RISK-HUNT3R

Institutions
  • AG Leist (In vitro Toxikologie und Biomedizin)
Publications
  Kranaster, Petra; Blum, Jonathan; Dold, Jeremias E.G.A.; Wittmann, Valentin; Leist, Marcel (2023): Use of metabolic glycoengineering and pharmacological inhibitors to assess lipid and protein sialylation on cells Journal of Neurochemistry ; 164 (2023), 4. - S. 481-498. - Wiley. - eISSN 1471-4159

Use of metabolic glycoengineering and pharmacological inhibitors to assess lipid and protein sialylation on cells

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Metabolic glycoengineering (MGE) has been developed to visualize carbohydrates on live cells. The method allows the fluorescent labeling of sialic acid (Sia) sugar residues on neuronal plasma membranes. For instance, the efficiency of glycosylation along neurite membranes has been characterized as cell health measure in neurotoxicology. Using human dopaminergic neurons as model system, we asked here, whether it was possible to separately label diverse classes of biomolecules and to visualize them selectively on cells. Several approaches suggest that a large proportion of Sia rather incorporated in non-protein components of cell membranes than into glycoproteins. We made use here of deoxymannojirimycin (dMM), a non-toxic inhibitor of protein glycosylation, and of N-butyl-deoxynojirimycin (NBdNM) a well-tolerated inhibitor of lipid glycosylation, to develop a method of differential labeling of sialylated membrane lipids (lipid-Sia) or sialylated N-glycosylated proteins (protein-Sia) on live neurons. The time resolution at which Sia modification of lipids/proteins was observable was in the range of few hours. The approach was then extended to several other cell types. Using this technique of 'target-specific MGE', we found that in dopaminergic or sensory neurons > 60% of Sia is lipid bound, and thus polysialic acid-neural cell adhesion molecule (PSA-NCAM) cannot be considered the major sialylated membrane component. Different from neurons, most Sia was bound to protein in HepG2 hepatoma cells or in neural crest cells. Thus, our method allows visualization of cell-specific sialylation processes for separate classes of membrane constituents.

Origin (projects)

  Neuhaus, Winfried; Reininger-Gutmann, Birgit; Rinner, Beate; Plasenzotti, Roberto; Wilflingseder, Doris; De Kock, Joery; Hartung, Thomas; Pallocca, Giorgia; Rovida, Costanza; Leist, Marcel (2022): The Current Status and Work of Three Rs Centres and Platforms in Europe Alternatives to Laboratory Animals : ATLA ; 50 (2022), 6. - S. 381-413. - Sage. - ISSN 0261-1929. - eISSN 2632-3559

The Current Status and Work of Three Rs Centres and Platforms in Europe

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The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Origin (projects)

  Holzer, Anna-Katharina; Karreman, Christiaan; Suciu, Ilinca; Furmanowsky, Lara-Seline; Wohlfarth, Harald; Loser, Dominik; Dirks, Wilhelm G.; Pardo González, Emilio; Leist, Marcel (2022): Generation of Human Nociceptor-Enriched Sensory Neurons for the Study of Pain-Related Dysfunctions Stem Cells Translational Medicine ; 11 (2022), 7. - S. 727-741. - Oxford University Press (OUP). - ISSN 2157-6564. - eISSN 2157-6580

Generation of Human Nociceptor-Enriched Sensory Neurons for the Study of Pain-Related Dysfunctions

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In vitro models of the peripheral nervous system would benefit from further refinements to better support studies on neuropathies. In particular, the assessment of pain-related signals is still difficult in human cell cultures. Here, we harnessed induced pluripotent stem cells (iPSCs) to generate peripheral sensory neurons enriched in nociceptors. The objective was to generate a culture system with signaling endpoints suitable for pharmacological and toxicological studies. Neurons generated by conventional differentiation protocols expressed moderate levels of P2X3 purinergic receptors and only low levels of TRPV1 capsaicin receptors, when maturation time was kept to the upper practically useful limit of 6 weeks. As alternative approach, we generated cells with an inducible NGN1 transgene. Ectopic expression of this transcription factor during a defined time window of differentiation resulted in highly enriched nociceptor cultures, as determined by functional (P2X3 and TRPV1 receptors) and immunocytochemical phenotyping, complemented by extensive transcriptome profiling. Single cell recordings of Ca2+-indicator fluorescence from >9000 cells were used to establish the "fraction of reactive cells" in a stimulated population as experimental endpoint, that appeared robust, transparent and quantifiable. To provide an example of application to biomedical studies, functional consequences of prolonged exposure to the chemotherapeutic drug oxaliplatin were examined at non-cytotoxic concentrations. We found (i) neuronal (allodynia-like) hypersensitivity to otherwise non-activating mechanical stimulation that could be blocked by modulators of voltage-gated sodium channels; (ii) hyper-responsiveness to TRPV1 receptor stimulation. These findings and several other measured functional alterations indicate that the model is suitable for pharmacological and toxicological studies related to peripheral neuropathies.

Origin (projects)

  Pallocca, Giorgia; Moné, Martijn J.; Kamp, Hennicke; Luijten, Mirjam; Van de Water, Bob; Leist, Marcel (2022): Next-generation risk assessment of chemicals - Rolling out a human-centric testing strategy to drive 3R implementation : The RISK-HUNT3R project perspective Alternatives to Animal Experimentation : ALTEX ; 39 (2022), 3. - S. 419-426. - Springer Spektrum. - eISSN 1868-596X

Next-generation risk assessment of chemicals - Rolling out a human-centric testing strategy to drive 3R implementation : The RISK-HUNT3R project perspective

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In many industrial sectors, there is a need for reliable ways to evaluate the safety of chemicals with methods anchored to human biology and pathology. For this purpose, many animal-free new approach methods (NAMs) have been developed and implemented in various stages of risk assessment. Now it is time to assemble individual NAMs into a comprehensive next-generation risk assessment (NGRA) strategy. The European Horizon 2020 RISK-HUNT3R project ("Risk assessment of chemicals integrating human-centric next-generation testing strategies promoting the 3Rs") has been designed to promote a combination of computational toxicology, in vitro toxicology, and systems biology. It is assumed that this approach will lead to faster and more accurate risk assessment procedures. The RISK-HUNT3R NGRA strategy will be developed to address the implementation of a comprehensive NAM toolbox into the regulatory framework. Critical conceptual approaches of the project include i) the integration of human-relevant data on biotransformation and elimination, ii) the translation of high-content mode-of-action datasets into predictions of adverse outcomes, iii) development of quantitative adverse outcome pathways (qAOPs), and iv) quantification of uncertainties associated with the predictions based on NGRA strategies. Many of the project steps will be used iteratively to generate datasets with sufficient quality and certainty for NGRA. Scientists and regulators will work together on case studies to evaluate NAMs' practical applicability and the strategies to combine information therefrom. Here we delineate how the strategy will be deployed to establish an overall NGRA framework for chemicals, pesticides, food additives, and drugs.

Origin (projects)

  Pallocca, Giorgia; Rovida, Costanza; Leist, Marcel (2022): On the usefulness of animals as a model system (part I) : Overview of criteria and focus on robustness Alternatives to Animal Experimentation : ALTEX ; 39 (2022), 2. - S. 347-353. - Springer Spektrum. - eISSN 1868-596X

On the usefulness of animals as a model system (part I) : Overview of criteria and focus on robustness

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Banning or reduction of the use of animals for laboratory experiments is a frequently-discussed societal and scientific issue. Moreover, the usefulness of animals needs to be considered in any decision process on the permission of specific animal studies. This complex issue is often simplified and generalized in the media around the question, "Are animals useful as a model?" To render an often emotional discussion about animal experimentation more rational, it is important to define "usefulness" in a structured and transparent way. To achieve such a goal, many sub-questions need to be asked, and the following aspects require clarification: (i) consistency of animal-derived data (robustness of the model system); (ii) scientific domain investigated (e.g., toxicology vs disease modelling vs therapy); (iii) measurement unit for "benefit" (inte-grating positive and negative aspects); (iv) benchmarking to alternatives; (v) definition of success criteria (how good is good enough); (vi) the procedure to assess benefit and necessity. This series of articles discusses the overall benchmarking process by specifying the six issues. The goal is to provide guidance on what needs to be clarified in scientific and political discussions. This framework should help in the future to structure available information, to identify and fill information gaps, and to arrive at rational decisions in various sub-fields of animal use. In part I of the series, we focus on the robustness of animal models. This describes the capacity of models to produce the same output/response when faced with the "same" input. Follow-up articles will cover the remaining usefulness aspects.

Origin (projects)

  Pallocca, Giorgia; Leist, Marcel (2022): On the usefulness of animals as a model system (part II) : Considering benefits within distinct use domains Alternatives to Animal Experimentation : ALTEX ; 39 (2022), 3. - S. 531-539. - Springer Spektrum. - ISSN 1868-596X. - eISSN 1868-8551

On the usefulness of animals as a model system (part II) : Considering benefits within distinct use domains

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In many countries, animal experiments can only be performed when their necessity has been demonstrated in a legal document. As the usefulness of animals in research is also a significant societal and political issue, criteria to structure debates and evaluations are needed. Here, background information is given on laboratory animal studies. Moreover, parameters that may be considered in judging their usefulness are suggested. The discussion is strictly focused on animals used as tools/test systems/models to provide information on humans. In this context, general features and performance characteristics of models are discussed. Examples are given for well-recognized criteria (e.g., robustness, relevance, predictivity) to judge the usefulness of predictive models. The main hypothesis put forward here is that a benefits evaluation (usefulness metrics) is only possible within sharply circumscribed "use domains". Examples are given for the research fields of drug and vaccine research, toxicology, disease pathogenesis, and basic biological research. Efficacy, safety, and quality studies are highlighted as "use domains" within the field of drug discovery and production. A further separation into individual diseases, drug targets or symptoms is suggested for, e.g., efficacy studies or pathophysiology. Finally, an outlook is given on the evaluation of model advantages and disadvantages to arrive at their "net benefit". Moreover, the need to compare the net benefits of animal models versus that of their alternatives is highlighted.

Origin (projects)

Funding sources
Name Project no. Description Period
ERC524/21
Further information
Period: 01.06.2021 – 31.05.2026