Lee Mey-Fann, Yang Kai-Jei, Wang Nancy M, Chiu Yung-Tsung, Chen Pei-Chih, Chen Yi-Hsing
Department of Medical Research Taichung Veterans General Hospital, Taichung, Taiwan; Department of Life Science, Tunghai University, Taichung, Taiwan.
Institute of Biotechnology, National Changhua University of Education, Changhua, Taiwan.
PLoS One. 2014 Mar 20;9(3):e91871. doi: 10.1371/journal.pone.0091871. eCollection 2014.
Forcipomyia taiwana (biting midge) allergy is the most prevalent biting insect allergy in Taiwan. An animal model corresponding to the human immuno-pathologic features of midge allergy is needed for investigating the mechanisms and therapies. This study successfully developed a murine model of Forcipomyia taiwana allergy.
BALB/c mice were sensitized intra-peritoneally with midge extract on days 0, 7, 14, 21 then intra-dermally on days 28, 31 and 35. Serum midge-specific IgE, IgG1, and IgG2a were measured every 14 days by indirect ELISA. The mice were challenged intradermally with midge extract at day 40 and then sacrificed. Proliferation and cytokine production of splenocytes after stimulation with midge extract were determined by MTT assay and ELISA, respectively. The cytokine mRNA expression in response to midge stimulation was analyzed by RT-PCR.
Serum IgE, total IgG, and IgG1 antibody levels against midge extract were significantly higher in the midge-sensitized mice than in the control mice. After the two-step sensitization, all mice in the midge-sensitized group displayed immediate itch and plasma extravasation reactions in response to challenge with midge extract. Skin histology from midge-sensitized mice showed marked eosinophil and lymphocyte infiltrations similar to that observed in humans. Stimulation of murine splenocytes with midge extract elicited significant proliferation, IL-4, IL-10, IL-13 and IFN-γ protein production, and up-regulation of mRNA in a dose-dependent manner in the midge-sensitized group, but not in the control group.
A murine model of midge bite allergy has been successfully developed using a two-step sensitization protocol. The sensitized mice have very similar clinical and immunologic reactions to challenge with midge proteins as the reactions of human to midge bites. This murine model may be a useful platform for future research and the development of treatment strategies for insect bite allergy.
台湾蠛蠓过敏是台湾最常见的叮咬昆虫过敏。需要一个与人蠓过敏免疫病理特征相对应的动物模型来研究其机制和治疗方法。本研究成功建立了台湾蠛蠓过敏的小鼠模型。
在第0、7、14、21天对BALB/c小鼠进行腹腔注射蠓提取物致敏，然后在第28、31和35天进行皮内注射。每14天通过间接ELISA检测血清中蠓特异性IgE、IgG1和IgG2a。在第40天对小鼠进行皮内注射蠓提取物攻击，然后处死。分别通过MTT法和ELISA法测定蠓提取物刺激后脾细胞的增殖和细胞因子产生。通过RT-PCR分析对蠓刺激的细胞因子mRNA表达。
蠓致敏小鼠血清中针对蠓提取物的IgE、总IgG和IgG1抗体水平显著高于对照小鼠。经过两步致敏后，蠓致敏组的所有小鼠在接受蠓提取物攻击时均表现出即刻瘙痒和血浆外渗反应。蠓致敏小鼠的皮肤组织学显示出明显的嗜酸性粒细胞和淋巴细胞浸润，类似于人类观察到的情况。用蠓提取物刺激小鼠脾细胞，在蠓致敏组中引起显著的增殖、IL-4、IL-10、IL-13和IFN-γ蛋白产生以及mRNA的剂量依赖性上调，而在对照组中则没有。
采用两步致敏方案成功建立了蠓叮咬过敏的小鼠模型。致敏小鼠对蠓蛋白攻击的临床和免疫反应与人对蠓叮咬的反应非常相似。该小鼠模型可能是未来昆虫叮咬过敏研究和治疗策略开发的有用平台。
