Experimental studies on endotoxin infusion in human : Evaluation of pharmacological immunomodulation by adenosine and nicotinamide

Introduction: Septicaemia and the systemic inflammatory response syndrome (SIRS), is a major cause of mortality among patients in the intensive care units. Inflammation and thrombosis involve multicellular phenomena with activation of leukocytes, platelets and endothelial cells resulting in synthesis and release of various mediators e.g. cytokines, nitric oxide (NO), endothelin and neuropeptide Y, complement factors and reactive oxygen species such as superoxide anions (02-) and its metabolites. Endotoxin (lipopolysaccharide) is a major stimulus for triggering the host response with the subsequent production of pro- and antiinflammatory cytokines such as tumor necrosis factor alpha (TNF-á) and IL- 10. Aim: The overall aim of this thesis was to study some response patterns of sepsis and potential modulations in an experimental model where human volunteers receive endotoxin. The work could thereby contribute to increased understanding of sepsis-related mechanisms and pharmacological interventions and hopefully attenuate the inflammatory response. Methods: The studies were performed using endotoxin (2-4 ng/kg BW) intravenously in healthy male volunteers and took place in the intensive care unit at Huddinge University Hospital. All together, 38 volunteers participated in 73 experiments, 32 received endotoxin at one or two occasions. We measured exhaled NO, plasma cGMP, endothelin, neuropeptide Y, haemostatic parameters, complement activation and nitrite/nitrate after endotoxin (E. Coli, Lot G 1) administration of 4 and 2 ng/kg BW respectively. Using doubleblind, cross-over design, we investigated the influence of the anti-inflammatory compounds adenosine (in a low continous, intravenous dose, 40 µg/kg/min) as well as nicotinamide (the amide derivate of vitamin B3, in a high oral dose, 4+4 g, 14 and 2 hours preceding the experiment), on inflammatory parameters after endotoxin. Results: Endotoxin provoked clinical signs of inflammation (fever, headache, increased heart rate and decreased diastolic blood pressure) over a period of 4-6 hours. There were no adverse responses. An early increase in orally exhaled NO concentration was observed and cGMP levels were increased. Cardiac output increased by 100%, systemic vascular resistance decreased by 50%. There was activation of the complement system as well as increased arterial endothelin-1 -like immunoreactivity (ET-1-LI) over time. NPY-like immunoreactivity (NPY-LI) remained unchanged. Endotoxin evoked in vivo activation of platelets, leukocytes and endothelial cells, as well as enhancing platelet-leukocyte aggregate formation and thrombin generation. A modest anti-inflammatory effect by adenosine treatment was observed in this study. Adenosine significantly attenuated the endotoxininduced IL-6 response whereas there was no clear effect on the TNF-á response. After oral nicotinamide treatment, there was no effect on the inflammatory parameters (TNF-á, IL-6, IL-8, IL-10 levels or exhaled NO) at the maximum dose that can be administered without inducing side-effects. Conclusion: In conclusion, the present human endotoxaemic model exhibited reproducible results, thereby providing a stable and safe model for randomised studies. Adenosine, in a low continuous intravenous dose, showed only modest immunomodulatory properties. Nicotinamide did not have any anti-inflammatory effects when a high oral dose was given to healthy volunteers receiving endotoxin

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

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