Manuel Garcia-Käufer

Cyclotides Suppress Human T-Lymphocyte Proliferation by an Interleukin 2-Dependent Mechanism

Cyclotides are a diverse and abundant group of ribosomally synthesized plant peptides containing a unique cyclic cystine-knotted topology that confers them with remarkable stability. Kalata B1, a representative member of this family of mini-proteins, has been found to inhibit the proliferation of human peripheral blood mononuclear cells. Analysis of T-cell proliferation upon treatment with chemically synthesized kalata B1 mutants revealed a region comprising inter-cysteine loops 1 and 2 of the cyclotide framework to be important for biological activity. Cytokine signaling analysis using an ‘active’ kalata B1 mutant [T20K], and the reference drug cyclosporin A (CsA) demonstrated that treatment of activated T-lymphocytes with these compounds decreased the expression of the interleukin-2 (IL-2) surface receptor as well as IL-2 cytokine secretion and IL-2 gene expression, whereas the ‘inactive’ kalata B1 mutant [V10K] did not cause any effects. The anti-proliferative activity of [T20K] kalata B1 was antagonized by addition of exogenous IL-2. Furthermore, treatment with [T20K] kalata B1 led to an initial reduction of the effector function, as indicated by the reduced IFN-γ and TNF-α production, but the levels of both cytokines stabilized over time and returned to their normal levels. On the other hand, the degranulation activity remained reduced. This indicated that cyclotides interfere with T-cell polyfunctionality and arrest the proliferation of immune-competent cells through inhibiting IL-2 biology at more than one site. The results open new avenues to utilize native and synthetically-optimized cyclotides for applications in immune-related disorders and as immunosuppressant peptides.

Chronic ecotoxic effects to Pseudomonas putida and Vibrio fischeri, and cytostatic and genotoxic effects to the hepatoma cell line (HepG2) of ofloxacin photo(cata)lytically treated solutions.

Ofloxacin (OFL), a broad-spectrum and widespread-used photolabile fluoroquinolone, is frequently found in treated wastewaters, aquatic and terrestrial ecosystems leading to increasing concern during the past decades regarding its effects to the environment and human health. The elimination of OFL and other xenobiotics by the application of advanced oxidation processes using photolytic (PL) and photocatalytic (PC) treatments seems promising. However, an integrated assessment scheme is needed, in which, not only the removal of the parent compound, but also the effects of the photo-transformation products (PTPs) are investigated. For this purpose, in the present study, a chronic ecotoxic assessment using representative bacteria of marine and terrestrial ecosystems and a cytostatic and genotoxic evaluation using hepatoma cell line were performed. PL and PC treatments of OFL were applied using UV radiation. The photo-transformation of OFL during the treatments was monitored by DOC measurements and UPLC-MS/MS analysis. The chronic ecotoxicity of OFL and treated samples was evaluated using Pseudomonas putida and Vibrio fischeri; whereas the cytostasis and genotoxicity were estimated by the cytokinesis-block micronucleus assay (CBMN). The main results suggest that photo-transformation of OFL took place during these treatments since the concentration of OFL decreased when the irradiation time increased, as quantified by UPLC-MS/MS analysis, and this was not coupled with an analogous DOC removal. Furthermore, nine compounds were identified as probable PTPs formed through piperazinyl dealkylation and decarboxylation. The ecotoxicity of treated solutions to the bacteria studied decreased while the cytostasis to the hepatoma cell line remained at low levels during both treatments. However, the genotoxicity to the hepatoma cell line demonstrated a different pattern in which treated samples induced a greater number of MNi for the 4-16 min of irradiation (p<0.05) during both treatments. After 64 min of irradiation, the effects decreased to non genotoxic levels (p<0.05). These findings suggest that UV radiation for various treatment processes (catalytic or not), such as disinfection, may create genotoxic by-products. Therefore, in relevant technical applications, the residence time during treatment should receive special attention.

Cell-Cycle Changes and Oxidative Stress Response to Magnetite in A549 Human Lung Cells

In a recent study, magnetite was investigated for its potential to induce toxic effects and influence signaling pathways. It was clearly demonstrated that ROS formation leads to mitochondrial damage and genotoxic effects in A549 cells. On the basis of these findings, we wanted to elucidate the origin of magnetite-mediated ROS formation and its influence on the cell cycle of A549 and H1299 human lung epithelial cells. Concentration- and size-dependent superoxide formation, measured by electron paramagnetic resonance (EPR), was observed. Furthermore, we could show that the GSH level decreased significantly after exposure to magnetite particles, while catalase (CAT) activity was increased. These effects were also dependent on particle size, albeit less pronounced than those observed with EPR. We were able to show that incubation of A549 cells prior to particle treatment with diphenyleneiodonium (DPI), a NADPH-oxidase (NOX) inhibitor, leads to decreased ROS formation, but this effect was not observed for the NOX inhibitor apocynin. Soluble iron does not contribute considerably to ROS production. Analysis of cell-cycle distribution revealed a pronounced sub-G1 peak, which cannot be linked to increased cell death. Western blot analysis did not show activation of p53 but upregulation of p21 in A549. Here, we were unexpectedly able to demonstrate that exposure to magnetite leads to p21-mediated G1-like arrest. This has been reported previously only for low concentrations of microtubule stabilization drugs. Importantly, the arrested sub-G1 cells were viable and showed no caspase 3/7 activation.

Traditionally used Veronica officinalis inhibits proinflammatory mediators via the NF-κB signalling pathway in a human lung cell line

ETHNOPHARMACOLOGICAL RELEVANCE Extracts from Veronica officinalis L. are traditionally used for the treatment of lung diseases; however, the effective compounds and the mode of action are still unknown. AIM OF THE STUDY Here we analyzed the effects of a standardized Veronica extract on genes expression and signalling protein production associated with the development of inflammatory lung diseases. MATERIAL AND METHODS The degranulation capacity of primary mast cells, as well as gene expression and release of inflammatory mediators from human lung epithelial cells (A549 cells) were analyzed in relation to the synthetic drugs azelastine and dexamethasone. Gene and protein expression of cyclooxygenase-2 were investigated by semi-quantitative RT-PCR and western blotting, respectively. The involvement of phosphorylated mitogen-activated protein kinases and NF-κB signaling in regulation of these molecules were characterized by western blotting and electrophoretic mobility shift assays. Characteristic extract components were identified by LC-MS and verminoside was quantified by HPLC analysis. RESULTS We demonstrated that Veronica officinalis has a small influence on the degranulation capacity of mast cells but rather inhibits gene and protein expression of the chemokine eotaxin in A549 lung epithelial cells, which is essential for recruitment of inflammatory-associated cells in lung diseases. Furthermore, release of the inflammatory mediator PGE(2) was diminished through inhibition of COX-2 expression via the NF-κB signaling pathway in TNF-α-activated A549 cells. Phytochemical analysis identified verproside and verminoside as the most abundant iridoid glycosides. CONCLUSION Our results are a contribution to explaining the observed anti-inflammatory effects of Veronica offcinalis extract on a molecular level. However, its clinical potency has at first to be proven in animals and subsequently in clinical trials.

Antigenotoxic action of isothiocyanate-containing mustard as determined by two cancer biomarkers in a human intervention trial.

Chemopreventive constituents in food plants, such as brassica-derived isothiocyanates (ITC), have been shown to be quite effective in the prevention of genotoxic DNA damage in cell culture models and carcinogenesis in laboratory animals. We have conducted a controlled intervention study with 14 participants (10 female, four male) using DNA damage and micronucleus formation as intermediate endpoints to assess the chemopreventive nature of mustard. For this trial, human volunteers were fed 20 g (25 mg total ITC) of mustard preparation, daily, for 4 days. Heparinized blood was collected by venipuncture and processed for the comet assay or the micronucleus test. A 3-day intervention with mustard led to a significant reduction in DNA damage and micronucleus formation induced by hydrogen peroxide or benzo(a)pyrene diolepoxide. Clinical liver parameters were unchanged by the intervention; however, cholesterol levels were significantly reduced. The results of this study indicate that consumption of low amounts of ITC-containing mustard quickly and effectively modulates cytoprotective factors in peripheral blood mononuclear cells and/or blood. The fact that these observations were confirmed by two cytogenetic biomarkers for cancer risk implies that even short-term intake of ITC-containing vegetables might indeed be associated with reduced cancer risk.

Cellular Uptake and Toxic Effects of Fine and Ultrafine Metal-Sulfate Particles in Human A549 Lung Epithelial Cells

Ambient airborne particulate matter is known to cause various adverse health effects in humans. In a recent study on the environmental impacts of coal and tire combustion in a thermal power station, fine crystals of PbSO(4) (anglesite), ZnSO(4)·H(2)O (gunningite), and CaSO(4) (anhydrite) were identified in the stack emissions. Here, we have studied the toxic potential of these sulfate phases as particulates and their uptake in human alveolar epithelial cells (A549). Both PbSO(4) and CaSO(4) yielded no loss of cell viability, as determined by the WST-1 and NR assays. In contrast, a concentration-dependent increase in cytotoxicity was observed for Zn sulfate. For all analyzed sulfates, an increase in the production of reactive oxygen species (ROS), assessed by the DCFH-DA assay and EPR, was observed, although to a varying extent. Again, Zn sulfate was the most active compound. Genotoxicity assays revealed concentration-dependent DNA damage and induction of micronuclei for Zn sulfate and, to a lower extent, for CaSO(4), whereas only slight effects could be found for PbSO(4). Moreover, changes of the cell cycle were observed for Zn sulfate and PbSO(4). It could be shown further that Zn sulfate increased the nuclear factor kappa-B (NF-κB) DNA binding activity and activated JNK. During our TEM investigations, no effect on the appearance of the A549 cells exposed to CaSO(4) compared to the nonexposed cells was observed, and in our experiments, only one CaSO(4) particle was detected in the cytoplasm. In the case of exposure to Zn sulfate, no particles were found in the cytoplasm of A549 cells, but we observed a concentration-dependent increase in the number and size of dark vesicles (presumably zincosomes). After exposure to PbSO(4), the A549 cells contained isolated particles as well as agglomerates both in vesicles and in the cytoplasm. Since these metal-sulfate particles are emitted into the atmosphere via the flue gas of coal-fired power stations, they may be globally abundant. Therefore, our study is of direct relevance to populations living near such power plants.

Viscum album neutralizes tumor-induced immunosuppression in a human in vitro cell model

Tumor cells have the capacity to secrete immunosuppressive substances in order to diminish dendritic cell (DC) activity and thereby escape from immune responses. The impact of mistletoe (Viscum album) extracts (VAE), which are frequently used as an additive anti-cancer therapy to stimulate the immune response, is still unknown. Using a human cellular system, the impact of two different VAE (VAEA + VAEI) on the maturation of human dendritic cells and on T cell function has been investigated using flow cytometry, automated fluorescence microscopy and cytokine bead array assays. Furthermore, we examined whether VAEI was able to counteract tumor-induced immunosuppression within this cellular system using a renal cancer cell model. The role of mistletoe lectin (ML) was analyzed using ML-specific antibodies and ML-depleted VAEI. VAEI and VAEA augmented the maturation of dendritic cells. VAEI abrogated tumor-induced immunosuppression of dendritic cells and both processes were partially mediated by ML since ML-depleted VAEI and ML-specific antibodies almost neutralized the rehabilitative effects of VAEI on DC maturation. Using these settings, co-culture experiments with purified CD4+ T cells had no influence on T cell proliferation and activation but did have an impact on IFN-γ secretion. The study provides a potential mode-of-action of VAE as an additive cancer therapy based on immunomodulatory effects. However, the impact on the in vivo situation has to be evaluated in further studies.

Cytotoxic and genotoxic responses of human lung cells to combustion smoke particles of Miscanthus straw, softwood and beech wood chips

Inhalation of particulate matter (PM) from residential biomass combustion is epidemiologically associated with cardiovascular and pulmonary diseases. This study investigates PM0.4-1 emissions from combustion of commercial Miscanthus straw (MS), softwood chips (SWC) and beech wood chips (BWC) in a domestic-scale boiler (40 kW). The PM0.4-1 emitted during combustion of the MS, SWC and BWC were characterized by ICP-MS/OES, XRD, SEM, TEM, and DLS. Cytotoxicity and genotoxicity in human alveolar epithelial A549 and human bronchial epithelial BEAS-2B cells were assessed by the WST-1 assay and the DNA-Alkaline Unwinding Assay (DAUA). PM0.4-1 uptake/translocation in cells was investigated with a new method developed using a confocal reflection microscope. SWC and BWC had a inherently higher residual water content than MS. The PM0.4-1 emitted during combustion of SWC and BWC exhibited higher levels of Polycyclic Aromatic Hydrocarbons (PAHs), a greater variety of mineral species and a higher heavy metal content than PM0.4-1 from MS combustion. Exposure to PM0.4-1 from combustion of SWC and BWC induced cytotoxic and genotoxic effects in human alveolar and bronchial cells, whereby the strongest effect was observed for BWC and was comparable to that caused by diesel PM (SRM 2 975), In contrast, PM0.4-1 from MS combustion did not induce cellular responses in the studied lung cells. A high PAH content in PM emissions seems to be a reliable chemical marker of both combustion efficiency and particle toxicity. Residual biomass water content strongly affects particulate emissions and their toxic potential. Therefore, to minimize the harmful effects of fine PM on health, improvement of combustion efficiency (aiming to reduce the presence of incomplete combustion products bound to PM) and application of fly ash capture technology, as well as use of novel biomass fuels like Miscanthus straw is recommended.

Immunomodulatory effects of metal salts at sub-toxic concentrations.

Because different metals are used in complementary medicine for the treatment of diseases related to a dysfunction of the immune system, this study aimed at determining the immunomodulatory potential of Pb(NO3)2, AuCl3, Cu(NO3)2, HgCl2, AgNO3, SnCl2, AsCl3 and SbCl3 at sub‐toxic concentrations and at assessing possible toxic side effects of low‐concentrated metal preparations. The influence of the metal salts on primary human mononuclear cells was analyzed by measuring cell viability using the water‐soluble tetrazolium salt assay, apoptosis and necrosis induction by annexin V/propidium iodide staining and proliferation by carboxyfluorescein diacetate succinimidyl ester staining and flow cytometry. Effects on T‐cell activation were assessed with CD69 and CD25 expression using flow cytometry whereas CD83, CD86 and CD14 expression was measured to evaluate the influence on dendritic cell maturation. Alterations of interleukin‐2 and interferon‐γ secretion were detected by enzyme‐linked immunosorbent assay and genotoxic effects were analyzed using the comet assay. At sub‐toxic concentrations retardation of T‐cell proliferation was caused by Pb(NO3)2, AuCl3 and Cu(NO3)2 and inhibitory effects on interleukin‐2 secretion were measured after incubation with Pb(NO3)2, AuCl3, Cu(NO3)2, HgCl2 and AsCl3. Cu(NO3)2 had immunosuppressive activity at dosages within the serum reference range for copper. All other metal salts showed effects at dosages above upper serum limits of normal. Therefore, only low‐concentrated copper preparations are promising to have immunomodulatory potential. Toxic side effects of metal preparations used in complementary medicine are improbable because upper limits of metals set in the drinking water ordinance are either not exceeded or the duration of their application is limited. Copyright

4-Methylthiobutyl isothiocyanate (Erucin) from rocket plant dichotomously affects the activity of human immunocompetent cells

Isothiocyanates (ITC) from the Brassicaceae plant family are regarded as promising for prevention and treatment of cancer. However, experimental settings consider their therapeutic action without taking into account the risk of unwanted effects on healthy tissues. In the present study we investigated the effects of Eruca sativa seed extract containing MTBITC (Erucin) and pure Erucin from rocket plant on healthy cells of the human immune system in vitro. Hereby, high doses of the plant extract as well as of Erucin inhibited cell viability of human lymphocytes via induction of apoptosis to comparable amounts. Non-toxic low concentrations of the plant extract and pure Erucin altered the expression of the interleukin (IL)-2 receptor but did not affect further T cell activation, proliferation and the release of the effector molecules interferon (IFN)-gamma and IL-2 of T-lymphocytes. However, the activity of NK-cells was significantly reduced by non-toxic concentrations of the plant extract and pure Erucin. These results indicate that the plant extract and pure Erucin interfere with the function of human T lymphocytes and decreases the activity of NK-cells in comparable concentrations. Long-term clinical studies with ITC-enriched plant extracts from Brassicaceae should take this into account.