Exoenzyme Y Contributes to End-Organ Dysfunction Caused by Pseudomonas aeruginosa Pneumonia in Critically Ill Patients: An Exploratory Study.

Pseudomonas aeruginosa is an opportunistic pathogen that causes pneumonia in immunocompromised and intensive care unit (ICU) patients. During host infection, P. aeruginosa upregulates the type III secretion system (T3SS), which is used to intoxicate host cells with exoenzyme (Exo) virulence factors. Of the four known Exo virulence factors (U, S, T and Y), ExoU has been shown in prior studies to associate with high mortality rates. Preclinical studies have shown that ExoY is an important edema factor in lung infection caused by P. aeruginosa, although its importance in clinical isolates of P. aeruginosa is unknown. We hypothesized that expression of ExoY would be highly prevalent in clinical isolates and would significantly contribute to patient morbidity secondary to P. aeruginosa pneumonia. A single-center, prospective observational study was conducted at the University of Alabama at Birmingham Hospital. Mechanically ventilated ICU patients with a bronchoalveolar lavage fluid culture positive for P. aeruginosa were included. Enrolled patients were followed from ICU admission to discharge and clinical P. aeruginosa isolates were genotyped for the presence of exoenzyme genes. Ninety-nine patients were enrolled in the study. ExoY was present in 93% of P. aeruginosa clinical isolates. Moreover, ExoY alone (ExoY+/ExoU-) was present in 75% of P. aeruginosa isolates, compared to 2% ExoU alone (ExoY-/ExoU+). We found that bacteria isolated from human samples expressed active ExoY and ExoU, and the presence of ExoY in clinical isolates was associated with end-organ dysfunction. This is the first study we are aware of that demonstrates that ExoY is important in clinical outcomes secondary to nosocomial pneumonia.

Histone-Complexed DNA Fragments Levels are Associated with Coagulopathy, Endothelial Cell Damage, and Increased Mortality after Severe Pediatric Trauma.

BACKGROUND: The release of damage-associated molecular pattern molecules in the extracellular space secondary to injury has been shown to cause systemic activation of the coagulation system and endothelial cell damage. We hypothesized that pediatric trauma patients with increased levels of histone-complexed DNA fragments (hcDNA) would have evidence of coagulopathy and endothelial damage that would be associated with poor outcomes. METHODS: We conducted a prospective observational study of 149 pediatric trauma patients and 62 control patients at two level 1 pediatric trauma centers from 2013 to 2016. Blood samples were collected upon arrival and at 24 h, analyzed for hcDNA, coagulation abnormalities, endothelial damage, and clinical outcome. Platelet aggregation was assessed with impedance aggregometry (Multiplate) and coagulation parameters were assessed by measuring prothrombin time ratio in plasma and the use of viscoelastic techniques (Rotational Thromboelastometry) in whole blood. RESULTS: The median age was 8.3 years, the median injury severity score (ISS) was 20, and overall mortality was 10%. Significantly higher levels of hcDNA were found on admission in patients with severe injury (ISS > 25), coagulopathy, and/or abnormal platelet aggregation. Patients with high hcDNA levels also had significant elevations in plasma levels of syndecan-1, suggesting damage to the endothelial glycocalyx. Finally, significantly higher hcDNA levels were found in non-survivors. CONCLUSION: hcDNA is released following injury and correlates with coagulopathy, endothelial glycocalyx damage, and poor clinical outcome early after severe pediatric trauma. These results indicate that hcDNA may play an important role in development of coagulation abnormalities and endothelial glycocalyx damage in children following trauma.

THE GLYCOCALYX AND TRAUMA: A REVIEW.

In the United States trauma is the leading cause of mortality among those under the age of 45, claiming approximately 192,000 lives each year. Significant personal disability, lost productivity, and long-term healthcare needs are common and contribute 580 billion dollars in economic impact each year. Improving resuscitation strategies and the early acute care of trauma patients has the potential to reduce the pathological sequelae of combined exuberant inflammation and immune suppression that can co-exist, or occur temporally, and adversely affect outcomes. The endothelial and epithelial glycocalyx has emerged as an important participant in both inflammation and immunomodulation. Constituents of the glycocalyx have been used as biomarkers of injury severity and have the potential to be target(s) for therapeutic interventions aimed at immune modulation. In this review, we provide a contemporary understanding of the physiologic structure and function of the glycocalyx and its role in traumatic injury with a particular emphasis on lung injury.

Detection of acute traumatic coagulopathy and massive transfusion requirements by means of rotational thromboelastometry: an international prospective validation study.

INTRODUCTION: The purpose of this study was to re-evaluate the findings of a smaller cohort study on the functional definition and characteristics of acute traumatic coagulopathy (ATC). We also aimed to identify the threshold values for the most accurate identification of ATC and prediction of massive transfusion (MT) using rotational thromboelastometry (ROTEM) assays. METHODS: In this prospective international multicentre cohort study, adult trauma patients who met the local criteria for full trauma team activation from four major trauma centres were included. Blood was collected on arrival to the emergency department and analyzed with laboratory international normalized ratio (INR), fibrinogen concentration and two ROTEM assays (EXTEM and FIBTEM). ATC was defined as laboratory INR >1.2. Transfusion requirements of ≥10 units of packed red blood cells within 24 hours were defined as MT. Performance of the tests were evaluated by receiver operating characteristic curves, and calculation of area under the curve (AUC). Optimal cutoff points were estimated based on Youden index. RESULTS: In total, 808 patients were included in the study. Among the ROTEM parameters, the largest AUCs were found for the clot amplitude (CA) 5 value in both the EXTEM and FIBTEM assays. EXTEM CA5 threshold value of ≤37 mm had a detection rate of 66.3% for ATC. An EXTEM CA5 threshold value of ≤40 mm predicted MT in 72.7%. FIBTEM CA5 threshold value of ≤8 mm detected ATC in 67.5%, and a FIBTEM CA5 threshold value ≤9 mm predicted MT in 77.5%. Fibrinogen concentration ≤1.6 g/L detected ATC in 73.6% and a fibrinogen concentration ≤1.90 g/L predicted MT in 77.8%. Patients with either an EXTEM or FIBTEM CA5 below the optimum detection threshold for ATC received significantly more packed red blood cells and plasma. CONCLUSIONS: This study confirms previous findings of ROTEM CA5 as a valid marker for ATC and predictor for MT. With optimum threshold for EXTEM CA5 ≤ 40 mm and FIBTEM CA5 ≤ 9 mm, sensitivity is 72.7% and 77.5% respectively. Future investigations should evaluate the role of repeated viscoelastic testing in guiding haemostatic resuscitation in trauma.

Minimal invasive management of traumatic transection of the vertebral artery.

Stab wounds to the neck can be potentially lethal. They are often associated with vascular injury of the carotid artery and jugular vein. Injury of the vertebral artery is rarely seen. The injury can vary from severe bleeding after transection with hemorrhage into the surrounding soft tissues of the neck to dissection and separation of the intimal lining from the subjacent media of an artery and subsequent occlusion of the vessel. We report a case of traumatic vertebral artery transection managed by minimal invasive balloon occlusion.

Coagulopathy after severe pediatric trauma.

Trauma remains the leading cause of morbidity and mortality in the United States among children aged 1 to 21 years. The most common cause of lethality in pediatric trauma is traumatic brain injury. Early coagulopathy has been commonly observed after severe trauma and is usually associated with severe hemorrhage and/or traumatic brain injury. In contrast to adult patients, massive bleeding is less common after pediatric trauma. The classical drivers of trauma-induced coagulopathy include hypothermia, acidosis, hemodilution, and consumption of coagulation factors secondary to local activation of the coagulation system after severe traumatic injury. Furthermore, there is also recent evidence for a distinct mechanism of trauma-induced coagulopathy that involves the activation of the anticoagulant protein C pathway. Whether this new mechanism of posttraumatic coagulopathy plays a role in children is still unknown. The goal of this review is to summarize the current knowledge on the incidence and potential mechanisms of coagulopathy after pediatric trauma and the role of rapid diagnostic tests for early identification of coagulopathy. Finally, we discuss different options for treating coagulopathy after severe pediatric trauma.

Protein C and acute inflammation: a clinical and biological perspective.

The protein C system plays an active role in modulating severe systemic inflammatory processes such as sepsis, trauma, and acute respiratory distress syndrome (ARDS) via its anticoagulant and anti-inflammatory properties. Plasma levels of activated protein C (aPC) are lower than normal in acute inflammation in humans, except early after severe trauma when high plasma levels of aPC may play a mechanistic role in the development of posttraumatic coagulopathy. Thus, following positive results of preclinical studies, a clinical trial (PROWESS) with high continuous doses of recombinant human aPC given for 4 days demonstrated a survival benefit in patients with severe sepsis. This result was not confirmed by subsequent clinical trials, including the recently published PROWESS-SHOCK trial in patients with septic shock and a phase II trial with patients with nonseptic ARDS. A possible explanation for the major difference in outcome between PROWESS and PROWESS-SHOCK trials is that lung-protective ventilation was used for the patients included in the recent PROWESS-SHOCK, but not in the original PROWESS trial. Since up to 75% of sepsis originates from the lung, aPC treatment may not have added enough to the beneficial effect of lung-protective ventilation to show lower mortality. Thus whether aPC will continue to be used to modulate the acute inflammatory response in humans remains uncertain. Because recombinant human aPC has been withdrawn from the market, a better understanding of the complex interactions between coagulation and inflammation is needed before considering the development of new drugs that modulate both coagulation and acute inflammation in humans.

Early coagulopathy is an independent predictor of mortality in children after severe trauma.

To determine whether early coagulopathy affects the mortality associated with severe civilian pediatric trauma, trauma patients younger than 18 years admitted to a pediatric intensive care unit from 2001 to 2010 were evaluated. Patients with burns, primary asphyxiation, preexisting bleeding diathesis, lack of coagulation studies, or transferred from other hospitals more than 24 h after injury were excluded. Age, sex, race, mechanism of injury, initial systolic blood pressure, Glasgow Coma Scale score, Injury Severity Score, prothrombin time, partial thromboplastin time, platelet count, and international normalized ratio were recorded. An arterial or venous blood gas was performed, if clinically indicated. Coagulopathy was defined as an international normalized ratio greater than 1.2. The primary outcome was in-hospital mortality. Secondary outcomes were lengths of intensive care unit and hospital stay. Eight hundred three patients were included in the study. Overall mortality was 13.4%. The incidence of age-adjusted hypotension was 5.4%. Early coagulopathy was observed in 37.9% of patients. High Injury Severity Score and/or hypotension were associated with early coagulopathy and higher mortality. Early coagulopathy was associated with a modest increase in mortality in pediatric trauma patients without traumatic brain injury (TBI). In contrast, the combination of TBI and early coagulopathy was associated with a fourfold increase in mortality in this patient population. Early coagulopathy is an independent predictor of mortality in civilian pediatric patients with severe trauma. The increase in mortality was particularly significant in patients with TBI either isolated or combined with other injuries, suggesting that a rapid correction of this coagulopathy could substantially decrease the mortality after TBI in pediatric trauma patients.

[Transfusion policy in trauma involving massive blood loss]. / Transfusiebeleid bij trauma met massaal bloedverlies.

Severe haemorrhage is a significant cause of death in trauma patients. In the case of massive blood loss a combination of coagulation defects, acidosis and hypothermia arise, which are accompanied by high morbidity and mortality rates unless properly corrected. Research in wounded military showed that a high ratio of fresh frozen plasma to packed red blood cells (FFP:PRBC) seemed to have a positive effect on survival. These studies do not provide a definition of the ideal ratio FFP:PRBC; the ratio in which a positive effect is seen varies from 1:1 to 1:3. Unnecessary FFP transfusions in trauma patients without imminent severe haemorrhage increase the risk of complications such as multi-organ failure and acute respiratory distress syndrome. Additional research is required into the accuracy of diagnosis of acute coagulation disorders.

Early release of high mobility group box nuclear protein 1 after severe trauma in humans: role of injury severity and tissue hypoperfusion.

INTRODUCTION: High mobility group box nuclear protein 1 (HMGB1) is a DNA nuclear binding protein that has recently been shown to be an early trigger of sterile inflammation in animal models of trauma-hemorrhage via the activation of the Toll-like-receptor 4 (TLR4) and the receptor for the advanced glycation endproducts (RAGE). However, whether HMGB1 is released early after trauma hemorrhage in humans and is associated with the development of an inflammatory response and coagulopathy is not known and therefore constitutes the aim of the present study. METHODS: One hundred sixty eight patients were studied as part of a prospective cohort study of severe trauma patients admitted to a single Level 1 Trauma center. Blood was drawn within 10 minutes of arrival to the emergency room before the administration of any fluid resuscitation. HMGB1, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, von Willebrand Factor (vWF), angiopoietin-2 (Ang-2), Prothrombin time (PT), prothrombin fragments 1+2 (PF1+2), soluble thrombomodulin (sTM), protein C (PC), plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA) and D-Dimers were measured using standard techniques. Base deficit was used as a measure of tissue hypoperfusion. Measurements were compared to outcome measures obtained from the electronic medical record and trauma registry. RESULTS: Plasma levels of HMGB1 were increased within 30 minutes after severe trauma in humans and correlated with the severity of injury, tissue hypoperfusion, early posttraumatic coagulopathy and hyperfibrinolysis as well with a systemic inflammatory response and activation of complement. Non-survivors had significantly higher plasma levels of HMGB1 than survivors. Finally, patients who later developed organ injury, (acute lung injury and acute renal failure) had also significantly higher plasma levels of HMGB1 early after trauma. CONCLUSIONS: The results of this study demonstrate for the first time that HMGB1 is released into the bloodstream early after severe trauma in humans. The release of HMGB1 requires severe injury and tissue hypoperfusion, and is associated with posttraumatic coagulation abnormalities, activation of complement and severe systemic inflammatory response.

Role of small GTPases and alphavbeta5 integrin in Pseudomonas aeruginosa-induced increase in lung endothelial permeability.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe pneumonia associated with airspace flooding with protein-rich edema in critically ill patients. The type III secretion system is a major virulence factor and contributes to dissemination of P. aeruginosa. However, it is still unknown which particular bacterial toxin and which cellular pathways are responsible for the increase in lung endothelial permeability induced by P. aeruginosa. Thus, the first objective of this study was to determine the mechanisms by which this species causes an increase in lung endothelial permeability. The results showed that ExoS and ExoT, two of the four known P. aeruginosa type III cytotoxins, were primarily responsible for bacterium-induced increases in protein permeability across the lung endothelium via an inhibition of Rac1 and an activation of the RhoA signaling pathway. In addition, inhibition of the alphavbeta5 integrin, a central regulator of lung vascular permeability, prevented these P. aeruginosa-mediated increases in albumin flux due to endothelial permeability. Finally, prior activation of the stress protein response or adenoviral gene transfer of the inducible heat shock protein Hsp72 also inhibited the damaging effects of P. aeruginosa on the barrier function of lung endothelium. Taken together, these results demonstrate the critical role of the RhoA/alphavbeta5 integrin pathway in mediating P. aeruginosa-induced lung vascular permeability. In addition, activation of the stress protein response with pharmacologic inhibitors of Hsp90 may protect lungs against P. aeruginosa-induced permeability changes.

Angiopoietin-2, marker and mediator of endothelial activation with prognostic significance early after trauma?

OBJECTIVE: To measure plasma levels of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and vascular endothelial growth factor (VEGF) early after trauma and to determine their clinical significance. BACKGROUND: Angiopoietins and VEGF play a central role in the physiology and pathophysiology of endothelial cells. Ang-2 has recently been shown to have pathogenetic significance in sepsis and acute lung injury. Little is known about the role of angiopoietins and VEGF early after trauma. METHODS: Blood specimens from consecutive major trauma patients were obtained immediately upon arrival in the emergency department and plasma samples assayed for Ang-1, Ang-2, VEGF, markers of endothelial activation, protein C pathway, fibrinolytic system, and complement. Base deficit was used as a measure of tissue hypoperfusion. Data were collected prospectively. RESULTS: Blood samples were obtained from 208 adult trauma patients within 30 minutes after injury before any significant fluid resuscitation. Plasma levels of Ang-2, but not Ang-1 and VEGF were increased and correlated independently with severity of injury and tissue hypoperfusion. Furthermore, plasma levels of Ang-2 correlated with markers of endothelial activation, coagulation abnormalities, and activation of the complement cascade and were associated with worse clinical outcome. CONCLUSIONS: Ang-2 is released early after trauma with the degree proportional to both injury severity and systemic hypoperfusion. High levels of Ang-2 were associated with an activated endothelium, coagulation abnormalities, complement activation, and worse clinical outcome. These data indicate that Ang-2 is a marker and possibly a direct mediator of endothelial activation and dysfunction after severe trauma.