喘息モデルの紹介

喘息は慢性気管支炎の疾患で、可変性気道閉塞、気道過敏症(AHR)及び気道炎などの特徴があります。喘息の主な臨床症状は呼吸の促迫、息切れ、咳やアレルゲンと接触後、粘液の分泌が多くなります。喘息の発病メカニズムは遺伝、エピジェネティクス及び環境要因によより引き起こされます。異なる病理改変の喘息臨床症状は気道上皮細胞及び好酸球、T細胞サブタイプなどの免疫応答に関与しているためです。特に、Th2細胞は高好酸球性喘息に重要な役割をしていると考えられており、IL-4、IL-5、およびIL-13のレベルは伴って上昇しています。

典型的な喘息マウスモデルは蛋白アルブミンで誘導する気管支炎モデルです。マウスに蛋白アルブミンを複数回の腹腔内注射によって感作され、その後、エアロゾル化卵白アルブミンの吸入によって刺激されます。喘息マウスモデルではIgE及び好酸球レベルが高くなり、組織染色で気道粘液の増加が見られ、炎症性白血球湿潤も増えます。バイオサイトジェンはOVA誘導の喘息マウスモデルを作製し、B-hIL4/IL4RAマウス(ヒトIL-4及びIL4受容体遺伝子をマウスの相当遺伝子に置き換える)の中で検証することによってヒトIL-4通路の喘息に対する治療薬物を評価します。

B-IL4/IL4Rマウスの作製とその表現型解析

喘息モデルの作製と検証 

急性喘息マウスモデルの作製方法


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B-hIL4/hIL4RA mice between 5 and 8 weeks of age were acclimatized for at least 1 week before initiation of sensitization and challenge with ovalbumin (OVA) in the creation of the acute murine model of asthma.


B-hIL4/IL4RAマウスを用いて、気管支肺胞液(BALF)中の好酸球検査のためのOVA喘息モデルの作製


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Upon ovalbumin sensitization and challenge, B-hIL4/hIL4RA mice showed a considerable increase of infiltration of leukocytes including eosinophils and neutrophils. This represented a clear allergic phenotype at day 26 in BALF when compared with mice not sensitized or challenged.



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Figure 1. BALF cell count

Mice (n=5/group) were sensitized and challenged with OVA. Twenty-four hours following the last challenge, BALF cells were collected and eosnophils were counted with flow cytometry.

喘息モデルマウスの気管支肺胞洗浄液(BALF)では好酸球免疫細胞の増加


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The proportion of BALF Immune cells in mouse asthma model 

BALF Immune cells were isolated from B-hIL4/hIL4RA mice (n=6). The proportion of eosinophils was analyzed by flow cytometry under the treatment of dupilumab (in house) before OVA challenge(G1 was not treatment with OVA). After treatment of dupilumab (in house), the expression level of inflammatory cells was much lower than the positive control in homozygous B-hIL4/hIL4RA mice.


ELISA方法でOVA特定的IgEを測定

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IgE production

Serum was collected at the study endpoint. IgE levels responded to OVA-specific antibody and total IgE levels were analyzed. The results show that the levels of IgE in mice treated with dupilumab (in house) is much lower than that in untreated mice.


喘息モデルH&E染色

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Airways from B-hIL4/hIL4RA mice exposed to PBS aerosols did not show any inflammation. OVA exposure resulted in a significant increase in peribronchial and perivascular inflammation both in C57BL/6 and B-hIL4/hIL4RA mice, as well as an increase in the level of mucus secretion. A reduction in inflammatory infiltrates and mucus secretion was observed in mice treated with dupilumab (in house).

B-hIL4/hIL4RAマウスモデルを用いて喘息薬物薬効の評価の実例

B-hIL4/hIL4RAマウスモデルでを用いて喘息薬物薬効の評価の実例


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All drugs were administered i.p.

The experimental design for OVA challenge asthma model in B-hIL4/hIL4RA mice and treatment with or without dupilumab.G1: Mice were challenged with PBS and treated without any drug. G2: Mice were challenged with OVA  and treated without any drug. G3: Mice were challenged with OVA and treated with dupilumab before challenged as a prophylactic treatment. G4: Mice were challenged with OVA and treated with dupilumab after challenged as a therapeutic treatment.

デュピルマブ(dupilumab)はB-hIL4/hIL4RAマウスのOVA誘発喘息モデルでの炎症細胞浸潤を除去します。

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BALF immune cells were isolated from homozygous B-hIL4/hIL4RA mice (n=7). The proportions of eosinophils subpopulations were determined by flow cytometry in B-hIL4/IL4RA mice treated with or without dupilumab. Prophylactic treatment before OVA challenge (G3) with dupilumab almost completely abolished the infiltration of eosinophils as opposed to untreated group (G2). Therapeutic treatment after OVA challenge (G4) with dupilumab could also obviously reduce the infiltration of eosinophils.

フローサイトメトリーの解析はデュピルマブ(dupilumab)の治療によりBALF中の炎症細胞を除去することを示しています。


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BALF immune cells were isolated from homozygous B-hIL4/hIL4RA mice (n=7). The number of CD45+ cells and eosinophils were determined by flow cytometry in B-hIL4/IL4RA mice treated with or without dupilumab. Prophylactic treatment before OVA challenge (G3) with dupilumab almost completely abolished the infiltration of eosinophils as opposed to untreated group (G2). Therapeutic treatment after OVA challenge (G4) with dupilumab could also obviously reduce the infiltration of eosinophils.

デュピルマブ(dupilumab)はOVAによって誘発された喘息マウスモデルのIgEを除去します。


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Serum were collected at the study endpoint. OVA-specific IgE antibody levels were analyzed. The results showed that IgE levels were dramatically decreased in the dupilumab treatment group compared with the untreated group.


H&E染色法ではデュピルマブ(dupilumab)の投与後、B-hIL4/IL4RAマウスの炎症減少を示しました。

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Airways from B-hIL4/hIL4Ra mice exposed to PBS aerosols (G1) [1] did not show any inflammation. OVA exposure resulted in a significant increase in peribronchial and perivascular inflammation in B-hIL4/hIL4RA mice,[2]  as well as an increase in the level of mucus secretion (G2). A reduction in inflammatory infiltrates and mucus secretion was observed in mice treated with dupilumab (G3, G4).

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