Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects. Current knowledge assembled at an international workshop at BfR, Germany.

Contact allergies are complex diseases, and one of the important challenges for public health and immunology. The German 'Federal Institute for Risk Assessment' hosted an 'International Workshop on Contact Dermatitis'. The scope of the workshop was to discuss new discoveries and developments in the field of contact dermatitis. This included the epidemiology and molecular biology of contact allergy, as well as the development of new in vitro methods. Furthermore, it considered regulatory aspects aiming to reduce exposure to contact sensitisers. An estimated 15-20% of the general population suffers from contact allergy. Workplace exposure, age, sex, use of consumer products and genetic predispositions were identified as the most important risk factors. Research highlights included: advances in understanding of immune responses to contact sensitisers, the importance of autoxidation or enzyme-mediated oxidation for the activation of chemicals, the mechanisms through which hapten-protein conjugates are formed and the development of novel in vitro strategies for the identification of skin-sensitising chemicals. Dendritic cell cultures and structure-activity relationships are being developed to identify potential contact allergens. However, the local lymph node assay (LLNA) presently remains the validated method of choice for hazard identification and characterisation. At the workshop the use of the LLNA for regulatory purposes and for quantitative risk assessment was also discussed.

[Assessment of the sensitizing potency of cosmetic ingredients and commodities. How will the ingredients of cosmetics and commodities be tested in Europe today and tomorrow?]. / Bewertung des sensibilisierenden Potenzials von Stoffen in Kosmetika und Bedarfsgegenständen. Wie werden Inhaltsstoffe von Kosmetika und Bedarfsgegenständen heute und morgen in Europa getestet?

Cosmetics and certain commodities are applied or used by consumers directly on the skin. Creams may remain on the skin for longer periods, hair is dyed multiple times per year, nickel ions can be released from studs and piercings in areas of skin damage or migrate from toy materials into the skin of children. Accordingly, using or handling such products always entails a risk for developing a contact allergy. Moreover, daily usage and repeated contacts to certain cosmetics and commodities might lead to repeated elicitation of contact eczema in people already sensitized against allergenic ingredients. Unfortunately, contact allergy is not curable. For the assessment of the allergenic potential of chemicals, only testing based on animal experiments was available in the past. In 2003, the 7(th) amendment of the Cosmetics Directive 76/768/EWG laid down a ban on animal testing of cosmetic ingredients and from 2013 a general marketing ban of such products as well. Therefore, the development and validation of non-animal methods for assessing the toxicological endpoint sensitization/allergenic potency of chemicals is a major task for the years ahead and remains equally a challenge for industry and regulatory agencies.

Selective induction of nerve growth factor and brain-derived neurotrophic factor by LPS and allergen in dendritic cells.

BACKGROUND: Neurotrophins are produced by various cells upon different stimuli and participate in the initiation and regulation of inflammation in various diseases including allergy and asthma, but little is known about the production and control of neurotrophins by dendritic cells (DCs). The aim of this study was to assess whether DCs produce the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and whether inflammatory stimuli or allergens are able to induce the production of neurotrophic factors. METHODS: Monocyte-derived dendritic cells (MoDCs) were generated from different donors. The neurotrophins NGF and BDNF were demonstrated by RT-PCR, Western blotting, flow cytometry analysis and fluorescence microscopy. MoDCs were cultured and stimulated with lipopolysaccharide (LPS) or allergen for 24 h. The supernatants and cells were collected. Measurement for NGF and BDNF was performed by ELISA. RESULTS: DCs express mRNA for the neurotrophins NGF and BDNF. Proteins were detectable by Western blot, FACS analysis and fluorescence microscopy. LPS led to an up-regulation of BDNF, while NGF was unaffected. Cell lysates demonstrated an increased amount of BDNF after stimulation with LPS or allergen, while NGF was not affected significantly. CONCLUSIONS: DCs are a source of neurotrophins. LPS selectively regulates the production of BDNF. Allergen stimulation leads to an LPS-independent regulation. This contributes to a complex involvement of neurotrophins in allergic diseases.

Evaluation and improvement of QSAR predictions of skin sensitization for pesticides.

In vivo skin sensitization assays have to be provided by applicants to the competent authorities in the European Union for the approval of active substances (AS) in pesticides. This study aimed to test the practicability of in silico predictions for AS by freely available (Q)SAR tools to evaluate their use as a time- and cost-effective alternative to animal testing in the context of the 3R concept. Predictions of skin sensitization for 48 selected sensitizing and non-sensitizing AS by the software programs CAESAR, Toxtree, OECD (Q)SAR Toolbox, CASE Ultra, Leadscope and SciQSAR were collected and compared. Different data evaluation methodologies (score definition, mean, weighted mean, threshold score definition) were applied to optimize the predictions. The calculation methods were internally cross-validated and further validated with an additional validation set of 80 AS. Although the presented calculation methodologies are not suitable as a stand-alone method, this study has shown weaknesses and strengths of some prominent (Q)SAR programs and diverse combinatorial options in the prediction of skin sensitization by pesticidal AS. The present study will help to foster discussions on in silico alternatives to animal testing in the pesticide area.

Antibody blocking of MHC II on human activated regulatory T cells abrogates their suppressive potential.

Natural regulatory CD4(+)CD25(+)Foxp3(+) T cells control peripheral immune responses. Freshly isolated regulatory T-cell populations are regarded as being unable to suppress the proliferation of strongly stimulated effector T cells. We now provide evidence that it is not the strength of the proliferative signal to effector T cells but activation and accessibility of regulatory T cells that determine whether suppression may occur. Human regulatory T cells were initially cocultured with allogeneic monocyte-derived dendritic cells for a short time and were then rendered accessible for effector T cells by removal of the dendritic cells. That way activated regulatory T cells effectively suppressed the proliferation of autologous effector T cells which was strongly driven by cell-sized Dynabeads coated with CD3/CD28 antibodies. Although regulatory T cells are known to display MHC II molecules and to upregulate their expression along with activation, a role of MHC II molecules in forming the contact to effector T cells was not yet envisaged. However, blocking of MHC II on activated regulatory T cells abrogated their suppressive potential. It should not be excluded that self-MHC molecules on physically accessible activated regulatory T cells arrange the contact to effector T cells.

Differential activation of dendritic cells by nerve growth factor and brain-derived neurotrophic factor.

BACKGROUND: Neurotrophins are involved in inflammatory reactions influencing several cells in health and disease including allergy and asthma. Dendritic cells (DCs) play a major role in the induction of inflammatory processes with an increasing role in allergic diseases as well. OBJECTIVE: The aim of this study was to investigate the influence of neurotrophins on DC function. METHODS: Monocyte-derived dendritic cells were generated from allergic and non-allergic donors. Neurotrophin receptors were demonstrated by western blotting, flow cytometry and fluorescence microscopy. Activation of small GTPases was evaluated by pull-down assays. DCs were incubated with nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and supernatants were collected for measurement of IL-4, IL-6, IL-10, IL-12p70, TNF-alpha and TGF-beta. RESULTS: Receptor proteins were detectable by western blot, fluorescence activated cell sorting analysis and fluorescence microscopy. Signalling after neurotrophin stimulation occurred in a ligand-specific pattern. NGF led to decreased RhoA and increased Rac activation, while BDNF affected RhoA and Rac activity in a reciprocal fashion. Cells of allergics released a significantly increased amount of IL-6, while for healthy subjects a significantly higher amount of IL-10 was found. CONCLUSION: These data indicate that DCs are activated by the neurotrophins NGF and BDNF by different pathways in a receptor-dependant manner. These cells then may initiate inflammatory responses based on allergic sensitization releasing preferred cytokines inducing tolerance or a T-helper type 2 response.

A loose-fit coculture of activated keratinocytes and dendritic cell-related cells for prediction of sensitizing potential.

Protection against contact allergy begins with the collection of reliable data about the sensitizing potential of chemicals. Today, the local lymph node assay (LLNA) in mice is widely used to identify sensitizing substances. For several reasons, an in vitro assay could be preferable to animal experiments. We propose an in vitro test for the detection of a sensitizing potential of a chemical composed of a single layer of human nondifferentiating keratinocytes and of allogenic floating monocytes which are cocultured in serum-free medium in the presence of a cytokine cocktail. Within days, the coculture develops to an allergen- sensitive system consisting of activated keratinocytes and of mobile dendritic cell-related cells (DC-related cell). The sensitizing potential can be determined by analyzing the expression of the dendritic cell maturation marker CD86. For the model contact allergens tested so far [trinitrobenzenesulfonic acid (TNBS), phenylendiamine, and 4-aminoacetanilide], the strength of the reaction was in concordance with results from the LLNA. Sensitivity of the assay allowed testing at concentrations without general cytotoxicity. Thus, a differentiation between allergens and irritants was possible. Regarding cytokine secretion, the assay distinguished between the allergen TNBS and the Toll-like receptor ligand lipopolysaccharide. The coculture can be set up from cryopreserved cells. The assay is easy to perform and reproducible. Donor-variance is negligible. This in vitro assay based on a loose-fit coculture is a reasonable approach to screen for the sensitizing potential of xenobiotics and might partially replace the LLNA and other animal tests.