Martijn D. de Kruif

Induction of IRAK-M Is Associated with Lipopolysaccharide Tolerance in a Human Endotoxemia Model

Recent in vitro and murine in vivo studies have identified several potential LPS tolerance factors. In this study, we describe the expression kinetics of these LPS tolerance factors in standardized human endotoxemia models using i.v. LPS bolus administration. Responsiveness to LPS as well as the expression of potential regulators of LPS signaling were determined in peripheral whole blood. Intravenous LPS administration (4 ng/kg) resulted in peak plasma levels of TNF-α at 1.5 h followed by subsequent peaks of the classic negative feedback inhibitors A20 and IL-10 at 2 and 3 h, respectively. Circulating blood monocyte counts decimated during the initial inflammatory response, but normalized in the period between 4 and 8 h post-LPS. The LPS response as determined by ex vivo TNF release per monocyte in whole blood was profoundly decreased at 6–8 h post-LPS injection despite cessation of A20 and IL-10 expression after 4 h. Analysis of MyD88short, IL-1R-associated kinase (IRAK)-1, IRAK-M, ST2, suppressor of cytokine signaling-1 and -3, SHIP-1, and MAP kinase phosphatase-1 expression indicated that the observed LPS tolerance was associated with decreased IRAK-1 and elevated IRAK-M expression in this human model. Interestingly, a lower dose of LPS (1 ng/kg) induced LPS tolerance accompanied with IRAK-M up-regulation but without depletion of IRAK-1. In vitro studies in whole blood showed that IRAK-M up-regulation by LPS is largely dependent on TNF-α. The observed rise of IRAK-M transcription in the human endotoxemia model appeared much greater compared with in vitro-stimulated whole blood. In conclusion, LPS tolerance in human endotoxemia models is associated with IRAK-M up-regulation.

In Vivo Lipopolysaccharide Exposure of Human Blood Leukocytes Induces Cross-Tolerance to Multiple TLR Ligands

In vitro and in vivo experiments in mice have shown that exposure of cells to the TLR4 ligand LPS induces tolerance toward a second exposure to LPS and induces cross-tolerance to certain other TLR ligands. Recently, we found that LPS tolerance in experimental human endotoxemia and Gram-negative sepsis is associated with elevated levels of IL-1R-associated kinase M, an intracellular negative regulator of MyD88-dependent TLR signaling. In the present study, we investigated whether in vivo exposure of humans to LPS induces tolerance in circulating leukocytes to other TLR agonists that rely either on MyD88- dependent or on MyD88-independent signaling. Analysis of TNF, IL-1β, IL-6, and IL-10 levels in whole blood demonstrated that leukocytes were hyporesponsive to ex vivo LPS restimulation 3–8 h after i.v. LPS injection (4 ng/kg). Reduced cytokine release during the same interval was also observed in whole blood further stimulated with MyD88-dependent ligands for TLR2, TLR5, and TLR7 or with whole bacteria. Strikingly, blood leukocytes were also tolerant to a ligand for TLR3, which signals solely through a MyD88-independent (Toll IL-1R domain-containing adaptor-inducing IFN-β (TRIF)-dependent) pathway. The hyporesponsiveness of leukocytes to TLR3 ligation was associated with reduced rather than increased levels of the recently identified TRIF inhibitor SARM. Taken together, these data indicate that systemic LPS challenge of human volunteers induces cross-tolerance to multiple TLR ligands that signal in a MyD88-dependent or MyD88-independent manner and suggest that LPS exposure of human blood leukocytes may hamper the inflammatory response to various microbial components.

Prednisolone Dose-Dependently Influences Inflammation and Coagulation during Human Endotoxemia

The effects of steroids on the outcome of sepsis are dose dependent. Low doses appear to be beneficial, but high doses do not improve outcome for reasons that are insufficiently understood. The effects of steroids on systemic inflammation as a function of dose have not previously been studied in humans. To determine the effects of increasing doses of prednisolone on inflammation and coagulation in humans exposed to LPS, 32 healthy males received prednisolone orally at doses of 0, 3, 10, or 30 mg (n = 8 per group) at 2 h before i.v. injection of Escherichia coli LPS (4 ng/kg). Prednisolone dose-dependently inhibited the LPS-induced release of cytokines (TNF-α and IL-6) and chemokines (IL-8 and MCP-1), while enhancing the release of the anti-inflammatory cytokine IL-10. Prednisolone attenuated neutrophil activation (plasma elastase levels) and endothelial cell activation (von Willebrand factor). Most remarkably, prednisolone did not inhibit LPS-induced coagulation activation, measured by plasma concentrations of thrombin-antithrombin complexes, prothrombin fragment F1+2, and soluble tissue factor. In addition, activation of the fibrinolytic pathway (tissue-type plasminogen activator and plasmin-α2-antiplasmin complexes) was dose-dependently enhanced by prednisolone. These data indicate that prednisolone dose-dependently and differentially influences the systemic activation of different host response pathways during human endotoxemia.

Effects on Coagulation and Fibrinolysis Induced by Influenza in Mice With a Reduced Capacity to Generate Activated Protein C and a Deficiency in Plasminogen Activator Inhibitor Type 1

Influenza infections increase the risk of diseases associated with a prothrombotic state, such as venous thrombosis and atherothrombotic diseases. However, it is unclear whether influenza leads to a prothrombotic state in vivo. To determine whether influenza activates coagulation, we measured coagulation and fibrinolysis in influenza-infected C57BL/6 mice. We found that influenza increased thrombin generation, fibrin deposition, and fibrinolysis. In addition, we used various anti- and prothrombotic models to study pathways involved in the influenza-induced prothrombotic state. A reduced capacity to generate activated protein C in TMpro/pro mice increased thrombin generation and fibrinolysis, whereas treatment with heparin decreased thrombin generation in influenza-infected C57Bl/6 mice. Thrombin generation was not changed in hyperfibrinolytic mice, deficient in plasminogen activator inhibitor type-1 (PAI-1−/−); however, increased fibrin degradation was seen. Treatment with tranexamic acid reduced fibrinolysis, but thrombin generation was unchanged. We conclude that influenza infection generates thrombin, increased by reduced levels of protein C and decreased by heparin. The fibrinolytic system appears not to be important for thrombin generation. These findings suggest that influenza leads to a prothrombotic state by coagulation activation. Heparin treatment reduces the influenza induced prothrombotic state.

Additional value of procalcitonin for diagnosis of infection in patients with fever at the emergency department

Objective: First, to determine whether procalcitonin (PCT) significantly adds diagnostic value in terms of sensitivity and specificity to a common set of markers of infection, including C-reactive protein (CRP), at the Emergency Department. Second, to create a simple scoring rule implementing PCT values. Third, to determine and compare associations of CRP and PCT with clinical outcomes. Design: The additional diagnostic value of PCT was determined using multiple logistic regression analysis. A score was developed to help distinguish patients with a culture-proven bacterial infection from patients not needing antibiotic treatment using 16 potential clinical and laboratory variables. The prognostic value of CRP and PCT was determined using Spearmans correlation and logistic regression. Setting: Emergency Department of a 310-bed teaching hospital. Patients: Patients between 18 and 85 years old presenting with fever to the Emergency Department. Interventions: None. Measurements and Main Results: A total of 211 patients were studied (infection confirmed, n = 73; infection likely, n = 58; infection not excluded, n = 46; no infection, n = 34). CRP and chills were the strongest predictors for the diagnosis of bacterial infection. After addition of PCT to these parameters, model fit significantly improved (p = .003). The resulting scoring rule (score = 0.01 * CRP + 2 * chills + 1 * PCT) was characterized by an AUC value of 0.83 (sensitivity 79%; specificity of 71%), which was more accurate than physician judgment or SIRS (systemic inflammatory response syndrome). PCT levels were significantly associated with admission to a special care unit, duration of intravenous antibiotic use, total duration of antibiotic treatment, and length of hospital stay, whereas CRP was related only to the latter two variables. Conclusions: These data suggest that PCT may be a valuable addition to currently used markers of infection for diagnosis of infection and prognosis in patients with fever at the Emergency Department.

The Hemostatic Balance in HIV-Infected Patients with and without Antiretroviral Therapy: Partial Restoration with Antiretroviral Therapy

The incidence of arterial and venous thrombosis in HIV-infected patients is increased compared to healthy controls. In this cross-sectional analysis we measured markers of endothelial cell activation, thrombin generation, fibrinolysis and anticoagulation combined with endogenous thrombin potential (ETP) and activated protein C sensitivity ratio (APCsr) as more global markers. We included 160 consecutive HIV-infected patients with a median age of 46 years (range, 27-77), of whom 92% were male, 74% Caucasian, 11% African American, 9% Hispanic, and 6% Asian. Homosexual contact was the main transmission mode. Seventy percent of patients were using combined antiretroviral therapy (cART). In 83% of patients laboratory markers outside the normal range for a non-HIV-infected population were observed. Significant lower levels of von Willebrand factor (vWF; p = 0.03), factor VIII (p < 0.0001), D-dimer (p = 0.01), and ETP (p = 0.01) were observed in HIV-infected patients on cART compared to patients not on cART. Significant lower levels of protein C (p = 0.05) and free protein S (p < 0.0001), and increased APCsr (p < 0.0001) were found in the HIV-infected patients not on cART. A single association was observed between raised levels of fibrinogen and use of a protease inhibitor (p = 0.002). No significant difference was observed in the percentage of patients with laboratory markers outside the normal range between patients using cART-regimens containing abacavir, stavudine, or didanosine and those with other nucleoside reverse transcriptase inhibitors. Although the prevalence of coagulation abnormalities was lower in HIV-infected patients using cART, a considerable proportion of HIV-infected patients on cART show endothelial cell activation and increased APCsr, suggestive of a persistent procoagulant state.

Prescription of physical activity is not sufficient to change sedentary behavior and improve glycemic control in type 2 diabetes patients

OBJECTIVE To assess the impact of personalized exercise prescription on habitual physical activity and glycemic control in sedentary, insulin treated type 2 diabetes patients during a 2-y intervention period. RESEARCH DESIGN AND METHODS 74 patients were randomized to the intervention (n=38) or control (n=36) group. The intervention group was stimulated to increase daily physical activity through regular, structured, and personalized exercise prescription by a physical therapist over the 2-y intervention period. RESULTS Physical activity levels at work or in leisure time were not modulated by the exercise prescription intervention. In accordance, no changes in body composition, glycemic control, medication use or risk factors for cardiovascular disease were observed. CONCLUSIONS Long-term behavioral intervention programs, providing individualized exercise prescription, are not sufficient to change sedentary behavior and/or improve glycemic control in insulin treated, type 2 diabetes patients.

Dobutamine does not influence inflammatory pathways during human endotoxemia

Objective:Catecholamines have anti-inflammatory and anticoagulant properties. Dobutamine is a synthetic catecholamine frequently used in patients with septic myocardial dysfunction. The objective was to determine whether a continuous infusion of dobutamine exerts immunomodulatory effects in healthy volunteers challenged with endotoxin. Design:Prospective, open-label study. Setting:Clinical research unit of a university hospital. Participants:Sixteen male healthy volunteers. Interventions:Volunteers received a constant infusion with dobutamine (10 &mgr;g·kg−1·min−1, n = 8) or physiologic saline (n = 8). All participants were challenged with a bolus injection of endotoxin prepared from Escherichia coli (4 ng/kg). Dobutamine infusion was commenced 1 hr before endotoxin challenge and was continued until 3 hrs thereafter. Measurements and Main Results:Dobutamine infusion was associated with an increase in mean arterial blood pressure (peak 122 ± 5 mm Hg) and heart rate (peak 84 ± 4 beats/min, both p < .05 vs. saline). Endotoxin injection induced the systemic release of cytokines (tumor necrosis factor-α, interleukins-6, -8, and -10) and secretory phospholipase A2, endothelial cell activation (increase in the plasma levels of soluble E-selectin and von Willebrand factor), activation of coagulation (increased plasma levels of soluble tissue factor, F1 + 2 prothrombin fragment, and thrombin-antithrombin complexes), and activation with subsequent inhibition of fibrinolysis (increased plasma concentrations of tissue-type plasminogen activator, plasminogen activator inhibitor type I, and plasmin-α2-antiplasmin complexes). None of these responses were influenced by dobutamine. Conclusions:Dobutamine, infused in a clinically relevant dose, does not influence inflammatory and coagulant pathways during human endotoxemia.

Differential dose-dependent effects of prednisolone on shedding of endothelial adhesion molecules during human endotoxemia.

Low dose prednisolone was shown to be beneficial in the treatment of the Acute respiratory distress syndrome (ARDS) and septic shock. One corticosteroid-induced effect, postulated to mediate corticosteroid-induced anti-inflammatory effects, is decreased expression of adhesion molecules on endothelial cells, thereby preventing leukocyte recruitment at inflammatory sites. The current study aimed to investigate the effect of increasing doses of prednisolone on the release of soluble adhesion molecules in healthy volunteers challenged with endotoxin. Therefore, 32 healthy, male volunteers received prednisolone orally at doses of 0mg, 3mg, 10mg or 30 mg at 2h before injection of endotoxin prepared from Escherichia coli (4 ng/kg) and levels of soluble E-selectin (sE-selectin), soluble VCAM-1 (sVCAM-1) and soluble ICAM-1 (sICAM-1) were measured. Levels of all markers were increased after induction of endotoxemia. Levels of sE-selectin were inhibited by a dose of 3mg prednisolone and levels of sVCAM-1 were decreased after a dose of 10mg. Maximal inhibition of both sE-selectin and sVCAM-1 levels was achieved by the highest dose of prednisolone 30 mg. Remarkably, prednisolone 3mg potentiated endotoxin-induced sVCAM-1 release. Levels of sICAM-1 were not affected by prednisolone. Together, the data suggest that prednisolone differentially and dose-dependently influences the release of soluble endothelial adhesion molecules during human endotoxemia.

Functional thrombomodulin deficiency causes enhanced thrombus growth in a murine model of carotid artery thrombosis

Abstract.Thrombomodulin (TM) bound thrombin initiates the protein C anticoagulant pathway and defects in TM result in enhanced coagulation. Recent studies suggest a role for TM in arterial vascular disease. In order to corroborate this association we studied arterial thrombus formation in mice with a functional TM defect. We used mice homozygous for a 404Glu-to-Pro mutation in the TM gene (TMpro/pro) and compared these with wildtype littermates in a model of FeCl3 induced carotid artery thrombosis. Time-to-occlusion (TTO) was assessed by arterial blood flow measurement, using a Doppler flow probe. Complete occlusion occurred in 8/10 (80%) TMpro/pro mice and in 3/11 (27%) littermate controls. Mean time to occlusion (TTO) [± SE] was 767 ± 196 s in the F2-TMpro/pro mice, versus 1507 ± 159 s in controls (p = 0.007, Mann Whitney U test). Histology and immunostaining for tissue factor did not reveal any differences in thrombus morphology or thrombogenicity between the two groups.These data confirm and extend the fiding that a functional deficiency in TM results in enhanced thrombus formation in a murine model of carotid artery thrombosis and support a role for TM defects in arterial thrombotic disease.