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The non-steroidal anti-inflammatory drug (NSAID) diclofenac (DCF) is an important environmental contaminant occurring in surface waters all over the world, because, after excretion, it is not adequately removed from wastewater in sewage treatment plants. To be able to monitor this pollutant, highly efficient analytical methods are needed, including immunoassays. In a medical research project, monoclonal antibodies against diclofenac and its metabolites had been produced. Based on this monoclonal anti-DCF antibody, a new indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed and applied for environmental samples. The introduction of a spacer between diclofenac and the carrier protein in the coating conjugate led to higher sensitivity. With a test midpoint of 3 μg L−1and a measurement range of 1–30 μg L−1, the system is not sensitive enough for direct analysis of surface water. However, this assay is quite robust against matrix influences and can be used for wastewater. Without adjustment of the calibration, organic solvents up to 5%, natural organic matter (NOM) up to 10 mg L−1, humic acids up to 2.5 mg L−1, and salt concentrations up to 6 g L−1NaCl and 75 mg L−1CaCl2are tolerated. The antibody is also stable in a pH range from 3 to 12. Cross-reactivity (CR) of 1% or less was determined for the metabolites 4′-hydroxydiclofenac (4′-OH-DCF), 5-hydroxydiclofenac (5-OH-DCF), DCF lactam, and other NSAIDs. Relevant cross-reactivity occurred only with an amide derivative of DCF, 6-aminohexanoic acid (DCF-Ahx), aceclofenac (ACF) and DCF methyl ester (DCF-Me) with 150%, 61% and 44%, respectively. These substances, however, have not been found in samples. Only DCF-acyl glucuronide with a cross-reactivity of 57% is of some relevance. For the first time, photodegradation products were tested for cross-reactivity. With the ELISA based on this antibody, water samples were analysed. In sewage treatment plant effluents, concentrations in the range of 1.9–5.2 μg L−1were determined directly, with recoveries compared to HPLC-MS/MS averaging 136%. Concentrations in lakes ranged from 3 to 4.4 ng L−1and were, after pre-concentration, determined with an average recovery of 100%.

The number of consumer products containing nanoparticles (NPs) experienced a rapid increase during the past decades. However, most studies of nanosafety have been conducted using only pure NPs produced in the laboratory, while the interactions with other ingredients in consumer products have rarely been considered so far. In the present study, we investigated such interactions—with a special focus on modern lifestyle products (MLPs) used by adolescents. An extensive survey was undertaken at different high schools all over Austria to identify MLPs that either contain NPs or that could come easily in contact with NPs from other consumer products (such as TiO2from sunscreens). Based on the results from a survey among secondary schools students, we focused on ingredients from Henna tattoos (2-hydroxy-1,4-naphtoquinone, HNQ, and p-phenylenediamine, PPD), fragrances (butylphenyl methylpropional, known as Lilial), cosmetics and skin-care products (four different parabens). As a cellular model, we decided to use neonatal normal human dermal fibroblasts (nNHDF), since skin contact is the main route of exposure for these compounds. TiO2NPs interacted with these compounds as evidenced by alterations in their hydrodynamic diameter observed by nanoparticle tracking analysis. Combinations of TiO2NPs with the different MLP components did not show altered cytotoxicity profiles compared to MLP components without TiO2NPs. Nevertheless, altered cellular glutathione contents were detected after incubation of the cells with Lilial. This effect was independent of the presence of TiO2NPs. Testing mixtures of NPs with other compounds from consumer products is an important approach to achieve a more reliable safety assessment.

Type I hypersensitivity reactions against environmental entities, i.e.allergens, are an ever growing problem, with increasing numbers of affected individuals in Western countries. In parallel, nanotechnology has been growing during the past decades, entering more and more areas of application in everyday life. Exposure to allergens may occur in a combined way with environmental particulate matter or (nano-)particles potentially abundant at the workplace. Such combinations may thus affect workers and consumers alike. The present study conceived a methodological and mechanistic approach based on recombinant allergen–SiO2nanoparticle conjugates and the novel hAELVi cell line as two well-defined model systems to investigate the effects of co-exposure towards allergens and nanoparticles. We applied pristine nanoparticles, allergen–nanoparticle conjugates and unbound allergen under submerged and air–liquid interface conditions. The uptake kinetics and mechanisms, cellular localization and resulting immune responses were studied in hAELVi cells, a cell line derived from human alveolar epithelium showing the characteristics of type I epithelial cells. Modification in the uptake kinetics as well as an increase in pro-inflammatory cyto-/chemokine expression was observed under submerged conditions, but much less under air–liquid interface conditions. While for mechanistic investigations the submerged culture system still proved suitable, more robust and cost-effective, these culture conditions showed considerable deviations in cellular susceptibility observed under more realistic conditions.

Nanotechnology is a fast growing field and already a multi-billion dollar market with numerous products available for consumers. TiO2and SiO2are by mass the most produced nanomaterials and, thus, of particular interest regarding their biological effects upon inhalation – especially in combination with other inhalable biomolecules such as allergens. We investigated the protein-binding capacity of these two nanomaterials and present detailed uptake profiles of protein-conjugated TiO2or SiO2nanoparticles (NPs) in A549 lung epithelial and THP-1 macrophage-like cells. TiO2and SiO2NPs (both with a hydrodynamic diameter of about 150 nm) are able to bind bovine serum albumin (BSA), green fluorescent protein (GFP) and the major birch pollen allergen Bet v 1 in substantial amounts that suggest protein monolayers around the particles. GFP-conjugated TiO2and SiO2NPs were taken up in A549 and THP-1 cells in a time-dependent manner as demonstrated by confocal laser scanning microscopy, flow cytometry and life cell imaging. By the aid of fluorescent proteins (GFP or fluorescently labeled Bet v 1) we found that both, the NPs and the conjugated proteins entered the cells. Using A549/THP-1 co-cultures, we showed that the majority of SiO2NPs is taken up by THP-1 cells; however, also A549 cells take up significant amounts of particles when co-cultured with THP-1 macrophage-like cells. The here discussed data provide valuable insights into protein (allergen) delivery by NPs at the lung epithelial barrier.

Allergic diseases are among the most prevalent immune-mediated diseases, with incidences of well over 20% of the population in industrialized countries. Nevertheless, the question of whether allergies should be considered as a risk factor for exposure to nanomaterials has rarely been addressed. Experimental studies have attempted to improve allergy therapy by combining allergens with various nanomaterials, which may lead to a beneficial re-direction of the immune system. Binding to nanoparticles may for allergens - as for other proteins - lead to structural changes or modification of other properties. We assess here the state of knowledge about biological effects deriving from the combination of nanoparticles with allergens.

Background: Mugwort pollen is an important allergen source in hay fever and pollen-related food allergy. Little is known about the clinical relevance of the major mugwort allergen Art v 1 and its importance in allergy. Objective: In this study we aimed to investigate the allergenicity of mugwort extract compared with the allergenicity of native (n)Art v 1 and recombinant (r)Art v 1, one major allergen of mugwort, in vivo and in vitro. Methods: Thirty-two patients allergic to mugwort and 10 control subjects were investigated by means of skin prick and nasal provocation testing with different concentrations of mugwort extract, nArt v 1, and rArt v 1. nArt v 1 was purified from aqueous mugwort extract, and rArt v 1 was cloned, expressed in Escherichia coli, and then purified. The in vitro allergenicity was measured by means of ImmunoCAP, ELISA, ELISA-inhibition experiments, and T-cell proliferation assays. Results: nArt v 1 and rArt v 1 were able to elicit positive in vivo and in vitro reactions. The IgE-binding capacity, as determined by means of ELISA, was slightly higher for nArt v 1 than for rArt v 1, and both allergens were able to induce T-cell proliferation in sensitized patients. However, rArt v 1 elicited a reduced response in skin and nasal provocation tests compared with nArt v 1. Compared with mugwort extract, both nArt v 1 and rArt v 1 showed lower sensitivity in patients with mugwort allergy in vivo. Conclusions: Art v 1, either in its native or recombinant form, is able to induce allergic reactions in patients with mugwort allergy. rArt v 1 induced comparable humoral and cell-mediated responses in vitro but showed reduced in vivo allergenicity compared with biochemically purified nArt v 1. (J Allergy Clin Immunol 2003;111:1328-36.)

Background: Propyphenazone (1,2–dihydro-1,5-dimethyl-4-(1-methylethyl)-2-phenyl-3H-pyrazol-3-one; PP) is a nonsteroidal anti-inflammatory drug frequently used as mild analgesic medicament. It belongs to the chemical group of pyrazolones. Severe adverse reactions to PP are frequent and have generally been regarded as pseudoallergic or intolerance reactions. Presently, there are no useful in vitro test systems available for the detection of antibodies directed against analgesic drugs. Objective: The purpose of this study was to unequivocally demonstrate that IgE-mediated Type I allergy is the main mechanism leading to immediate-type adverse reactions to the analgesic drug PP. Methods: We investigated 53 young adult patients with adverse reactions to PP. All patients developed symptoms suggestive of IgE-mediated anaphylaxis within 30 minutes after intake of a painkiller containing PP. Patients were subjected to skin tests (prick test and intracutaneous test). In addition, a novel ELISA system was developed to prove the existence of specific IgE antibodies in patients' sera. Results: In 44 of 53 (83%) patients, skin tests showed typical wheal and flare reactions. Significant amounts of PP-specific serum IgE was detected in 31 of 53 (58%) of the serum samples. Moreover, in 7 of 9 patients with skin test negative results, PP-specific IgE could be detected. The assay was PP-specific because only PP, but no other pyrazolone derivative (antipyrine, aminophenazone, or metamizol), was able to inhibit IgE-binding in the system. Conclusion: Propyphenazone is a sensitizing agent in susceptible individuals and can elicit IgE-mediated anaphylaxis. By using skin tests and our ELISA system we were able to confirm Type I allergy in 51 of 53 (96%) patients in this study.

The retroviral oncogene p3k(v-p3k) of avian sarcoma virus 16 (ASV16) codes for the catalytic subunit of phosphoinositide (PI) 3-kinase, p110α. The v-P3k protein is oncogenic in vivoand in vitro; its cellular counterpart, c-P3k, lacks oncogenicity. Fusion of viral Gag sequences to the amino terminus of c-P3k activates the transforming potential. Activation can also be achieved by the addition of a myristylation signal to the amino terminus or of a farnesylation signal to the carboxyl terminus of c-P3k. A mutated myristylation signal was equally effective; it also caused a strong increase in the kinase activity of P3k. Mutations that inactivate lipid kinase activity abolish oncogenicity. The transforming activity of P3k is correlated with the ability to induce activating phosphorylation in Akt. Point mutations and amino-terminal deletions recorded in v-P3k were shown to be irrelevant to the activation of oncogenic potential. Interactions of P3k with the regulatory subunit of PI 3-kinase, p85, or with Ras are not required for transformation. These results support the conclusion that the oncogenicity of P3k depends on constitutive lipid kinase activity. Akt is an important and probably essential downstream component of the oncogenic signal from P3k.

We have described recently the construction of a defective vaccinia virus (VV) lacking the essential D4R open reading frame and have shown furthermore the selection of a complementing cell line providing the essential D4R gene product. The D4R gene belongs to the group of early transcribed vaccinia genes preventing a virus defective in D4R from entering into the intermediate and late phase of replication under noncomplementing conditions. Here we show that this property, which is unique among the group of so called nonreplicating poxviruses, is helpful for the production of (secretable) recombinant human proteins. Recombinant VV based on a D4R-defective parental strain expressing cDNAs coding for the human blood coagulation factors VII and XI produced significantly more recombinant protein than the corresponding recombinants based on wild-type VV. Moreover, the complementing cell line RK-D4R-44.20 was a more effective production cell system for both vD4 and wild-type VV recombinants compared to wild-type RK-13 cells. Surprisingly, recombinant human factor VII was more efficiently produced with the defective vaccinia recombinant even under noncomplementing conditions, suggesting that persistence of the early phase of vaccinia replication in combination with a delayed host shutoff is advantageous for the overproduction of certain recombinant proteins using the VV expression system.

DF-1 is a continuous cell line of chicken embryo fibroblasts. The cells are free of endogenous sequences related to avian sarcoma and leukosis viruses and have normal fibroblastic morphology. DF-1 cells support the replication of avian retroviruses; diverse oncogenes induce foci of oncogenic transformation on monolayers of DF-1, and avian leukosis viruses of envelope subgroups B, D, and C induce cell death and form plaques. The new cell line will greatly facilitate studies on oncogenic transformation and cell killing by avian viruses.

In late summer, pollen grains originating from Compositaeweeds (e.g., mugwort, ragweed) are a major source of allergens worldwide. Here, we report the isolation of a cDNA clone coding for Art v 1, the major allergen of mugwort pollen. Sequence analysis showed that Art v 1 is a secreted allergen with an N‐terminal cysteine‐rich domain homologous to plant defensins and a C‐terminal proline‐rich region containing several (Ser/Ala)(Pro)2–4repeats. Structural analysis showed that some of the proline residues in the C‐terminal domain of Art v 1 are posttranslationally modified by hydroxylation and O‐glycosylation. The O‐glycans are composed of 3 galactoses and 9–16 arabinoses linked to a hydroxyproline and represent a new type of plant O‐glycan. A 3‐D structural model of Art v 1 was generated showing a characteristic “head and tail” structure. Evaluation of the antibody binding properties of natural and recombinant Art v 1 produced in Escherichia colirevealed the involvement of the defensin fold and posttranslational modifications in the formation of epitopes recognized by IgE antibodies from allergic patients. However, posttranslational modifications did not influence T‐cell recognition. Thus, recombinant nonglycosylated Art v 1 is a good starting template for engineering hypoallergenic vaccines for weed‐pollen therapy.