Development of an animal model of opticospinal form of multiple sclerosis using the HLA-DP5 transgenic mice.

1.多发性硬化症患者中新自身抗原hsp105的鉴定为了在日本多发性硬化症患者中发现新的自身抗原，我们用8例多发性硬化症患者的血清对脊髓来源的cDNA文库进行了免疫筛选(每例患者筛选5-10×10~(-5)个克隆)。五个阳性克隆中的一个是热休克蛋白105(Hsp105)，它在大脑中表达最丰富。然后我们检测了MS患者对hsp105的免疫应答，发现：(1)MS患者抗hsp105抗体的频率高于正常对照组；(2)HSP105在MS皮损和EAE中的表达增强；(3)仅MS患者外周血中的CD4~+CD45RO^+T细胞对hsp105的反应显著增强；(4)ELISPOT试验显示，MS患者和健康对照组IL-10对hsp105的反应均显著高于IL-4和干扰素-γ，而IL-10的反应明显低于正常对照组。这些发现提示人类对hsp105的免疫反应，而多发性硬化症患者的反应减弱。此外，我们还研究了hsp105在小鼠体内的免疫原性，发现(5)人和自体小鼠均不能诱发EAE，(6)用pHSP105免疫的小鼠表现出明显的EAE加重。在日本，OS-MS的易感性与HLADPB1*0501等位基因相关。将人类白细胞抗原-DPA1*02022/pDOI-6基因片段与人类白细胞抗原-DPA1*02022/pDOI-6基因片段共注射到受精卵(C57BL/6)中，获得转基因小鼠。通过尾部DNA的PCR分析，对得到的小鼠进行了转基因整合测试。目前，来自三个独立创建者的转基因系被检测了人类白细胞抗原-DPA1*02022和人类白细胞抗原-DPB1*0501RNA在胸腺和脾中的表达。

1.Identification of a novel autoantigen hsp105 in patients, with multiple sclerosisTo discover novel autoantigens in Japanese patients with multiple sclerosis(MS), we immunoscreened spinal cord-derived cDNA library with sera from 8 MS patients(5-10×10^5 clones screened in each patient). One of five positive clones was heat shock protein 105(hsp105), which is expressed most abundantly in the brain. We then examined immune responses to hsp105 in MS patients and found that (1)the frequency of anti-hsp105 IgG antibody increased in MS patients than in controls, (2)hsp105 expression was enhanced in the MS lesions and EAE, (3)significant proliferation of CD4^+CD45RO^+T cell to hsp105 was noted only in MS patients, (4)in the ELISPOT assay, IL-10 response to hsp105 was significantly higher than IL-4 and IFN-gamma responses to hsp105 in both MS patients and healthy controls, however the IL-10 response was significantly lower as compared with controls. These findings suggest the immune response to hsp105 in human and that the response is attenuated in MS patients. Furthermore, we studied immunogenicity of hsp105 in mice and found that (5)immunization with either human or autologous mouse hsp105 did not induce EAE, (6)mice vaccinated with pHSP105showed marked exacerbation of EAE. In future, we are going to analyze the mechanism of exacerbation of EAE by pHSP105.2.Animal model of opticospinal form of multiple sclerosisIn Japan, susceptibility to OS-MS is associated with the HLA-DPB1*0501 allele. HLA-DP5(DPA1*02022/DPB1*0501) transgenic mice were generated by co-injection of the HLA-DPA1*02022/pDOI-6 fragment and HLA-DPB1*0501/pDOI-6 fragment into fertilized mouse eggs(C57BL/6). The resulting mice were tested for integration of the transgene by PCR analysis of tail DNA. Now, transgenic lines derived from three independent founders are tested for expression of HLA-DPA1*02022 RNA and HLA-DPB1*0501 RNA in thymus and spleen.