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Pros and Cons of the C3H HeJ versus the Humanized Mouse Model

In order to probe the efficacy of new immune-intervention strategies in alopecia areata, the field can now choose from two mutually complementary mouse models.

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Pros and Cons of the C3H HeJ versus the Humanized Mouse Model

  1. 1. Skin Research Laboratory, The B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel Human Scalp Skin Xenotransplants in Androgenetic AlopeciaPre-clinical research in Vivo: Pros and Cons of the C3H/HeJ versus Humanized Mouse Model Human skin grafts on animal models Amos Gilhar, M.D.
  2. 2. In our opinion both models should be usedBochenska K et al. J. Mol. Sci. 2017 Murine models of Psoriasis According to the literature there are 11 models for vitiligo 19 for Atopic Dermatitis, more than 20 for psoriasis but 2 for Alopecia Areata 1. The C3H/HeJ is used widely, whereas 2. The humanized mice (no NK , T and B cells) is used in only one Lab.
  3. 3. Normal Human scalp skin/SCID* mice injected with IL-2 enriched PBMCs C3H/HeJ with skin graft- induced hair loss Animal Model +Regrowth <3% Sudden hair loss with temporarily hair regrowth. ++Spontaneous hair growth ++Mononuclear infiltrate around and within the hair bulb. ++Peri- and intrafollicular- CD8+ T-cells +NANKG2D and NKG2D- ligands ++Major histocompatibility complex (MHC) I and II -+The hair loss pattern is categorize to focal and diffuse pattern Table 1 AA criteria in the animal models Similarities and differences between the C3H/HeJ & humanized mouse model Gilhar A et al Autoimmun Rev. 2016
  4. 4. C3H/HeJ with skin graft-induced hair loss AA humanized mouse model Easy to use Housed together. Do not require specific pathogen-free conditions. Technical expertise is required, complicated Convenient and cheap Relatively inexpensive, convenient model of AA. Expensive Grooming induced hair loss Grooming and scratching behaviors None Potential for pre-clinical drug screening With limited value due to murine-specific disease pathology. Can be used widely due to its reliance on healthy human donor tissue and human disease pathology Disadvantages The histological feature is not specific for AA: The inflammatory cells are also observed above the hair bulb, CD4+ cells dominants over CD8 + cells CTLA4 polymorphisms, is not adequately represented. MICA is strikingly absent in mice (only 27% amino-acid identity with human MICA). Difficulty in obtaining sufficient clinical material Require a pathogen-free environment for maintenance Absence of genetic background Advantages Has yielded tremendous insight into the pathogenesis and treatment of AA Specific histological feature: around and within the hair bulb as in human AA CD8+ cells dominants over CD4 + cells as in human AA
  5. 5. Differences between non-conventional T cells populations among human versus mice should be taken into account when using mice as preclinical models of human disease. There are several distinct subsets of γδ T cells in mice and humans, but mouse and human subsets notably have different TCR use, antigen reactivity and patterns of tissue homing Godfrey et al. Nature Immunology, 2015 Mouse models have failed to account for the natural diversity in human immune responses. As a result, insights gained in the lab may be lost in translation Konrad Buscher et al. Nature Communications, 2017 Differences between immune system of human & mice There is growing concern that laboratory mice do not reflect relevant aspects of the human immune system, which may account for failures to translate disease treatments Beura et al. Nature , 2016 from bench to bedside
  6. 6. Foxp3 Vδ2TCR ‫ע‬37% Increased level of γδTreg γ/δTregs γδTregs are the predominant regulatory T cells and have more potent immunosuppressive activity than CD4+ or CD8+ Tregs Hu G, et al. Oncoimmunology 2017 Gu Y, et al. J Immunol Methods 2014 Feng Y, et al. Nature 2015 Hu y, et al. Hematol Oncol. 2017 Humanized model TGF-β INF-γ Gilhar et al. unpublished High levels of TGF-β and low of IFN-ƴ in γδTreg Humanized model Human AA Human Scalp Skin Humanized AA model Green –Keratin 15 Normal HF of normal scalp skin AA HF Presence of γ/δTregs among stem cells in the bulge area
  7. 7. High levels of intracellular IL-10 & TGF-β in γδTreg Co-culture of CD8+NKG2D+ cells with γδTreg cells Human HF organ culture Effect of IL-10 and TGFβ on HFs co-culture with CD8/NKG2D cells IFN-γTGF-β %Catagen Control CD8/ 71% 44% %43%43 p<0.01 CD8/NKG2D cells TGFβ IL-10 TGFβ/ IL10
  8. 8. Induction of psoriatic skin by ILC3+ cells Normal Skin Graft l Psoriatic Induced Graft Keren ….Gilhar et al . JACI , 2018 PSORIASIS CAN BE INDUCED by pure ILC3 without the involvement TH17 Normal scalp skin AA scalp skin graft Normal scalp skin graft AA scalp skin Normal scalp skin N o r m a l s Increased number of ILC1 in AA patients A study to determine the role of ILC1 in AA
  9. 9. Kv1.3 blocker, PAP-1 suppresses AA development in human skin grafts Ecopic HLA-DR Non - responder graft Responder graft No R No DR Gilhar et al. J Invest Dermatol. 2013 Kv1.3 blockers preferentially suppress autoreactive CCR7− effector memory T cells Additional Therapeutic Targets for AA Need to be Identified and Explored Gilhar, Keren. Paus . Lancet- accepted for publication The Humanized mice can be used as a pre clinical drug screening
  10. 10. Schafer PH, et al . Br J Pharmacol. 2010 Apremilast in a model of psoriasis Preclinical modeling of atopic dermatitis Apremilast on Humanized Mice for Psoriasis and Atopic Dermatitis Remission of Established Atopic Dermatitis Predicting the exact efficacy of apremilast as was observed later on in clinical trials
  11. 11. A comparative study: Therapeutic Effect of Apremilast versus Tofacitinib Mice were randomly divided into three groups: (i) Control (0.5% methylcellulose, bid) (n=5) (ii) Treated orally with Apremilast (5 mg/kg/day,bid) (n=5). (iii) Treated orally with Tofacininib (10 mg/kg, bid) (n=5). Control Apremilast Tofacitinib Therapeutic effect Apremilast Versus Tofacitinib A comparative study Treatments of Apremilast and Tofacitinib were given to humanized mice with ongoing alopecia from day 45 after injections of NKG2D enriched cells, till day 105. . Tofacitini b Control Gilhar et al. unpublished
  12. 12. Agent 1 Montarolo et al. demonstrated protective but not therapeutic effect in EAE mice. PLOS ONE, 2014 Protective but not Therapeutic Effect of Apremilat in Mice Preventive Non-therapeutic Potent enough Treatment Not potent enough t
  13. 13. Increased number of NKT/IL-10 in humanized AA mice following treatment with α-GalCer IL-10 IL-10 IL-10 IL-10 Double staining Double staining NKT cells Double staining NKT cells NKT cells NKT cells Double staining Ghraieb … Gilhar , J Autoimmun. 2018
  14. 14. MeannumberofIL10+/NKT+cells (per0.5mm2) AA patient Human alopecia areata AA likeNormal skin Humanized mouse model AA like/GalCer treatment Healthy volunteer p=0.05 P<0.05 P<0.05 P<0.005 Mean number of NKT10 cells in lesional areas of AA patients and humanized mouse model α-GalCer Induced Expansion of NKT10 Ghraieb … Gilhar , J Autoimmun. 2018 o
  15. 15. Normal scalp skin before induction of hair loss Induction of AA skinday 60))graft Hair growth on AA induced graft /rIL-10 day day105))treatment Therapeutic Effect of IL-10 and α-Gal-Cer in AA mouse Model Hair growth on AA induced graft /α-Gal-Cer Ghraieb … Gilhar , J Autoimmun. 2018
  16. 16. Meanhairnumber(pergraft) Before injection 30-40d after injection 50-60d after treatment Treatment starts Treatment ends 16-30d after stopping treatment Before injection 50- 60d of treatment Treatment starts Treatment ends Normal scalp skin grafts bearing hair AA-Induction Therapeutic effect of α-GalCer Reappearance of AA 30-40d after injection 16-30 after stopping treatment Reappearance of AA Following Sopping Treatment With α-GalCer Reappearance of AA conclusion The study demonstrated the role of NKT and NKT10 in AA Ghraieb … Gilhar , J Autoimmun. 2018 Ghraieb … Gilhar , J Autoimmun. 2018 The regulatory effect is achieved not only by Tregs
  17. 17. Our very preliminary experiments demonstrated •Effects of Kv1.3 blocker and α-GaLcer (NKT10) •A significant difference between therapeutic effect of Tofacitinib VS Apremilast •Non-conventional T cells may play a role in AA and thus may serve as future therapeutic targets Summary
  18. 18. Laboratory Staff Aviad Keren , PhD Nadia Smirnov , B A Natalia Kaplun, M D Gil Kaufman, PhD Rimma Laufer, MD Amal Ghraieb , MSc Prof. Y. Ullmann Rambam Medical Center, Israel Prof. A. Shemer Sheba Medical Center, Israel Prof. Y. Refaeli, University of Colorado, Denver Prof. M. David Beilinson Medical Center, Israel Prof. R. Paus University of Manchester,UK & University of Miami, USA Nira Goldstein, BA Prof. A. Schrum, PhD Columbia, Missouri

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In order to probe the efficacy of new immune-intervention strategies in alopecia areata, the field can now choose from two mutually complementary mouse models.

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