Mannan activates tissue native and IgE-sensitized mast cells to proinflammatory response and chemotaxis in TLR4-dependent manner.

Mast cells take part in host defense against microorganisms as they are numerous at the portal of infection, exert several essential mechanisms of pathogen destruction, and they express pattern recognition receptors. Accumulating evidence indicates that these cells are involved in the control and clearance of bacterial, viral, or parasitic infections, but much less is known about their contribution in defense against fungi. The study was aimed to establish whether mannan, which comprises an outermost layer and major structural constituent of the fungal cell wall, may directly stimulate tissue mast cells to the antifungal response. Our findings indicate that mannan activates mast cells isolated from the rat peritoneal cavity to initiate the proinflammatory response. We found that mannan stimulates mast cells to release histamine and to generate cysteinyl leukotrienes, cytokines (IFN-γ, GM-CSF, TNF), and chemokines (CCL2, CCL3). It also increased the mRNA expression of various cytokines/chemokines. We also documented that mannan strongly activates mast cells to generate reactive oxygen species and serves as a potent chemoattractant for these cells. Furthermore, we established that mannan-induced activity of mast cells is mediated via TLR4 with the involvement of the spleen tyrosine kinase molecule. Taking together, our results clearly support the idea that mast cells act as sentinel cells and crucially determine the course of the immune response during fungal infection. Additionally, presented data on IgE-coated mast cells suggest that exposure to fungal mannan could influence the severity of IgE-dependent diseases, including allergic ones.

Atrazine biodegradation by mycoinsecticide Metarhizium robertsii: Insights into its amino acids and lipids profile.

Atrazine, is one of major concern pesticides contaminating agricultural areas and ground water. Its microbial biodegradation seems to be the most efficient in terms of economic and environmental benefits. In the present work the cometabolic biodegradation of atrazine by the fungus Metarhizum robertsii IM 6519 during 10-day batch cultures was characterized. The herbicide was transformed to several hydroxy-, dechlorinated or dealkylated metabolites with the involvement of cytochrome P450 monooxygenases. The obtained metabolomics data revealed that atrazine induced oxidative stress (increased the levels of L-proline, L-ornithine, L-arginine, GABA and L-methionine), disruptions of the carbon and nitrogen metabolism (L-aspartic acid, L-asparagine, L-tyrosine, L-threonine, L-isoleucine, L-phenylalanine, 1-methyl-L-histidine, L-tryptophan, L-valine, L-alanine, O-phospho-L-serine, L-sarcosine or L-lysine) and caused an increase in the membrane fluidity (a rise in the phosphatidylcholines/phosphatidylethanolamines (PC/PE) ratio together with the growth of the taurine level). The increased level of hydroxyl derivatives of linoleic acid (9-HODE and 13-HODE) confirmed that atrazine induced lipid peroxidation. The presented results suggesting that M. robertsii IM 6519 might be applied in atrazine biodegradation and may bring up the understanding of the process of triazine biodegradation by Metarhizum strains.

Adipocytokines leptin and adiponectin function as mast cell activity modulators.

A growing body of data indicates that adipocytokines, including leptin and adiponectin, are critical components not only of metabolic regulation but also of the immune system, mainly by influencing the activity of cells participating in immunological and inflammatory processes. As mast cells (MCs) are the key players in the course of those mechanisms, this study aimed to evaluate the impact of leptin and adiponectin on some aspects of MC activity. We documented that in vivo differentiated mature tissue MCs from the rat peritoneal cavity express a receptor for leptin (OB-R), as well as receptors for adiponectin (AdipoR1 and AdipoR2). We established that leptin, but not adiponectin, stimulates MCs to release of histamine as well as to generation of cysteinyl leukotrienes (cysLTs) and chemokine CCL2. We also found that both adipocytokines affect mRNA expression of various cytokines/chemokines. Leptin and adiponectin also activate MCs to produce reactive oxygen species. Moreover, we documented that leptin significantly augments the surface expression of receptors for cysLTs, i.e. CYSLTR1, CYSLTR2, and GPR17 on MCs, while adiponectin increases only GPR17 expression, and decreases CYSLTR2. Finally, we showed that both adipocytokines serve as potent chemoattractants for MCs. In intracellular signaling in MCs activated by leptin Janus-activated kinase 2, phospholipase C, phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK1/2), and p38 molecules play a part whereas the adiponectin-induced activity of MCs is mediated through PI3K, p38, and ERK1/2 pathways. Our observations that leptin and adiponectin regulate MC activity might indicate that adipocytokines modulate the different processes in which MCs are involved.

Leptin stimulates tissue rat mast cell pro-inflammatory activity and migratory response.

OBJECTIVE: The aim of this study was to determine whether leptin, a member of the adipocytokines involved in immune and inflammatory response regulation, may influence some aspects of mast cell biology. MATERIALS AND METHODS: Experiments were done in vitro on fully mature tissue rat mast cells isolated from the peritoneal cavity, and leptin was used at concentrations 0.001-100 ng/ml. The effect of leptin on mast cell degranulation (histamine release assay), intracellular Ca2+ level (fluorimetry), pro-inflammatory mediator release (ELISA technique), surface receptor expression (flow cytometry and confocal microscopy), and migration (Boyden microchamber assay) was estimated. RESULTS: Leptin was found to stimulate mast cells to degranulation and histamine release. It induced the intracellular Ca2+ increase, as well. In response to leptin stimulation, mast cells generated and released cysLTs and chemokine CCL3. Leptin-induced upregulation of CYSLTR1 and CYSLTR2 surface expression was observed. Moreover, this adipocytokine stimulated mast cells to migratory response, even in the absence of extracellular matrix (ECM) proteins. CONCLUSIONS: Our observations clearly documented that leptin promotes the pro-inflammatory activity of mast cells, and it thereby engages these cells in the inflammatory processes.

Cathelicidin LL-37 Affects Surface and Intracellular Toll-Like Receptor Expression in Tissue Mast Cells.

Undoubtedly, mast cells take part in host defense against microorganisms as they are numerous at the portal of infection, they release many proinflammatory and antimicrobial mediators, and they express pattern recognition receptors, such as TLRs. These receptors play a key role in recognition and binding molecules associated with microorganisms and molecules associated with damage. Cathelicidins exhibit direct antimicrobial activities against a broad spectrum of microbes by perturbing their cell membranes. Accumulating evidence suggests a role for these molecules in supporting cell activation. We examined the impact of human cathelicidin LL-37 on tissue mast cell TLR expression and distribution. Depending on context, we show that LL-37 stimulation resulted in minor to major effects on TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9 expression. Confocal microscopy analysis showed that, upon stimulation, TLRs may translocate from the cell interior to the surface and conversely. FPR2 and EGFR inhibitors reduced the increase in expression of selected receptors. We also established that LL-37 acts as a powerful inducer of CCL3 and ROS generation. These results showed that in response to LL-37, mast cells enhance the capability to detect invading pathogens by modulation of TLR expression in what may be involved FPR2 or EGFR molecules.

Biogenic nanosilver synthesized in Metarhizium robertsii waste mycelium extract - As a modulator of Candida albicans morphogenesis, membrane lipidome and biofilm.

Due to low efficacy of classic antimicrobial drugs, finding new active preparations attracts much attention. In this study an innovative, cost-effective and environmentally friendly method was applied to produce silver nanoparticles (AgNPs) using filamentous fungi Metarhizium robertsii biomass waste. It was shown that these NPs possess prominent antifungal effects against C. albicans, C. glabrata and C. parapsilosis reference strains. Further detailed studies were performed on C. albicans ATCC 90028. AgNPs kill curve (CFU method and esterase-mediated reduction of fluorescein diacetate); fractionally inhibitory concentration index (FICI) with fluconazole (FLC); effect on fungal cell membrane permeability (propidium iodide (PI) staining), membrane lipids profile (HPLC-MS), yeast morphotypes and intracellular reactive oxygen species level (H2DCFDA probe) were investigated. Anti-adhesive and anti-biofilm properties of AgNPs (alone and in combination with FLC) were also tested. Biosafety of AgNPs use was assessed in vitro in cytotoxicity tests against L929 fibroblasts, pulmonary epithelial A549 cell line, and red blood cells. Significant reduction in the viability of yeast cells treated with AgNPs was shown within 6 h. The proportion of C. albicans PI-positive cells increased in a dose and time-dependent manner. Changes in the qualitative and quantitative profile of cell membrane lipids, including significant decline in the quantity of most phospholipid species containing C18:2 and an increase in the amount of phospholipids containing C18:1 acyl species were observed after yeast exposure to AgNPs. CLSM images showed an enhancement in ROS intracellular accumulation in C. albicans treated with biogenic nanosilver. C. albicans transformation from yeast to hyphal forms was also reduced. AgNPs decreased adhesion of yeast to abiotic surfaces, as well as acted synergistically with FLC against sessile population. At fungicidal and fungistatic concentrations, they were non-toxic to mammalian cells. Obtained results confirm suitability of our "green synthesis" method to produce AgNPs with therapeutic potential against fungal infections.

Estrogen-mediated protection of the organotin-degrading strain Metarhizium robertsii against oxidative stress promoted by monobutyltin.

Dibutyltin (DBT) is a global pollutant characterized by pro-oxidative properties. The fungal strain Metarhizium robertsii can eliminate high levels of DBT efficiently. In this study, induction of oxidative stress as well as its alleviation through the application of natural estrogens during the elimination of DBT by M. robertsii were evaluated. During the first 24 h of incubation, the initial concentration of DBT (20 mg l-1) was reduced to 3.1 mg l-1, with simultaneous formation of a major byproduct - monobutyltin (MBT). In the presence of estrone (E1) or 17ß-estradiol (E2), the amounts of dibutyltin residues in the fungal cultures were found to be approximately 2-fold higher compared to cultures without estrogens, which was associated with the simultaneous utilization of the compounds by cytochrome P450 enzymes. On the other hand, MBT levels were approximately 2.5 times lower in the fungal cultures with the addition of one of the estrogens. MBT (not DBT) promotes the generation of O2-, H2O2, and NO at levels 65.89 ± 18.08, 4.04 ± 3.62, and 27.92 ± 1.95, respectively. Superoxide dismutase and catalase activities did not show any response of the M. robertsii strain against the overproduction of superoxide anion and hydrogen peroxide. Application of E1 as well as E2 ensured non-enzymatic defense against nitrosative and oxidative stress through scavenging of nitrogen and oxygen reactive species, and limited their levels from 1.5-fold to 21-fold, depending on the used estrogen.

Cytotoxic activity of genistein-8-C-glucoside form Lupinus luteus L. and genistein against human SK-OV-3 ovarian carcinoma cell line.

Genistein belongs to isoflavones, which are a subclass of flavonoids, a large group of polyphenolic compounds widely distributed in plants. Numerous in vitro studies suggest that isoflavones, particularly genistein, have both chemopreventive and chemotherapeutic potential in multiple tumor types. However, the molecular and cellular mechanisms of genistein effects on human ovarian cancer cells are still little known. In the present study, we investigated anticancer activity of genistein and its natural glucoside, genistein-8-C-glucoside isolated from flowers of Lupinus luteus L. We examined the effects of the two isoflavones alone or in combination on cultured human SK-OV-3 ovarian carcinoma cells. The cells were exposed to genistein and genistein-8-C-glucoside at various concentrations (1-90 µM) for 24 and 48 h. The cytotoxic and apoptotic properties of compounds were studied by the colorimetric 3-[4,5-2-yl]-2-5-diphenyltetrazolium bromide assay and the acridine orange/ethidium bromide staining technique. The morphological features of SK-OV-3 cells were examined by Nomarski differential interference contrast combined with a confocal laser scanning microscope. The level of ROS was evaluated with fluorescence probes: dichlorofluorescein-diacetate by flow cytometry. Changes in mitochondrial membrane potential were determined using 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide. Genistein-treatment and genistein-8-C-glucoside-treatment resulted in the inhibition of cell proliferation, induction of apoptotic cell death and loss of mitochondrial membrane potential. The present data provide the first evidence in vitro that genistein-8-C-glucoside and combination genistein-genistein-8-C-glucoside could be a potential chemotherapeutic candidate for ovarian cancer therapy.

Poly(Propylene Imine) Dendrimers and Amoxicillin as Dual-Action Antibacterial Agents.

Besides acting as antimicrobial compounds, dendrimers can be considered as agents that improve the therapeutic effectiveness of existing antibiotics. In this work we present a new approach to using amoxicillin (AMX) against reference strains of common Gram-negative pathogens, alone and in combination with poly(propylene imine) (PPI) dendrimers, or derivatives thereof, in which 100% of the available hydrogen atoms are substituted with maltose (PPI 100%malG3). The concentrations of dendrimers used remained in the range non-toxic to eukaryotic cells. The results indicate that PPI dendrimers significantly enhance the antibacterial effect of amoxicillin alone, allowing antibiotic doses to be reduced. It is important to reduce doses of amoxicillin because its widespread use in medicine could lead to the development of bacterial resistance and environmental pollution. This is the first report on the combined antibacterial activity of PPI surface-modified maltose dendrimers and amoxicillin.

Efficient alachlor degradation by the filamentous fungus Paecilomyces marquandii with simultaneous oxidative stress reduction.

The acceleration of alachlor degradation by Paecilomyces marquandii under controlled and optimized conditions of fungal cultivation in liquid batches was observed (by ca. 20% in comparison to the flask cultures). Acidic environment and oxygen limitation resulted in deterioration of herbicide elimination. Efficient xenobiotic degradation did not correlate with free radicals formation, but some conditions of bioreactor cultivation such as neutral pH and oxygen enriched atmosphere (pO2⩾30%) caused a decrease in the reactive oxygen species (ROS) accumulation in mycelia. The changes in the glutathione (GSH) and ascorbic acid (AA) levels, also in the dismutase (SOD) and catalase (CAT) activities showed active response of the tested fungus against alachlor induced oxidative stress. These results will contribute to the improvement of chloroacetanilides elimination by fungi and extend the knowledge concerning oxidative stress induction and fungal cellular defense.