggravates lung injury caused by mechanical ventilation.

Innate immunity pathways are found to play an important role in ventilator-induced lung injury. We analyzed pulmonary expression of Toll-like receptor 2 (TLR2) in humans and mice and determined the role of TLR2 in the pathogenesis of ventilator-induced lung injury in mice. Toll-like receptor 2 gene expression was analyzed in human bronchoalveolar lavage fluid (BALF) cells and murine lung tissue after 5 h of ventilation. In addition, wild-type (WT) and TLR2 knockout (KO) mice were ventilated with either lower tidal volumes (VT) of 7 mL/kg with positive end-expiratory pressure (PEEP) or higher VT of 15 mL/kg without PEEP for 5 h. Spontaneously breathing mice served as controls. Total protein and immunoglobulin M levels in BALF, neutrophil influx into the alveolar compartment, and interleukin 6 (IL-6), IL-1ß, and keratinocyte-derived chemokine concentrations in lung tissue homogenates were measured. We observed enhanced TLR2 gene expression in BALF cells of ventilated patients and in lung tissue of ventilated mice. In WT mice, ventilation with higher VT without PEEP resulted in lung injury and inflammation with higher immunoglobulin M levels, neutrophil influx, and levels of inflammatory mediators compared with controls. In TLR2 KO mice, neutrophil influx and IL-6, IL-1ß, and keratinocyte-derived chemokine were enhanced by this ventilation strategy. Ventilation with lower VT with PEEP only increased neutrophil influx and was similar in WT and TLR2 KO mice. In summary, injurious ventilation enhances TLR2 expression in lungs. Toll-like receptor 2 deficiency does not protect lungs from ventilator-induced lung injury. In contrast, ventilation with higher VT without PEEP aggravates inflammation in TLR2 KO mice.

The receptor for advanced glycation end products in ventilator-induced lung injury.

BACKGROUND: Mechanical ventilation (MV) can cause ventilator-induced lung injury (VILI). The innate immune response mediates this iatrogenic inflammatory condition. The receptor for advanced glycation end products (RAGE) is a multiligand receptor that can amplify immune and inflammatory responses. We hypothesized that RAGE signaling contributes to the pro-inflammatory state induced by MV. METHODS: RAGE expression was analyzed in lung brush and lavage cells obtained from ventilated patients and lung tissue of ventilated mice. Healthy wild-type (WT) and RAGE knockout (KO) mice were ventilated with relatively low (approximately 7.5 ml/kg) or high (approximately 15 ml/kg) tidal volume. Positive end-expiratory pressure was set at 2 cm H2O during both MV strategies. Also, WT and RAGE KO mice with lipopolysaccharide (LPS)-induced lung injury were ventilated with the above described ventilation strategies. In separate experiments, the contribution of soluble RAGE, a RAGE isoform that may function as a decoy receptor, in ventilated RAGE KO mice was investigated. Lung wet-to-dry ratio, cell and neutrophil influx, cytokine and chemokine concentrations, total protein levels, soluble RAGE, and high-mobility group box 1 (HMGB1) presence in lung lavage fluid were analyzed. RESULTS: MV was associated with increased RAGE mRNA levels in both human lung brush samples and lung tissue of healthy mice. In healthy high tidal volume-ventilated mice, RAGE deficiency limited inflammatory cell influx. Other VILI parameters were not affected. In our second set of experiments where we compared RAGE KO and WT mice in a 2-hit model, we observed higher pulmonary cytokine and chemokine levels in RAGE KO mice undergoing LPS/high tidal volume MV as compared to WT mice. Third, in WT mice undergoing the LPS/high tidal volume MV, we observed HMGB1 presence in lung lavage fluid. Moreover, MV increased levels of soluble RAGE in lung lavage fluid, with the highest levels found in LPS/high tidal volume-ventilated mice. Administration of soluble RAGE to LPS/high tidal volume-ventilated RAGE KO mice attenuated the production of inflammatory mediators. CONCLUSIONS: RAGE was not a crucial contributor to the pro-inflammatory state induced by MV. However, the presence of sRAGE limited the production of pro-inflammatory mediators in our 2-hit model of LPS and high tidal volume MV.

Serum levels of N-terminal proB-type natriuretic peptide in mechanically ventilated critically ill patients--relation to tidal volume size and development of acute respiratory distress syndrome.

BACKGROUND: Serum levels of N-terminal proB-type natriuretic peptide (NT-proBNP) are elevated in patients acute respiratory distress syndrome (ARDS). Recent studies showed a lower incidence of acute cor pulmonale in ARDS patients ventilated with lower tidal volumes. Consequently, serum levels of NT-proBNP may be lower in these patients. We investigated the relation between serum levels of NT-proBNP and tidal volumes in critically ill patients without ARDS at the onset of mechanical ventilation. METHODS: Secondary analysis of a randomized controlled trial of lower versus conventional tidal volumes in patients without ARDS. NT-pro BNP were measured in stored serum samples. Serial serum levels of NT-pro BNP were analyzed controlling for acute kidney injury, cumulative fluid balance and presence of brain injury. The primary outcome was the effect of tidal volume size on serum levels of NT-proBNP. Secondary outcome was the association with development of ARDS. RESULTS: Samples from 150 patients were analyzed. No relation was found between serum levels of NT-pro BNP and tidal volume size. However, NT-proBNP levels were increasing in patients who developed ARDS. In addition, higher levels were observed in patients with acute kidney injury, and in patients with a more positive cumulative fluid balance. CONCLUSION: Serum levels of NT-proBNP are independent of tidal volume size, but are increasing in patients who develop ARDS.

Pre-treatment with allopurinol or uricase attenuates barrier dysfunction but not inflammation during murine ventilator-induced lung injury.

INTRODUCTION: Uric acid released from injured tissue is considered a major endogenous danger signal and local instillation of uric acid crystals induces acute lung inflammation via activation of the NLRP3 inflammasome. Ventilator-induced lung injury (VILI) is mediated by the NLRP3 inflammasome and increased uric acid levels in lung lavage fluid are reported. We studied levels in human lung injury and the contribution of uric acid in experimental VILI. METHODS: Uric acid levels in lung lavage fluid of patients with acute lung injury (ALI) were determined. In a different cohort of cardiac surgery patients, uric acid levels were correlated with pulmonary leakage index. In a mouse model of VILI the effect of allopurinol (inhibits uric acid synthesis) and uricase (degrades uric acid) pre-treatment on neutrophil influx, up-regulation of adhesion molecules, pulmonary and systemic cytokine levels, lung pathology, and regulation of receptors involved in the recognition of uric acid was studied. In addition, total protein and immunoglobulin M in lung lavage fluid and pulmonary wet/dry ratios were measured as markers of alveolar barrier dysfunction. RESULTS: Uric acid levels increased in ALI patients. In cardiac surgery patients, elevated levels correlated significantly with the pulmonary leakage index. Allopurinol or uricase treatment did not reduce ventilator-induced inflammation, IκB-α degradation, or up-regulation of NLRP3, Toll-like receptor 2, and Toll-like receptor 4 gene expression in mice. Alveolar barrier dysfunction was attenuated which was most pronounced in mice pre-treated with allopurinol: both treatment strategies reduced wet/dry ratio, allopurinol also lowered total protein and immunoglobulin M levels. CONCLUSIONS: Local uric acid levels increase in patients with ALI. In mice, allopurinol and uricase attenuate ventilator-induced alveolar barrier dysfunction.