Introduction In vivo animal models for Mast cell research: 1.Guinea Pig 2.Humanized Mice 3.Zebrafish Aim: comparison of established versus new in vivo models Group 2: Animal models for mast cell research Trainer: Katerina Tiligada

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

P: Pyrilamine - H 1 antagonist M: Montelukast - cysLT inhibitor I: Indomethacin – COX inhibitor - NSAID Treatment with pyrilamine in combination with montelukast or/and indomethacin reduced EAR

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

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

Differentiation of myeloid and lymphoid cells in hu-HSC NOG IL-3/GM-Tg mice

Differentiation of myeloid and lymphoid cells in hu-HSC NOG IL-3/GM-Tg mice

Extravasation of Evans blue dye in the skin of hu-HSC NOG IL-3/GM-Tg mice

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

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

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

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

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

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 

Difficulties in in vivo studies: validation protocols; different markers, triggers, agents, etc Need for harmonization & standardization of the essential protocols/methodologies for validation of novel in 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/basophil phenotypes & behaviour): need for a (minimal) list of characteristics and comparison between species Group 2: Animal models for mast cell research Trainer: Katerina Tiligada Points following discussion