Search

Background: Biosampling studies in critically ill patients traditionally involve bedside collection of samples followed by local processing (ie. centrifugation, aliquotting, and freezing) and storage. However, community hospitals, which care for the majority of Canadian patients, often lack the infrastructure for local processing and storage of specimens. A potential solution is a "simplified" biosampling protocol whereby blood samples are collected at the bedside and then shipped to a central site for processing and storage. One potential limitation of this approach is that delayed processing may alter sample characteristics., Objective: To determine whether delays in blood sample processing affect the stability of cytokines (IL-6, TNF, IL-10, IFN-γ), chemokines (IL-8, IP-10, MCP-1, MCP-4, MIP-1α, MIP-1β), cell-free DNA (cfDNA) (released by dying cells), and blood clotting potential in human blood samples., Methods: Venous blood was collected into EDTA and citrate sample tubes and stored at room temperature (RT) or 4°C for progressive intervals up to 72 hours, prior to processing. Plasma cytokines and chemokines were quantified using single or multiplex immunoassays. cfDNA was measured using Picogreen DNA Quantification. Blood clotting potential was measured using a thrombin generation assay., Results: Blood samples were collected from 9 intensive care unit (ICU) patients and 7 healthy volunteers. Admission diagnoses for the ICU patients included sepsis, trauma, ruptured abdominal aortic aneurysm, intracranial hemorrhage, gastrointestinal bleed, and hyperkalemia. After pre-processing delays of up to 72 hours at RT or 4°C, no significant changes were observed in plasma cytokines, chemokines, cfDNA, or thrombin formation., Conclusions: Delayed sample processing for up to 72 hours at either RT or 4°C did not significantly affect cytokines, chemokines, cfDNA, or blood clotting potential in plasma samples from healthy volunteers and ICU patients. A "simplified" biosampling protocol is a feasible solution for conducting biosampling research at hospitals without local processing capacity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gyorffy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Despite older adults being more vulnerable to sepsis, most preclinical research on sepsis has been conducted using young animals. This results in decreased scientific validity since age is an independent predictor of poor outcome. In this study, we explored the impact of aging on the host response to sepsis using the fecal-induced peritonitis (FIP) model developed by the National Preclinical Sepsis Platform (NPSP)., Methods: C57BL/6 mice (3 or 12 months old) were injected intraperitoneally with rat fecal slurry (0.75 mg/g) or a control vehicle. To investigate the early stage of sepsis, mice were culled at 4 h, 8 h, or 12 h to investigate disease severity, immunothrombosis biomarkers, and organ injury. Mice received buprenorphine at 4 h post-FIP. A separate cohort of FIP mice were studied for 72 h (with buprenorphine given at 4 h, 12 h, and then every 12 h post-FIP and antibiotics/fluids starting at 12 h post-FIP). Organs were harvested, plasma levels of Interleukin (IL)-6, IL-10, monocyte chemoattract protein (MCP-1)/CCL2, thrombin-antithrombin (TAT) complexes, cell-free DNA (CFDNA), and ADAMTS13 activity were quantified, and bacterial loads were measured., Results: In the 12 h time course study, aged FIP mice demonstrated increased inflammation and injury to the lungs compared to young FIP mice. In the 72 h study, aged FIP mice exhibited a higher mortality rate (89%) compared to young FIP mice (42%) (p < 0.001). Aged FIP non-survivors also exhibited a trend towards elevated IL-6, TAT, CFDNA, CCL2, and decreased IL-10, and impaired bacterial clearance compared to young FIP non-survivors., Conclusion: To our knowledge, this is the first study to investigate the impact of age on survival using the FIP model of sepsis. Our model includes clinically-relevant supportive therapies and inclusion of both sexes. The higher mortality rate in aged mice may reflect increased inflammation and worsened organ injury in the early stage of sepsis. We also observed trends in impaired bacterial clearance, increase in IL-6, TAT, CFDNA, CCL2, and decreased IL-10 and ADAMTS13 activity in aged septic non-survivors compared to young septic non-survivors. Our aging model may help to increase the scientific validity of preclinical research and may be useful for identifying mechanisms of age-related susceptibility to sepsis as well as age-specific treatment strategies., (© 2024. The Author(s).)

Background: Preclinical sepsis models have been criticized for their inability to recapitulate human sepsis and suffer from methodological shortcomings that limit external validity and reproducibility. The National Preclinical Sepsis Platform (NPSP) is a consortium of basic science researchers, veterinarians, and stakeholders in Canada undertaking standardized multi-laboratory sepsis research to increase the efficacy and efficiency of bench-to-bedside translation. In this study, we aimed to develop and characterize a 72-h fecal-induced peritonitis (FIP) model of murine sepsis conducted in two independent laboratories. The experimental protocol was optimized by sequentially modifying dose of fecal slurry and timing of antibiotics in an iterative fashion, and then repeating the experimental series at site 1 and site 2., Results: Escalating doses of fecal slurry (0.5-2.5 mg/g) resulted in increased disease severity, as assessed by the modified Murine Sepsis Score (MSS). However, the MSS was poorly associated with progression to death during the experiments, and mice were found dead without elevated MSS scores. Administration of early antibiotics within 4 h of inoculation rescued the animals from sepsis compared with late administration of antibiotics after 12 h, as evidenced by 100% survival and reduced bacterial load in peritoneum and blood in the early antibiotic group. Site 1 and site 2 had statistically significant differences in mortality (60% vs 88%; p < 0.05) for the same dose of fecal slurry (0.75 mg/g) and marked differences in body temperature between groups., Conclusions: We demonstrate a systematic approach to optimizing a 72-h FIP model of murine sepsis for use in multi-laboratory studies. Alterations to experimental conditions, such as dose of fecal slurry and timing of antibiotics, have clear impact on outcomes. Differences in mortality between sites despite rigorous standardization warrants further investigations to better understand inter-laboratory variation and methodological design in preclinical studies., (© 2023. The Author(s).)

Protein C is a vitamin K dependant protein in plasma that plays an essential role in regulating the coagulation cascade and inflammatory response. As a result of its importance in these roles, it has been suggested as a biomarker for prognosis of patients affected by sepsis. Sepsis is a dysregulated host response to an infection that is the leading cause of mortality in U.S. hospitals and results in the highest cost of hospitalization. It was found that protein C concentration in non-surviving sepsis patients is significantly lower (1.8 μg mL -1 ) than in survivors and healthy patients who have a protein C concentration of 3.9-5.9 μg mL -1 . Current methods for diagnosing sepsis rely on expensive immunoassays or functional assays that require multiple steps for isolation and activation of protein C. We demonstrate in this paper a low cost, single step assay for detection of protein C in blood plasma. This was done by combining isoelectric gates with barium-immobilized metal affinity trapping. The electric field was optimized for use with immobilized metal affinity using COMSOL simulation. The integrated device was tested with samples containing buffered protein C, protein C in the presence of high concentration bovine serum albumin and alpha 1-proteinase inhibitor, and in blood plasma with spiked protein C. The stability of the measured values was tested by monitoring the intensity of a mixture of protein C with BSA and A1PI every minute to determine that measurement after 40 minutes was optimal. The results showed that the device could be used to distinguish a reduction in protein C from 4.46 μg mL -1 to 1.96 μg mL -1 with greater than 98% confidence in plasma making it suitable for sepsis prognosis.

Background: Extracellular histones exert cytotoxic and procoagulant effects which contribute to immunothrombosis in vascular diseases such as sepsis. Heparin has been shown to neutralize the pathologic effects of histones in vitro and in animal models., Objectives: To compare the effectiveness of unfractionated heparin (UFH), low-molecularweight heparin (LMWH), Vasoflux (lacks anticoagulant activity), and fondaparinux in neutralizing the cytotoxic and procoagulant activities of histones METHODS: Binding affinities between heparin variants and histone subunits were determined by Bio-layer Interferometry. The ability of heparin variants to diminish the cytotoxic and procoagulant effects of histones was studied by treating endothelial cells or monocytic THP-1 cells with histones ± heparin variants., Results: Unfractionated heparin, LMWH, and Vasoflux bind histone subunits with high affinities (K d <1 pM-66.7 nM) whereas fondaparinux exhibited a low affinity (K d of 3.06 µM-81.1 mM). UFH, LMWH, and Vasoflux neutralize histone-mediated cytotoxicity as well as monocytic procoagulant activity (as assessed by cell surface tissue factor and phosphatidylserine). In contrast, fondaparinux has no effect on these activities. All four heparin variants reverse histone-mediated impairment of APC generation in a dose-dependent manner., Conclusions: The ability of heparin to neutralize the cytotoxic and procoagulant effects of histones require heparin fragments >1.7 kDa and is independent of the antithrombin-binding pentasaccharide. In contrast, the ability of heparin to neutralize histone-mediated impairment of APC generation is independent of size and anticoagulant activity. These findings suggest that heparin variants may have differential therapeutic potential in vascular diseases associated with elevated levels of histones., (© 2022 International Society on Thrombosis and Haemostasis.)

Importance: Coronavirus disease 2019 patients have an increased risk of thrombotic complications that may reflect immunothrombosis, a process characterized by blood clotting, endothelial dysfunction, and the release of neutrophil extracellular traps. To date, few studies have investigated longitudinal changes in immunothrombosis biomarkers in these patients. Furthermore, how these longitudinal changes differ between coronavirus disease 2019 patients and noncoronavirus disease septic patients with pneumonia are unknown., Objectives: In this pilot observational study, we investigated the utility of immunothrombosis biomarkers for distinguishing between coronavirus disease 2019 patients and noncoronavirus disease septic patients with pneumonia. We also evaluated the utility of the biomarkers for predicting ICU mortality in these patients., Design Setting and Participants: The participants were ICU patients with coronavirus disease 2019 ( n = 14), noncoronavirus disease septic patients with pneumonia ( n = 19), and healthy age-matched controls ( n = 14)., Main Outcomes and Measures: Nine biomarkers were measured from plasma samples (on days 1, 2, 4, 7, 10, and/or 14). Analysis was based on binomial logit models and receiver operating characteristic analyses., Results: Cell-free DNA, d-dimer, soluble endothelial protein C receptor, protein C, soluble thrombomodulin, fibrinogen, citrullinated histones, and thrombin-antithrombin complexes have significant powers for distinguishing coronavirus disease 2019 patients from healthy individuals. In comparison, fibrinogen, soluble endothelial protein C receptor, antithrombin, and cell-free DNA have significant powers for distinguishing coronavirus disease 2019 from pneumonia patients. The predictors of ICU mortality differ between the two patient groups: soluble thrombomodulin and citrullinated histones for coronavirus disease 2019 patients, and protein C and cell-free DNA or fibrinogen for pneumonia patients. In both patient groups, the most recent biomarker values have stronger prognostic value than their ICU day 1 values., Conclusions and Relevance: Fibrinogen, soluble endothelial protein C receptor, antithrombin, and cell-free DNA have utility for distinguishing coronavirus disease 2019 patients from noncoronavirus disease septic patients with pneumonia. The most important predictors of ICU mortality are soluble thrombomodulin/citrullinated histones for coronavirus disease 2019 patients, and protein C/cell-free DNA for noncoronavirus disease pneumonia patients. This hypothesis-generating study suggests that the pathophysiology of immunothrombosis differs between the two patient groups., (Copyright © 2021 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine.)

Background: Excessive production of neutrophil extracellular traps (NETs) in sepsis contributes to vascular occlusion by acting as a scaffold and stimulus for thrombus formation. Removal of extracellular DNA, the major structural component of NETs, by DNase I may reduce host injury., Objectives: (1) To determine how heparin variants (unfractionated heparin, enoxaparin, Vasoflux, and fondaparinux) affect DNase I activity, (2) to measure temporal changes in circulating DNA and DNase I in septic patients., Methods: DNA–histone complexes were treated with DNase I ± heparin variants and visualized via agarose gels. We compared the ability of DNase I ± heparin variants to digest NETs released by phorbol 12-myristate 13-acetate-stimulated neutrophils versus DNA–histone complexes released by necrotic HEK293 cells. Plasma DNA and DNase I levels were measured longitudinally in 76 septic patients., Results: Heparin enhances DNase I-mediated digestion of DNA–histone complexes in a size-dependent manner that does not require the antithrombin-binding region. In contrast, DNase I alone was able to degrade the DNA–histone component of NETs presumably due to peptidylarginine deiminase 4 (PAD4)-mediated histone citrullination that weakens DNA–histone interactions. In purified systems, PAD4 treatment of DNA–histone complexes enhanced the ability of DNase I to degrade histone-bound DNA. In septic patients, endogenous DNase I levels remained persistently low over 28 days, and there were no significant correlations between DNA and DNase I levels., Conclusion: Heparin enhances DNA-mediated digestion of DNA–histone complexes in a size-dependent manner that is independent of its anticoagulant properties. Citrullination of histones by PAD4 renders DNA–histone complexes susceptible to DNase I digestion. Endogenous DNase I levels are persistently decreased in septic patients, which supports the potential utility of DNase I as a therapy for sepsis., Competing Interests: The authors report no conflicts of interest.

Background: Immunothrombosis and coagulopathy in the lung microvasculature may lead to lung injury and disease progression in coronavirus disease 2019 (COVID-19). We aim to identify biomarkers of coagulation, endothelial function, and fibrinolysis that are associated with disease severity and may have prognostic potential., Methods: We performed a single-center prospective study of 14 adult COVID-19(+) intensive care unit patients who were age- and sex-matched to 14 COVID-19(-) intensive care unit patients, and healthy controls. Daily blood draws, clinical data, and patient characteristics were collected. Baseline values for 10 biomarkers of interest were compared between the three groups, and visualized using Fisher's linear discriminant function. Linear repeated-measures mixed models were used to screen biomarkers for associations with mortality. Selected biomarkers were further explored and entered into an unsupervised longitudinal clustering machine learning algorithm to identify trends and targets that may be used for future predictive modelling efforts., Results: Elevated D-dimer was the strongest contributor in distinguishing COVID-19 status; however, D-dimer was not associated with survival. Variable selection identified clot lysis time, and antigen levels of soluble thrombomodulin (sTM), plasminogen activator inhibitor-1 (PAI-1), and plasminogen as biomarkers associated with death. Longitudinal multivariate k-means clustering on these biomarkers alone identified two clusters of COVID-19(+) patients: low (30%) and high (100%) mortality groups. Biomarker trajectories that characterized the high mortality cluster were higher clot lysis times (inhibited fibrinolysis), higher sTM and PAI-1 levels, and lower plasminogen levels., Conclusions: Longitudinal trajectories of clot lysis time, sTM, PAI-1, and plasminogen may have predictive ability for mortality in COVID-19., (© 2021 International Society on Thrombosis and Haemostasis.)

Background: Between 75% and 80% of patients with sepsis arrive in the hospital through the emergency department. Early diagnosis is important to alter patient prognosis, but currently, there is no reliable biomarker. The innate immune response links inflammation and coagulation. Several coagulation -related biomarkers are associated with poor prognosis in the ICU. The role of coagulation biomarkers to aid in early sepsis diagnosis has not previously been investigated. The objective of our study is to determine the individual or combined accuracy of coagulation and inflammation biomarkers with standard biochemical tests to diagnose adult septic patients presenting to the emergency department., Methods: in the Emergency Department is a prospective, observational cohort study with a target enrolment of 250 suspected septic patients from two Canadian emergency departments. The emergency physicians will enroll patients with suspected sepsis. Blood samples will be collected at two time points (initial presentation and 4 hr following). Patients will be adjudicated into septic, infected, or not infected status in accordance with the Sepsis-3 definitions. Patient demographics, cultures, diagnosis, and biomarkers will be reported using descriptive statistics. Optimal cut off values with sensitivity and specificity for each biomarker will be determined using C-statistics to distinguish between septic and nonseptic patients. Stepwise multiple logistic regression analysis with exclusion of nonsignificant covariates from the final model will be used to establish a panel of biomarkers., Conclusions: Our protocol describes the processes and methods for a pragmatic observational biomarker study in the emergency department. This study will seek to determine the potential diagnostic importance of early coagulation abnormalities to identify additional tools for sepsis diagnosis., Competing Interests: The authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2021 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine.)

Background: A limitation of diagnostic scoring systems for disseminated intravascular coagulation (DIC) is that once DIC is identified, it may be in a state of irreversible deterioration., Objectives: To identify hemostatic markers that can identify the pre-DIC state., Methods: This was a multi-center observational study of 357 septic patients. The incidence of DIC was determined using the International Society on Thrombosis and Haemostasis (ISTH) DIC Score. Markers of interest include components of the DIC score: protein C (PC), antithrombin (AT), and citrullinated histones (H3Cit), which is a marker of NETosis., Results: Out of 357 sepsis patients, 236 patients did not develop DIC (without-DIC), 79 patients had DIC on Day 1 (overt-DIC), and 42 patients developed DIC after Day 1 (pre-DIC). Compared to without-DIC patients, pre-DIC patients had decreased platelet count, increased international normalized ratio (INR), decreased PC and AT, and increased H3Cit. In contrast, D-dimer and fibrinogen levels did not differ between pre-DIC and without-DIC patients. Using receiver operating characteristics (ROC) analysis, we found that platelet count and INR in combination with PC and AT could discriminate pre-DIC from without-DIC. The area under the curve in the ROC analysis was 0.83 (95% confidence interval, 0.76 to 0.89)., Conclusion: Our study suggests that platelets and INR in combination with PC and AT can identify the pre-DIC state in septic patients. In contrast, D-dimer increased and fibrinogen decreased in the late (ie, overt) stages of DIC. Our data also suggest that NETosis contributes to the onset of DIC in sepsis., (© 2020 International Society on Thrombosis and Haemostasis.)

Introduction: Cell-free DNA (CFDNA) is the major structural component of neutrophil extracellular traps (NETs). CFDNA contributes to the prothrombotic potential of NETs by promoting thrombin generation and inhibiting fibrinolysis. Patients with venous thromboembolism (VTE) have elevated circulating nucleosomes (i.e. DNA-histone complexes). In this study, we investigated if CFDNA contributes to a procoagulant and an antifibrinolytic state in patients with unprovoked VTE., Materials and Methods: Plasma samples from patients with a first episode of unprovoked VTE were obtained from the D-Dimer Optimal Duration Study (DODS). We measured CFDNA plasma levels in 263 patients while on warfarin and 1-month after stopping. Thrombin generation assays and clot lysis assays were measured in patients after stopping warfarin. Comparisons were made with healthy controls., Results: CFDNA levels in VTE patients who stopped warfarin (5.53 μg/mL; 95%CI: 5.34-5.72) were higher than during warfarin therapy (3.11 μg/mL; 95%CI: 2.98-3.25; p < .001), and higher than in healthy controls (2.77 μg/mL; 95%CI: 2.42-3.11; p < .001). VTE patients had a procoagulant state as evidenced by a shorter lag time (30.8 min; 95%CI: 29.2-32.4) compared to controls (48.2 min; 95%CI :41.0-55.5; p < .001) and a greater endogenous thrombin potential (2656 nM∗min; 95%CI: 2479-2836) compared to healthy controls (1198 nM ∗ min; 95%CI: 793-1603). There was a higher proportion of clots generated from patient plasma that were resistant to lysis (43.7%) compared to healthy controls (46.3%; p < .05). CFDNA levels were not associated with enhanced thrombin generation or impaired fibrinolysis in VTE patients., Conclusion: CFDNA levels are elevated in patients with unprovoked VTE but do not correlate with the procoagulant or anti-fibrinolytic properties of patient plasma. This study suggests that additional factors in addition to CFDNA may contribute to the pathogenesis of VTE., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objectives: Epigenetic alterations are an important regulator of gene expression in health and disease; however, epigenetic data in sepsis are lacking. To demonstrate proof of concept and estimate effect size, we performed the first epigenome-wide methylation analysis of whole blood DNA samples from a cohort of septic and nonseptic critically ill patients., Design: A nested case-control study using genomic DNA isolated from whole blood from septic (n = 66) and nonseptic (n = 68) critically ill patients on "Day 1" of ICU admission. Methylation patterns were identified using Illumina 450K arrays with percent methylation expressed as β values. After quality control, 134 participants and 414,818 autosomal cytosine-phosphate-guanine sites were used for epigenome-wide methylation analyses., Setting: Tertiary care hospitals., Subjects: Critically ill septic and nonseptic patients., Interventions: Observational study., Measurements and Main Results: A total of 668 differentially methylated regions corresponding to 443 genes were identified. Known sepsis-associated genes included complement component 3; angiopoietin 2; myeloperoxidase; lactoperoxidase; major histocompatibility complex, class I, A; major histocompatibility complex, class II, isotype DR β I; major histocompatibility complex, class I, C; and major histocompatibility complex, class II, isotype DQ β I. When compared with whole blood gene expression data from seven external datasets containing septic and nonseptic patients, 81% of the differentially methylated region-associated genes were differentially expressed in one or more datasets and 31% in three or more datasets. Functional analysis showed enrichment for antigen processing and presentation, methyltransferase activity, cell adhesion, and cell junctions. Analysis by weighted gene coexpression network analysis revealed DNA comethylation modules that were associated with clinical traits including severity of illness, need for vasopressors, and length of stay., Conclusions: DNA methylation marks may provide important causal and potentially biomarker information in critically ill patients with sepsis.

To determine if a set of time-varying biological indicators can be used to: 1) predict the sepsis mortality risk over time and 2) generate mortality risk profiles., Design: Prospective observational study., Setting: Nine Canadian ICUs., Subjects: Three-hundred fifty-six septic patients., Interventions: None., Measurements and Main Results: Clinical data and plasma levels of biomarkers were collected longitudinally. We used a complementary log-log model to account for the daily mortality risk of each patient until death in ICU/hospital, discharge, or 28 days after admission. The model, which is a versatile version of the Cox model for gaining longitudinal insights, created a composite indicator (the daily hazard of dying) from the "day 1" and "change" variables of six time-varying biological indicators (cell-free DNA, protein C, platelet count, creatinine, Glasgow Coma Scale score, and lactate) and a set of contextual variables (age, presence of chronic lung disease or previous brain injury, and duration of stay), achieving a high predictive power (conventional area under the curve, 0.90; 95% CI, 0.86-0.94). Including change variables avoided misleading inferences about the effects of day 1 variables, signifying the importance of the longitudinal approach. We then generated mortality risk profiles that highlight the relative contributions among the time-varying biological indicators to overall mortality risk. The tool was validated in 28 nonseptic patients from the same ICUs who became septic later and was subject to 10-fold cross-validation, achieving similarly high area under the curve., Conclusions: Using a novel version of the Cox model, we created a prognostic tool for septic patients that yields not only a predicted probability of dying but also a mortality risk profile that reveals how six time-varying biological indicators differentially and longitudinally account for the patient's overall daily mortality risk., Competing Interests: Drs. Liaw, Dwivedi, and Medeiros received support for article research from the Canadian Institutes of Health Research (CIHR). Drs. Liaw and Medeiros disclosed government work. Dr. Fox-Robichaud’s institution received funding from CIHR, CIHR/Natural Sciences and Engineering Research Council of Canada, and Hamilton Academic Hospital Fund. Dr. Dodek’s institution received funding from McMaster University. Dr. Winston received grant support from the Alberta Lung Association and the Canadian Intensive Care Foundation. Dr. Lellouche received compensation for patient inclusions in the study, and he disclosed he is a co-founder, administrator, and consultant of Oxynov, R&D company. Dr. Marshall received patient recruitment fees per CIHR grant, and he received funding from Data Monitoring Committee, Asahi Kasei Pharmaceuticals and Baxter. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2019 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine.)

Background: Circulating cell-free DNA (cfDNA) may contribute to the pathophysiology of post-injury inflammation and coagulation in trauma. However, the source and mechanism of release of cfDNA in trauma is not well understood. One potential source of cfDNA is from Neutrophil Extracellular Traps (NETs), released by activated neutrophils during the process of NETosis. The primary objective of our study was to determine if cfDNA has prognostic utility in trauma. The secondary objective of this study was to determine the source of cfDNA in trauma compared to sepsis., Methods: We studied trauma patients from two prospective observational cohort studies: the DNA as a Prognostic Marker in ICU Patients (DYNAMICS) study and the Endotoxin in Polytrauma (ENPOLY) study. We also studied septic patients from the DYNAMICS study. Citrated plasma samples were collected longitudinally from the patients (days 1 to 7). The following molecules were measured in the plasma samples: cfDNA, protein C (PC), myeloperoxidase (MPO) (a marker of neutrophil activation), citrullinated Histone H3 (H3Cit, a marker of NETosis), cyclophilin A (a marker of necrosis), and caspase-cleaved K18 (a marker of apoptosis)., Results: A total of 77 trauma patients were included (n = 38 from DYNAMICS and n = 39 from ENPOLY). The median age was 49 years; 27.3% were female, and mortality was 16.9% at 28 days. Levels of cfDNA were elevated compared to healthy values but not significantly different between survivors and non-survivors. There was a positive correlation between MPO and cfDNA in septic patients (r = 0.424, p < 0.001). In contrast, there was no correlation between MPO and cfDNA in trauma patients (r = - 0.192, p = 0.115). Levels of H3Cit, a marker of NETosis, were significantly elevated in septic patients compared to trauma patients (p < 0.01) while apoptosis and necrosis markers did not differ between the two groups., Conclusion: Our studies suggest that the source and mechanism of release of cfDNA differ between trauma and sepsis patients. In sepsis, cfDNA is likely primarily released by activated neutrophils via the process of NETosis. In contrast, cfDNA in trauma appears to originate mainly from injured or necrotic cells. Although cfDNA is elevated in trauma and sepsis patients compared to healthy controls, cfDNA does not appear to have prognostic utility in trauma patients., Trial Registration: ClinicalTrials.gov Identifier: NCT01355042 . Registered May 17, 2011.

Background: Despite increasing ethical standards for conducting animal research, death is still often used as an endpoint in mouse sepsis studies. Recently, the Murine Sepsis Score (MSS), Mouse Clinical Assessment Score for Sepsis (M-CASS), and Mouse Grimace Scale (MGS) were developed as surrogate endpoint scoring systems for assessing pain and disease severity in mice. The objective of our study was to compare the effectiveness of these scoring systems and monitoring of body temperature for predicting disease progression and death in the cecal ligation and puncture (CLP) sepsis model, in order to better inform selection of surrogate endpoints for death in experimental sepsis., Methods: C57Bl/6J mice were subjected to control sham surgery, or moderate or severe CLP sepsis. All mice were monitored every 4 h for surrogate markers of death using modified versions of the MSS, M-CASS, and MGS scoring systems until 24 h post-operatively, or until endpoint (inability to ambulate) and consequent euthanasia., Results: Thirty percent of mice subjected to moderate severity CLP reached endpoint by 24 h post-CLP, whereas 100% undergoing severe CLP reached endpoint within 20 h. Modified MSS, M-CASS, and MGS scores all increased, while body temperature decreased, in a time-dependent and sepsis severity-dependent manner, although modified M-CASS scores showed substantial variability. Receiver operating characteristic curves demonstrate that the last recorded body temperature (AUC = 0.88; 95% CI 0.77-0.99), change in body temperature (AUC = 0.89; 95% CI 0.78-0.99), modified M-CASS (AUC = 0.93; 95% CI 0.85-1.00), and modified MSS (AUC = 0.95; 95% CI 0.88-1.01) scores are all robust for predicting death in CLP sepsis, whereas modified MGS (AUC = 0.78; 95% CI 0.63-0.92) is less robust., Conclusions: The modified MSS and body temperature are effective markers for assessing disease severity and predicting death in the CLP model, and should thus be considered as valid surrogate markers to replace death as an endpoint in mouse CLP sepsis studies.

Lipoteichoic acid (LTA) and lipopolysaccharide (LPS) are bacterial lipids that stimulate pro-inflammatory cytokine production, thereby exacerbating sepsis pathophysiology. Proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates uptake of cholesterol by downregulating hepatic lipoprotein receptors, including low-density lipoprotein (LDL) receptor (LDLR) and possibly LDLR-related protein-1 (LRP1). PCSK9 also negatively regulates Gram-negative LPS uptake by hepatocytes, however this mechanism is not completely characterized and mechanisms of Gram-positive LTA uptake are unknown. Therefore, our objective was to elucidate the mechanisms through which PCSK9 regulates uptake of LTA and LPS by investigating the roles of lipoproteins and lipoprotein receptors. Here we show that plasma PCSK9 concentrations increase transiently over time in septic and non-septic critically ill patients, with highly similar profiles over 14 days. Using flow cytometry, we demonstrate that PCSK9 negatively regulates LDLR-mediated uptake of LTA and LPS by HepG2 hepatocytes through an LDL-dependent mechanism, whereas LRP1 and high-density lipoprotein do not contribute to this uptake pathway. Bacterial lipid uptake by hepatocytes was not associated with cytokine production or hepatocellular injury. In conclusion, our study characterizes an LDL-dependent and LDLR-mediated bacterial lipid uptake pathway regulated by PCSK9, and provides evidence in support of PCSK9 inhibition as a potential therapeutic strategy for sepsis.

Introduction: When thrombin is bound to thrombomodulin (TM), it becomes a potent activator of protein C (PC) and thrombin-activable fibrinolysis inhibitor (TAFI). Activation of PC is enhanced when PC is bound to the endothelial protein C receptor (EPCR). Activated protein C (APC) inhibits thrombin generation while activated TAFI (TAFIa) attenuates fibrinolysis. To determine the impact of diminished EPCR function on thrombin generation and fibrinolysis we generated cells that expressed TM and a variant of EPCR (R96C) that does not bind PC., Methods: To determine the impact of EPCR on the generation of APC and TAFIa and how this affects thrombin generation and fibrinolysis we performed thrombin generation and clot lysis assays in the presence of cells expressing wild-type TM and EPCR (WT cells) or wild-type TM and the R96C variant of EPCR (R96C cells)., Results: In the presence of R96C cells, thrombin generation in normal plasma is increased, as a result of impaired PC activation when compared to WT cells. In addition, clot lysis is delayed in normal plasma in the presence of R96C cells, despite no increase in TAFI activation. In PC deficient plasma, clot lysis is delayed in the presence of WT and R96C cells as a result of increased TAFI activation., Conclusions: We demonstrate that impaired EPCR function can be detected by thrombin generation and clot lysis assays on cells expressing TM and EPCR. We also demonstrated that deficiency in EPCR has procoagulant effects that lead to a delay in clot lysis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Lung cancer is the second leading type of cancer, with venous thromboembolism being the second leading cause of death. Studies have shown increased levels of microparticles and cell-free DNA (CFDNA) in cancer patients, which can activate coagulation through extrinsic and intrinsic pathways, respectively. However, the impact of lung cancer chemotherapy on microparticle and/or CFDNA generation is not completely understood. The aim of the study was to study the effects of platinum-based chemotherapeutic agents on generation of procoagulant microparticles and CFDNA in vitro and in vivo. Microparticles were isolated from chemotherapy-treated monocytes, human umbilical vein endothelial cells, or cancer cells. Tissue factor (TF) and phosphatidylserine levels were characterized and thrombin/factor Xa generation assays were used to determine microparticle procoagulant activity. CFDNA levels were isolated from cell supernatants and plasma. A murine xenograft model of human lung carcinoma was used to study the procoagulant effects of TF microparticles and CFDNA in vivo. In vitro, platinum-based chemotherapy induced TF/phosphatidylserine microparticle shedding from A549 and A427 lung cancers cells, which enhanced thrombin generation in plasma in a FVII-dependent manner. CFDNA levels were increased in supernatants of chemotherapy-treated neutrophils and plasma of chemotherapy-treated mice. TF microparticles were elevated in plasma of chemotherapy-treated tumour-bearing mice. Plasma CFDNA levels are increased in chemotherapy-treated tumour-free mice and correlate with increased thrombin generation. In tumour-bearing mice, chemotherapy increases plasma levels of CFDNA and TF/phosphatidylserine microparticles. Platinum-based chemotherapy induces the shedding of TF/phosphatidylserine microparticles from tumour cells and the release of CFDNA from host neutrophils.

Objectives: Sepsis is characterized by systemic activation of inflammatory and coagulation pathways in response to infection. Recently, it was demonstrated that histones released into the circulation by dying/activated cells may contribute to sepsis pathology. Although the ability of extracellular histones to modulate the procoagulant activities of several cell types has been investigated, the influence of histones on the hemostatic functions of circulating monocytes is unknown. To address this, we investigated the ability of histones to modulate the procoagulant potential of THP-1 cells and peripheral blood monocytes, and examined the effects of plasmas obtained from septic patients to induce a procoagulant phenotype on monocytic cells., Methods/results: Tissue factor (TF) activity assays were performed on histone-treated THP-1 cells and blood monocytes. Exposure of monocytic cells to histones resulted in increases in TF activity, TF antigen, and phosphatidylserine exposure. Histones modulate the procoagulant activity via engagement of Toll-like receptors 2 and 4, and this effect was abrogated with inhibitory antibodies. Increased TF activity of histone-treated cells corresponded to enhanced thrombin generation in plasma determined by calibrated automated thrombography. Finally, TF activity was increased on monocytes exposed to plasma from septic patients, an effect that was attenuated in plasma from patients receiving unfractionated heparin (UFH)., Conclusions: Our studies suggest that increased levels of extracellular histones found in sepsis contribute to dysregulated coagulation by increasing TF activity of monocytes. These procoagulant effects can be partially ameliorated in sepsis patients receiving UFH, thereby identifying extracellular histones as a potential therapeutic target for sepsis treatment.

Introduction: Proprotein convertase subtilisin/kexin type 9 (PCSK9) targets lipoprotein receptors for degradation, thereby reducing hepatic lipid clearance. PCSK9 inhibition reduces mortality in septic mice, presumably through increased hepatic clearance of pathogen lipids due to increased lipoprotein receptor concentrations. However, PCSK9 overexpression in vivo has not been studied in sepsis. Therefore, this study aimed to evaluate the effects of differential PCSK9 expression on systemic infection, inflammation, and coagulation in sepsis., Methods: Wild-type, PCSK9 knockout (KO), and transgenic (Tg) mice that overexpress PCSK9 were subjected to sham surgery or cecal ligation and puncture (CLP). Bacterial loads were measured in lungs, peritoneal cavity fluid, and blood. Organ pathology was assessed in lungs, liver, and kidneys. Lung myeloperoxidase activity, and plasma concentrations of alanine aminotransferase (ALT), creatinine, cell-free DNA (cfDNA), protein C, thrombin-antithrombin (TAT) complexes, interleukin (IL)-6, and IL-10 were also measured 6 h postoperatively. Morbidity was assessed for 16 h following CLP., Results: Overexpression of PCSK9 in mice increased liver and kidney pathology, plasma IL-6, ALT, and TAT concentrations during sepsis, whereas PCSK9 KO mice exhibited reduced bacterial loads, lung and liver pathology, myeloperoxidase activity, plasma IL-10, and cfDNA during CLP-induced sepsis. All septic mice had reduced plasma levels of protein C, but the protein C ratio relative to normal was significantly decreased in PCSK9 Tg mice. Dyspnea, cyanosis, and overall grimace scores were greatest in septic mice overexpressing PCSK9, whereas PCSK9 KO mice retained core body temperature during sepsis., Conclusion: These findings demonstrate that PCSK9 deficiency confers protection against systemic bacterial dissemination, organ pathology, and tissue inflammation, particularly in the lungs and liver, while PCSK9 overexpression exacerbates multi-organ pathology as well as the hypercoagulable and pro-inflammatory states in early sepsis.

The opportunistic pathogen Pseudomonas aeruginosa causes a wide range of infections in multiple hosts by releasing an arsenal of virulence factors such as pyocyanin. Despite numerous reports on the pleiotropic cellular targets of pyocyanin toxicity in vivo, its impact on erythrocytes remains elusive. Erythrocytes undergo an apoptosis-like cell death called eryptosis which is characterized by cell shrinkage and phosphatidylserine (PS) externalization; this process confers a procoagulant phenotype on erythrocytes as well as fosters their phagocytosis and subsequent clearance from the circulation. Herein, we demonstrate that P. aeruginosa pyocyanin-elicited PS exposure and cell shrinkage in erythrocyte while preserving the membrane integrity. Mechanistically, exposure of erythrocytes to pyocyanin showed increased cytosolic Ca(2+) activity as well as Ca(2+) -dependent proteolytic processing of μ-calpain. Pyocyanin further up-regulated erythrocyte ceramide abundance and triggered the production of reactive oxygen species. Pyocyanin-induced increased PS externalization in erythrocytes translated into enhanced prothrombin activation and fibrin generation in plasma. As judged by carboxyfluorescein succinimidyl-ester labelling, pyocyanin-treated erythrocytes were cleared faster from the murine circulation as compared to untreated erythrocytes. Furthermore, erythrocytes incubated in plasma from patients with P. aeruginosa sepsis showed increased PS exposure as compared to erythrocytes incubated in plasma from healthy donors. In conclusion, the present study discloses the eryptosis-inducing effect of the virulence factor pyocyanin, thereby shedding light on a potentially important mechanism in the systemic complications of P. aeruginosa infection., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Objectives: Sepsis is characterized by systemic activation of inflammation and coagulation in response to infection. In sepsis, activated neutrophils extrude neutrophil extracellular traps composed of cell-free DNA (CFDNA) that not only trap pathogens but also provide a stimulus for clot formation. Although the effect of CFDNA on coagulation has been extensively studied, much less is known about the impact of CFDNA on fibrinolysis. To address this, we (1) investigated the relationship between CFDNA levels and fibrinolytic activity in sepsis and (2) determined the mechanisms by which CFDNA modulates fibrinolysis., Approach and Results: Plasma was collected from healthy and septic individuals, and CFDNA was quantified. Clot lysis assays were performed in plasma and purified systems, and lysis times were determined by monitoring absorbance. Clot morphology was assessed using scanning electron microscopy. Clots formed in plasma from septic patients containing >5 µg/mL CFDNA were dense in structure and resistant to fibrinolysis, a phenomenon overcome by deoxyribonuclease addition. These effects were recapitulated in control plasma supplemented with CFDNA. In a purified system, CFDNA delayed fibrinolysis but did not alter tissue-type plasminogen activator-induced plasmin generation. Using surface plasmon resonance, CFDNA bound plasmin with a Kd value of 4.2±0.3 µmol/L, and increasing concentrations of CFDNA impaired plasmin-mediated degradation of fibrin clots via the formation of a nonproductive ternary complex between plasmin, CFDNA, and fibrin., Conclusions: Our studies suggest that the increased levels of CFDNA in sepsis impair fibrinolysis by inhibiting plasmin-mediated fibrin degradation, thereby identifying CFDNA as a potential therapeutic target for sepsis treatment., (© 2015 American Heart Association, Inc.)

A rapid and accurate method to identify severe sepsis patients at high risk of death is critically needed for clinical practice. In a recent study, the concentration of cell-free DNA (cfDNA) in blood was found to be a prognostic indicator for ICU mortality in patients with severe sepsis. However, current DNA quantification techniques are time-consuming and involve extensive sample preparation. In this paper, we demonstrate a low-cost microfluidic device capable of rapid quantification of cfDNA in a small droplet (<10 μl) of blood plasma and whole blood in 5 min using only electrical power. The cfDNA in samples is selectively labeled by PicoGreen and is extracted and concentrated by electrophoresis into a gel by application of a DC potential of 9 V. This device has potential as a prognostic tool for early and rapid assessment of septic patients.

Purpose: Cell-free DNA (CFDNA) is elevated in sepsis and correlates with mortality. This DNA may come from nuclear, mitochondrial, or bacterial sources. Cell-free DNA from all three sources may play a pathogenic role in sepsis via activation of coagulation through the contact pathway, whereas CpG motifs on bacterial and mitochondrial DNA may additionally stimulate inflammatory responses via Toll-like receptor 9. This study elucidates the relative effects of nuclear, mitochondrial, and bacterial DNA on inflammatory and procoagulant pathways with relevance to sepsis., Methods: DNA was extracted from plasma of septic patients and control subjects, and nuclear and mitochondrial CFDNA concentrations were measured by quantitative polymerase chain reaction. Viability of primary cultured human neutrophils was measured by flow cytometry for phosphatidyl serine exposure and cell permeability to propidium iodide. Continuous thrombin generation was measured with a fluorogenic substrate (Technothrombin, Vienna, Austria). Interleukin 6 secretion was measured by enzyme-linked immunosorbent assay. Platelet activation was measured by flow cytometry for P-selectin and activated αIIbβ3., Results: Mitochondrial DNA and nuclear DNA were elevated in plasma from septic patients compared with control subjects. Both mitochondrial and bacterial DNA prolonged neutrophil viability. Bacterial DNA increased neutrophil interleukin 6 secretion, but mitochondrial and nuclear DNA did not. Nuclear, mitochondrial, and bacterial DNA increased thrombin generation in platelet-poor plasma to a similar degree in a FXI- and FXII-dependent manner, indicating dependence on the intrinsic pathway of coagulation. Independently of coagulation, DNA from all three sources was capable of causing activation of platelet integrin αIIbβ3., Conclusions: Cell-free DNA from nuclear, mitochondrial, and bacterial sources have varying proinflammatory effects, although all three have similar procoagulant and platelet-stimulating potential. The pathophysiological effects of CFDNA in sepsis may vary with the source of DNA.

Sepsis is characterized by systemic activation of coagulation and inflammation in response to microbial infection. Although cell-free DNA (cfDNA) released from activated neutrophils has antimicrobial properties, it may also exert harmful effects by activating coagulation and inflammation. The authors aimed to determine whether deoxyribonuclease (DNase) administration reduces cfDNA levels, attenuates coagulation and inflammation, suppresses organ damage, and improves outcome in a cecal ligation and puncture (CLP) model of polymicrobial sepsis. Healthy C57Bl/6 mice were subjected to CLP, a surgical procedure involving two punctures of the ligated cecum, or sham surgery (no ligation/puncture). Mice were given DNase or saline by intraperitoneal injection 2, 4, or 6 h after surgery. Two hours after treatment, organs were harvested and plasma levels of cfDNA, interleukin-6 (IL-6), IL-10, thrombin-antithrombin complexes, lung myeloperoxidase, creatinine, alanine transaminase, and bacterial load were quantified. Survival studies were also performed. The CLP-operated mice had rapid time-dependent elevations in cfDNA that correlated with elevations in IL-6, IL-10, and thrombin-antithrombin complexes and had organ damage in the lungs and kidneys. Administration of DNase at 2 h after CLP resulted in increased IL-6 and IL-10 levels and organ damage in the lungs and kidneys. In contrast, DNase administration at 4 or 6 h after CLP resulted in reduced cfDNA and IL-6 levels, increased IL-10, and suppressed organ damage and bacterial dissemination. Deoxyribonuclease administration every 6 h after CLP also rescued mice from death. Our studies are the first to demonstrate that delayed but not early administration of DNase may be protective in experimental sepsis.

The interaction of protein C (PC) with the endothelial PC receptor (EPCR) enhances activated PC (APC) generation. The physiological importance of EPCR has been demonstrated in EPCR knockout mice which show early embryonic lethality due to placental thrombosis. In order to study the role of EPCR independent of PC interaction, we generated an EPCR point mutation knock-in mouse (EPCR(R84A/R84A)) which lacks the ability to bind PC/APC. EPCR(R84A/R84A) mice are viable and reproduce normally. In response to thrombotic challenge with factor Xa/phospholipids, EPCR(R84A/R84A) mice generate more thrombin, less APC, and show increased fibrin deposition in lungs and heart compared with wild-type (WT) mice. EPCR(R84A/R84A) mice challenged with lipopolysaccharide generate less APC, more interleukin-6, and show increased neutrophil infiltration in the lungs compared with WT controls. Interestingly, EPCR(R84A/R84A) mice develop splenomegaly as a result of bone marrow (BM) failure. BM transplant experiments suggest a role for EPCR on hematopoietic stem cells and BM stromal cells in modulating hematopoiesis. Taken together, our studies suggest that impaired EPCR/PC-binding interactions not only result in procoagulant and proinflammatory effects, but also impact hematopoiesis., (© 2015 by The American Society of Hematology.)

Objective: Activation of neutrophils by microbial or inflammatory stimuli results in the release of neutrophil extracellular traps (NETs) that are composed of DNA, histones, and antimicrobial proteins. In purified systems, cell-free DNA (CFDNA) activates the intrinsic pathway of coagulation, whereas histones promote thrombin generation through platelet-dependent mechanisms. However, the overall procoagulant effects of CFDNA/histone complexes as part of intact NETs are unknown. In this study, we examined the procoagulant potential of intact NETs released from activated neutrophils. We also determined the relative contribution of CFDNA and histones to thrombin generation in plasmas from patients with sepsis., Approach and Results: NETs released from phorbyl myristate-activated neutrophils enhance thrombin generation in platelet-poor plasma. This effect was DNA dependent (confirmed by DNase treatment) and occurred via the intrinsic pathway of coagulation (confirmed with coagulation factor XII- and coagulation factor XI-depleted plasma). In platelet-rich plasma treated with corn trypsin inhibitor, addition of phorbyl myristate-activated neutrophils increased thrombin generation and shortened the lag time in a toll-like receptor-2- and toll-like receptor-4-dependent mechanism. Addition of DNase further augmented thrombin generation, suggesting that dismantling of the NET scaffold increases histone-mediated, platelet-dependent thrombin generation. In platelet-poor plasma samples from patients with sepsis, we found a positive correlation between endogenous CFDNA and thrombin generation, and addition of DNase attenuated thrombin generation., Conclusions: These studies examine the procoagulant activities of CFDNA and histones in the context of NETs. Our studies also implicate a role for the intrinsic pathway of coagulation in sepsis pathogenesis., (© 2014 American Heart Association, Inc.)

Objective: The interaction of protein C (PC) with the endothelial PC receptor (EPCR) enhances activated PC generation. We performed targeted gene sequencing of the PC gene (PROC) and EPCR genes (PROCR) in patients with unprovoked venous thromboembolism (VTE) to determine whether mutations that impair PC-EPCR interactions are associated with an increased risk of VTE., Approach and Results: We sequenced exon 3 of PROC and exons 2 and 3 of PROCR (the exons that encode the protein-protein binding domains of PC and EPCR) in 653 patients with unprovoked VTE and in 627 healthy controls. Five single nucleotide variants, each in individual patients, were identified that result in abnormal PC (Arg9Cys, Val34Met, and Arg-1Cys) or abnormal EPCR proteins (Arg96Cys and Val170Leu). We did not detect any nonsynonymous coding variants in the controls. When the PC variants were expressed in human embryonic kidney 293 cells, all exhibited decreased synthesis, and 2 of the variants had reduced capacity for activated PC generation. When expressed on the surface of human embryonic kidney 293 cells, the EPCR variants showed reduced affinity for fluorescently labeled PC. In addition, the previously reported EPCR A3 haplotype, which promotes cellular shedding of EPCR, is over-represented in the patient group (P=0.001)., Conclusions: This is the first targeted DNA sequencing analysis of PROC and PROCR in a large group of patients with unprovoked VTE. Our data suggest that mutations that impair PC-EPCR interactions may be associated with an increased risk of VTE.

Introduction: Although sepsis is the leading cause of death in noncoronary critically ill patients, identification of patients at high risk of death remains a challenge. In this study, we examined the incremental usefulness of adding multiple biomarkers to clinical scoring systems for predicting intensive care unit (ICU) mortality in patients with severe sepsis., Methods: This retrospective observational study used stored plasma samples obtained from 80 severe sepsis patients recruited at three tertiary hospital ICUs in Hamilton, Ontario, Canada. Clinical data and plasma samples were obtained at study inclusion for all 80 patients, and then daily for 1 week, and weekly thereafter for a subset of 50 patients. Plasma levels of cell-free DNA (cfDNA), interleukin 6 (IL-6), thrombin, and protein C were measured and compared with clinical characteristics, including the primary outcome of ICU mortality and morbidity measured with the Multiple Organ Dysfunction (MODS) score and Acute Physiology and Chronic Health Evaluation (APACHE) II scores., Results: The level of cfDNA in plasma at study inclusion had better prognostic utility than did MODS or APACHE II scores, or the biomarkers measured. The area under the receiver operating characteristic (ROC) curves for cfDNA to predict ICU mortality is 0.97 (95% CI, 0.93 to 1.00) and to predict hospital mortality is 0.84 (95% CI, 0.75 to 0.94). We found that a cfDNA cutoff value of 2.35 ng/μl had a sensitivity of 87.9% and specificity of 93.5% for predicting ICU mortality. Sequential measurements of cfDNA suggested that ICU mortality may be predicted within 24 hours of study inclusion, and that the predictive power of cfDNA may be enhanced by combining it with protein C levels or MODS scores. DNA-sequence analyses and studies with Toll-like receptor 9 (TLR9) reporter cells suggests that the cfDNA from sepsis patients is host derived., Conclusions: These studies suggest that cfDNA provides high prognostic accuracy in patients with severe sepsis. The serial data suggest that the combination of cfDNA with protein C and MODS scores may yield even stronger predictive power. Incorporation of cfDNA in sepsis risk-stratification systems may be valuable for clinical decision making or for inclusion into sepsis trials.

Transforming growth factor beta (TGFbeta) has been known to play a role in anterior subcapsular cataract (ASC) formation and posterior capsule opacification (PCO), both of which are fibrotic pathologies of the lens. Several models have been utilized to study ASC formation, including the TGFbeta1 transgenic mouse model and the ex-vivo rat lens model. A distinct characteristic of ASC development within these models includes the formation of isolated fibrotic plaques or opacities which form beneath the lens capsule. A hallmark feature of ASC formation is the epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) into myofibroblasts. Recently, the matrix metalloproteinases (MMPs) have been implicated in the formation of these cataracts through their involvement in EMT. In the present study, we sought to further investigate the role of MMPs in subcapsular cataract formation in a time course manner, through the examination of gene expression and morphological changes which occur during this process. RT-QPCR and immunohistochemical analysis was carried out on lenses treated with TGFbeta for a period of 2, 4 and 6 days. Laser capture microdissection (LCM) was utilized to specifically isolate cells within the plaque region and cells from the adjacent epithelium in lenses treated for a 6 day period. Multilayering of LECs was observed as early as day 2, which preceded the presence of alpha smooth muscle actin (alpha-SMA) immunoreactivity that was evident following 4 days of treatment with TGFbeta. A slight reduction in E-cadherin mRNA was detected at day 2, although this was not significant until the day 4 time point. Importantly, our results also indicate an early induction of MMP-9 mRNA following 2 days of TGFbeta treatment, whereas MMP-2 was found to be upregulated at the later 4 day time point. Further experiments using FHL 124 cells show an induction in MMP-2 protein levels following treatment with recombinant MMP-9. Together these findings suggest an upstream role for MMP-9 in ASC formation.

Keratocytes, also known as fibroblasts, are mesencyhmal-derived cells of the corneal stroma. These cells are normally quiescent, but they can readily respond and transition into repair phenotypes following injury. Cytokines and other growth factors that provide autocrine signals for stimulating wound responses in resident cells are typically presented by platelets at the site of an injury. However, due to the avascular nature of the cornea many of the environmental cues are derived from the overlying epithelium. Corneal epithelial-keratocyte cell interactions have thus been extensively studied in numerous in vivo corneal wound healing settings, as well as in in vitro culture models. Exposure to the different epithelial-derived factors, as well as the integrity of the epithelial substratum, are factors known to impact the keratocyte response and determine whether corneal repair will be regenerative or fibrotic in nature. Finally, the recent identification of bone-marrow derived stem cells in the corneal stroma suggests a further complexity in the regulation of the keratocyte phenotype following injury.

The pleotropic morphogen transforming growth factor-beta (TGFbeta) plays an important role in the development of fibrotic pathologies, including anterior subcapsular cataracts (ASCs). ASC formation involves increased proliferation and transition of lens epithelial cells into myofibroblasts, through epithelial-mesenchymal transformation that results in opaque plaques beneath the lens capsule. In this study, we used a previously established TGFbeta-induced rat cataract model to explore the role of matrix metalloproteinases (MMPs) in ASC formation. Treatment of excised rat lenses with TGFbeta resulted in enhanced secretion of MMP-2 and MMP-9. Importantly, co-treatment with two different MMP inhibitors (MMPIs), the broad spectrum inhibitor GM6001 and an MMP-2/9-specific inhibitor, suppressed TGFbeta-induced ASC changes, including the epithelial-mesenchymal transformation of lens epithelial cells. Using an anti-E-cadherin antibody, we revealed that conditioned media from lenses treated with TGFbeta contained a 72-kd E-cadherin fragment, indicative of E-cadherin shedding. This was accompanied by attenuated levels of E-cadherin mRNA. Conditioned media from lenses co-treated with TGFbeta and MMPIs exhibited attenuated levels of the E-cadherin fragment compared with those from TGFbeta-treated lenses. Together, these findings demonstrate that TGFbeta-induced E-cadherin shedding in the lens is mediated by MMPs and that suppression of this phenomenon might explain the mechanism by which MMPIs inhibit ASC plaque formation.

Purpose: The present study was undertaken to create a conditional knockout of AP-2alpha in the corneal epithelium., Methods: A line of mice expressing Cre-recombinase specifically in the early lens placode was crossed with mice in which the AP-2alpha allele is flanked by two loxP sites. The resultant Le-AP-2alpha mutants exhibited a targeted deletion of AP-2alpha in lens placode derivatives, including the differentiating corneal epithelium., Results: The Le-AP-2alpha mutant mice were viable and had a normal lifespan. The adult corneal epithelium exhibited a variation in the number of stratified epithelial layers, ranging from 2 to 10 cell layers. A substantial decrease in expression of the cell-cell adhesion molecule, E-cadherin, was observed in all layers of the Le-AP-2alpha mutant corneal epithelium. The basement membrane, or Bowman's layer, was thinner in the mutant cornea and in many regions was discontinuous. These defects corresponded with altered distribution of laminin and entactin, and to a lesser degree, type IV collagen. The Le-AP-2alpha mutant cornea also exhibited stromal defects, including disrupted organization of the collagen lamellae and accumulation of fibroblasts beneath the epithelium that showed increased immunoreactivity for proliferating cell nuclear antigen (PCNA), alpha-smooth muscle actin (alpha-SMA), p-Smad2, and TGF-beta2., Conclusions: In the absence of AP-2alpha, the corneal epithelium exhibits altered cell adhesion and integrity and defects in its underlying basement membrane. These defects likely caused the alterations in the corneal stroma.