Critical evaluation of nitric oxide as an immuno-modulator in humans

Introduction: Nitric oxide (NO) has been shown to possess anti-inflammatoryproperties. In a porcine endotoxin model the combination of intravenously (iv) administrated glucocorticoid (gc) and inhaled NO (iNO) compared to gc or iNO given separately, blunted the inflammatory response in vital organs. The ischemia and reperfusion (I/R) syndrome has an essential role regarding the pathogenesis in a number of clinical conditions. Animal and human studies have shown favourable effects with exogenously administrated NO in different I/R models. Aim: The overall aim of this thesis was to study the potential of inhaled NO as amodulator of the inflammatory response in humans. In study I-III two different concentration of iNO in combination with gc was studied and evaluated using clinical parameters and cytokines in plasma (study I-II) as well as microparticles in plasma (study III). In study IV we evaluated whether knee surgery in spinal anesthesia during tourniquet could be used as a model to study I/R, and if so, if this could be attenuated by iNO. Methods: In study I-III an endotoxin model was employed in 30 healthy human volunteers participating in 60 experiments. Study III is based on samples taken separately in study II. The studies were double-blind, cross-over and randomised with regard to iNO and placebo (nitrogen, N2), i.e. every volunteer had iv endotoxin (2 ng/kg) and iv gc (2 mg/kg) combined with iNO in one experiment (iNO/gc) and withplacebo (placebo/gc) in the other. In study I endotoxin was given before gc and iNO(30 ppm lasting for 5 hours), while in study II iNO (80 ppm, lasting for 7.5 hours) wasinitiated first, followed by endotoxin and gc. Clinical symptoms were recorded and blood samples collected. In study IV patients consecutively submitted to kneearthroplasty in spinal anaesthesia were included. As a standard procedure a tourniquet was used to create a bloodless surgical field. The patients were randomised into three groups (n=15). Groups 1 and 3 were either receiving iNO 80 ppm or placebo throughout the entire operation, whereas group 2 received iNO 80 ppm just in the beginning and in the end of the operation, hence, no iNO during the period with activated tourniquet. Blood samples and muscle biopsies were collected during theoperation. Adhesion molecules before and after the ischemic period were analysed. Results: In study I and II endotoxin elicited typical flulike symptoms e.g. headache and fever as well as activation of cytokine levels. In study III there was an increase in platelet and monocyte microparticles (MP) while endothelial derived MP were unaltered. Also, platelet derived MP positive to CD40L and monocyte MP positive to HMGB1 showed an early increase. There was no difference between the two treatments (iNO/gc and placebo/gc) in study I-III. In study IV no signs of endothelial cell activation or inflammatory response neither systemically nor locally in adjacent muscle were seen. Conclusions: The present human endotoxaemic model exhibited reproducible results, thereby providing a stable and safe model for randomized studies. The combination of intravenously administered gc and iNO, elicited no anti-inflammatory effect. The endotoxin infusion in healthy volunteers resulted in an increase in plasma cytokines as well as in microparticles released from platelets and from monocytes but not from theendothelium. In patients undergoing knee arthroplasty in spinal anesthesia, the ischemia/reperfusion created by a tourniquet did not cause any signs of endothelial cell activation or inflammatory response

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

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