Objective
The aim of the project is to develop an in vitro method for detection of sensitizing compounds. The final objective of the project is to validate an alternative method to in vivo sensitization and reduce the number of living animals in predictive tests.
Allergic contact dermatitis is induced by substances present in our environment; household and industrial products, drugs, cosmetics, jewellery, plants etc. Most new molecules, especially those that may enter in contact with skin, have to be tested as pure compounds or in the form of a manufactured product to assess their innocuity. The only available and reliable methods to determine the allergenic potential of a product are laboratory animal experimental sensitizations. These studies require considerable numbers of guinea pigs or mice. The scientific community has become aware that it is necessary to reduce the use of animals where possible by alternative in vitro studies. The objective of the project is thus to develop a total in vitro system using cultured epidermal cells. Initial objectives of this continuing project included:
determination of optimal conditions of epidermal cell preparation respectively from guinea pigs, human and murine skinny cytokine production assay and or cytokine messenger ribonucleic acid (mRNA) analysis;
preparation and or purification of natural and synthetic allergenic or nonallergenic compounds.
Participating laboratories approached the objectives through the following investigations:
purification of commercially available haptens and synthesis of sensitizers and nonsensitizers;
optimization of epidermal cell preparation;
determination of optimal conditions for pulse contact of epidermal cells with xenobiotics;
measurement of IL-6 and TNFalpha secretion by bulk epidermal cells;
cytokine (IL-1, IL-6, TNFalpha) induction of epidermal cells pulsed with haptens;
cytokine induction and mRNA analysis on cultured keratinocytes after contact with haptens;
endocytic activity of Langerhans cells after contact with haptens.
In the first phase of this project optimal conditions for in vitro testing of soluble and insoluble xenobiotics on bulk epidermal cells were defined.
A predictive in vitro test for detection of sensitizing compounds has been developed and preliminary results are available.
A panel of known allergenic, irritant and non-allergenic compounds will be tested on bulk epidermal cells, enriched Langerhans cells and endothelial cells of mice, guinea pig and man in in vitro conditions. Each team will use the same preparation of xenobiotics (purified or synthesized by one of the contractors). Data will be gathered on three complementary cellular systems with parallel experiments. The murine system is a reference in immunology, most existing in vivo data were generated in the guinea pig and finally human cells have been chosen for clinical relevance and final validation of the method.
We will focus on early morphological, biochemical and functional modifications of keratinocytes, Langerhans cells (epidermal antigen-presenting cells) and endothelial cells, that occur specifically with sensitizing molecules. Cell surface markers (class II and adhesion molecules) expression, as well as cytokine (IL-1, IL-6, IL-8, TNFalpha, GM-CSF) production and requirement will be studied in murine, guinea pig and human cellular systems. In some cases ultrastructural (electron microscopy) analysis will be implemented. Conditions for optimal in vitro haptenization and antigen-presentation to naive T cells (functional analysis) in these three cellular systems will also be defined.
So far in vitro sensitization has remained difficult due to technical pitfalls (insolubility of most haptens in physiological media) and gaps in our knowledge of hapten processing. Therefore hapten-cystein or hapten-S-peptide conjugates (which could generate the original hapten in situ through biochemical transformation) as well as hapten hydrophobic peptide conjugates (which could associate directly to class II molecules and be presented to T cells) will be prepared and tested as hapten substitutes.
Sharing of reciprocal findings will enable us to progress quickly and remove many bottle-necks that still exist for development of an alternative method to laboratory animals sensitization.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences biochemistry biomolecules
- natural sciences physical sciences optics microscopy electron microscopy
- medical and health sciences basic medicine immunology
- natural sciences mathematics pure mathematics mathematical analysis functional analysis
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Coordinator
34082 Montpellier
France
The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.