Dual Effect of the Extract from the Fungus Coriolus Versicolor on Lipopolysaccharide-Induced Cytokine Production in RAW 264.7 Macrophages Depending on the Lipopolysaccharide Concentration

Purpose: Extract from the fungus Coriolus versicolor (CV) is classified as an immunological response modifier. Previously, we have shown that this extract induces interleukin 6 (IL-6)-related extension of lipopolysaccharide (LPS)-induced fever. This study investigated the effect of CV extract on the production of pro-inflammatory cytokines and the expression of components of signal transduction pathways leading to the secretion of cytokines from RAW 264.7 macrophages stimulated with different doses of LPS. Methods: RAW 264.7 cells were stimulated with CV extract alone or co-treated with CV extract and LPS. The level of IL-6 and tumour necrosis factor α (TNF-α) in the culture media was measured using ELISA. Protein expression of Toll-like receptor (TLR) 4, phosphorylated IκB (p-IκB), CD14 glycoprotein and phospho-phosphatidylinositol 3-kinase (p-PI3K) was evaluated using Western blot. The effects of TLR4, nuclear factor κB (NF-κB) and p-PI3K on cytokine secretion were estimated using inhibitors: TAK-242, JSH-23 and Y294002. Results: CV extract itself stimulates the secretion of IL-6 and TNF-α and increases the expression of TLR4, p-IκB and p-PI3K. The presence of CV extract during the treatment of cells with lower concentrations of LPS (10 and 100 ng/mL) increases the cytokine production. Co-stimulation of cells with CV extract and LPS at a higher dose (500 ng/mL) decreases the secretion of cytokines. This effect is related to the changes in the expression of TLR4, CD14 glycoprotein, p-IκB and p-PI3K. Conclusion: This is the first report showing that the CV extract-induced production of cytokines is mediated by the PI3K signalling pathway. This extract acts antagonistically or additively with LPS on the production of IL-6 and TNF-α, depending on the LPS concentration. Our results are helpful for illustrating the mechanisms for the immunostimulatory effect of CV extract in inflammatory processes

Population genetic structure of Juniperus phoenicea (Cupressaceae) in the western Mediterranean Basin: gradient of diversity on a broad geographical scale

Introduction, Material and Methods: The genetic structure and diversity of ten natural populations of Juniperus phoenicea L. from the western part of the species range have been studied using random amplified polymorphic DNA (RAPD) markers. Results and discussion: Among 10 analyzed primers only 3 reproduced consistently across successful PCR reactions and gave 45 loci. The percentage of polymorphic loci (P) and Nei’s heterozygosity (H e ) have average values of 64.9% and 0.177. The average expected heterozygosity of particular populations positively correlate with latitude and negatively with altitude (τ = 0.556, P = 0.025; τ = −0.494, P = 0.047, respectively). The proportion of genetic variation contributed by the differences between populations was low (G ST  =  0.056). The gene flow (N m) has an average value of 4.2, and was higher in subsp. turbinata (7.3) than in subsp. phoenicea (4.1). Significant proportion of the variation (ΦST = 0.106) was attributable to differences among populations, as revealed in analysis of molecular variance analysis of pair-wise RAPD distances. No evidence for isolation by distance was detected in Mantel test on genetic (ΦST) and geographic distances. European populations differed at a higher level from the African, subsp. phoenicea from turbinata (3.97% and 3.14% of total variance, respectively). The significant level of differences between European and African populations can result from (1) the earlier divergence and considerably low level of gene flow between them, or (2) a different mutation rate within population of different continent. Conclusion: The results suggest rather local forest economy with J. phoenicea, without seed exchange on large distance.The study was partly sponsored by the Polish Committee for Scientific Research, grant no. 2P04C 030 26 and partly by the Institute of Dendrology. The collection of plant material was made possible thanks to the cooperation between CSIC (Spanish Research Council) and PAS (Polish Academy of Sciences).Peer Reviewe

Taxonomic and geographic variation of the Pinus mugo complex on chloroplast microsatellite markers

16 p., tablas, figuras -- Contiene material suplementario -- Postprint del artículo publicado en Systematics and Biodiversity. Versión revisada y corregidaThe high mountain plants of Central and Southern Europe survived the glacial periods in the same mountain ridges, but at lower altitudes and possibly covering larger areas than during interglacials. This implies a high level of species differentiation between isolated mountain ridges. Pinus mugo complex, which includes P. mugo s.s. (Alps, Sudetes, Carpathians, Dynaric Alps, and Rhodopes), P. uncinata (Pyrenees and Alps), and P. uliginosa (Sudetes and neighbouring mountain ridges) is a good group to examine such a scenario. We screened 44 populations across the geographic range of the complex, using 10 cpSSR markers to study (1) taxonomic relations among P. mugo s.s., P. uncinata, and P. uliginosa and (2) genetic and phylogeographic structure in P. mugo s.s. and P. uncinata. Allelic combinations of 87 size variants produced a total of 757 haplotypes. Haplotypic diversity was high and similar in every species (0.997, 0.986 and 0.991, respectively). The highest divergence between haplotypes was observed in P. uliginosa (= 10.29). The AMOVA revealed that most of the overall genetic variation is explained by the within-population component (FST = 0.121, RST = 0.206) and by the geography (FCT = 0.056, RCT = 0.083). The differentiation between P. mugo s.s., P. uncinata, and P. uliginosa is explained by about 5% (P<0.001) of the total variation. Vicariant gene pools for the complex were identified in the Pyrenees, the Alps with the Tatra Mts, the Sudetes, and the East and South Carpathians along with the Balkan Mountains. The phylogeographic structure was observed in P. mugo s.l., P. mugo s.s., and P. uncinata. Results support the separate taxonomic status of P. uncinata and P. mugo s.s. and possible hybrid origin of P. uliginosa.This work was supported by the Polish Ministry of Sciences [grant number 2P04C 018 29], [grant number 2P06L 04628], [grant number 6P04G 06016], [grant number NN303 360535]; Institute of Dendrology of Polish Academy of Sciences [statutory activity].Peer reviewe

Extract from the Coriolus versicolor Fungus as an Anti-Inflammatory Agent with Cytotoxic Properties against Endothelial Cells and Breast Cancer Cells

Chronic inflammation is a well-recognised tumour-enabling component, which includes bioactive molecules from cells infiltrating the tumour microenvironment and increases the risk of cancer progression. Since long-term use of the currently available anti-inflammatory drugs used in cancer therapy causes numerous side effects, the aim of this study was to investigate the effect of an extract isolated from the Coriolus versicolor fungus (CV extract) on HUVEC endothelial cells and MCF-7 breast cancer cells in a pro-inflammatory microenvironment mimicked by lipopolysaccharide (LPS). The cells were simultaneously stimulated with the LPS and CV extract. After co-treatment, the cell viability, generation of reactive oxygen species (ROS), wound-healing assay, production of the pro-inflammatory and pro-angiogenic factors (interleukin (IL) 6, IL-8, and metalloproteinase (MMP) 9)), as well as expression of Toll-like receptor (TLR) 4 and phosphorylated I&kappa;B (p-I&kappa;B) were evaluated. The results showed that the CV extract inhibited IL-6, IL-8, and MMP-9 production by the LPS-stimulated cells. This effect was accompanied by a decrease in TLR4 and p-I&kappa;B expression. The CV extract also had anti-migratory properties and induced a cytotoxic effect on the cells that was enhanced in the presence of LPS. The observed cytotoxicity was associated with an increase in ROS generation. We conclude that the CV extract possesses cytotoxic activity against cancer cells and endothelial cells and has the ability to inhibit the expression of the pro-tumorigenic factors associated with inflammation

Fever-range whole body hyperthermia leads to changes in immune-related genes and miRNA machinery in Wistar rats

AbstractObjective Fever is defined as a rise in body temperature upon disease. Fever-range hyperthermia (FRH) is a simplified model of fever and a well-established medical procedure. Despite its beneficial effects, the molecular changes induced by FRH remain poorly characterized. The aim of this study was to investigate the influence of FRH on regulatory molecules such as cytokines and miRNAs involved in inflammatory processes.Methods We developed a novel, fast rat model of infrared-induced FRH. The body temperature of animals was monitored using biotelemetry. FRH was induced by the infrared lamp and heating pad. White blood cell counts were monitored using Auto Hematology Analyzer. In peripheral blood mononuclear cells, spleen and liver expression of immune-related genes (IL-10, MIF and G-CSF, IFN-γ) and miRNA machinery (DICER1, TARBP2) was analyzed with RT-qPCR. Furthermore, RT-qPCR was used to explore miRNA-155 levels in the plasma of rats.Results We observed a decrease in the total number of leukocytes due to lower number of lymphocytes, and an increase in the number of granulocytes. Furthermore, we observed elevated expressions of DICER1, TARBP2 and granulocyte colony-stimulating factor (G-CSF) in the spleen, liver and PBMCs immediately following FRH. FRH treatment also had anti-inflammatory effects, evidenced by the downregulation of pro-inflammatory macrophage migration inhibitor factor (MIF) and miR-155, and the increased expression of anti-inflammatory IL-10.Conclusion FRH affects the expression of molecules involved in inflammatory processes leading to alleviated inflammation. We suppose these effects may be miRNAs-dependent and FRH can be involved in therapies where anti-inflammatory action is needed