Albert Duschl
University of Salzburg, Dept. of Molecular Biology, Faculty Member
A role of IgE antibodies in cancer surveillance has been implicated for a long time. Studies dealing with IgE antibodies directly targeted to tumor antigens have shown marked anticancer effects mediated by this antibody class. Thus, the... more
A role of IgE antibodies in cancer surveillance has been implicated for a long time. Studies dealing with IgE antibodies directly targeted to tumor antigens have shown marked anticancer effects mediated by this antibody class. Thus, the basic function of IgE antibodies may be to control tumor growth. Thus far, cancer-specific IgE has only been applied passively. Consequently, the aim
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A one-step purification method for halorhodopsin was developed. Functional proteoliposomes were prepared from this preparation using cholate, which is removed by dialysis in the presence of asolectin or the polar halobacterial lipids.... more
A one-step purification method for halorhodopsin was developed. Functional proteoliposomes were prepared from this preparation using cholate, which is removed by dialysis in the presence of asolectin or the polar halobacterial lipids. Light-induced outward directed transport of chloride by halorhodopsin was followed by measuring passive proton efflux in the presence of uncoupler; initial rates and extents amounted to significant fractions of values obtained for halorhodopsin-containing cell envelope vesicles. The transport activity was much higher when cholate rather than octyl glucoside was used in the reconstitution. Since CD spectra in cholate but not in octyl glucoside showed band-splitting in the visible region, suggestive of exciton interaction between halorhodopsin monomers, the reconstitution may depend on an aggregate state of the halorhodopsin. The rate constants for three thermal steps in the halorhodopsin photocycle were greatly reduced in the detergent-solubilized sampl...
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Pharaonis halorhodopsin is a light-driven transport system for chloride, similarly to the previously described halorhodopsin, but we find that it transports nitrate as effectively as chloride. We studied the photoreactions of the... more
Pharaonis halorhodopsin is a light-driven transport system for chloride, similarly to the previously described halorhodopsin, but we find that it transports nitrate as effectively as chloride. We studied the photoreactions of the purified, detergent-solubilized pharaonis pigment with a gated multichannel analyzer. At a physiological salt concentration (4 M NaCl), the absorption spectra and rate constants of rise and decay for intermediates of the photocycle were similar to those for halorhodopsin. In buffer containing nitrate, halorhodopsin exhibits a second, truncated photocycle; this difference in the photoreaction of the pigment occurs when an anion is bound in such a way as to preclude transport. As expected from the lack of anion specificity in the transport, the photocycle of pharaonis halorhodopsin was nearly unaffected by replacement of chloride with nitrate. All presumed buried positively charged residues, which might play a role in anion binding, are conserved in the two p...
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We cloned and sequenced the gene coding for the polypeptide of a halorhodopsin in Natronobacterium pharaonis (named here pharaonis halorhodopsin). Peptide sequencing of cyanogen bromide fragments, and immunoreactions of the protein and... more
We cloned and sequenced the gene coding for the polypeptide of a halorhodopsin in Natronobacterium pharaonis (named here pharaonis halorhodopsin). Peptide sequencing of cyanogen bromide fragments, and immunoreactions of the protein and synthetic peptides derived from the COOH-terminal gene sequence, confirmed that the open reading frame is the structural gene for the pharaonis halorhodopsin polypeptide. The flanking DNA sequences, as well as those for other bacterial rhodopsins, were compared to previously proposed archaebacterial consensus sequences. In pairwise comparisons of the open reading frame with DNA sequences for bacterio-opsin and halo-opsin from Halobacterium halobium, silent divergences (mutations/nucleotide at codon positions which do not result in amino acid changes) were calculated. These indicate very considerable evolutionary distance between each pair of genes. In spite of this, the three protein sequences show extensive similarities, indicating strong selective p...
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Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin... more
Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c+ dendritic cells (DCs) with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002-2 ng/ml). We show that CD1c+ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated with high CD14 e...
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IL-31, a member of the IL-6 protein family, is one of the latest additions to the list of T cell-derived cytokines. Th2 cells are regarded as a main source of IL-31, which is produced in response to stimulation by IL-4. Because the... more
IL-31, a member of the IL-6 protein family, is one of the latest additions to the list of T cell-derived cytokines. Th2 cells are regarded as a main source of IL-31, which is produced in response to stimulation by IL-4. Because the development of Th9 cells also requires IL-4 as a polarizing cytokine, the current study investigates IL-31 production in human Th9 cells compared with Th2 cells. We found that, although Th9 cells were able to release IL-31 during the first weeks of in vitro polarization, no IL-31 was detected in Th9 cultures after a final restimulation in the absence of polarizing cytokines. We further show that TGF-β, which is required to obtain Th9 cells in vitro, potently inhibits the release of IL-31 from Th2 cells, whereas IL-33, a cytokine associated with Th2-mediated inflammation, synergizes with IL-4 in inducing IL-31 secretion. To analyze the molecular mechanisms underlying the induction of IL-31, EMSAs, reporter gene assays, and small interfering RNA-based silen...
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Many toxicological studies on silica nanoparticles (NPs) have been reported, however, the literature often shows various conclusions concerning the same material. This is mainly due to a lack of sufficient NPs characterization as... more
Many toxicological studies on silica nanoparticles (NPs) have been reported, however, the literature often shows various conclusions concerning the same material. This is mainly due to a lack of sufficient NPs characterization as synthesized as well as in operando. Many characteristics of NPs may be affected by the chemistry of their surroundings and the presence of inorganic and biological moieties. Consequently, understanding the behavior of NPs at the time of toxicological assay may play a crucial role in the interpretation of its results.The present study examines changes in properties of differently functionalized fluorescent 50 nm silica NPs in a variety of environments and assesses their ability to absorb proteins from cell culture medium containing either bovine or human serum. The colloidal stability depending on surface functionalization of NPs, their concentration and time of exposure was investigated in water, standard biological buffers, and cell culture media by dynami...
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In nanotoxicology, the exact role of particle shape, in relation to the composition, on the capacity to induce toxicity is largely unknown. We investigated the toxic and immunotoxic effects of silver wires (length: 1.5 - 25 μm; diameter... more
In nanotoxicology, the exact role of particle shape, in relation to the composition, on the capacity to induce toxicity is largely unknown. We investigated the toxic and immunotoxic effects of silver wires (length: 1.5 - 25 μm; diameter 100 - 160 nm), spherical silver nanoparticles (30 nm) and silver microparticles (<45 μm) on alveolar epithelial cells (A549). Wires and nanoparticles were synthesized by wet-chemistry methods and extensively characterized. Cell viability and cytotoxicity were assessed and potential immunotoxic effects were investigated. To compare the effects on an activated and a resting immune system, cells were stimulated with rhTNF-α or left untreated. Changes in intracellular free calcium levels were determined using calcium imaging. Finally, ion release from the particles was assessed by ICP-MS and the effects of released ions on cell viability and cytotoxicity were tested. No effects were observed for the spherical particles, whereas the silver wires signif...
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Allergy prevalence has increased in industrialized countries. One contributing factor could be pollution, which can cause nitration of allergens exogenously (in the air) or endogenously (in inflamed lung tissue). We investigated the... more
Allergy prevalence has increased in industrialized countries. One contributing factor could be pollution, which can cause nitration of allergens exogenously (in the air) or endogenously (in inflamed lung tissue). We investigated the impact of nitration on both the structural and immunological behavior of the major birch pollen allergen Bet v 1.0101 to determine whether nitration might be a factor in the increased incidence of allergy. Bet v 1.0101 was nitrated with tetranitromethane. Immune effects were assessed by measuring the proliferation of specific T-cell lines (TCLs) upon stimulation with different concentrations of nitrated and unmodified allergen, and by measurement of cytokine release of monocyte-derived dendritic cells (moDCs) and primary DCs (primDCs) stimulated with nitrated versus unmodified allergen. HPLC-MS, crystallography, gel electrophoresis, amino acid analysis, size exclusion chromatography and molecular dynamics simulation were performed to characterize structu...
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Some organophosphate insecticides have immunomodulating capacities, but it is unknown whether different compounds within this class affect the immune system to the same extent. In this in vitro study, human immortalized T-lymphocytes or... more
Some organophosphate insecticides have immunomodulating capacities, but it is unknown whether different compounds within this class affect the immune system to the same extent. In this in vitro study, human immortalized T-lymphocytes or bronchial epithelial cells were treated with diazinon or chlorpyrifos in the absence or presence of cellular stress factors, thereby mimicking a stimulated immune system. Cytotoxicity was determined and cytokine release or cytokine-promoter studies were performed to study immunomodulatory effects of these chemicals, whereby the same concentrations of chlorpyrifos and diazinon were used. Results showed that chlor- pyrifos was cytotoxic at concentrations &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;/= 250 muM, whereas diazinon was not toxic at concentrations up to 1 mM. The immunomodulatory effects of these two compounds were similar for most cytokine promoters tested and induction of cellular stress enhanced these effects. The results were compared to data obtained with blood mononuclear cells, which confirmed the results of stably transfected cell lines, but refer to a higher sensitivity of primary cells. In conclusion, these two pesticides act in a different manner on cell viability and on some immune parameters, but cell viability was not linked to immunomodulation. The results also imply that healthy and diseased individuals are differentially affected by these pollutants.
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The most commonly used assay to quantitate the response of peripheral T-cells upon stimulation with growth factors is determination of incorporated [3H]TdR. We compared this test to three other methods: 1. direct counting of cells with a... more
The most commonly used assay to quantitate the response of peripheral T-cells upon stimulation with growth factors is determination of incorporated [3H]TdR. We compared this test to three other methods: 1. direct counting of cells with a Coulter type counter as reference assay, 2. a colorimetric assay using the tetrazolium dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium (MTT), which is a cheap and increasingly popular non-radioactive method and 3. incorporation of the thymidine analog 5-bromo-2&#39;-deoxyuridine detection with a monoclonal antibody on cytospins. Primary human PHA-blasts from greater than 30 healthy individuals were stimulated with IL-2, IL-4 and IL-7 and assayed with up to four different methods. We discuss the advantages and disadvantages of the assays used and the effects of differences between cell preparations. We observed no significant variations between individuals for the dose dependence, but the relative efficiency of IL-4 compared to IL-2 and IL-7 was variable. This was probably due to the slower response observed upon stimulation with this factor.
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Chlorobenzene is a volatile organic compound that is used as a solvent in many industrial settings and has been shown to be related with irritations of the respiratory tract. Exposure to chlorobenzene induces the release of monocyte... more
Chlorobenzene is a volatile organic compound that is used as a solvent in many industrial settings and has been shown to be related with irritations of the respiratory tract. Exposure to chlorobenzene induces the release of monocyte chemoattractant protein 1 (MCP-1) by lung epithelial cells, a chemokine involved in inflammatory reactions. To characterize the underlying mechanisms we investigated the influence of chlorobenzene on the activation of two intracellular signalling pathways: the nuclear factor-kappa B (NF-kappa B) and the p38 mitogen-activated protein kinase (MAPK) pathways. Human lung epithelial cells (A549) were stimulated with tumor necrosis factor (TNF)-alpha in the presence or absence of specific inhibitors of NF-kappaB or the p38 MAP kinase and exposed to chlorobenzene using an air-liquid cell culture system. Exposure of lung epithelial cells to chlorobenzene resulted in an activation of NF-kappa B and p38 MAP kinase and a release of the chemokine MCP-1. In the presence of IKK-NBD, a specific NF-kappa B inhibitor, or the inhibitors of the p38 MAP kinase SB 203580 and SB 202190, the chlorobenzene-related MCP-1 release was suppressed, suggesting an involvement of both pathways in the chlorobenzene induced expression of MCP-1. Our data show that the release of MCP-1 following chlorobenzene exposure is dependent on the NF-kappa B and MAPK pathways.
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BackgroundNanoparticle (NPs) functionalization has been shown to affect their cellular toxicity. To study this, differently functionalized silver (Ag) and gold (Au) NPs were synthesised, characterised and tested using lung epithelial cell... more
BackgroundNanoparticle (NPs) functionalization has been shown to affect their cellular toxicity. To study this, differently functionalized silver (Ag) and gold (Au) NPs were synthesised, characterised and tested using lung epithelial cell systems.MethodsMonodispersed Ag and Au NPs with a size range of 7 to 10 nm were coated with either sodium citrate or chitosan resulting in surface charges from ¿50 mV to +70 mV. NP-induced cytotoxicity and oxidative stress were determined using A549 cells, BEAS-2B cells and primary lung epithelial cells (NHBE cells). TEER measurements and immunofluorescence staining of tight junctions were performed to test the growth characteristics of the cells. Cytotoxicity was measured by means of the CellTiter-Blue ® and the lactate dehydrogenase assay and cellular and cell-free reactive oxygen species (ROS) production was measured using the DCFH-DA assay.ResultsDifferent growth characteristics were shown in the three cell types used. A549 cells grew into a co...
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Single-walled carbon nanotubes have gained enormous popularity due to a variety of potential applications which will ultimately lead to increased human and environmental exposure to these nanoparticles. This study was carried out in order... more
Single-walled carbon nanotubes have gained enormous popularity due to a variety of potential applications which will ultimately lead to increased human and environmental exposure to these nanoparticles. This study was carried out in order to evaluate the inflammatory response of immortalised and primary human lung epithelial cells (A549 and NHBE) to single-walled carbon nanotube samples (SWCNT). Special focus was placed
Research Interests: Cytokines, Cell Culture, Cell line, Humans, Cell Viability, and 12 moreLung, Anti-inflammatory agents, Epithelial cells, Time Factors, Tumor necrosis factor-alpha, Cell Proliferation, Recombinant Proteins, Cell Survival, Environmental Exposure, Nanotubes Carbon, Down-Regulation, and Inflammatory response
Diesel engine emission particle filters are often placed at exhaust outlets to remove particles from the exhaust. The use of filters results in the exposure to a reduced number of nanometer-sized particles, which might be more harmful... more
Diesel engine emission particle filters are often placed at exhaust outlets to remove particles from the exhaust. The use of filters results in the exposure to a reduced number of nanometer-sized particles, which might be more harmful than the exposure to a larger number of micrometer-sized particles. An in vitro exposure system was established to expose human alveolar epithelial cells to freshly generated exhaust. Computer simulations were used to determine the optimal flow characteristics and ensure equal exposure conditions for each well of a 6-well plate. A selective particle size sampler was used to continuously deliver diesel soot particles with different particle size distributions to cells in culture. To determine, whether the system could be used for cellular assays, alterations in cytokine production and cell viability of human alveolar A549 cells were determined after 3h on-line exposure followed by a 21-h conventional incubation period. Data indicated that complete diesel engine emission slightly affected pre-stimulated cells, but naive cells were not affected. The fractions containing large or small particles never affected the cells. The experimental set-up allowed a reliable exposure of the cells to the complete exhaust fraction or to the fractions containing either large or small diesel engine emission particles.
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Styrene is a volatile organic compound (VOC) that is widely used as a solvent in many industrial settings. Chronic exposure to styrene can result in irritation of the mucosa of the upper respiratory tract. Contact of styrene with... more
Styrene is a volatile organic compound (VOC) that is widely used as a solvent in many industrial settings. Chronic exposure to styrene can result in irritation of the mucosa of the upper respiratory tract. Contact of styrene with epithelial cells stimulates the expression of a variety of inflammatory mediators, including the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1). To characterise the underlying mechanisms of the induction of inflammatory signals by styrene, we investigated the influence of this compound on the induction of oxidative stress and the activation of the nuclear factor-kappa B (NF-kappaB) signalling pathway in human lung epithelial cells (A549). The results demonstrate that styrene-induced MCP-1 expression, as well as the expression of the oxidative stress marker glutathione S-transferase (GST), is associated with a concentration dependent pattern of NF-kappaB activity. An inhibitor of NF-kappaB, IKK-NBD, and the anti-inflammatory antioxidant N-acetylcysteine (NAC) were both effective in suppressing styrene-induced MCP-1 secretion. In addition, NAC was capable of inhibiting the upregulation of GST expression. Our findings suggest that the activation of the NF-kappaB signalling pathway by styrene is mediated via a redox-sensitive mechanism.
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Research Interests: Cytokines, Cell Culture, Cell line, Humans, Cell Viability, and 12 moreLung, Anti-inflammatory agents, Epithelial cells, Time Factors, Tumor necrosis factor-alpha, Cell Proliferation, Recombinant Proteins, Cell Survival, Environmental Exposure, Nanotubes Carbon, Down-Regulation, and Inflammatory response
Research Interests: Carbon, Carbon Nanotubes, Nanomaterials, Carbon Nanotube, Oxidative Stress, and 18 moreCell Culture, Transition-Metal Oxides, Dispersion, In Vitro, Humans, Reactive Oxygen Species, Lung Surfactant, Lung, Specific surface area, Exposure, Biological systems, Epithelial cells, Occupational Exposure, Bronchial Epithelial Cells, Carbon Black, Culture Media, Pulmonary Surfactant, and Nanotubes Carbon
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Research Interests: Engineering, Physics, Chemistry, Biology, Inflammation, and 22 moreAir pollution, Medicine, Multidisciplinary, Protein Stability, Food Allergy, Mice, Female, Animals, Nitrogen, Tandem Mass Spectrometry, Allergens, Immunoglobulin E, PLoS one, High Pressure Liquid Chromatography, Chip, Amino Acid Sequence, Smog, Murine Model, Epitopes, Tyrosine, immunoglobulin G, and Inflammatory response
Abstract Nickel, zinc, and copper oxide nanoparticles (NiONP, ZnONP, and CuONP) and their aqueous extracts (AEs) were applied to A549 lung epithelial cells to determine the cytotoxicity, IL-8 production, and activation of transcription... more
Abstract Nickel, zinc, and copper oxide nanoparticles (NiONP, ZnONP, and CuONP) and their aqueous extracts (AEs) were applied to A549 lung epithelial cells to determine the cytotoxicity, IL-8 production, and activation of transcription factors. Nanoparticles (NPs) and their AEs were also instilled into rat lungs to evaluate acute and chronic inflammatory effects. In vitro AEs had specific effects; for example NiOAE had no effect and ZnOAE affected all parameters measured. NPs themselves all had cytotoxic effects but only ZnONP and CuONP impacted pro-inflammatory endpoints. The inflammatory cells in the BAL were also different from AEs and NPs with ZnONP and CuONP recruiting eosinophils and neutrophils whilst ZnOAE and CuOAE elicited only mild neutrophilic inflammation that had resolved by four weeks. NiONP recruited neutrophils only whilst NiOAE did not cause any inflammation. Understanding differences in the toxic role of the ionic components of metal oxide NPs will contribute to full hazard identification and characterisation.
Research Interests: Nanotechnology, Metal Nanoparticles, Nanotoxicology, Zinc Oxide, Humans, and 12 moreCopper, Animals, Nickel, Lung, Cattle, Neutrophils, Eosinophils, Rats, Oxides, Cell Survival, Ions, and Interleukin
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This study describes immunomodulatory effects of small inorganic nanoparticles and the impact of experimental design therein. Gold, cobalt oxide and iron oxide nanoparticles were synthesized in solution under conditions that ensured... more
This study describes immunomodulatory effects of small inorganic nanoparticles and the impact of experimental design therein. Gold, cobalt oxide and iron oxide nanoparticles were synthesized in solution under conditions that ensured monodispersed and stable particles in solvents ...