Effect of selected fluorinated drugs in a "ringing" gel on rheological behaviour and skin permeation.

The purpose of the present study was to investigate the influence of different drugs exhibiting different solubility on the viscoelastic properties and on the skin diffusion profile of a ringing gel. In a preliminary rheology study with the placebo gel predominating elastic properties were confirmed and a temperature influence was indicated. Fluconazole, fludrocortisone-acetate, flumethasone-pivalate, flutamide and flufenamic-acid each 1% (w/w) were incorporated into the preparation and oscillatory measurements were performed at temperatures of 25, 28, 32 and 37 degrees C. In all drug containing formulations a high elastic G' value predominated the viscous G' value. The highest G' value could be obtained with the incorporated flumethasone-pivalate. Additionally in almost all cases the G' values decreased with increasing temperature compared to the placebo gel. Additionally in vitro standard diffusion experiments using Franz-type cells and porcine skin were performed. Following rank order of the cumulative drug release after 48 h was obtained: fluconazole>flufenamic-acid>flumethasone-pivalate>flutamide>fludrocortisone-acetate. Furthermore an excellent chemical stability of all incorporated drugs was confirmed over 10 weeks.     

On the effects of Fusarium toxin contaminated wheat and wheat chaff on nutrient utilisation and turnover of deoxynivalenol and zearalenone in vitro (Rusitec).

The objective of the present study was to investigate the effects of Fusarium toxin contaminated wheat and wheat chaff (mycotoxin diet) on nutrient degradability and the metabolism of the mycotoxins deoxynivalenol (DON) and zearalenone (ZON) using the rumen simulations technique (Rusitec). A 6 day application period with control wheat and wheat chaff (control diet) was followed by an 8 day sampling phase. During this time three fermenters received the mycotoxin diet (64.9 mg DON/kg dry matter (DM) and 500 microg ZON/kg DM) and the remaining fermenters served as the controls (1.0mg DON/kg DM and 6 microg ZON/kg DM). Feed residues of the bags and samples of the effluent liquids were pooled per fermenter during the last 8 days of the experiment. Additionally, effluents of the mycotoxin fermenters were taken 6, 12 and 24h after the morning feeding on the first day of the sampling phase. The degradation of organic matter (OM; P<0.05), neutral detergent fibre (NDF; P<0.01) and protein (P<0.001) were increased by administration the Fusarium contaminated diet which was accompanied by an increased ammonia concentration (P<0.01) and increased butyrate (P<0.01), isobutyrate (P<0.01) and isovalerate (P<0.05) values of the mycotoxin effluents in relation to the controls. High proportions of ingested DON of 90% (85-93%) and ingested ZON of 93% (80-104%) were recovered at the pooled feed residues and effluents in form of DON and de-epoxy DON, and ZON and alpha-ZOL after administering the Fusarium toxin contaminated feed. While adsorption of DON as DON and de-epoxy DON in the feed particles was only minor (5%), a higher amount of 38% of ingested ZON was recovered as ZON and alpha-ZOL at the feed residues. The total recovery of DON plus de-epoxy DON in effluents as a percentage of DON intake reached 8%, 9% and 22% of ingested DON at 6, 12 and 24h after application of the contaminated diet the first time, whereby the recovery of de-epoxy DON as percentage of DON intake was only 5% at 24h. Concentrations of ZON and metabolites were lower than detection limits in the time dependent effluent samples.     

IFN-gamma-enhanced allergen penetration across respiratory epithelium augments allergic inflammation

BACKGROUND: Respiratory allergen contact is the critical event in the elicitation and boosting of allergen-specific immune responses, as well as in the induction of immediate and late inflammatory reactions. OBJECTIVE: We sought to investigate the influence of various factors of allergic inflammation on the integrity and barrier function of respiratory epithelium for allergens. METHODS: We cultured the human bronchial epithelial cell line 16HBE14o- in a transwell culture system as a surrogate of intact respiratory epithelium and used purified iodine 125-labeled recombinant major birch pollen allergen (rBet v 1) to study the extent, kinetics, and factors influencing transepithelial allergen penetration. RESULTS: Culture supernatants from activated allergen-specific T H 1 clones decreased transepithelial resistance. A screening of various factors (histamine, IFN-gamma, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-8, IL-12, and TNF-alpha) identified IFN-gamma as a potent factor capable of reducing epithelial barrier properties and enhancing transepithelial allergen penetration. Increased submucosal allergen concentrations caused by IFN-gamma-mediated reduction of epithelial barrier function provoked a more than 7-fold augmentation of histamine release from sensitized basophils. CONCLUSION: These results demonstrate that the T H 1 cell-derived cytokine IFN-gamma facilitates allergen penetration through the respiratory epithelium and thereby can aggravate allergic inflammation.     

Suggestions for the assessment of the allergenic potential of genetically modified organisms

The prevalence of allergic diseases has been increasing continuously and, accordingly, there is a great desire to evaluate the allergenic potential of components in our daily environment (e.g., food). Although there is almost no scientific evidence that genetically modified organisms (GMOs) exhibit increased allergenicity compared with the corresponding wild type significant concerns have been raised regarding this matter. In principle, it is possible that the allergenic potential of GMOs may be increased due to the introduction of potential foreign allergens, to potentially upregulated expression of allergenic components caused by the modification of the wild type organism or to different means of exposure. According to the current practice, the proteins to be introduced into a GMO are evaluated for their physiochemical properties, sequence homology with known allergens and occasionally regarding their allergenic activity. We discuss why these current rules and procedures cannot predict or exclude the allergenicity of a given GMO with certainty. As an alternative we suggest to improve the current evaluation by an experimental comparison of the wild-type organism with the whole GMO regarding their potential to elicit reactions in allergic individuals and to induce de novo sensitizations. We also recommend that the suggested assessment procedures be equally applied to GMOs as well as to natural cultivars in order to establish effective measures for allergy prevention.     

Recombinant allergens promote expression of CD203c on basophils in sensitized individuals

BACKGROUND: Traditionally, the diagnosis of type I allergies is based on clinical data, skin test results, and laboratory test results with allergen extracts. During the past few years, several attempts have been made to refine diagnostic assays in clinical allergy by introducing recombinant allergens and novel markers of IgE-dependent cell activation. OBJECTIVES: We have identified the ectoenzyme CD203c as a novel basophil antigen that is upregulated on IgE receptor cross-linkage. In this study we applied CD203c and a panel of recombinant allergens to establish a novel basophil test that allows for a reliable quantification of IgE-dependent responses at the effector cell level. METHODS: Patients allergic to birch (Bet v 1, n = 15; Bet v 2, n = 8) and grass (Phl p 1, n = 15; Phl p 2, n = 10; Phl p 5, n = 14) pollen allergens, as well as 10 nonallergic donors, were examined. Basophils were exposed to various concentrations of recombinant allergens for 15 minutes and then examined for expression of CD203c by means of flow cytometry. CD203c upregulation was correlated with the increase in CD63. RESULTS: Exposure to recombinant allergens resulted in a dose-dependent increase in expression of CD203c on peripheral blood basophils in sensitized individuals, whereas no increase was seen in healthy control subjects. The effects of the recombinant allergens on CD203c expression were also time dependent. There was a good correlation between allergen-induced upregulation of CD203c and upregulation of CD63 (R = 0.76). CONCLUSION: Flow cytometric quantitation of CD203c on blood basophils exposed to recombinant allergens is a useful approach to determine the allergic state in sensitized individuals and represents a basis for a sensitive novel allergy test.     

Sensitization to human milk

Background Allergy to milk is one of the earliest manifestations of IgE‐mediated allergies and affects about 2.5% of newborn children. Several reports indicate that milk‐allergic patients may be sensitized also to human milk proteins. Objective To analyse the specificity and possible biological relevance of IgE reactivity to human milk antigens in milk‐allergic patients. Methods The specificity of IgE reactivity to cow's milk and human milk antigens was analysed with sera from milk‐allergic children and adults by IgE immunoblotting. IgE cross‐reactivity between milk antigens was studied by immunoblot inhibition experiments. That IgE reactivity to human milk antigens is not due to alloreactivity or due to the transmission of foreign antigens into mother's milk was demonstrated through the analysis of milk samples from genetically unrelated mothers before and after intake of dietary milk products. The biological relevance of IgE reactivity to human milk was confirmed by skin testing. Results IgE antibodies to human milk were found in more than 80% of the tested milk‐allergic patients. Cross‐reactive IgE‐reactive human antigens such as α‐lactalbumin and non‐cross‐reactive human milk antigens were identified. Immediate‐type skin reactions could be elicited with human milk samples in patients with IgE reactivity to human milk. Conclusion IgE reactivity to human milk in milk‐allergic patients can be due to cross‐ sensitization and genuine sensitization to human milk and may cause allergic symptoms. IgE‐mediated sensitization to human milk is common in milk‐allergic patients and may require diagnostic testing and monitoring.     

Characterization of wild-type recombinant Bet v 1a as a candidate vaccine against birch pollen allergy

BACKGROUND: We describe the production in Escherichia coli as a recombinant protein of clinical grade wild-type Bet v 1a (rBet v 1a), to be used as a candidate vaccine against birch pollen allergy. METHODS: This recombinant protein was purified by hydrophobic interaction and ion exchange chromatography and characterized by SDS-PAGE, immunoprint and circular dichroism in parallel with natural Bet v 1 (nBet v 1) purified from a birch pollen extract. We also compared rBet v 1 and nBet v 1 for their capacity to induce histamine release from basophils and to stimulate T lymphocyte proliferation. RESULTS: rBet v 1a appears in SDS-PAGE as an 18-kDa monomeric protein, whereas purified nBet v 1 comprises a mixture of isoforms (resolving as three distinct bands and six spots after 1-dimensional and 2-dimensional electrophoresis, respectively). Both recombinant and natural purified Bet v 1 molecules are recognized by IgE from birch pollen-allergic patients as well as anti-Bet v 1 murine monoclonal antibodies, suggesting that the recombinant protein is correctly folded in a native configuration. Circular dichroism analysis confirmed that the two Bet v 1 molecules exhibit similar 3-dimensional structures, even if rBet v 1a appears more compact and stable in thermodenaturation/renaturation experiments. Both rBet v 1 and nBet v 1 induce the degranulation of sensitized basophils and proliferation of Bet v 1-specific T lymphocytes in a similar manner. CONCLUSIONS: On the basis of these structural and biological properties, rBet v 1a is a valid candidate vaccine against birch pollen allergy, currently evaluated in humans.