1. Group 2: Animal models for mast cell research Trainer: Katerina Tiligada Introduction In vivo animal models for Mast cell research: • Guinea Pig • Humanized Mice • Zebrafish Aim: comparison of established versus new in vivo models

2. Background: insufficiency of mouse models due to differences between human and mice MC distribution, mediator release and phenotype Aim: to evaluate a combination treatment in an animal model of early allergic reaction in the lung by using the established guinea pig model

3. Treatment with pyrilamine in combination with montelukast or/and indomethacin reduced EAR P: Pyrilamine- H1 antagonist M: Montelukast - cysLT inhibitor I: Indomethacin – COX inhibitor - NSAID

4. Summary The sensitized guinea pig is a well accepted and validated model for the study of pharmacological approaches related to allergy treatment Limitations of the study Methodological shortcomings (e.g. no dose-response-curve, no justification for the selection of drugs to be investigated) Limited novel information

5. Background: lack of mice model which reflects human allergy response Aim: To generate a humanized mouse strain that develops human granulocytes and mast cells

6. Differentiation of myeloid and lymphoid cells in hu-HSC NOG IL-3/GM-Tgmice

7. Differentiation of myeloid and lymphoid cells in hu-HSC NOG IL-3/GM-Tgmice

8. Extravasation of Evans blue dye in the skin of hu-HSC NOG IL-3/GM-Tgmice

9. Summary The first human IL-3 and GM-CSF producing mice able to develop human granulocytes and mast cells A new model which enables the research on human cell in vivo Limitations of the study Lack of cell-cell interactions of the same species origin Mast cells can differentiate depending on the (micro)environment

10. Zebrafish is a robust model for studying vertebrate hematopoiesis • Presence of mast cells, basophils and eosinophils • Orthologs of toll-like receptors (TLRs) and myd88 • Structural and functional similarities between zebrafish and mammalian MCs Aim: Investigation of the FcεRI-mediated MC response upon stimulation

11. Release of tryptase is induced by C48/80 and A. salmonicida while being inhibited by ketotifen

12. Release of tryptase is induced by anti-IgE:DNP complex

13. Summary Zebrafish represents a potential new model for studying MC-mediated responses in vivo Limitationsof the study Functional studies to support the preliminary findings not conducted Inhibition of the IgE-mediated pathway not investigated Experimental settings are not specific for zebrafish

14. Overall summary • The attempts of calibration & development of suitable models are continuous.Overall, we are slowly getting closer to (patho)physiological relevance • Functional and behavioral studies on MCs are being researched in few models, each one being suitable for a limited range of research questions/hypotheses • No universal model is available; depending on the question to be answered, acceptable models have to be selected Preliminary data Validated Guinea pig  Humanized mice  Zebrafish 

15. Group 2: Animal models for mast cell research Trainer: Katerina Tiligada Points following discussion • Difficulties in in vivo studies: validation protocols; different markers, triggers, agents, etc • Need for harmonization & standardization of the essential protocols/methodologies for validation of novelin vivo models in various species: consideration of strain, gender, age differences/variations; visibility of negative results is essential • In vivo drug administration: consideration of strain-related differences, ligand specificity, ligand-directed signaling, threshold/baseline/placebo responses, dose-response curves, selection of the appropriate control drug • Inter/intra species cell-cell interaction and immune responses in in vivo models (particularly concerning Tg & humanised models) • Comparative definition of mast cell/basophil (rodent models are different from human, including MC/basophilphenotypes & behaviour): need for a (minimal) list of characteristics and comparison between species