Lars Hareng

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, while we established a new protocol 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, beta-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 precursors was 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.

Cytokine induction by Gram-positive bacteria

Despite similar clinical relevance of Gram-positive and Gram-negative infections, immune activation by Gram-positive 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.

Comparison of the skin sensitizing potential of unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and the guinea pig maximization test (GPMT)

The skin sensitization potential of eight unsaturated and one saturated lipid (bio)chemicals was tested in both the LLNA and the GPMT to address the hypothesis that chemicals with unsaturated carbon-carbon double bonds may result in a higher number of unspecific (false positive) results in the LLNA compared to the GPMT. Seven substances (oleic acid, linoleic acid, linolenic acid, undecylenic acid, maleic acid, squalene and octinol) gave clear positive results in the LLNA (stimulation index (SI)> or = 3) and thus would require labelling as skin sensitizer. Fumaric acid and succinic acid gave clearly negative results. In the GPMT, besides some sporadic skin reactions, reproducible skin reactions indicating an allergic response were found in a few animals for four test substances. Based on the GPMT results, only undecylenic acid would have to be classified and labelled as a skin sensitizer according to the European Dangerous Substance Directive (67/548/EEC) (results for linoleic acid were inconclusive), while the other seven test substances would not require labelling. Possible mechanisms for unspecific skin cell stimulation and lymph node responses are discussed. In conclusion, the suitability of the LLNA for unsaturated compounds bearing structural similarity to the tested substances should be carefully considered and the GPMT should remain available as an accepted test method for skin sensitization hazard identification.

Induction and regulation of endogenous granulocyte colony-stimulating factor formation

Abstract Granulocyte colonystimulating factor (GCSF) is one of the most prominent endogenous proteins in broad clinical use. While its biological and clinical effects are relatively well studied, little is known about its endogenous formation in health and disease. However, such knowledge is crucial to decide in which situations GCSF should be applied efficiently in the clinic, i.e. when endogenous production does not suffice. The dramatic changes induced by GCSF in the differential blood cell count are directly immunomodulatory, strengthening the innate defence by multiplying neutrophilic granulocytes. A multitude of further immunomodulatory effects contribute to the regulation of the concerted host defence. In this review, following a short introduction into the biology of GCSF, the available data on endogenous formation in a number of animal models and human diseases is compiled. The cellular sources and inducers of GCSF formation are reviewed and the regulation of GCSF expression on both the transcriptional and translational level are discussed. The emerging understanding of the role and regulation of endogenous GCSF formation opens up possibilities to define therapeutic windows as well as targets for diagnostics or drug development. Lastly, the modulation of GCSF formation by various pharmacological agents alerts to putative side effects of these drug treatments.

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.

Activation of Neutrophils and Inhibition of the Proinflammatory Cytokine Response by Endogenous Granulocyte Colony-Stimulating Factor in Murine Pneumococcal Pneumonia

Granulocyte colony-stimulating factor (G-CSF) is considered to improve host defense during infection, via increased recruitment of and enhanced performance of neutrophils and subsequent inhibition of potentially harmful proinflammatory mediators. The present study sought to determine the role of endogenous G-CSF in host defense against pneumococcal pneumonia. Patients with unilateral community-acquired pneumonia demonstrated elevated concentrations of G-CSF in bronchoalveolar lavage fluid obtained from the infected, but not from the contralateral, site. Treatment of mice with pneumococcal pneumonia with an anti-G-CSF antibody reduced neutrophil counts in lung tissue and diminished CD11b expression on pulmonary neutrophils but increased the lung concentrations of tumor necrosis factor- alpha, interleukin-1 beta, and cytokine-induced neutrophil chemoattractant. Treatment with anti-G-CSF did not influence the outgrowth of pneumococci in lungs, the dissemination of the infection, or survival in murine pneumonia. During pneumococcal pneumonia, G-CSF is produced locally at the site of the infection, where it exerts both pro- and anti-inflammatory effects.

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 the role of lipopolysaccharide 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 and muramyl dipeptide isolated from Staphylococcus aureus to activate primary cultures of rat microglia. Exposure of pure microglial cultures to lipoteichoic acid triggered a significant time‐ and dose‐dependent production of pro‐inflammatory cytokines (tumour‐necrosis factor‐α, interleukin‐1β, interleukin‐6) and nitric oxide. Muramyl dipeptide strongly and selectively potentiated lipoteichoic acid‐induced inducible nitric oxide synthase expression and nitric oxide production. However, it did not have any significant influence on the production of pro‐inflammatory cytokines. As bacterial components are recognised by the innate immunity through Toll‐like receptors (TLRs) we showed that lipoteichoic acid was recognised in microglia by the TLR2 and lipopolysaccharide by the TLR4, as cells isolated from mice lacking TLR2 or TLR4 did not produce pro‐inflammatory cytokines and nitric oxide upon lipoteichoic acid or lipopolysaccharide stimulation, respectively. Lipoteichoic acid‐induced glia activation was mediated by p38 and ERK1/2 MAP kinases, as pretreatment with inhibitor of p38 or ERK1/2 decreased lipoteichoic acid‐induced cytokine release, iNOS mRNA expression and nitric oxide production. The observed pro‐inflammatory response induced by lipoteichoic acid‐activated microglia could play a major role in the inflammatory response of CNS induced by Gram‐positive bacteria.

Embryotoxicity hazard assessment of cadmium and arsenic compounds using embryonic stem cells

The Embryonic Stem Cell Test (EST) has been successfully validated as an in vitro method for detecting embryotoxicity, showing a good overall test accuracy of 78% [Genschow, E., Spielmann, H., Scholz, G., Seiler, A., Brown, N., Piersma, A., Brady, M., Clemann, N., Huuskonen, H., Paillard, F., Bremer, S., Becker, K., 2002. The ECVAM international validation study on in vitro embryotoxicity tests: results of the definitive phase and evaluation of prediction models. European Centre for the Validation of Alternative Methods. Altern. Lab. Anim. 30, 151-176]. Methylmercury was the only strong in vivo embryotoxicant falsely predicted as non-embryotoxic making the metal the most significant outlayer [Genschow, E., Spielmann, H., Scholz, G., Pohl, I., Seiler, A., Clemann, N., Bremer, S., Becker, K., 2004. Validation of the Embryonic Stem Cell Test in the international ECVAM validation study on three in vitro embryotoxicity tests. Altern. Lab. Anim. 32, 209-244]. The misclassification of methylmercury and the potential environmental exposure to developmental toxic heavy metals promoted our investigation of whether the EST applicability domain covers cadmium and arsenic compounds. The EST misclassified cadmium, arsenite and arsenate compounds as non-embryotoxic, even when including arsenic metabolites (methylarsonate, methylarsonous and dimethylarsinic). The reasons were the lack of higher cytotoxicity towards embryonic stem cells as compared to more mature cells (3T3 fibroblasts) or the absence of inhibition of cardiac differentiation by specific mechanisms rather than general cytotoxicity. Including EST data on heavy metals from the literature (lithium, methylmercury, trivalent chromium and hexavalent chromium) revealed that the test correctly predicted the embryotoxic potential of three out of the seven heavy metals, indicating an insufficient predictivity for such metals. Refinement of the EST prediction model and inclusion of additional toxicological endpoints could expand the applicability domain and enhance the predictive power of the test.

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 quantification of 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 that 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. The comparison demonstrated higher sensitivity to chronotropic effects of the beta-adrenoceptor agonist isoprenaline, and hence implied that more embryonic stem cells underwent differentiation into beta-adrenoceptor-responding cardiomyocytes, in the experimental setup applied for movie analysis than in the setup used for extracellular electric field potential recordings. Our study indicates that the movie analysis method may have potential to be optimized for screening in early drug discovery, aiming to identify cardiac drug candidates or to alert for adverse effects on heart functionality or embryonic heart development.