Anne Soop

Differential release of matrix metalloproteinase‐9 and nitric oxide following infusion of endotoxin to human volunteers

Background: Roughly 400 000 cases of sepsis occur every year in the United States only and this is associated with a very high mortality. Bacterial lipopolysaccharide (LPS) triggers systemic inflammatory reactions in sepsis. However, down‐stream cellular cascade initiated by LPS is still being elucidated. Nitric oxide (NO) and matrix metalloproteinases‐2 and ‐9 (MMP‐2 and MMP‐9) are known to be induced by LPS. We have investigated the release of NO, MMP‐2 and MMP‐9 following infusion of LPS to volunteers.

Complement activation, endothelin-1 and neuropeptide Y in relation to the cardiovascular response to endotoxin-induced systemic inflammation in healthy volunteers.

Background:  Endotoxin is a major stimulus for triggering the host response in septicaemia. The pathophysiology of sepsis involves activation of the vascular endothelium and leukocytes, resulting in the release of various mediators, e.g. cytokines, nitric oxide (NO), endothelin (ET‐1) and complement factors. We evaluated the blood levels of complement activation, ET‐1 and neuropeptide Y (NPY) in parallel with the haemodynamic and oxygen transport response during human experimental endotoxemia.

Adenosine treatment attenuates cytokine interleukin-6 responses to endotoxin challenge in healthy volunteers.

Anti-inflammatory effects of adenosine were evaluated in two randomized, double-blind, placebo-controlled studies. In one study healthy male volunteers received no endotoxin (adenosine study, n = 10), in the other intravenous endotoxin (4 ng/kg, endotoxin study n = 11) was given. All subjects were treated with adenosine infusion (40 &mgr;g/kg/min) and placebo (saline) infusion in a crossover design. Heart rate, body temperature, blood pressure and plasma cytokines (tumor necrosis factor [TNF]-&agr;, interleukin [IL]-6, IL-10, and soluble TNF receptors I and II), nitric oxide oxidation products, nitrite and nitrate, as well as superoxide anions were determined. There were no significant changes of any measured parameter after adenosine treatment alone. Endotoxin elicited clinical signs of an inflammatory reaction, prominent release of all cytokines and O2− synthesis by neutrophils (N-formyl-methionin-leucyl-phenylalanin-stimulated cells measured by cytochrome C reduction). The plasma IL-6 response to endotoxin was attenuated by adenosine, as IL-6 increased from 0.9 (0.8–1.6) to 1345 (743–1906) pg/mL (median; 25–75th percentiles) with adenosine infusion, and from 0.8 (0.5–1) to 1,959 (1,344–2,505) pg/mL with placebo (P = 0.0065). There was no significant influence of adenosine infusion on the other variables examined. In conclusion, systemic adenosine infusion counteracts the release of IL-6 in healthy volunteers, indicating an anti-inflammatory effect of adenosine which should be further explored.

Exhaled NO and plasma cGMP increase after endotoxin infusion in healthy volunteers.

Nitric oxide (NO) is believed to be involved in the pathophysiology of sepsis. This study evaluated the activity of the NO pathway in a human endotoxin model. At baseline and after endotoxin, on-line measurements of exhaled NO (eNO) were made using a chemiluminescence technique with a single-breath method. NO-free air was inhaled prior to exhalation against a resistance. NO in orally and nasally exhaled air and in rectal gas was investigated. Plasma nitrite, nitrate, and guanosine 3′, 5′-monophosphate (cGMP) and the events after diclophenac administration were also studied. Endotoxin infusion resulted in tachycardia and fever. An early increase in oral eNO concentration was observed and oral eNO decreased after diclophenac administration. NO exhaled nasally, NO in rectum gas and nitrite/nitrate levels remained unchanged over the study period. cGMP increased after 4 h. These findings suggest an early increase in nitric oxide production from the lungs, probably due to increased activity of the constitutive nitric oxide synthase upon endotoxin stimulation. In contrast, nitric oxide production in the upper airways, measured as nasally exhaled nitric oxide and nitric oxide in rectal gas, remained unchanged. Further studies will elucidate if exhaled nitric oxide is a valuable marker of sepsis-induced lung injury and if monitoring of treatment is possible.

Nitric oxide inhalation and glucocorticoids as combined treatment in human experimental endotoxemia

Objective:Inhaled nitric oxide and glucocorticoids as a combination therapy may attenuate endotoxin-induced inflammatory responses in humans as indicated by levels of cytokines and clinical signs. Since other authors have shown that combined inhaled nitric oxide and steroids improved the histologic damage both in pulmonary and systemic organs in a porcine endotoxin model, we examined if an anti-inflammatory interaction could be demonstrated in humans. Design:Double-blind, crossover, placebo-controlled randomized study. Setting:The intensive care unit in a university hospital. Subjects:Fifteen healthy white volunteers (4 women, 11 men). Interventions:Endotoxin (2 ng/kg) was administered intravenously. Thirty minutes thereafter the volunteers were given glucocorticoids (2 mg/kg) intravenously and inhaled nitric oxide 30 ppm or placebo (nitrogen) administered through a nasal cannula. Blood samples and clinical signs were collected before and up to 5.5 hrs after the endotoxin infusion. Measurements and Main Result:Following endotoxin body temperature and heart rate increased significantly compared with baseline. There were no differences observed between the treatments. Endotoxin challenge also markedly elevated the plasma levels of tumor necrosis factor-α, interleukin (IL)-6, IL-10, and IL1-ra concentrations during the study period. No difference between placebo/glucocorticoids and inhaled nitric oxide/glucocorticoids treatment was seen in the cytokine response. Conclusions:In a human experimental inflammatory model using endotoxin, inhaled nitric oxide and glucocorticoids in low doses given after the endotoxin challenge did not modify the inflammatory cascade as monitored in this study.

Nicotinamide does not influence cytokines or exhaled NO in human experimental endotoxaemia.

This study examined the hypothesis that nicotinamide could attenuate endotoxin‐induced inflammatory responses in humans as indicated by levels of cytokines and nitric oxide. Ten healthy male volunteers participated in a randomised, double‐blind, cross‐over design with regard to the effects of nicotinamide. The volunteers received orally 4 g nicotinamide or placebo at 14 h and at 2 h preceding the experiment (total dose of 8 g). Endotoxin (E. coli, 2 ng/kg), was administered intravenously. Blood samples and haemodynamic data were collected prior to and up to 6 h after the endotoxin infusion. Orally exhaled NO was measured hourly. Following endotoxin, body temperature increased from baseline 36·3 ± 0·09°C to a maximum of 38·0 ± 0·1°C for all (mean ± SEM, P < 0·001) and heart rate increased from 59 ± 1·9 to 87·0 ± 2·6 beats/min after 3 h (mean ± SEM, P < 0·001). Endotoxin challenge also markedly elevated the TNF‐α, IL‐6, IL‐8 and IL‐10 concentrations (P < 0·001 versus baseline for all) during the study period. Orally exhaled NO also increased (P < 0·01) compared to baseline. Nicotinamide treatment did not influence the patterns of cytokine and NO response to endotoxin.

Immunomodulation by a combination of nitric oxide and glucocorticoids in a human endotoxin model

Background: Inflammatory reactions arise in reaction to a variety of pathogenic insults. The combination of inhaled nitric oxide (iNO) and glucocorticoids (GC) may attenuate endotoxin‐induced inflammatory responses. It has been shown that the combination of iNO (30 p.p.m.) and steroids blunted the inflammatory response in a porcine endotoxin model, but not in humans. Therefore, we investigated whether a clinically ‘maximal’ dose of iNO in combination with GC could modulate the systemic inflammatory response in a human endotoxin model.

Local wound and systemic coagulation/fibrinolysis responses in hip arthroplasty: Influence of allogeneic and autologous blood transfusion

22 patients undergoing elective hip arthroplasty were studied. In 12 patients, a closed-loop autotransfusion system, without anticoagulant, was used and 10 had an ordinary wound drainage allowing repeated blood sampling from the wound. Plasma concentrations of antithrombin (AT), fibrin, soluble (SF) and fibrin D-dimer were determined preoperatively, 3, 8, and 24 hours after starting surgery. Wound drainage blood had increased concentrations of SF and fibrin D-dimer and decreased concentrations of AT compared to reference values and systemic concentrations in patients. Plasma concentrations of SF, fibrin D-dimer and AT did not differ between patients receiving retrieved blood and those receiving stored red blood cell concentrates (RBCs). Patients receiving blood transfusions had lower AT concentrations at 8 hours after starting surgery than those not receiving such a transfusion.

Inflammation and thrombin generation cause increased thrombin activatable fibrinolysis inhibitor levels in experimental human endotoxemia.

The thrombin activatable fibrinolysis inhibitor (TAFI) is a 58-kDa carboxypeptidase that is synthesized in the liver and circulates in the plasma as a zymogen, pro-TAFI. The principal mediator of TAFI activation is the thrombin–thrombomodulin complex. The active form of TAFI attenuates fibrinolysis by removing C-terminal lysine and arginine residues from partially degraded fibrin. This inhibits plasminogen binding and subsequently plasmin formation in the fibrin-rich clot [1,2].

Circulating H3Cit is elevated in a human model of endotoxemia and can be detected bound to microvesicles

Early diagnosis of sepsis is crucial since prompt interventions decrease mortality. Citrullinated histone H3 (H3Cit), released from neutrophil extracellular traps (NETs) upon binding of platelets to neutrophils following endotoxin stimulation, has recently been proposed a promising blood biomarker in sepsis. Moreover, microvesicles (MVs), which are released during cell activation and apoptosis and carry a variety of proteins from their parental cells, have also been shown to be elevated in sepsis. In a randomized and placebo-controlled human model of endotoxemia (lipopolysaccharide injection; LPS), we now report significant LPS-induced elevations of circulating H3Cit in 22 healthy individuals. We detected elevations of circulating H3Cit by enzyme-linked immunosorbent assay (ELISA), as well as bound to MVs quantified by flow cytometry. H3Cit-bearing MVs expressed neutrophil and/or platelet surface markers, indicating platelet-neutrophil interactions. In addition, in vitro experiments revealed that H3Cit can bind to phosphatidylserine exposed on platelet derived MVs. Taken together; our results demonstrate that NETs can be detected in peripheral blood during endotoxemia by two distinct H3Cit-specific methods. Furthermore, we propose a previously unrecognized mechanism by which H3Cit may be disseminated throughout the vasculature by the binding to MVs.