Effect of dental composite dust on human gingival keratinocytes.

OBJECTIVE: The aim was to investigate the effect of particles released during grinding of dental composites on human gingival keratinocytes (HGK). METHODS: Specimens from Filtek™ Supreme XTE and ceram.x® universal were prepared and ground to dust. The dust was filtered (≤ 5 µm) and the particle size distribution was examined using NANO-flex®-180° dynamic light scattering (DLS). Suspensions at five concentrations (3, 10, 30, 100 and 300 µg/mL) were prepared using keratinocyte growth medium (KGM). These suspensions, as well as a positive (CuO) and a negative control (KGM) were added to HGK. The cells treated with Filtek™ Supreme XTE suspensions were analyzed by real-time monitoring using RTCA iCELLigence™. In addition, light and scanning electron microscopic images of the exposed cells were taken. Indirect immunofluorescence staining was performed to detect the extracellular matrix protein fibronectin. RESULTS: In distilled water, DLS showed similar particles' range (171.9 nm- 2.7 µm) for both composites. In saliva, larger particles were detected (Filtek™ Supreme XTE: 243 nm-6,5 µm; ceram.x® universal: 204 nm- 4,6 µm). iCELLigence™ revealed similar results of cell growth parameters for HGK incubated with composite dust (≤ 5 µm) at different concentrations. The microscopic images indicated unaltered cell structures and formation of large agglomerates with high particle concentration (> 100 µg/mL). Exposure to composite dust resulted in upregulation of fibronectin expression. SIGNIFICANCE: Grinding of dental composite materials generates dust particles of different sizes. The particle size distribution seems to be more influenced by the suspending medium than the material itself. While cell growth of HGK seem not to be affected by the particles, an upregulation of fibronectin in the intercellular space concomitant by increasing particle concentration may indicate an increase of cell migration/mobility.

Method development for the intraoral release of nanoparticles from dental restorative materials.

OBJECTIVE: The aim of this study was the development of a novel in-vitro method to evaluate the intraoral release of wear particles with a diameter< 1 µm from dental restorative materials. METHODS: Test fixtures for a dual-axis chewing simulator (CS-4.8, SD Mechatronik, Feldkirchen-Westerham, Germany), consisting of three components to mount the specimens and a solvent (distilled water) as well as a zirconia antagonist to transfer the masticatory forces onto the specimen was developed. Ceram.x Spectra™ ST HV (CS) and Filtek™ Supreme XTE (FS) specimens (n = 3) were fixed into the mounts and immersed in 25 ml solvent. All specimens were subjected to 500.000 wear cycles with a load of 49 N. The particle size distribution of the suspensions were examined by dynamic light scattering (DLS). The collected particles were characterised by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS). For wear quantification, the surfaces of the specimens were photo-optically scanned and the wear was measured. For the statistical analysis, one-way ANOVA and post-hoc Scheffé tests were applied. RESULTS: DLS showed particle diameters< 1 µm (CS: 18.06 nm-1.64 µm, FS: 72.30 nm-2.31 µm). SEM/EDS indicated an association between the detected elements and the materials' composition. FS showed significantly higher volume loss (p = 0.007) and maximum depth of the wear profile (p = 0.005) than CS, but no significant differences in the surface loss (p = 0.668). SIGNIFICANCE: The novel method is able to detect material dependent particles to the size of nanoscale after in-vitro abrasion.

Cytotoxic and inflammatory response of human lung epithelial cells A549 to particles released from dental restorative materials during dry and wet grinding.

OBJECTIVES: The aim was to evaluate the release of particles from dental materials during wet and dry grinding and test their effects on human lung epithelia cells in-vitro. METHODS: Four dental restorative materials were used: two composites [Ceram.x® universal (Dentsply Sirona) and Filtek™ Supreme XTE (3 M)], one ceramic [VITABLOCS® Mark II (VITAy)] and a ceramic-resin material [Lava™ Ultimate (3 M)]. Material samples were ground to powder under standardized wet and dry conditions in an isolated dental room. During grinding, the particle concentrations were measured with LAS and CPC. Baseline values were measured before grinding. The particles' size was evaluated using DLS and SEM. Water was used as control. The cytotoxicity and inflammatory response of the lung cells (A549) after exposure to different concentrations (1, 3, 10, 30, 100, 300 µg/mL) of the generated dust were analyzed with LDH, WST-1 and ELISA. RESULTS: LAS and CPC revealed a high concentration of particles< 10 µm and< 1 µm respectively, into the air. Particles showed high tendency to agglomerate. DLS showed particle size distribution between 150 nm and 18 µm independently of the material composition. All materials induced significant effects (p < 0.05) on the cell membrane integrity and viability of the A549 cells. Only the ceramic particles showed a significant increase in hydroxyl radical formation at low concentrations (p < 0.05), for both wet and dry conditions. All materials except ceramic, induced a significant release of IL-8 in A549 cells at 300 µg / mL (p < 0.05). SIGNIFICANCE: Wet and dry grinding of dental materials result in release of ultrafine and fine particulate matter into the air. The in-vitro findings on the cellular response of lung cells to generated dust indicate a potential risk for human health due inhalation of the released particles. The use of water-cooling seems to be beneficial resulting in reduced release of particles compared to dry grinding.

Impact of Nanocomposite Combustion Aerosols on A549 Cells and a 3D Airway Model.

The use of nanomaterials incorporated into plastic products is increasing steadily. By using nano-scaled filling materials, thermoplastics, such as polyethylene (PE), take advantage of the unique properties of nanomaterials (NM). The life cycle of these so-called nanocomposites (NC) usually ends with energetic recovery. However, the toxicity of these aerosols, which may consist of released NM as well as combustion-generated volatile compounds, is not fully understood. Within this study, model nanocomposites consisting of a PE matrix and nano-scaled filling material (TiO2, CuO, carbon nano tubes (CNT)) were produced and subsequently incinerated using a lab-scale model burner. The combustion-generated aerosols were characterized with regard to particle release as well as compound composition. Subsequently, A549 cells and a reconstituted 3D lung cell culture model (MucilAir™, Epithelix) were exposed for 4 h to the respective aerosols. This approach enabled the parallel application of a complete aerosol, an aerosol under conditions of enhanced particle deposition using high voltage, and a filtered aerosol resulting in the sole gaseous phase. After 20 h post-incubation, cytotoxicity, inflammatory response (IL-8), transcriptional toxicity profiling, and genotoxicity were determined. Only the exposure toward combustion aerosols originated from PE-based materials induced cytotoxicity, genotoxicity, and transcriptional alterations in both cell models. In contrast, an inflammatory response in A549 cells was more evident after exposure toward aerosols of nano-scaled filler combustion, whereas the thermal decomposition of PE-based materials revealed an impaired IL-8 secretion. MucilAir™ tissue showed a pronounced inflammatory response after exposure to either combustion aerosols, except for nanocomposite combustion. In conclusion, this study supports the present knowledge on the release of nanomaterials after incineration of nano-enabled thermoplastics. Since in the case of PE-based combustion aerosols no major differences were evident between exposure to the complete aerosol and to the gaseous phase, adverse cellular effects could be deduced to the volatile organic compounds that are generated during incomplete combustion of NC.

Rosemary has immunosuppressant activity mediated through the STAT3 pathway.

OBJECTIVE: In Europe extracts of Rosmarinus officinalis were traditionally used for the treatment of rheumatic diseases. We investigated the capacity of standardized aqueous extracts of Rosmarinus officinalis on human primary lymphocyte function in vitro, as activated lymphocytes are an important mediator of rheumatic diseases. METHODS: Lymphocyte proliferation was measured using membrane-permeable dye carboxyfluorescein diacetate succinimidyl ester (CFSE). Apoptosis was analysed by surface staining of phosphatidylserine (annexin V-assay) and necrosis was analysed by staining with propidium iodide. Modification of cell activity was detected by surface staining of CD69 and CD25. The activity of STAT3 in T-lymphocytes was determined by intracellular staining of STAT3 molecules. All endpoints were analyzed by using flow cytometry. The Rosmarinus officinalis extract was investigated at concentrations of 0.05-25 mg/mL. Analysis of the extract was performed using HPLC methods. RESULTS: Rosmarinus officinalis inhibited proliferation of human lymphocytes and CD4+ T-cells in a dose-dependent manner (3.1-25 mg/mL) through induction of apoptosis. The intracellular signalling pathway STAT3 in T-cells, but not NF-kappaB and ERK1/2 in T- and B-cells was inhibited in a dose-dependent manner by Rosmarinus officinalis (0.2-6.2 mg/mL). Rosmanol, carnosolic acid, carnosol and trans-caffeic acid were tested in the same cellular models as the crude extract. From these, only trans-caffeic acid inhibited lymphocyte proliferation and STAT3 (30-100 µg/mL). Trans-caffeic acid was found in the extract in a concentration of 14.7 µg/mL. CONCLUSIONS: We conclude that an immunosuppressive effect of Rosmarinus officinalis is mostly due to the effect of trans-caffeic acid. It results in inhibition of the activity of STAT3 causing induction of apoptosis and inhibition of proliferation of T-lymphocytes.

Characterization and in vitro biological effects of ambient air PM10 from a rural, an industrial and an urban site in Sulaimani City, Iraq.

High urban atmospheric pollution is caused by economic and industrial growth, especially in developing countries. The objective of this study was to assess possible relationships between in vitro effects on human alveolar epithelial cells of source-related dust types collected at Sulaimani City (Iraq), and to determine their mineralogical and chemical composition. A passive sampler was used to collect dust particles at a rural, an industrial and an urban sampling site during July and August 2014. The samples were size-fractionated by a low-pressure impactor to obtain respirable dust with aerodynamic diameters of less than 10 µm. The dust was mainly composed of quartz and calcite. Chrysotile fibres (white asbestos) were also found at the urban site. Dust from the industrial and urban sites triggered cytotoxic and genotoxic effects in the cells, whereas only minor effects were observed for the sample from the rural site.

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 © 2016 John Wiley & Sons, Ltd.

Constituents from oak bark (Quercus robur L.) inhibit degranulation and allergic mediator release from basophils and mast cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Oak bark has been used since ancient times in Europaen ethnomedicine because of its adstringent, antimicrobial and hemostatic features, e.g. as a remedy for the treatment of wounds and skin diseases. PURPOSE: Oak bark tannins are considered as bioactive natural products, interacting with surface proteins of mucous membranes and might be beneficial for the treatment of allergic diseases. This study investigated the effect of an oak bark decoction (OBD) and isolated tannin fractions on the degranulation capacity and cytokine/chemokine release from rat basophilic cells and human mast cells in vitro, which are essential for the initiation of early- and late-phase allergic reactions. METHODS AND METHODS: By chromatographic separation on Sephadex® LH-20 high- and low-molecular weight tannins were separated from OBD and the tannin composition analyzed by HPLC(DAD)-MSn. Then, the OBD and its fractions were tested in degranulation (ß-hexosaminidase activity) of allergen-specific-activated basophilic cells in a photometric assay. RESULTS: The OBD and the high-molecular tannin fraction showed a dose-dependent inhibition of cell degranulation. Furthermore, the OBD and particularly its high molecular weight tannin fraction exhibited an inhibitory activity on the IL-8-, IL-6- and TNF-α-secretion from stimulated human mast cells, detected and quantified by ELISA. CONCLUSION: The OBD and its high-molecular weight tannins revealed an impact on allergic mediator release of basophilic cells and human mast cells and thereby provide a rationale for the topical treatment with OBD preparations.

Effects of Inonotus hispidus Extracts and Compounds on Human Immunocompetent Cells.

Inonotus hispidus is used as a traditional medicine in China. Previous investigations revealed promising immunomodulatory activity of fruit body extracts of I. hispidus. Bioactivity-guided fractionation showed that hispolon and hispidin were active substances.In this study, we analysed the effects of I. hispidus extract and selected constituents on different types of human immune cells and investigated the potential of I. hispidus extract as a medicinal mushroom. The influence of I. hispidus extract on activity and maturation of human T cells, purified natural killer cells, and dendritic cells was analysed using cytometric-based surface marker expression. The cell division characteristics of the activated T cells were assessed by membrane permeable dye, and the function of natural killer cells was investigated by a degranulation CD107a assay. Apoptosis induction was assessed by surface staining of phosphatidylserine, and camptothecin and cyclosporine A were used individually as controls. Phytochemical analysis, using TLC chromatograms and HPLC analysis, was conducted to characterise the I. hispidus extract. I. hispidus extract increased the activation and diminished the proliferation of activated human T cells in the presence of apoptosis. Natural killer cell activity and function were dose-dependently increased. Surface marker expression of dendritic cells demonstrated that I. hispidus extract has the potential to induce maturation. TLC and HPLC analyses showed that the extract contained hispidin and hispolon. Investigations using hispidin and hispolon demonstrated similar, albeit noncongruent, results with extracts on measured parameters.The results indicate that extracts from I. hispidus and their constituents, hispidin and hispolon, interfere with the function of multiple immune cells, thus providing a rationale for their potential as a medicinal mushroom.

Immunomodulatory effects of preparations from Anthroposophical Medicine for parenteral use.

BACKGROUND: Preparations from anthroposophical medicine (AM) are clinically used to treat inflammatory disorders. We wanted to investigate effects of a selection of AM medications for parenteral use in cell-based systems in vitro. METHODS: Colchicum officinale tuber D3, Mandragora D3, Rosmarinus officinale 5% and Bryophyllum 5% were selected for the experiments. Induction of apoptosis and necrosis (human lymphocytes and dendritic cells [DCs]) and proliferation of lymphocytes as well as maturation (expression of CD14, CD83 and CD86) and cytokine secretion (IL-10, IL12p70) of DCs were analyzed. Furthermore, proliferation of allogeneic human T lymphocytes was investigated in vitro in coculture experiments using mature DCs in comparison to controls. RESULTS: The respective preparations did not induce apoptosis or necrosis in lymphocytes or DCs. Lymphocyte proliferation was dose-dependently reduced by Colchicum officinale tuber D3 while the viability was unchanged. Rosmarinus officinale 5%, but not the other preparations, dose-dependently inhibited the maturation of immature DCs, reduced secretion of IL-10 and IL-12p70 and slightly inhibited proliferation of allogeneic CD4(+) T-lymphocytes in coculture experiments with DCs. CONCLUSION: The selected preparations from AM for parenteral use are nontoxic to lymphocytes and DCs. Rosmarinus officinale 5% has immunosuppressive properties on key functions of the immune system which propose further investigation.

Investigations on sediment toxicity of German rivers applying a standardized bioassay battery.

River sediments may contain a huge variety of environmental contaminants and play a key role in the ecological status of aquatic ecosystems. Contaminants adsorbed to sediments and suspended solids may contribute directly or after remobilization to an adverse ecological and chemical status of surface water. In this subproject of the joint research project DanTox, acetonic Soxhlet extracts from three German river sediments from the River Rhine (Altrip and Ehrenbreitstein with moderate contamination) and River Elbe (Veringkanal Hamburg heavily contaminated) were prepared and redissolved in dimethyl sulfoxide (DMSO). These extracts were analyzed with a standard bioassay battery with organisms from different trophic levels (bacteria, algae, Daphnia, fish) as well as in the Ames test and the umuC test for bacterial mutagenicity and genotoxicity according to the respective OECD and ISO guidelines. In total, 0.01% (standard) up to 0.25% (only fish embryo test) of the DMSO sediment extract was dosed to the test systems resulting in maximum sediment equivalent concentrations (SEQ) of 2 up to 50 g l(-1). The sediment of Veringkanal near Hamburg harbor was significantly more toxic in most tests compared to the sediment extracts from Altrip and Ehrenbreitstein from the River Rhine. The most toxic effect found for Veringkanal was in the algae test with an ErC50 (72 h) of 0.00226 g l(-1) SEQ. Ehrenbreitstein and Altrip samples were about factor 1,000 less toxic. In the Daphnia, Lemna, and acute fish toxicity tests, no toxicity at all was found at 2 g l(-1) SEQ. corresponding to 0.01% DMSO. Only when increasing the DMSO concentration the fish embryo test showed a 22-fold higher toxicity for Veringkanal than for Ehrenbreitstein and Altrip samples, while the toxicity difference was less evident for the Daphnia test due to the overlaying solvent toxicity above 0.05% dimethyl sulfoxide (DMSO). The higher toxicities observed with the Veringkanal sample are supported by the PAH and PCB concentrations analyzed in the sediments. The sediment extracts of Altrip and Veringkanal were mutagenic in the Ames tester strain TA98 with metabolic activation (S9-mix). The findings allow a better ecotoxicological characterization of the sediments extensively analyzed in all subprojects of the DanTox project (e.g., Garcia-Kaeufer et al. Environ Sci Pollut Res. doi: 10.1007/s11356-014-3894-4 , 2014; Schiwy et al. Environ Sci Pollut Res. doi: 10.1007/s11356-014-3185-0 , 2014; Hollert and Keiter 2015). In the absence of agreed limit values for sediment extracts in standard tests, further data with unpolluted reference sediments are required for a quantitative risk assessment of the investigated polluted sediments.

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.

Differential cytotoxic properties of Helleborus niger L. on tumour and immunocompetent cells.

ETHNOPHARMACOLOGICAL RELEVANCE: In Romanian folk medicine, Helleborus niger L. is used for the treatment of rheumatoid arthritis or viral infections and in complementary therapy, especially in anthroposophic medicine (AM), where the plant is administered as an adjuvant to treat malignant diseases. In the present study, we investigated the differential cytotoxic effects of H. niger on human tumour and healthy cells of the human immune system in vitro. MATERIAL AND METHODS: Protoanemonin and saponins, as significant constituents of H. niger extracts, were quantified in five individual batches using validated HPLC methods. Further, the impact of H. niger on proliferation capacity (MTT assay) as well as on apoptosis and necrosis induction in a panel of tumour cell lines and human lymphocytes (combined annexin V and propidium iodide staining) was monitored. In addition, NK cell function (degranulation-CD107a assay and IFN-gamma secretion) was also investigated since these immunocompetent cells are important for the control of malignancies within the human body. RESULTS: Extracts of H. niger induced proliferation inhibition not only of lymphoblastic leukaemia cells (MOLT4; IC50: 171 µg/mL) but also of myosarcoma (SK-UT-1b; IC50: 304 µg/mL) and melanoma cells (HT-144; IC50: 569 µg/mL) due to the induction of apoptosis. Purified T cells or NK cells were significantly affected through the presence of high H. niger concentrations while bulk lymphocytes were not affected. NK cells' anti-tumour functions expressed by degranulation capacity as well as IFN-y production were unaffected by the presence of the H. niger extract. Since protoanemonin and saponins have been reported in the literature to exert cytotoxic effects, their content was also determined. CONCLUSIONS: H. niger extracts exhibit differential cytotoxicity towards tumour cell lines and healthy human T- and NK-cells.

Equisetum arvense (common horsetail) modulates the function of inflammatory immunocompetent cells.

BACKGROUND: In Europe, extracts of Equisetum arvense (common horsetail) have a long tradition in the treatment of inflammatory disorders. To understand the molecular basis for its use, we investigated the immunomodulatory capacity of a standardized commercially available common horsetail extract on human primary lymphocyte function in vitro. METHODS: The standardized extract of Equisetum arvense was phytochemically characterized. Effects on proliferation, viability and activity of mitogen-activated human lymphocytes were assessed in comparison to cyclosporine A using annexin V/propidium iodide staining assays and flow cytometry-based surface receptor characterization, respectively. Intracellular levels of effector molecules (IL-2, IFN-γ and TNF-α) were analyzed with cytokine assays. RESULTS: T cell proliferation was inhibited dose dependently by the Equisetum extract without induction of apoptosis or necrosis. This effect was mediated through inhibition of lymphocyte activation, specifically by diminishing CD69 and IL-2 surface receptor expression and intracellular IL-2 production. Furthermore, treatment with Equisetum arvense inhibited effector functions, as indicated by reduced production of IFN-γ and TNF-α. CONCLUSIONS: The data indicate that the used extract of Equisetum arvense interferes with the polyfunctionality of immunocompetent cells thereby providing an anti-inflammatory mode-of-action.

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.

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.

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.