Session A7: Organ Studies II (Neuro, Embryo, and Immuno)

Chairs: Ellen Silbergeld (USA) and Sandra Coecke (Italy)

A7: Validation Strategies for Newly Developed Technologies in Neurotoxicology
S. Coecke, J. Gartlon, E. van Vliet, S. Bernardini, and C. Eskes. ECVAM, Institute for Health and Consumer Protection, European Commission Joint Research Centre, 21020 Ispra (Va), Italy. Sandra.coecke@jrc.it.

Up to now, no in vitro methods for evaluating the neurotoxic hazard of a chemical have been validated. The current guidelines from the OECD for the Assessment of Neurotoxic Effects of Chemicals (guidelines 418 and 419 from 1995, guideline 424 from 1997) are based on in vivo studies. Due to the widely-recognized shortfalls in this current whole-animal approach for valuating the safety of chemicals and the recent White Paper on a Future Chemicals Policy of the European Commission, alternative approaches to conventional neurotoxicity testing are urgently needed.

A commonly recommended strategy for the evaluation of the neurotoxic potential of chemicals is the use of a tiered approach. Such an approach would offer the possibility of the reliable and relevant detection of neurotoxic potential in a scientific, economical, and ethical manner.

Due to the introduction of some novel in vitro systems, including the use of genetically engineered neuronal cell lines for specific mechanistically relevant endpoints and of aggregating brain cell cultures and their integration into batteries of tests and tiered testing strategies, a scientifically based human chemicals risk assessment can now be provided, and animal use can be reduced.

A7: An In Vitro System to Assess Neuronal Migration
Ellen K. Silbergeld and Jennifer B. Sass. Johns Hopkins University, Baltimore, MD, USA. esilber@aol.com.

Neuronal migration is a critical state in neurobiological development, essential to attaining the mature cytoarchitecture and synaptic connections of the central nervous system. Prenatal exposures to agents such as methylmercury and radiation exert profound effects on motor function and cognition through impaired neuronal migration. However, to date there have been no in vitro methods that permit quantitation of neuronal migration in terms of actual distance moved by individual cells. We have developed a system, utilizing primary cultures of mouse cerebellum, in which the movement of neurons can be tracked by photomicrography over long periods of culture. Agents can be added to the culture system to identify potential developmental neurotoxicants. The method permits the determination of initiation of movement, as well as distance moved, which appear to be under separate control. The inflammatory cytokines TNF-a and interleukin-6 increase granule cell movement in vitro, without affecting the number of cells that move. Methylmercury (as low as 0.1 µM) inhibits both distance and percentage of moving cells. Research supported by grants from the Bressler Research Fund and the Cure Autism Now Foundation.

A7: Improvement of the Embryonic Stem Cell Test by Establishing Molecular Endpoints of Tissue-Specific Development
Andrea Seiler, A. Visan, I. Pohl, R. Buesen, E. Genschow, and H. Spielmann. Center for Documentation and Evaluation of Alternative Methods to Animal Experiments (ZEBET), 12277 Berlin, Germany. seiler.zebet@bgvv.de.

This study was performed to improve an in vitro approach for developmental toxicity that is based on blastocyst-derived pluripotent embryonic stem (ES) cells of the mouse (cell line D3), the so-called embryonic stem cell test (EST). In the EST the capacity of ES cells to differentiate into contracting cardiomyocytes is used to assess the embryotoxic potential of test compounds. In a joint project with major German pharmaceutical companies, we are currently attempting to expand the EST protocol by establishing molecular endpoints of differentiation (e.g., cardiac, neuronal, and chondrogenic) in cultured ES cells. We have studied the expression of tissue-specific proteins in ES cell cultures in the presence of embryotoxic chemicals by immunofluorescent antibody techniques, e.g. FACS analysis. The other groups are focusing on endogenous gene expression in early development by RT-PCR methods or the DNA microarray technique. The results obtained recently by studying the express of cardiac-specific proteins under the influence of the test compound employing intercellular flow cytometry for quantification will be presented.

A7: A Humanized In Vitro High-Throughput Immunotechnology Platform

The platform is used for producing low allergenic proteins and for improving vaccines and therapeutic and diagnostics proteins. Animal usage is reduced ten-fold. In Silico Epitope Mapping: A peptide library is expressed on phages and enriched for clones that bind specifically to target-specific antibody. The resulting antibody-binding peptides are analyzed and epitope patterns are identified. Finally, computer modeling is used for determining surface residues of the target protein that match to the epitope patterns. A computer program for mapping linear and conformational B-cell epitopes is available. Protein Modification: The target protein is modified by specifically altering the amino acid sequence in order to change the surface properties. A computer program for automated optimization of protein engineering strategies is available. In vitro screening: The efficacy of the introduced modifications is assessed by in vitro assays. One assay focuses on changes in the antibody-binding capacity of the variants as compared to the unmodified protein. This assay is used to identify potential candidates among a large number of variants. A second assay involves human lung epithelial cells, and relates in vivo allergenicity of the protein with cytokine profiles produced by these cells upon stimulation with protein. This assay is used as a confirmation assay.