Workgroup Report: Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity into International Hazard and Risk Assessment Strategies

This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19–21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work

Hazard assessment of methylmercury toxicity to neuronal induction in embryogenesis using human embryonic stem cells

Pluripotent human embryonic stem cell (hESC) lines can to some extent mimic in vitro the development of the embryo, providing the scientific rationale for the use of these cells to establish tests for toxicity to embryogenesis. Such humanised in vitro tests have potential to improve human hazard prediction by avoiding interspecies differences.We explored the potential of a hESC-based assay for detection of toxicity to neuronal induction in embryonic development. Neuronal precursor differentiation was performed according to a previous publication, whilewe established a newprotocol for maturation of the precursors into neuron-like cells. Appearance of neuronal derivatives was demonstrated by real-time PCR, showing up-regulation of several neuronal marker genes, and immunohistochemistry, demonstrating the appearance of neurofilament medium polypeptide, -tubulin III and microtubule-associated protein 2 positive cells. In order to assess whether the hESC model could detect chemically induced developmental toxicity, we exposed the differentiating cells to methylmercury (MeHg) causing structural developmental abnormalities in the brain. Two separate exposure intervals were used to determine the effects of MeHg on neuronal precursor formation and their further maturation, respectively. The formation of precursorswas sensitive to MeHg in non-cytotoxic concentrations, as the expression of several neuronal mRNA markers changed. In contrast, non-cytotoxic MeHg concentrations did not effect the mRNA marker expression in matured cells, indicating that neuronal precursor formation is more sensitive to MeHg than later stages of neuronal differentiation. Overall, our experiments demonstrate that the hESC assay can provide alerts for the adverse effects of MeHg on neuronal induction.JRC.DDG.I.3-In-vitro method

Embryotoxicity Hazard Assessment of Methylmercury and Chromium Using Embryonic Stem Cells

The embryonic stem cell test (EST) has been scientifically validated (2001) as an in vitro embryotoxicity test, showing a good overall test accuracy of 78%. Methylmercury (MeHg) was the most significant outlayer identified, as the metal was the only strong in vivo embryotoxicant falsely predicted to be non-embryotoxic. The EST misclassification of MeHg, and the potential environmental exposure and developmental toxic hazards of heavy metals gave us the rationale to investigate whether the EST can correctly predict the embryotoxic potential of two heavy metals different from MeHg. The EST correctly classified trivalent chromium to be non-embryotoxic and hexavalent chromium to be embryotoxic, while we confirmed the misclassification of MeHg. MeHg causes developmental abnormalities in the brain. We therefore aimed to improve the in vitro prediction of MeHg embryotoxicity by including a neuronal ES cell differentiation assay. Differentiation of neuronal-like cells was demonstrated by real-time PCR experiments, showing up-regulation of several neuronal marker genes, and immunohistochemistry, demonstrating the appearance of nestin, neurofilament medium polypeptide, -tubulin III and microtubule-associated protein 2 (Mtap2) positive cells.We identified Mtap2 mRNA expression as a sensitive toxicological endpoint for MeHg-induced neuronal embryotoxicity, as Mtap2 mRNA was down-regulated in the presence of non-cytotoxic concentrations of MeHg. Noticeably, several other neuronal marker genes were unaffected by MeHg and Mtap2 expression was not affected until day 14 of differentiation. This implies that the total neuronal-like cell number was unchanged and that the down-regulation of Mtap2 expression reflects neuron-specific toxicity, i.e. instability of the neuron-specific microtubules, and arrest of the neuronal maturation. The fact, that most marker genes were unaffected by MeHg, stresses the importance of including an array of marker genes. In conclusion, our results imply that inclusion of additional target tissues and refinement of the current prediction model may enhance the predictive power of the EST.JRC.I.2-Validation of biomedical testing method

Entwicklung von in vitro-Tests in Embryotoxizitaets- Screening

Abstract is not availableJRC.I.2-Validation of biomedical testing method

G-CSF: Boosting endogenous production - a new strategy?

Granulocyte colony-stimulating factor (G-CSF) has been in clinical use for over a decade. Its main applications are in adjunctive medication to chemotherapy and in mobilizing stem cells for bone marrow transplantation. However. it has additional effects in that it primes neutrophilic granulocytes for improved host defense, and reduces the release of pro-inflammatory cytokines. These effects have prompted trials for numerous other indications. New research into the production and regulation of G-CSF in health and disease may now enable tailored strategies to induce or boost G-CSF formation. Similarly. new forms of application may increase its effectiveness

Highly Purified Lipoteichoic Acid Induced Pro-inflammatory Signalling in Primary Culture of Rat Microglia through Toll-like Receptor 2: Selective Potentiation of Nitric Oxide Production by Muramyl Dipeptide

In contrast to lipopolysaccharide (LPS) from Gram-negative bacteria, the role of Gram-positive bacterial components in inducing inflammation in the CNS remains controversial. We studied the potency of highly purified lipoteichoic acid (LTA) and muramyl dipeptide isolated from Staphylococcus aureus to activate primary cultures of rat microglia. Exposure of pure microglial cultures to LTA triggered a significant time- and dose-dependent production of proinflammatory cytokines (TNF-α, IL-1β, IL-6) and nitric oxide (NO). Muramyl dipeptide strongly and selectively potentiated LTA-induced iNOS expression and NO production. However, it did not have any significant influence on the production of proinflammatory cytokines. As bacterial components are recognized by the innate immunity through Toll-like receptors (TLRs) we showed that LTA was recognized in microglia by the TLR2, while LPS by the TLR4, since cells isolated from mice lacking TLR2 or TLR4 did not produce proinflammatory cytokines and nitric oxide upon LTA or LPS stimulation, respectively. LTA-induced glia activation was mediated by p38 and ERK1/2 MAP kinases, since pre-treatment with inhibitor of p38 or ERK1/2 decreased LTA-induced cytokine release, iNOS mRNA expression and NO production. The observed proinflammatory response induced by LTA-activated microglia could play a major role in the inflammatory response of CNS induced by Gram-positive bacteria.JRC.I.2-Validation of biomedical testing method

Digital Movie Analysis for Quantification of Beating Frequencies, Chronotropic Effects and Beating Areas in Cardiomyocyte Cultures

Software, named Cardio Analyser, was developed for digital movie analysis of beating frequencies, drug induced chronotropic effects and beating areas of contracting cardiomyocyte cultures. A major novelty of the software is the introduction of automated noise filtering and automated movie analysis of beating frequencies and areas of contracting cardiomyocyte cultures. The software was based on the observation that the intensity of light transmitted through a contractive tissue changes periodically in a way which correlates with the contractions. We provided proof of principle for the method by derivation of relevant data from movies of multicellular cardiomyocyte cultures derived from embryonic stem cells. Moreover, we compared the data to equivalent results obtained by extracellular electric field potential recordings and demonstrated higher sensitivity to chronotropic effects of isoprenaline and better cardiac differentiation of ES cells in the experimental set-up applied for movie analysis. Our study indicates that the movie analysis method may have potential to be optimised for screening in early drug discovery, aiming to identify potential cardiac drug candidates or to alerts for adverse effects on heart functionality or embryonic heart development.JRC.I.2-In-vitro Toxicolog

Cytokine induction by Gram-positive bacteria

Despite similar clinical relevance of Gram-positive and Gram-negative infections, immune activation by Grampositive bacteria is by far less well understood than immune activation by Gram-negative bacteria. Our group has made available highly purified lipoteichoic acids (LTA) as a key Gram-positive immunostimulatory component. We have characterized the reasons for lower potency of LTA compared to Gram-negative lipopolysaccharide (LPS), identifying lack of IL-12/IFNgamma induction as a general characteristic of TLR2 agonists, and need for presentation of LTA on surfaces for enhanced immunostimulatory potency, as major aspects. Aspects of chemokine induction, where LTA is more potent than LPS, have been addressed. Furthermore, novel complement and plant defence activation, as well as CD36 as a new LTA receptor, were identified. The bacterial costimuli and modulators of LTA inducible responses are being investigated: LTA isolated from so far 16 bacterial species, although different in structure, behave remarkably similar while whole live and killed bacteria differ with regard to the pattern of induced responses. The purification and characterization of the respective components of the bacterial cell wall has begun

Lipoteichoic acid from Staphylococcus aureus is a potent stimulus for neutrophil recruitment

Lipoteichoic acid (LTA) is a major immunostimulatory principle of Gram-positive bacteria. Intranasal application of LTA from S. aureus to mice resulted in greatly increased neutrophil and macrophage counts in the bronchoalveolar lavage as well as increased levels of the chemokine KC. The potential of highly pure, bioactive LTA from S. aureus to induce neutrophil recruitment and activation was investigated further in the human system.Although neutrophils expressed the key known receptors, CD14, TLR2 and TLR6, LTA did not induce or prime neutrophils for oxidative burst, or release of chemokines, bactericidal permeability-increasing protein or myeloperoxidase. However, LTA induced a strong release of the chemoattractants LTB4, IL-8, C5a, MCP-1 and the colony-stimulating factor G-CSF in whole blood comparable to stimulation with the same concentration of LPS (S. abortus equi). Further, the cytokine and chemoattractant pattern induced by LTA correlated well with that induced by live S. aureus of the same strain.LTA does not appear to activate neutrophils directly, but is a strong stimulus for the recruitment of phagocytes to the site of infection