Immunomodulatory effects in vitro of vitamin K antagonist acenocoumarol.

Neopterin production and tryptophan breakdown by indoleamine 2,3-dioxygenase (IDO) are induced within cell-mediated (=Th1-type) immune response, and in patients with coronary artery disease, serum neopterin and the kynurenine to tryptophan ratio (Kyn/Trp) are significantly predictive for cardiovascular and total mortality. To examine the potential impact of vitamin K-antagonist acenocoumarol (Sintrom) on the inflammatory response we investigated its effect on freshly isolated peripheral blood mononuclear cells (PBMC) from healthy donors, on myelomonocytic THP1-Blue and on intestinal Caco-2 cells in vitro. PBMC were incubated with increasing doses of acenocoumarol, and after 30 min either left unstimulated or stimulated with the mitogen phytohemagglutinin (PHA). Concentrations of neopterin, tryptophan, kynurenine, interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α) were measured in supernatants of PBMC after 48 h. Caco-2 cells were stimulated with IFN-γ and Kyn/Trp was used as readout. In THP1-Blue cells, the induction of NF-κB dependent reporter gene expression upon stimulation with lipopolysaccharide (LPS) was determined as an indicator of pro-inflammatory response. Upon stimulation, all measured immune response markers increased significantly compared to unstimulated cells. Acenocoumarol had no effect in unstimulated cells but in PHA-stimulated PBMC tryptophan breakdown and the formation of neopterin, as well as IFN-γ and TNF-α, were dose-dependently suppressed at concentrations as low as 10 µg/ml. Likewise, acenocoumarol dose-dependently inhibited tryptophan breakdown in IFN-γ stimulated Caco-2 cells. Interestingly, NF-κB expression was super-induced in the LPS treated cells. Data suggest that the immunomodulatory capacity of acenocoumarol contributes to its therapeutic efficacy.

Decline of exhaled isoprene in lung cancer patients correlates with immune activation.

Isoprene (2-methylbuta-1,3-diene) represents a precursor molecule of isoprenoids (steroids, terpens), and available data suggest that isoprene is related to cholesterol biosynthesis. Breath concentrations of isoprene have been reported to be altered in a number of clinical conditions. However, the physiological meaning of isoprene changes has not yet been established. Utilizing proton-transfer-mass spectroscopy, we analyzed isoprene concentrations (m/z 69, tentatively identified as isoprene) in breath samples in Tedlar bags collected from 79 lung cancer patients (23 females, 56 males). Results were compared to the concentrations of immune activation marker neopterin (ELISA, BRAHMS, Hennigsdorf, Germany), lipid parameters (routine enzymology) and C-reactive protein (CRP). Isoprene concentrations were median 92.5 ppb (25th-75th percentile: 79-131 ppb). There was no relationship with staging, grading or age, but isoprene concentrations correlated significantly with total cholesterol (rs = 0.281, p < 0.01) and LDL cholesterol (rs = 0.236, p < 0.05). There was no significant relationship between exhaled isoprene concentrations and HDL cholesterol (rs = 0.048), triglycerides (rs = 0.164) and CRP (rs = -0.115; all not significant). A significant inverse correlation existed between isoprene and neopterin concentrations (rs = -0.215, p < 0.05); the latter also correlated with total cholesterol (rs = -0.343, p = 0.001), HDL cholesterol (rs = -0.273, p = 0.01), LDL cholesterol (rs = -0.236, p < 0.05) and CRP (rs = 0.230, p < 0.05) but not with triglycerides (rs = 0.035, not significant). Results suggest that immune activation might play a role in the decline of isoprene which is probably related to lipid metabolic changes. Interestingly, similar relationships between elevated neopterin and decreased lipid concentrations have been reported earlier in other clinical conditions, e.g. in patients with HIV-1 infection.

In vitro testing for anti-inflammatory properties of compounds employing peripheral blood mononuclear cells freshly isolated from healthy donors.

INTRODUCTION: Inflammation is crucially involved in a variety of diseases like autoimmune syndromes, cardiovascular and neurodegenerative disorders, cancer, sepsis and allograft rejection. METHODS: Freshly isolated human peripheral blood mononuclear cells (PBMCs) are used as a screening assay for anti-inflammatory properties of compounds. Determinations of neopterin production by ELISA and of tryptophan degradation by HPLC are used as read-outs. Results are compared with further markers of immune response and oxidative stress. RESULTS: Phytohaemagglutinin induced significant tryptophan degradation and neopterin formation in PBMC, which correlated with IFN-γ, TNF-α, soluble cytokine receptors and isoprostane-8. Addition of vitamin C and E suppressed the responses dose-dependently. DISCUSSION: The determination of tryptophan degradation and neopterin production in PBMC reflects various pro- and anti-inflammatory cascades that are of relevance also in patients. It constitutes a robust and reliable approach to screen anti-inflammatory or immunosuppressive drugs and may improve throughput, speed and cost-effectiveness in drug discovery.