No abstract

Citrullinated histone H3 (H3Cit) is a central player in the neutrophil release of nuclear chromatin, known as neutrophil extracellular traps (NETs). NETs have been shown to elicit harmful effects on the host, and were recently proposed to promote tumor progression and spread. Here we report significant elevations of plasma H3Cit in patients with advanced cancer compared with age-matched healthy individuals. These elevations were specific to cancer patients as no increase was observed in severely ill and hospitalized patients with a higher non-malignant comorbidity. The analysis of neutrophils from cancer patients showed a higher proportion of neutrophils positive for intracellular H3Cit compared to severely ill patients. Moreover, the presence of plasma H3Cit in cancer patients strongly correlated with neutrophil activation markers neutrophil elastase (NE) and myeloperoxidase (MPO), and the inflammatory cytokines interleukin-6 and -8, known to induce NETosis. In addition, we show that high levels of circulating H3Cit strongly predicted poor clinical outcome in our cohort of cancer patients with a 2-fold increased risk for short-term mortality. Our results also corroborate the association of NE, interleukin-6 and -8 with poor clinical outcome. Taken together, our results are the first to unveil H3Cit as a potential diagnostic and prognostic blood marker associated with an exacerbated inflammatory response in patients with advanced cancer.

There is an emerging interest in the diverse functions of neutrophil extracellular traps (NETs) in a variety of disease settings. However, data on circulating NETs rely largely upon surrogate NET markers such as cell-free DNA, nucleosomes, and NET-associated enzymes. Citrullination of histone H3 by peptidyl arginine deiminase 4 (PAD4) is central for NET formation, and citrullinated histone H3 (H3Cit) is considered a NET-specific biomarker. We therefore aimed to optimize and validate a new enzyme-linked immunosorbent assay (ELISA) to quantify the levels of H3Cit in human plasma. A standard curve made of in vitro PAD4-citrullinated histones H3 allows for the quantification of H3Cit in plasma using an anti-histone antibody as capture antibody and an anti-histone H3 citrulline antibody for detection. The assay was evaluated for linearity, stability, specificity, and precision on plasma samples obtained from a human model of inflammation before and after lipopolysaccharide injection. The results revealed linearity and high specificity demonstrated by the inability of detecting non-citrullinated histone H3. Coefficients of variation for intra- and inter-assay variability ranged from 2.1 to 5.1% and from 5.8 to 13.5%, respectively, allowing for a high precision. Furthermore, our results support an inflammatory induction of a systemic NET burden by showing, for the first time, clear intra-individual elevations of plasma H3Cit in a human model of lipopolysaccharide-induced inflammation. Taken together, our work demonstrates the development of a new method for the quantification of H3Cit by ELISA that can reliably be used for the detection of NETs in human plasma.

Muscle sympathetic nerve activity (MSA; peroneal nerve) and arterial and femoral venous plasma norepinephrine (NE) were studied in 10 volunteers at rest, during a relaxation procedure (RELAX), and during two mental challenges, a word identification test (WIT) and a color word test (CWT). [3H]NE infusions were used to assess NE spillover to and clearance from plasma. Net NE overflow from the leg was calculated. RELAX reduced MSA and femoral venous NE concentrations. CWT increased blood pressure, cardiac output (thermodilution), and calf flow and reduced systemic vascular resistance. Responses to WIT were less marked. CWT increased MSA by 25%, femoral venous NE concentrations by 25%, and NE overflow from the leg by 26% at 3 min. Fractional epinephrine and [3H]NE extractions were flow related and decreased during CWT. The arterial contribution to femoral venous NE (about half) increased by 10% during CWT. Arterial NE levels and spillover increased, but NE clearance was unchanged. Femoral venous NE concentrations and NE spillover (not based on flow measurements) and regional NE overflow correlated with MSA. Thus NE concentrations in plasma reflect spillover rather than clearance at rest and during mental challenge. Biochemical and neurophysiological indexes of sympathetic activity correlate when assessed in the same region. Mental stress increases sympathetic activity in leg muscle.

8 subjects were exposed to the Stroop mental performance test in a design with alternating hourly periods of rest and stress. During each period one urine sample and several venous plasma samples were obtained. Heart rate responded rapidly to initiation and termination of the stress exposure with increases and decreases respectively. Both urinary and plasma adrenaline increased significantly during stress. The plasma response was immediate and sustained. Neither urinary, nor plasma noradrenaline were significantly increased by the test. Plasma noradrenaline, however, increased significantly on termination of the exposure to stress. It was suggested that the latter effect may be due to muscle sympathetic nerve activity decreasing during stress and increasing following stress. The sample-to-sample variation was more than 20% of the mean for both catecholamines, indicating the need for frequent sampling to reliably reflect plasma levels. The mean intraindividual plasma/urine correlation was r = 0.70 (p less than 0.001) for adrenaline and r = 0.40 (p less than 0.05) for noradrenaline. When only resting periods were considered, no significant correlations remained, apparently due to a reduced range of variation and accompanying reduced signal-to-noise ratio. It is concluded that both urinary and plasma adrenaline may be useful in the evaluation of changes in sympatho-adrenal activity during stress.