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A new technology for fluorescent "cell chip" immunotoxicity testing

Objectif

Immunotoxicity is usually assessed employing well-defined animal models. Usage of transformed immortalized cell lines of desired phenotype is one of possible alternative for animal testing. There are evidences showing that at least certain types of immunotoxicity like these leading to hypersensitivity and autoimmunity are associated with modulation of expression of cytokine genes in immune or non-immune cells. This proposal outlines the development of technology for detecting xenobiotics-mediated modulation of cytokine gene expression in vitro. Specialized reporter cell lines will be prepared by genetic modifications to instantly detect signals modulating expression of cytokine genes. These cell lines will be tested and become a part of the cell chip. The prototype of a cell chip will be pre-validated as a system for testing immunotoxicity, and might be incorporated into a battery of screening tests.
We have developed a new system for in vitro immunotoxicity testing, which employs changes in cytokine expression observed in vitro as an endpoint indicating potential for perturbation of the immune system in vivo. To this end a 30 different of DNA constructs designed in such a way that expression of reporter fluorescence protein depends of regulatory sequences derived from different cytokine genes were generated. These DNA construct including reporter vectors for IL-2, IFN-g, IL-4, IL-1b, TNF-a, and b-actin were employed for genetic modification of cell lines and resultede in development of a large collection of reporter cell lines. Upregulation of EGFP-mediated fluorescence upon stimulation with cell specific stimuli was observed in multiple reporter cell lines derived from lymphocytes, mast cells, keratinocytes and macrophages. Furthermore, this upregulation of EGFP expression was paralleled with upregulation of endogenous cytokine expression. Morphological and functional features of selected cell lines expressing EGFP under the control of cytokine promoters were compared with maternal cell lines and this comparison showed that critical functional features of the maternal cell lines were preserved in EGFP expressing cells (Fig. 2). The prototype of "Fluorescent Cell Chip" (FCC) was assembled based on selected reporter cell line and tested. In testing of the prototype of FCC chemicals with known immunotoxic activities mediated compound-specific pattern of inhibition and activation of reporter gene expression.

Several immunosuppressive substances known to inhibit functions of immune cells in vitro and in vivo specifically and in dose dependent manner decreased fluorescence of cytokine reporter cells. These effects were consistent with known mechanisms of their action and previous observations of their effect on cytokine expression in lymphocytes. Some of tested allergens such as Der p-mite and TDI generated specific pattern of changes in fluorescence level reflecting the immunomodulatory properties of these substances observed in vivo. These data suggest that the particular set of reporter cell line employed in the prototype of FCC is able to detect immunomodulatory and especially immunosupressive activities of certain xenobiotics. Thus the prototype of the "Fluorescent Cell Chip" has demonstrated potential for application as a predictive screening test for immunomodulatory activities of chemicals. The major advantage of this approach is the possibility to apply this test in high throughput screening of high number of compounds for their well-defined biological activity. The usefulness of this approach for practical testing will be revealed following further analysis of multiple chemical compounds of known activity in vivo.

Appel à propositions

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Régime de financement

CSC - Cost-sharing contracts

Coordinateur

INTERNATIONAL INSTITUTE OF MOLECULAR AND CELL BIOLOGY
Contribution de l’UE
Aucune donnée
Adresse
ks. Trojdena 4
WARSZAWA
Pologne

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Coût total
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Participants (5)