Commentaries on viewpoint : physiology and fast marathons

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Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants

Organ-specific in vitro toxicity assays are often highly sensitive, but they lack specificity. We evaluated here examples of assay features that can affect test specificity, and some general procedures are suggested on how positive hits in complex biological assays may be defined. Differentiating human LUHMES cells were used as potential model for developmental neurotoxicity testing. Forty candidate toxicants were screened, and several hits were obtained and confirmed. Although the cells had a definitive neuronal phenotype, the use of a general cell death endpoint in these cultures did not allow specific identification of neurotoxicants. As alternative approach, neurite growth was measured as an organ-specific functional endpoint. We found that neurite extension of developing LUHMES was specifically inhibited by diverse compounds such as colchicine, vincristine, narciclasine, rotenone, cycloheximide, or diquat. These compounds reduced neurite growth at concentrations that did not compromise cell viability, and neurite growth was affected more potently than the integrity of developed neurites of mature neurons. A ratio of the EC50 values of neurite growth inhibition and cell death of >4 provided a robust classifier for compounds associated with a developmental neurotoxic hazard. Screening of unspecific toxicants in the test system always yielded ratios <4. The assay identified also compounds that accelerated neurite growth, such as the rho kinase pathway modifiers blebbistatin or thiazovivin. The negative effects of colchicine or rotenone were completely inhibited by a rho kinase inhibitor. In summary, we suggest that assays using functional endpoints (neurite growth) can specifically identify and characterize (developmental) neurotoxicants

Automated image processing to quantify neurite growth in Luhmes human neuronal precursor cells

Chemicals that specifically inhibit human neurite outgrowth pose a hazard to the developing nervous system. Identifying such chemicals remains a major challenge in biological research. In response to the need for more efficient methods to identify potential developmental neurotoxicants, an image processing framework is presented that allows to automatically quantify neurite growth in LUHMES human neuronal precursor cells. For this purpose, a H-33342 staining is used in order to identify the outline of the nucleus of each neuronal cell. Based on this outline, a region growing approach is performed that expands the soma until an intensity threshold is reached, which allows to quantify the number of cells with neurites. The results demonstrate that our image processing framework can rapidly quantify chemical effects on neurite outgrowth. Concentration-response data for neurite outgrowth allows for the determination of the specificity of chemical effects on developing neuronal cells. Further studies will examine the utility of the approach for other cell-based assays of neurite outgrowth

DNA vaccination with CD25 protects rats from adjuvant arthritis and induces an antiergotypic response

Ab’s to the α-chain of the IL-2 receptor (anti-CD25) are used clinically to achieve immunosuppression. Here we investigated the effects of DNA vaccination with the whole CD25 gene on the induction of rat adjuvant arthritis. The DNA vaccine protected the rats and led to a shift in the cytokine profile of T cells responding to disease target antigens from Th1 to Th2. The mechanism of protection was found to involve the induction of an antiergotypic response, rather than the induction of anti-CD25 Ab’s. Antiergotypic T cells respond to activation molecules, ergotopes, expressed on syngeneic activated, but not resting, T cells. CD25-derived peptides function as ergotopes that can be recognized by the antiergotypic T cells. Antiergotypic T cells taken from control sick rats did not proliferate against activated T cells and secreted mainly IFN-γ. In contrast, antiergotypic cells from CD25-DNA–protected rats proliferated against activated T cells and secreted mainly IL-10. Protective antiergotypic T cells were found in both the CD4(+) and CD8(+) populations and expressed α/β or γ/δ T cell receptors. Antiergotypic α/β T cells were MHC restricted, while γ/δ T cells were MHC independent. Thus, CD25 DNA vaccination may induce protection from autoimmunity by inducing a cytokine shift in both the antiergotypic response and the response to the antigens targeted in the disease