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.

Lipopolysaccharide Alters Motivated Behavior in a Monetary Reward Task: a Randomized Trial.

Inflammation-induced sickness is associated with a large set of behavioral alterations; however, its motivational aspects remain poorly explored in humans. The present study assessed the effect of lipopolysaccharide (LPS) administration at a dose of 2 ng/kg of body weight on motivation in 21 healthy human subjects in a double-blinded, placebo (saline)-controlled, cross-over design. Incentive motivation and reward sensitivity were measured using the Effort Expenditure for Rewards Task (EEfRT), in which motivation for high-effort/high-reward trials vs low-effort/low-reward trials are manipulated by variations in reward magnitude and probability to win. Because of the strong interactions between sleepiness and motivation, the role of sleepiness was also determined. As expected, the probability to win predicted the choice to engage in high-effort/high-reward trials; however, this occurred at a greater extent after LPS than after saline administration. This effect was related to the level of sleepiness. Sleepiness increased motivation to choose the high-effort/high-reward mode of response, but only when the probability to win was the highest. LPS had no effect on reward sensitivity either directly or via sleepiness. These results indicate that systemic inflammation induced by LPS administration causes motivational changes in young healthy subjects, which are associated with sleepiness. Thus, despite its association with energy-saving behaviors, sickness allows increased incentive motivation when the effort is deemed worthwhile.

Intrinsic functional connectivity of insular cortex and symptoms of sickness during acute experimental inflammation.

Task-based fMRI has been used to study the effects of experimental inflammation on the human brain, but it remains unknown whether intrinsic connectivity in the brain at rest changes during a sickness response. Here, we investigated the effect of experimental inflammation on connectivity between areas relevant for monitoring of bodily states, motivation, and subjective symptoms of sickness. In a double-blind randomized controlled experiment, 52 healthy volunteers were injected with 0.6ng/kg LPS (lipopolysaccharide) or placebo, and participated in a resting state fMRI experiment after approximately 2h 45min. Resting state fMRI data were available from 48 participants, of which 28 received LPS and 20 received placebo. Bilateral anterior and bilateral posterior insula sections were used as seed regions and connectivity with bilateral orbitofrontal and cingulate (anterior and middle) cortices was investigated. Back pain, headache and global sickness increased significantly after as compared to before LPS, while a non-significant trend was shown for increased nausea. Compared to placebo, LPS was followed by increased connectivity between left anterior insula and left midcingulate cortex. This connectivity was significantly correlated to increase in back pain after LPS and tended to be related to increased global sickness, but was not related to increased headache or nausea. LPS did not affect the connectivity from other insular seeds. In conclusion, the finding of increased functional connectivity between left anterior insula and middle cingulate cortex suggests a potential neurophysiological mechanism that can be further tested to understand the subjective feeling of malaise and discomfort during a sickness response.

CD40L expression in plasma of volunteers following LPS administration: A comparison between assay of CD40L on platelet microvesicles and soluble CD40L.

CD40 ligand (CD40L) is a transmembrane protein that is mainly expressed on activated T cells and platelets. This protein, however, may also be shed from cells and circulate in the blood in a soluble form. "Soluble CD40L" has attracted interest as a biomarker as it can interact with CD40 and elicit cellular responses involved in the pathophysiology of various thrombotic and inflammatory conditions. As platelets can release microvesicles following activation, we investigated the expression of CD40L on circulating microvesicles as well as CD40L in plasma, in an experimental model of inflammation in healthy volunteers (i.e., intravenous lipopolysaccharide administration). We studied CD40L quantified as CD40L-positive platelet microvesicles by flow cytometry, and as CD40L in plasma ("soluble CD40L") by an ELISA. Results of these studies showed that levels of CD40L exposed on platelet microvesicles were significantly increased after lipopolysaccharide administration. ELISA measurements of CD40L in plasma ("soluble CD40L") did not show any significant increase in plasma levels over time. Separation of soluble and vesicle-bound CD40L by high-speed centrifugation indicated that the ELISA can also detect CD40L on microvesicles, as a trend toward increased concentrations were observed in the pellet of high-speed centrifuged samples (i.e., in samples in which microvesicles are enriched). Together, these findings suggest that platelet microvesicles are a source of CD40L in the circulation and that CD40L exposure on platelet microvesicles increases following experimentally induced inflammation. Our data also suggest that determining levels of CD40L on microvesicles in plasma samples may provide a more sensitive detection of changes in CD40L expression than measurement of "soluble CD40L" in plasma with an ELISA. In addition, information regarding the cellular source of CD40L can be obtained with a flow cytometry-based microvesicle assay in a way not possible with an ordinary ELISA.

No signs of inflammation during knee surgery with ischemia: a study involving inhaled nitric oxide.

Nitric oxide donors and inhaled nitric oxide (iNO) may decrease ischemia/reperfusion injury as reported in animal and human models. We investigated whether the attenuation of reperfusion injury, seen by others, in patients undergoing knee arthroplasty could be reproduced when patients had spinal anesthesia. 45 consecutive patients were randomized into three groups (n = 15). Groups 1 and 3 were receiving iNO 80 ppm or placebo (nitrogen, N2) throughout the entire operation, and group 2 only received iNO in the beginning and at the end of the operation. Blood samples were collected before surgery, at the end of the surgery, and 2 hours postoperatively. Muscle biopsies were taken from quadriceps femoris muscle before and after ischemia. There were no increases in plasma levels of soluble adhesion molecules: ICAM, VCAM, P-selectin, E-selectin, or of HMGB1, in any of the groups. There were low numbers of CD68+ macrophages and of endothelial cells expression of ICAM, VCAM, and P-selectin in the muscle analyzed by immunohistochemistry, prior to and after ischemia. No signs of endothelial cell activation or inflammatory response neither systemically nor locally could be detected. The absence of inflammatory response questions this model of ischemia/reperfusion, but may also be related to the choice of anesthetic method EudraCTnr.

The scent of disease: human body odor contains an early chemosensory cue of sickness.

Observational studies have suggested that with time, some diseases result in a characteristic odor emanating from different sources on the body of a sick individual. Evolutionarily, however, it would be more advantageous if the innate immune response were detectable by healthy individuals as a first line of defense against infection by various pathogens, to optimize avoidance of contagion. We activated the innate immune system in healthy individuals by injecting them with endotoxin (lipopolysaccharide). Within just a few hours, endotoxin-exposed individuals had a more aversive body odor relative to when they were exposed to a placebo. Moreover, this effect was statistically mediated by the individuals' level of immune activation. This chemosensory detection of the early innate immune response in humans represents the first experimental evidence that disease smells and supports the notion of a "behavioral immune response" that protects healthy individuals from sick ones by altering patterns of interpersonal contact.

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-alpha, 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.

Bedside monitoring of coagulation activation after challenging healthy volunteers with intravenous endotoxin.

INTRODUCTION: In the intensive care, many patients are at risk of developing procoagulant changes leading to severe coagulopathy and disseminated intravascular coagulation (DIC). Close monitoring of the coagulation is therefore crucial. In this study, we assess a novel bedside instrument based on free oscillating rheometry, measuring the degree of coagulation activity as clotting onset time, COT. COT measurements are conducted in whole blood, and thus reflect the overall hemostasis. MATERIALS AND METHODS: Nine healthy volunteers were subjected to intravenous injection of 2 ng/kg endotoxin. To quantify the activation of the coagulation system, COT was assessed along with prothrombin fragment 1 + 2 (F1 + 2) and activated partial thromboplastin time (aPTT). RESULTS: Baseline COT was 10.6 +/- 3.6 min. Three hours after endotoxin injection, maximal body temperature and heart rate were registered. At this time, an activation of the coagulation was observed as a significant decrease in COT to 8.0 +/- 1.0 min, a decrease in aPTT and increased levels of F1 + 2. COT levels, body temperature and heart rate subsequently normalized towards initial levels. F1 + 2 however continued to increase throughout the study. CONCLUSIONS: COT is a rapid test, performed in less than 15 min. COT was superior to F1+ 2 in monitoring the endotoxin-induced activation of the coagulation, since COT covariated with clinical symptoms, whereas F1 + 2 did not. This is, to our knowledge, the first time an overall coagulation monitoring method has been assessed bedside to detect endotoxin-induced activation of the coagulation. The COT test shows promising results, but further studies are needed to reveal its potential in the intensive care monitoring arsenal.

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 microg/kg/min) and placebo (saline) infusion in a crossover design. Heart rate, body temperature, blood pressure and plasma cytokines (tumor necrosis factor [TNF]-alpha, 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.