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Contenuto archiviato il 2024-04-15

ALTERED IMMUNE GENE EXPRESSION AS A NEW APPROACH TO IN VITRO IMMUNOTOXICOLOGICAL SCREENING

Obiettivo

OBTAIN A PROCEDURE FOR EVALUATING IN VITRO OF CERTAINS IMMUNOTOXIC EFFECTS OF CHEMICAL SUBSTANCES.
Molecular biological analysis at the level of cytokine gene expression has been studied as a method for determining the immunomodulatory potential of drugs and industrial chemicals. Studies were carried out to establish whether a correlation existed between conventional functional assays and the analysis of cytokine messenger ribonucleic acid (mRNA) levels in specific immune cell populations (primarily macrophages and T lymphocytes). Effects of well defined pharmaceutical compounds known to enhance or suppress immune responses in vivo were determined in in vitro cell culture systems, or, in some instances, in cells obtained after in vivo exposure. In vitro experiments showed that 3 immunomodulatory agents, biostim, cyclosporin A and gelonin, modulated the expression of specific immune genes, which was in keeping with alterations in production of cytokines as assessed in parallel experiments. The immunopotentiating effect of biostim on macrophage populations at both the mRNA and protein levels was clearly demonstrated in both systems. The expression of mRNA for inteurleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6 and tumour necrosis factor alpha (TNF-alpha) could be elevated by concentrations as low as 1-10 pg/ml, whereas functional assays were 100-fold less sensitive. An easy to perform and economical functional assay for IL-1 activity, however, was a great improvement over the conventionally performed assay system. The immunosuppressive effects of cyclosporin A on T-lymphocyte activity was clearly evident in both functional and molecular biological assays; the latter was 10-fold more sensitive and changes were detected at an earlier time point. Correlations between molecular biological and functional assays have thus been established.
THE EFFECTS OF THE ADMINISTRATION OF SPECIFIC IMMUNODEPRESSIVE AGENTS INCLUDING DAUNORUBICIN, HYDROCORTISONE, CYCLOSPORIN A, MAFOSFAMIDE, SURAMIN AND GELONIN ON A SERIES OF IN VIVO AND EX VIVO IMMUNE PARAMETERS WILL BE EVALUATED IN C3H MICE AT THE MNI. IN VIVO TESTS WILL INCLUDE FOR EXAMPLE IL-1 RELEASE BY PERITONEAL MACROPHAGES AND QUANTITATIVE MHC EXPRESSION BY PURIFIED SPENIC T CELLS, WHILST MIXED LYMPHOCYTE REACTIONS AND MACROPHAGE MEDIATED CYTOTOXICITY ASSAYS WILL BE PERFORMED EX VIVO.IN ADDITION, IN VIVO DERIVED SUBCELLULAR FRACTIONS WILL BE PREPARED FROM VARIOUS CELL POPULATIONS FOR SPECIFIC M-RNA AND PROTEIN ANALYSIS AT BIBRA.THE DATA SO GENERATED WILL PROVIDE A DETAILED PROFILE OF THE GROSS IN VIVO IMMUNOTOXICOLOGICAL EFFECTS OF THESE AGENTS WHICH WILL BE RELATED TO THEIR INDUCED ALTERATIONS ON THE LEVEL OF EXPRESSION OF DEFINED IMMUNOREGULATORY GENES FOR EXAMPLE IL-1, IL-2 AND THEIR RESPECTIVE RECEPTORS.

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Coordinatore

British Industrial Biological Research Association (BIBRA)
Contributo UE
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Indirizzo
Woodmansterne Road
SM5 4DS Carshalton
Regno Unito

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