病毒感染与多发性硬化的临床和实验室研究
|
摘要
第一部分
多发性硬化患者血清和脑脊液中常见病毒抗体／抗原的检测
[目的] 检测多发性硬化患者(MS)血清和脑脊液(CSF)中几种常见的可能与MS发病有关的病毒抗体和／或抗原的情况，并与其他神经科疾病患者(OND)和非神经科疾病患者(NND)的结果进行比较分析。
[方法] 多发性硬化组(MS组)65例，均符合Poser诊断标准。男23例，女42例；年龄18～62岁，平均42.5±6.82岁；病程1～20年，平均3.6±8.4年。其中复发缓解型40例、复发进展型9例、原发进展型7例、继发进展型9例，本组的MS患者均在发作期，住院前2个月未用糖皮质激素和免疫抑制剂。其他神经科疾病组(OND组)71例，男30例，女41例；年龄17～65岁，平均45.4±7.34岁；其中运动神经元病20例、帕金森病5例、重症肌无力3例、周围神经病19例、头痛8例、癫痫12例、神经症4例。非神经科疾病组(NND组)42例，为妇科、泌尿外科和血管外科疾病住院的手术腰麻患者。男18例，女24例；年龄18～70岁，平均48.2±5.02岁。每例患者取静脉血3ml，脑脊液2ml。用酶联免疫吸附法(ELISA)检测三组每例患者血清和CSF中乙型肝炎病毒(HBV)HBsAg、HBsAb、HBeAg、HBeAb和HbcAb；EB病毒(EBV)早期抗原(EA，包含p138和p54两种早期蛋白质)、壳抗原(VCA)、核抗原(EBNA复合物)的IgG抗体；人类疱疹病毒6(HHV-6)的IgG抗体；单纯疱疹病毒Ⅰ型(HSV-I)的IgG和IgM抗体；丙型肝炎病毒(HCV)的抗体。统计学检验用X~2检验和Fisher精确检验。
[结果] 1)MS组血清HBV感染的阳性人数为50人，感染率76.92％；OND组阳性30人，感染率42.25％；NND组阳性20人，感染率47.62％。MS组的HBV总感染率高于OND组和NND组，有统计学差异(均P＜0.05)。血清HBsAb，HBeAg的阳性率和HBsAg，HBeAg，HbcAb同时阳性的比例，MS组都高于OND组和NND组，有统计学差异(均P＜0.05)。2)MS组血清EB病毒EA IgG抗体阳性30例(46.15％)，OND组阳性13例(18.31％)，NND组阳性4例(9.52％)，三组比较，MS组阳性率显著高于OND组和NND组，有统计学差异(均P＜0.05)。EB病毒EBNA复合物和VCA的IgG抗体的阳性率三组无显著性差异(均P＞0.05)。3)三组血清中HHV—6 IgG抗体阳性率无统计学差异(均p＞0.05)。4)血清中HSV-I IgG抗体三组无统计学差异(均P＞0.05)。MS组HSV-I IgM抗体有53例阳性，阳性率为81.53％；OND组有30例阳性，阳性率42.25％；NND有22例阳性，阳性率为52.38％。MS组阳性率高于OND组和NND组，有统计学差
异(均P＜0.05)。5)三组血清中HCV抗体的测定结果无统计学差异(均P＞0.05)。6)在脑脊液中所有病毒抗体／抗原的结果，三组均无统计学差异(均P＞0.05)。
[结论] 1)MS组与其他两组比较，血清中HBV总感染率，HBsAb，HBeAg的阳性率和血清HBsAg，HBeAg，HBcAb同时阳性(俗称大三阳)的比例显著高，有统计学差异(均P＜0.05)。说明MS组HBV感染率高，部分病人病毒活跃复制。2)MS组EBV的EA IgG抗体阳性率升高，与OND组和NND组比较，有统计学差异(均P＜0.05)。提示发作期的MS患者常有活动性的EBV感染。3)MS组血清中HSV-I IgM抗体阳性率明显高于其他两组(均P＜0.05)，提示大部分发作期MS患者有新近的HSV-I感染或潜伏的HSV-I的重新活化。
第二部分 合成病毒多肽诱导实验性自身免疫性脑脊髓炎(EAE)的研究
一．用生物信息学方法选择病毒多肽抗原
[目的] 用生物信息学(Bioinformatics)的方法检索与多发性硬化发病可能相关的几种常见病毒的氨基酸序列与神经髓鞘主要成分—髓鞘碱性蛋白(myelin basic
protein，MBP)是否有相同或相似的序列，并明确具体氨基酸顺序，为分子模拟学说提供理论依据。
[方法] 选择SWISS-PROT蛋白质序列数据库，网址为http://www.expasy.ch/sprot/sprot-top.html，NCBI蛋白质序列数据库，网址为http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=protein。蛋白质序列同源性分析数据库网址为http://www.ncbi.nlm.nih.gov/BLAST；http://us.expasy.org/tools/blast/。MHC Ⅱ类分子结合位点预测：http://www.imtech.res.in/raghava/propred/。选定大鼠、豚鼠和人的MBP作为比对序列。
[结果] 通过综合考虑，筛选出4种与多发性硬化发病最可能相关的病毒肽段。它们是乙型肝炎病毒多聚酶肽段，氨基酸序列为GCYGSLPQSHIIQKIKECFR；JC病毒大T抗原肽段，氨基酸序列为HICKGFQCFKKPRTPPPK；EB病毒DNA多聚酶肽段，氨基酸序列为TGGVYHFVKKHVHES；人类疱疹病毒-6[U70]碱性核酸外切酶肽段，氨基酸序列为IVERETRGQSENPLWHALRR。
二．合成病毒多肽诱导实验性自身免疫性脑脊髓炎模型(EAE)免疫学分析的体外实验
[目的] 研究4条合成病毒肽是否带有与豚鼠MBP68-86相似的抗原表位，即具有与MBP68-86相似的免疫反应性(抗原性)。
[方法] 4条病毒多肽由吉尔生化(上海)有限公司合成，纯度98％，氨基酸序列由高效液相色谱仪分析和纯化，质谱仪检测确认。10只Lewis雌性大鼠，购于北京维通利华动物技术有限公司，6-8周龄，体重170-190克，随机分为两组。5只Lewis大鼠用豚鼠MBP68-86免疫，5只Lewis大鼠用牛MBP免疫。免疫后第9天处死大鼠，制备脾脏和淋巴结细胞悬液。用豚鼠MBP68-86／牛MBP和4条病毒多肽分别刺激两组大鼠的脾脏和淋巴结细胞，并设无抗原刺激为对照组(共6组)。用Griess试剂法测定各组脾脏细胞分泌一氧化氮(NO)的水平；用3H-TdR法测定各组淋巴细胞的增殖反应；用ELISA双抗体夹心法测定各组细胞因子IFN-γ的分泌量；用ELISA间接法检测MBP68-86／MBP诱导抗体和合成肽的交叉反应性。用统计软件SPSS12.0对数据进行统计分析和处理。
[结果] 1) 豚鼠MBP68-86和牛MBP都能在体外很好地刺激其对应免疫大鼠的脾细胞分泌NO。与其他组相比，JC病毒肽也能在体外刺激MBP68-86免疫大鼠的脾细胞分泌NO，72小时与对照组相比，有统计学差异(P＜0.05)。对MBP免疫大鼠脾细胞的刺激作用，各病毒肽和对照组相比，无统计学差异(P＞0.05)。2) 淋巴细胞增殖反应的结果显示，与对照组相比，JC病毒肽和豚鼠MBP68-86相似，在体外对MBP68-86免疫的大鼠淋巴结细胞有良好的刺激增殖作用，统计学检验有显著性差异(P＜0.05)。但JC病毒肽在体外对MBP免疫大鼠的淋巴结细胞无刺激作用。3) 4种病毒多肽中只有JC病毒肽可以在体外很好地刺激MBP68-86免疫大鼠的脾细胞和淋巴结细胞分泌细胞因子IFN—γ，与对照组比较有统计学的显著差异(P＜0.05)。4种病毒多肽在体外刺激MBP免疫大鼠的脾细胞和淋巴结细胞分泌IFN-γ的能力都较弱。4)MBP68-86和MBP免疫大鼠的血清抗体与EB病毒肽和乙肝病毒肽的交叉反应较强，与JC病毒肽和HHV-6病毒肽的交叉反应较弱。
[结论] 1) JC病毒肽能诱导豚鼠MBP68-86免疫大鼠的脾细胞分泌NO，淋巴细胞增殖和细胞因子IFN—γ分泌，提示JC肽可能较其他多肽具有与MBP68-86更相似的免疫反应性。2) 4条合成病毒肽与豚鼠MBP68-86诱导的抗体间交叉反应性的结果显示，JC病毒肽与豚鼠MBP68-86诱导的抗体间交叉反应性低。这提示JC病毒肽可能带有与豚鼠MBP68-86相似的T细胞表位，而不带有相似的B细胞表位。3) 考虑到JC病毒肽在氨基酸组成上与豚鼠MBP68-86不尽相同，我们推测这可能与效应T细胞识别的表位扩展和(或)相似表位氨基酸组成的复杂多样性有关。
三．合成病毒多肽诱导实验性自身免疫性脑脊髓炎模型(EAE)的行为学，免疫学和病理学分析的体内实验
[目的] 研究用与髓鞘碱性蛋白(MBP)有相似序列的四条病毒多肽免疫Lewis
大鼠后能否诱导出行为学、免疫学和病理学方面表现典型的EAE模型，并探讨其可能的发生机制。
[方法] 35只Lewis雌性大鼠，购于北京维通利华动物技术有限公司，6-8周龄，体重160-180克，随机分为A、B、C、D、E、F、G七组，每组5只。A组为阴性对照组，用TB 2mg+IFA 200μl(两者混合即CFA)每只大鼠双后足垫各皮下注射100μl；B组是阳性对照组，用豚鼠MBP68-86 25μg+TB2mg+IFA200μl免疫；C组是用牛MBP 100μg+TB2mg+IFA200μl免疫；D组是用乙肝多聚酶肽段免疫，每只鼠肽段250μg+TB2mg+IFA200μl；E组是用JC病毒大T抗原肽段免疫，每只鼠肽段250μg+TB 2mg+IFA 200μl；F组是用EBV DNA多聚酶肽段免疫，每只鼠肽段250μg+TB 2mg+IFA 200μl；G组是用HHV-6 U70碱性核酸外切酶肽段免疫，每只鼠肽段250μg+TB 2mg+IFA 200μl。EAE的评分标准按国际通用标准，免疫当天及以后每天称体重，并进行评分。免疫后第15天，处死大鼠。取腰段脊髓，制成冰冻切片。制备淋巴结细胞悬液。用豚鼠MBP68-86和PBS(作为对照组)分别刺激7组的淋巴结细胞，用3H-TdR法测定各组T淋巴细胞对MBP68-86的增殖反应；用ELISA双抗体夹心法检测各组淋巴细胞自发性和MBP68-86刺激下的IFN-γ和IL-10分泌；用ELISA间接法，测定每条合成肽与豚鼠MBP68-86及牛MBP诱导抗体相互间的交叉反应性。用HE染色对A组-G组35只大鼠脊髓切片进行脊髓细胞浸润的评分；用免疫组织化学方法对浸润细胞分类；用ELISA间接法测定18例MS患者和8例NND患者脑脊液中抗4条合成病毒肽的抗体。用统计软件SPSS12.0对数据进行统计分析和处理。
[结果] 1)EAE模型诱导的情况和评分：至免疫后第13天B组有4只鼠，E组有1只鼠出现临床症状。B组3只鼠均为完全性双后肢瘫痪，评分是3分，1只鼠为尾巴肌张力完全丧失，评分是1分；E组1只鼠为尾巴肌张力完全丧失，评分是1分。到第15天，E组另有1只鼠出现可疑尾巴张力降低，评分0.5分。B组和E组大鼠平均体重也显著下降，与CFA组相比，均有统计学差异(P＜0.05)。2)T淋巴细胞的增殖反应的测定：与CFA组比较，MBP68-86在体外有意义地刺激MBP68-86免疫组和JC病毒肽免疫组大鼠的淋巴结细胞增殖，有统计学差异(P＜0.05)。3)细胞因子IFN-γ的测定：B组(豚鼠MBP68-86免疫)和E组(JC病毒肽免疫)在豚鼠MBP 68-86刺激下，释放的IFN-γ量最大，两组与CFA组比较有统计学差异(P＜0.05)。4)在有或无MBP 68-86刺激下，细胞因子IL-10的水平在各组间无显著性差异。5)MBP68-86免疫大鼠的血清与四条病毒肽之间无明显的交叉反应性。6)HE染色脊髓浸润细胞评级示A组、C组、D组、F组和G组均无明显浸润细胞增加。B组和E组发病鼠脊髓内有大量浸润细胞，而且血管周围浸润明显，血管套形成。7)免疫组织化学浸润细胞分类显示A组、C组、D组、F组和G组大鼠腰髓片均无明显T细胞和巨噬细胞浸润，小胶质细
胞呈静止态无激活形，B组(MBP 68-86)和E组(JC病毒肽)发病大鼠的腰髓片显示与EAE评分相关的不同程度的T细胞和巨噬细胞浸润及小胶质细胞激活。8)在18例多发性硬化患者和8例非神经科疾病患者脑脊液中四条合成病毒肽抗体检测的结果示，两组组间和组内都无统计学差异。
[结论] 1)从动物行为学(EAE评分，体重)，免疫学(T细胞增殖反应，细胞因子IFN-γ的测定)，病理学(HE染色炎性细胞浸润情况，免疫组织化学炎性细胞分类)等三方面来分析，本课题用与MBP68-86有相似氨基酸序列的JC病毒肽在敏感鼠中诱导出了EAE模型，只是JC病毒肽免疫大鼠的发病率和发病严重程度均明显低于豚鼠MBP 68-86免疫的阳性对照组。提示JC病毒肽具有与豚鼠MBP68-86相似的致病原性或致脑炎性。2)抗体交叉反应性的结果提示JC病毒肽与豚鼠MBP 68-86诱导的抗体的交叉反应性并不高，与前一部分结论一致，再次提示JC病毒肽可能具有与MBP68—86相似的T细胞表位，而缺乏与MBP68—86相似的B细胞表位。3)本课题的研究结果支持病毒的分子模拟学说在MS发病中可能有一定的作用。
Objective To evaluate the positive rates of antibodies and antigens of common MS-related viruses in sera and cerebrospinal fluid (CSF) of MS patients, and compared the results with those in other neurological patients(OND) and non-neurological patients(NND).
Methods 65 multiple sclerosis patients, 71 other neurological diseases patients and 42 non-neurological disease patients were included and divided into three groups. The diagnostic criteria of MS was according to Poser's rule. There were 23 male patients and 42 female patients in the MS group, aged between 18-62, average at 42.5 ±6.82. The disease course were 1-20 years, average at 3.6 ± 8.4. Among them, 40 cases were of relapsing and remitting MS, 9 were relapsing and progressive MS, 7 were primary progressive MS and 9 were secondary progressive MS. All the MS patients were in their relapse periods. Steroids and immunosuppression therapy were not administrated for two months before they were hospitalized. There were 30 male and 41 female patients in the OND group, with the age between 17-65, average at 45.4 ±7.34. Among them, 20 cases were of motor neuron diseases, 5 were Parkinson disease, 3 were myasthenia gravis, 19 were peripheral neuropathies, 8 were headache, 12 were epilepsy and 4 were neurosis. There were 18 male and 24 female patients in NND group. They were of gynecological, urological and peripheral vascular diseases. Each patient was taken 3ml blood and 2ml CSF for testing. ELISA was applied for determine HBsAg, HBsAb, HBeAg, HBeAb and HBcAb of hepatitis B virus, IgG antibodies of EA, VCA and EBNA of EB virus, IgG antibody of HHV-6 virus, IgG and IgM antibodies of HSV-I virus and antibody of hepatitis C virus in serum and CSF of every patient. The results were analyzed and compared among three groups using X~2test and Fisher's exact test.
Results 1) In sera, the overall HBV infection rate, HBsAb and HBeAg positive rates and the rate with HBsAg, HBeAg and HBcAb positive simultaneously were statistically higher in MS group than those in other two groups (P<0.05). 2) In sera,
the positive rate(46.15%) of EA IgG antibody of EB virus was significantly elevated in MS group, which was statistically different compared to the other two groups(18.31%, 9.52%, P＜0.05). There were no statistical significances of IgG seropositive rates of EBNA and VCA in all three groups. 3) There were no statistical significances of IgG antibodies of HHV-6 among three groups in sera. 4) There were no significant differences of IgG antibodies of HSV-I in sera in three groups. While positive rate of IgM antibodies of HSV-I was significantly elevated (81.53%) in MS group, which were statistically different compared to the other two groups (42.25%, 52.38%, P<0.05). 5) There were no statistical significances of antibodies of HCV in sera in three groups. 6) There were no statistical differences of positive rates of antibodies/antigens of all viruses in CSF.
Conclusions 1) The results of HBV infection in MS group infer that there were more patients with active HBV infection and the virus was in the replication stage. 2) The elevation of EA IgG antibodies of EB virus mean there were acute or chronic infection of EBV. Compared with the other two groups, more MS patients were in the state of EBV reactivation according to our results. 3) There were more MS patients with recent HSV-I infection or HSV-I reactivation as IgM antibodies of HSV-I were significantly elevated in MS group. It may be a trigger in MS relapse.
Part two
Research of induction of experimental autoimmune encephalomyelitis (EAE) by synthetic viral peptides 1. Selection of IMS-related viral peptides using bioinformatics technology
Objective To search amino acid sequence similarities between MS-related viruses and myelin basic protein (MBP), the major component of nerve myelin and identified the detailed sequence using the technique of bioinformatics, which may provide possible evidence for molecular mimicry hypothesis.
Methods The database we used are as follows: SWISS-PROT protein database, http.//www.expasy.ch/sprot/sprot-top.htm1, NCBI protein database
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=protein, http://www.ncbi.nlm.nih.gov/BLAST; http://us.expasy.org/tools/blast/.
The database of MHC II binding site: http://www.imtech.res.in/raghava/propred/. The amino acid sequence of rat MBP, guinea pig MBP and human MBP are searched and compared with MS-related viruses sequences.
Results The amino acid sequences of four common MS-related viruses peptides were identified according the above database search. They were peptides of HBV Polymerase protein (amino acid sequence are GC YGSLPQSHIIQKIKECFR), Large T protein of JC virus (HICKGFQCFKKPRTPPPK), EB virus DNA polymerase (TGGVYHFVKKHVHES) and Alkaline exonuclease [U70] of Human herpesvirus 6 (IVERETRGQSENPLWHALRR).
2. In vitro immunological study of induction of experimental autoimmune encephalomyelitis by synthetic viral peptides
Objective To evaluate if four synthetic viral peptides have similar epitopes with guinea pig MBP 68-86, that is , similar antigenicity with guinea pig MBP 68-86. Methods Four viral peptides with the purity of 98% were synthesized by Shanghai Ji'er Biochemical Company. The amino acid sequence of four peptides were analyzed and purified by HPLC and verified by MS. Ten Lewis female rats (from Beijing weitonglihua animal company), 6-8 weeks old, weighing 170-190g, were randomized into two groups. Five rats were immunized subcutaneously in hind footpads with guinea pig MBP 68-86 (plus mycobacterium tuberculosis and IFA), while the other five were immunized with bovine MBP(plus mycobacterium tuberculosis and IFA). The rats were sacrificed at day 9 post immunization (p.i.) . Spleen and lymph node cell suspension were prepared. The spleen and lymph node cells of two groups were stimulated with guinea pig MBP 68-86/bovine MBP and four synthetic viral peptides respectively, or with no stimulation as control. The NO level secreted by spleen cells were measured by Griess reaction. The lymphcytes proliferation assay were determined by 3H-TdR method. ELISA (sandwich method) were applied to measure cytokine IFN- γ and ELISA (indirect method) were used to examine the cross-reactivities between the antibodies induced by MBP 68-86/MBP and four synthetic peptides.
Results 1) Guinea pig MBP 68-86 and bovine MBP could stimulate the rat spleen cells effectively to produce NO they immunized respectively in vitro. The spleen cells of the rats immunized with guinea pig MBP 68-86 could also be stimulated by JC virus peptide to produce more NO, which was statistically different compared to
control(P<0.05). The NO production of rats spleen cells immunized with MBP were not of much differences under four viral peptides' stimulation. 2) As to the results of lymphocyte proliferation assay, the lymphocytes of the rats immunized with guinea pig MBP 68-86 responded significantly to both guinea pig MBP 68-86 and JC virus peptide in vitro. Compared with the control, there were statistically difference(P<0.05). The lymphocytes of the rats immunized with MBP responded poorly to JC virus peptide. 3) Spleen and lymph node cells of the rats immunized with MBP 68-86 could produce high levels of IFN- γ upon MBP 68-86 and JC virus peptide stimulations, with statistically difference(P<0.05) compared to the control group. Spleen and lymph node cells of the rats immunized with MBP could not generate high levels of IFN- γ upon stimulation with either of the four viral peptides. 4) The sera antibodies in the rats immunized with MBP 68-86 and MBP had stronger cross-reactivities with EB and HBV peptides than those with JC and HHV-6 peptides. Conclusions 1) The results of NO secretion by spleen cells, lymphocytes proliferation assay and IFN- γ production all revealed that JC virus peptide might have more similar antigenieity with guinea pig MBP 68-86 than the other three viral peptides. 2) JC virus peptide had weak cross-reactivity with the antibody induced by guinea pig MBP 68-86. This inferred that JC virus peptide might have similar T cell epitopes with guinea pig MBP 68-86, but not B cell epitopes. 3) As the amino acid sequence of JC virus were not exactly the same sequence of MBP68-86, it might contribute to T cell epitope spreading and /or the complexity of amino acide compositon of similar epitopes.
3. In vivo behavioral, immunological and pathological study of induction of experimental autoimmune encephalomyelitis by synthetic viral peptides
Objectives To observe if Lewis rats immunized with four viral peptides with amino acid sequence similarities with MBP could develop experimental autoimmune encephalomyelitis (EAE) with typical behavioral, immunological and pathological manifestations. The possible mechanisms were discussed.
Methods Induction of EAE: 35 Lewis female rats (from Beijing weitonglihua animal company), 6-8 weeks old, weighing 160-180g, were randomized into 7 groups (A, B, C, D, E, F and G). Every group consisted of 5 rats. The rats in group A were immunized subcutaneously in hind footpads with IFA 200μl and mycobacterium
tuberculosis 2 mg (the mixture was CFA). The rats in group B were immunized with inoculum containing 25μg guinea pig MBP 68-86, TB 2mg and IFA 200μl. The rats in group C were immunized with 100μg bovine MBP, TB 2mg and IFA 200μl. The rats in group D were immunized with 250μg peptide of HBV polymerase protein, TB 2mg and IFA 200μl. The rats in group E were immunized with 250μg peptide of large T protein of JC virus, TB 2mg and IFA 200μl. The rats in group F were immunized with 250μg peptide of DNA polymerase of EB virus, TB 2mg and IFA 200μl. The rats in group G were immunized with 250 μg peptide of alkaline exonuclease [U70] of HHV-6, TB 2mg and IFA 200μl. EAE clinical scores were graded according to international criteria. All rats were sacrificed at day 15 post immunization. Frozen sectons of lumbar spinal cord were obtained and lymph node cells' suspension were prepared. The lymph node cells of 7 groups were stimulated in vitro with guinea pig MBP 68-86 and PBS (as control), respectively. The lymphcytes proliferation assay with or without MBP 68-86 presence were determined by 3H-TdR method. ELISA (sandwich method) were applied to measure cytokine IFN-γ and IL-10 production by lymphcytes of each group with or without MBP 68-86 stimulation. ELISA (indirect method) were used to examine the cross-reactivities between the antibodies induced by MBP 68-86/MBP and four synthetic peptides. Hematoxylin-erosin-stained (HE) spinal cord sections of each rat were examined for inflammatory cell infiltration. Immunohistochemistry methods were used to classify the infiltration cells. ELISA (indirect method) were applied to detect the antibodies to four synthetic viral peptides in 18 MS patients and 8 NND patients.
Results 1) EAE induction: 4 rats in group B and 2 rats in group E developed clinical symptoms of EAE around day 13-15 post immunization. 3 rats in group B were scored 3 and 1 rat was scored 1. 1 rat in group E were scored 1 and the other was scored 0.5. The mean body weight of rats in group B and E reduced significantly compared to CFA group (P<0.05). 2) The T cell proliferation assay revealed that lymph node cells of rats in group B and E responded vigorously to MBP 68-86 with statistically significance(P<0.05). 3) Lymph node cells of the rats immunized with MBP 68-86 and JC virus peptide could produce high levels of IFN- γ upon MBP 68-86 stimulation, with statistically significance(P<0.05) compared to CFA group. 4) There were not much differences in IL-10 production with or without MBP 68-86 presence. 5) There were not obvious cross-reactivities among sera antibodies of the rats immunized with MBP 68-86 and four viral peptides. 6) There were no obvious
inflammatory cells in HE stained spinal cord sections of rats of group A, C, D, F and G. Extensive inflammatory cells in parenchyma and perivascular cuffing were found in rats with clinical EAE symptoms of group B and E. 7) Immunohistochemistry findings indicated there were no obvious T cell and macrophage infiltration in spinal cord sections of group A, C, D, F and G. And microglial cells were not activated in these groups. While large amounts of T cells and macrophages were found, with microglial cells' activation in spinal cord sections of EAE rats in group B and E. 8) There were no statistically differences of CSF antibodies to four synthetic viral peptides between 18 MS patients' group and 8 NND patients' group. Conclusions 1) EAE model were successfully induced using JC virus peptide which shared similar amino acid sequence with MBP according to animal behavioral, immunological and pathological evidences. JC virus peptide was encephalitogenic. But compared with guinea pig 68-86 immunized group, the incidence of EAE was lower and disease severity were milder in JC virus immunized group. 2) JC virus peptide had weak cross-reactivity with the antibody induced by guinea pig MBP 68-86, which inferred JC virus peptide might have similar T cell epitopes with guinea pig MBP 68-86, but not B cell epitopes. This is consistant with the results from above part. 3) Our research provided some evidence for molocular mimicry hypothesis in pathogenesis of MS.
多发性硬化患者血清和脑脊液中常见病毒抗体／抗原的检测
[目的] 检测多发性硬化患者(MS)血清和脑脊液(CSF)中几种常见的可能与MS发病有关的病毒抗体和／或抗原的情况，并与其他神经科疾病患者(OND)和非神经科疾病患者(NND)的结果进行比较分析。
[方法] 多发性硬化组(MS组)65例，均符合Poser诊断标准。男23例，女42例；年龄18～62岁，平均42.5±6.82岁；病程1～20年，平均3.6±8.4年。其中复发缓解型40例、复发进展型9例、原发进展型7例、继发进展型9例，本组的MS患者均在发作期，住院前2个月未用糖皮质激素和免疫抑制剂。其他神经科疾病组(OND组)71例，男30例，女41例；年龄17～65岁，平均45.4±7.34岁；其中运动神经元病20例、帕金森病5例、重症肌无力3例、周围神经病19例、头痛8例、癫痫12例、神经症4例。非神经科疾病组(NND组)42例，为妇科、泌尿外科和血管外科疾病住院的手术腰麻患者。男18例，女24例；年龄18～70岁，平均48.2±5.02岁。每例患者取静脉血3ml，脑脊液2ml。用酶联免疫吸附法(ELISA)检测三组每例患者血清和CSF中乙型肝炎病毒(HBV)HBsAg、HBsAb、HBeAg、HBeAb和HbcAb；EB病毒(EBV)早期抗原(EA，包含p138和p54两种早期蛋白质)、壳抗原(VCA)、核抗原(EBNA复合物)的IgG抗体；人类疱疹病毒6(HHV-6)的IgG抗体；单纯疱疹病毒Ⅰ型(HSV-I)的IgG和IgM抗体；丙型肝炎病毒(HCV)的抗体。统计学检验用X~2检验和Fisher精确检验。
[结果] 1)MS组血清HBV感染的阳性人数为50人，感染率76.92％；OND组阳性30人，感染率42.25％；NND组阳性20人，感染率47.62％。MS组的HBV总感染率高于OND组和NND组，有统计学差异(均P＜0.05)。血清HBsAb，HBeAg的阳性率和HBsAg，HBeAg，HbcAb同时阳性的比例，MS组都高于OND组和NND组，有统计学差异(均P＜0.05)。2)MS组血清EB病毒EA IgG抗体阳性30例(46.15％)，OND组阳性13例(18.31％)，NND组阳性4例(9.52％)，三组比较，MS组阳性率显著高于OND组和NND组，有统计学差异(均P＜0.05)。EB病毒EBNA复合物和VCA的IgG抗体的阳性率三组无显著性差异(均P＞0.05)。3)三组血清中HHV—6 IgG抗体阳性率无统计学差异(均p＞0.05)。4)血清中HSV-I IgG抗体三组无统计学差异(均P＞0.05)。MS组HSV-I IgM抗体有53例阳性，阳性率为81.53％；OND组有30例阳性，阳性率42.25％；NND有22例阳性，阳性率为52.38％。MS组阳性率高于OND组和NND组，有统计学差
异(均P＜0.05)。5)三组血清中HCV抗体的测定结果无统计学差异(均P＞0.05)。6)在脑脊液中所有病毒抗体／抗原的结果，三组均无统计学差异(均P＞0.05)。
[结论] 1)MS组与其他两组比较，血清中HBV总感染率，HBsAb，HBeAg的阳性率和血清HBsAg，HBeAg，HBcAb同时阳性(俗称大三阳)的比例显著高，有统计学差异(均P＜0.05)。说明MS组HBV感染率高，部分病人病毒活跃复制。2)MS组EBV的EA IgG抗体阳性率升高，与OND组和NND组比较，有统计学差异(均P＜0.05)。提示发作期的MS患者常有活动性的EBV感染。3)MS组血清中HSV-I IgM抗体阳性率明显高于其他两组(均P＜0.05)，提示大部分发作期MS患者有新近的HSV-I感染或潜伏的HSV-I的重新活化。
第二部分 合成病毒多肽诱导实验性自身免疫性脑脊髓炎(EAE)的研究
一．用生物信息学方法选择病毒多肽抗原
[目的] 用生物信息学(Bioinformatics)的方法检索与多发性硬化发病可能相关的几种常见病毒的氨基酸序列与神经髓鞘主要成分—髓鞘碱性蛋白(myelin basic
protein，MBP)是否有相同或相似的序列，并明确具体氨基酸顺序，为分子模拟学说提供理论依据。
[方法] 选择SWISS-PROT蛋白质序列数据库，网址为http://www.expasy.ch/sprot/sprot-top.html，NCBI蛋白质序列数据库，网址为http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=protein。蛋白质序列同源性分析数据库网址为http://www.ncbi.nlm.nih.gov/BLAST；http://us.expasy.org/tools/blast/。MHC Ⅱ类分子结合位点预测：http://www.imtech.res.in/raghava/propred/。选定大鼠、豚鼠和人的MBP作为比对序列。
[结果] 通过综合考虑，筛选出4种与多发性硬化发病最可能相关的病毒肽段。它们是乙型肝炎病毒多聚酶肽段，氨基酸序列为GCYGSLPQSHIIQKIKECFR；JC病毒大T抗原肽段，氨基酸序列为HICKGFQCFKKPRTPPPK；EB病毒DNA多聚酶肽段，氨基酸序列为TGGVYHFVKKHVHES；人类疱疹病毒-6[U70]碱性核酸外切酶肽段，氨基酸序列为IVERETRGQSENPLWHALRR。
二．合成病毒多肽诱导实验性自身免疫性脑脊髓炎模型(EAE)免疫学分析的体外实验
[目的] 研究4条合成病毒肽是否带有与豚鼠MBP68-86相似的抗原表位，即具有与MBP68-86相似的免疫反应性(抗原性)。
[方法] 4条病毒多肽由吉尔生化(上海)有限公司合成，纯度98％，氨基酸序列由高效液相色谱仪分析和纯化，质谱仪检测确认。10只Lewis雌性大鼠，购于北京维通利华动物技术有限公司，6-8周龄，体重170-190克，随机分为两组。5只Lewis大鼠用豚鼠MBP68-86免疫，5只Lewis大鼠用牛MBP免疫。免疫后第9天处死大鼠，制备脾脏和淋巴结细胞悬液。用豚鼠MBP68-86／牛MBP和4条病毒多肽分别刺激两组大鼠的脾脏和淋巴结细胞，并设无抗原刺激为对照组(共6组)。用Griess试剂法测定各组脾脏细胞分泌一氧化氮(NO)的水平；用3H-TdR法测定各组淋巴细胞的增殖反应；用ELISA双抗体夹心法测定各组细胞因子IFN-γ的分泌量；用ELISA间接法检测MBP68-86／MBP诱导抗体和合成肽的交叉反应性。用统计软件SPSS12.0对数据进行统计分析和处理。
[结果] 1) 豚鼠MBP68-86和牛MBP都能在体外很好地刺激其对应免疫大鼠的脾细胞分泌NO。与其他组相比，JC病毒肽也能在体外刺激MBP68-86免疫大鼠的脾细胞分泌NO，72小时与对照组相比，有统计学差异(P＜0.05)。对MBP免疫大鼠脾细胞的刺激作用，各病毒肽和对照组相比，无统计学差异(P＞0.05)。2) 淋巴细胞增殖反应的结果显示，与对照组相比，JC病毒肽和豚鼠MBP68-86相似，在体外对MBP68-86免疫的大鼠淋巴结细胞有良好的刺激增殖作用，统计学检验有显著性差异(P＜0.05)。但JC病毒肽在体外对MBP免疫大鼠的淋巴结细胞无刺激作用。3) 4种病毒多肽中只有JC病毒肽可以在体外很好地刺激MBP68-86免疫大鼠的脾细胞和淋巴结细胞分泌细胞因子IFN—γ，与对照组比较有统计学的显著差异(P＜0.05)。4种病毒多肽在体外刺激MBP免疫大鼠的脾细胞和淋巴结细胞分泌IFN-γ的能力都较弱。4)MBP68-86和MBP免疫大鼠的血清抗体与EB病毒肽和乙肝病毒肽的交叉反应较强，与JC病毒肽和HHV-6病毒肽的交叉反应较弱。
[结论] 1) JC病毒肽能诱导豚鼠MBP68-86免疫大鼠的脾细胞分泌NO，淋巴细胞增殖和细胞因子IFN—γ分泌，提示JC肽可能较其他多肽具有与MBP68-86更相似的免疫反应性。2) 4条合成病毒肽与豚鼠MBP68-86诱导的抗体间交叉反应性的结果显示，JC病毒肽与豚鼠MBP68-86诱导的抗体间交叉反应性低。这提示JC病毒肽可能带有与豚鼠MBP68-86相似的T细胞表位，而不带有相似的B细胞表位。3) 考虑到JC病毒肽在氨基酸组成上与豚鼠MBP68-86不尽相同，我们推测这可能与效应T细胞识别的表位扩展和(或)相似表位氨基酸组成的复杂多样性有关。
三．合成病毒多肽诱导实验性自身免疫性脑脊髓炎模型(EAE)的行为学，免疫学和病理学分析的体内实验
[目的] 研究用与髓鞘碱性蛋白(MBP)有相似序列的四条病毒多肽免疫Lewis
大鼠后能否诱导出行为学、免疫学和病理学方面表现典型的EAE模型，并探讨其可能的发生机制。
[方法] 35只Lewis雌性大鼠，购于北京维通利华动物技术有限公司，6-8周龄，体重160-180克，随机分为A、B、C、D、E、F、G七组，每组5只。A组为阴性对照组，用TB 2mg+IFA 200μl(两者混合即CFA)每只大鼠双后足垫各皮下注射100μl；B组是阳性对照组，用豚鼠MBP68-86 25μg+TB2mg+IFA200μl免疫；C组是用牛MBP 100μg+TB2mg+IFA200μl免疫；D组是用乙肝多聚酶肽段免疫，每只鼠肽段250μg+TB2mg+IFA200μl；E组是用JC病毒大T抗原肽段免疫，每只鼠肽段250μg+TB 2mg+IFA 200μl；F组是用EBV DNA多聚酶肽段免疫，每只鼠肽段250μg+TB 2mg+IFA 200μl；G组是用HHV-6 U70碱性核酸外切酶肽段免疫，每只鼠肽段250μg+TB 2mg+IFA 200μl。EAE的评分标准按国际通用标准，免疫当天及以后每天称体重，并进行评分。免疫后第15天，处死大鼠。取腰段脊髓，制成冰冻切片。制备淋巴结细胞悬液。用豚鼠MBP68-86和PBS(作为对照组)分别刺激7组的淋巴结细胞，用3H-TdR法测定各组T淋巴细胞对MBP68-86的增殖反应；用ELISA双抗体夹心法检测各组淋巴细胞自发性和MBP68-86刺激下的IFN-γ和IL-10分泌；用ELISA间接法，测定每条合成肽与豚鼠MBP68-86及牛MBP诱导抗体相互间的交叉反应性。用HE染色对A组-G组35只大鼠脊髓切片进行脊髓细胞浸润的评分；用免疫组织化学方法对浸润细胞分类；用ELISA间接法测定18例MS患者和8例NND患者脑脊液中抗4条合成病毒肽的抗体。用统计软件SPSS12.0对数据进行统计分析和处理。
[结果] 1)EAE模型诱导的情况和评分：至免疫后第13天B组有4只鼠，E组有1只鼠出现临床症状。B组3只鼠均为完全性双后肢瘫痪，评分是3分，1只鼠为尾巴肌张力完全丧失，评分是1分；E组1只鼠为尾巴肌张力完全丧失，评分是1分。到第15天，E组另有1只鼠出现可疑尾巴张力降低，评分0.5分。B组和E组大鼠平均体重也显著下降，与CFA组相比，均有统计学差异(P＜0.05)。2)T淋巴细胞的增殖反应的测定：与CFA组比较，MBP68-86在体外有意义地刺激MBP68-86免疫组和JC病毒肽免疫组大鼠的淋巴结细胞增殖，有统计学差异(P＜0.05)。3)细胞因子IFN-γ的测定：B组(豚鼠MBP68-86免疫)和E组(JC病毒肽免疫)在豚鼠MBP 68-86刺激下，释放的IFN-γ量最大，两组与CFA组比较有统计学差异(P＜0.05)。4)在有或无MBP 68-86刺激下，细胞因子IL-10的水平在各组间无显著性差异。5)MBP68-86免疫大鼠的血清与四条病毒肽之间无明显的交叉反应性。6)HE染色脊髓浸润细胞评级示A组、C组、D组、F组和G组均无明显浸润细胞增加。B组和E组发病鼠脊髓内有大量浸润细胞，而且血管周围浸润明显，血管套形成。7)免疫组织化学浸润细胞分类显示A组、C组、D组、F组和G组大鼠腰髓片均无明显T细胞和巨噬细胞浸润，小胶质细
胞呈静止态无激活形，B组(MBP 68-86)和E组(JC病毒肽)发病大鼠的腰髓片显示与EAE评分相关的不同程度的T细胞和巨噬细胞浸润及小胶质细胞激活。8)在18例多发性硬化患者和8例非神经科疾病患者脑脊液中四条合成病毒肽抗体检测的结果示，两组组间和组内都无统计学差异。
[结论] 1)从动物行为学(EAE评分，体重)，免疫学(T细胞增殖反应，细胞因子IFN-γ的测定)，病理学(HE染色炎性细胞浸润情况，免疫组织化学炎性细胞分类)等三方面来分析，本课题用与MBP68-86有相似氨基酸序列的JC病毒肽在敏感鼠中诱导出了EAE模型，只是JC病毒肽免疫大鼠的发病率和发病严重程度均明显低于豚鼠MBP 68-86免疫的阳性对照组。提示JC病毒肽具有与豚鼠MBP68-86相似的致病原性或致脑炎性。2)抗体交叉反应性的结果提示JC病毒肽与豚鼠MBP 68-86诱导的抗体的交叉反应性并不高，与前一部分结论一致，再次提示JC病毒肽可能具有与MBP68—86相似的T细胞表位，而缺乏与MBP68—86相似的B细胞表位。3)本课题的研究结果支持病毒的分子模拟学说在MS发病中可能有一定的作用。
Objective To evaluate the positive rates of antibodies and antigens of common MS-related viruses in sera and cerebrospinal fluid (CSF) of MS patients, and compared the results with those in other neurological patients(OND) and non-neurological patients(NND).
Methods 65 multiple sclerosis patients, 71 other neurological diseases patients and 42 non-neurological disease patients were included and divided into three groups. The diagnostic criteria of MS was according to Poser's rule. There were 23 male patients and 42 female patients in the MS group, aged between 18-62, average at 42.5 ±6.82. The disease course were 1-20 years, average at 3.6 ± 8.4. Among them, 40 cases were of relapsing and remitting MS, 9 were relapsing and progressive MS, 7 were primary progressive MS and 9 were secondary progressive MS. All the MS patients were in their relapse periods. Steroids and immunosuppression therapy were not administrated for two months before they were hospitalized. There were 30 male and 41 female patients in the OND group, with the age between 17-65, average at 45.4 ±7.34. Among them, 20 cases were of motor neuron diseases, 5 were Parkinson disease, 3 were myasthenia gravis, 19 were peripheral neuropathies, 8 were headache, 12 were epilepsy and 4 were neurosis. There were 18 male and 24 female patients in NND group. They were of gynecological, urological and peripheral vascular diseases. Each patient was taken 3ml blood and 2ml CSF for testing. ELISA was applied for determine HBsAg, HBsAb, HBeAg, HBeAb and HBcAb of hepatitis B virus, IgG antibodies of EA, VCA and EBNA of EB virus, IgG antibody of HHV-6 virus, IgG and IgM antibodies of HSV-I virus and antibody of hepatitis C virus in serum and CSF of every patient. The results were analyzed and compared among three groups using X~2test and Fisher's exact test.
Results 1) In sera, the overall HBV infection rate, HBsAb and HBeAg positive rates and the rate with HBsAg, HBeAg and HBcAb positive simultaneously were statistically higher in MS group than those in other two groups (P<0.05). 2) In sera,
the positive rate(46.15%) of EA IgG antibody of EB virus was significantly elevated in MS group, which was statistically different compared to the other two groups(18.31%, 9.52%, P＜0.05). There were no statistical significances of IgG seropositive rates of EBNA and VCA in all three groups. 3) There were no statistical significances of IgG antibodies of HHV-6 among three groups in sera. 4) There were no significant differences of IgG antibodies of HSV-I in sera in three groups. While positive rate of IgM antibodies of HSV-I was significantly elevated (81.53%) in MS group, which were statistically different compared to the other two groups (42.25%, 52.38%, P<0.05). 5) There were no statistical significances of antibodies of HCV in sera in three groups. 6) There were no statistical differences of positive rates of antibodies/antigens of all viruses in CSF.
Conclusions 1) The results of HBV infection in MS group infer that there were more patients with active HBV infection and the virus was in the replication stage. 2) The elevation of EA IgG antibodies of EB virus mean there were acute or chronic infection of EBV. Compared with the other two groups, more MS patients were in the state of EBV reactivation according to our results. 3) There were more MS patients with recent HSV-I infection or HSV-I reactivation as IgM antibodies of HSV-I were significantly elevated in MS group. It may be a trigger in MS relapse.
Part two
Research of induction of experimental autoimmune encephalomyelitis (EAE) by synthetic viral peptides 1. Selection of IMS-related viral peptides using bioinformatics technology
Objective To search amino acid sequence similarities between MS-related viruses and myelin basic protein (MBP), the major component of nerve myelin and identified the detailed sequence using the technique of bioinformatics, which may provide possible evidence for molecular mimicry hypothesis.
Methods The database we used are as follows: SWISS-PROT protein database, http.//www.expasy.ch/sprot/sprot-top.htm1, NCBI protein database
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=protein, http://www.ncbi.nlm.nih.gov/BLAST; http://us.expasy.org/tools/blast/.
The database of MHC II binding site: http://www.imtech.res.in/raghava/propred/. The amino acid sequence of rat MBP, guinea pig MBP and human MBP are searched and compared with MS-related viruses sequences.
Results The amino acid sequences of four common MS-related viruses peptides were identified according the above database search. They were peptides of HBV Polymerase protein (amino acid sequence are GC YGSLPQSHIIQKIKECFR), Large T protein of JC virus (HICKGFQCFKKPRTPPPK), EB virus DNA polymerase (TGGVYHFVKKHVHES) and Alkaline exonuclease [U70] of Human herpesvirus 6 (IVERETRGQSENPLWHALRR).
2. In vitro immunological study of induction of experimental autoimmune encephalomyelitis by synthetic viral peptides
Objective To evaluate if four synthetic viral peptides have similar epitopes with guinea pig MBP 68-86, that is , similar antigenicity with guinea pig MBP 68-86. Methods Four viral peptides with the purity of 98% were synthesized by Shanghai Ji'er Biochemical Company. The amino acid sequence of four peptides were analyzed and purified by HPLC and verified by MS. Ten Lewis female rats (from Beijing weitonglihua animal company), 6-8 weeks old, weighing 170-190g, were randomized into two groups. Five rats were immunized subcutaneously in hind footpads with guinea pig MBP 68-86 (plus mycobacterium tuberculosis and IFA), while the other five were immunized with bovine MBP(plus mycobacterium tuberculosis and IFA). The rats were sacrificed at day 9 post immunization (p.i.) . Spleen and lymph node cell suspension were prepared. The spleen and lymph node cells of two groups were stimulated with guinea pig MBP 68-86/bovine MBP and four synthetic viral peptides respectively, or with no stimulation as control. The NO level secreted by spleen cells were measured by Griess reaction. The lymphcytes proliferation assay were determined by 3H-TdR method. ELISA (sandwich method) were applied to measure cytokine IFN- γ and ELISA (indirect method) were used to examine the cross-reactivities between the antibodies induced by MBP 68-86/MBP and four synthetic peptides.
Results 1) Guinea pig MBP 68-86 and bovine MBP could stimulate the rat spleen cells effectively to produce NO they immunized respectively in vitro. The spleen cells of the rats immunized with guinea pig MBP 68-86 could also be stimulated by JC virus peptide to produce more NO, which was statistically different compared to
control(P<0.05). The NO production of rats spleen cells immunized with MBP were not of much differences under four viral peptides' stimulation. 2) As to the results of lymphocyte proliferation assay, the lymphocytes of the rats immunized with guinea pig MBP 68-86 responded significantly to both guinea pig MBP 68-86 and JC virus peptide in vitro. Compared with the control, there were statistically difference(P<0.05). The lymphocytes of the rats immunized with MBP responded poorly to JC virus peptide. 3) Spleen and lymph node cells of the rats immunized with MBP 68-86 could produce high levels of IFN- γ upon MBP 68-86 and JC virus peptide stimulations, with statistically difference(P<0.05) compared to the control group. Spleen and lymph node cells of the rats immunized with MBP could not generate high levels of IFN- γ upon stimulation with either of the four viral peptides. 4) The sera antibodies in the rats immunized with MBP 68-86 and MBP had stronger cross-reactivities with EB and HBV peptides than those with JC and HHV-6 peptides. Conclusions 1) The results of NO secretion by spleen cells, lymphocytes proliferation assay and IFN- γ production all revealed that JC virus peptide might have more similar antigenieity with guinea pig MBP 68-86 than the other three viral peptides. 2) JC virus peptide had weak cross-reactivity with the antibody induced by guinea pig MBP 68-86. This inferred that JC virus peptide might have similar T cell epitopes with guinea pig MBP 68-86, but not B cell epitopes. 3) As the amino acid sequence of JC virus were not exactly the same sequence of MBP68-86, it might contribute to T cell epitope spreading and /or the complexity of amino acide compositon of similar epitopes.
3. In vivo behavioral, immunological and pathological study of induction of experimental autoimmune encephalomyelitis by synthetic viral peptides
Objectives To observe if Lewis rats immunized with four viral peptides with amino acid sequence similarities with MBP could develop experimental autoimmune encephalomyelitis (EAE) with typical behavioral, immunological and pathological manifestations. The possible mechanisms were discussed.
Methods Induction of EAE: 35 Lewis female rats (from Beijing weitonglihua animal company), 6-8 weeks old, weighing 160-180g, were randomized into 7 groups (A, B, C, D, E, F and G). Every group consisted of 5 rats. The rats in group A were immunized subcutaneously in hind footpads with IFA 200μl and mycobacterium
tuberculosis 2 mg (the mixture was CFA). The rats in group B were immunized with inoculum containing 25μg guinea pig MBP 68-86, TB 2mg and IFA 200μl. The rats in group C were immunized with 100μg bovine MBP, TB 2mg and IFA 200μl. The rats in group D were immunized with 250μg peptide of HBV polymerase protein, TB 2mg and IFA 200μl. The rats in group E were immunized with 250μg peptide of large T protein of JC virus, TB 2mg and IFA 200μl. The rats in group F were immunized with 250μg peptide of DNA polymerase of EB virus, TB 2mg and IFA 200μl. The rats in group G were immunized with 250 μg peptide of alkaline exonuclease [U70] of HHV-6, TB 2mg and IFA 200μl. EAE clinical scores were graded according to international criteria. All rats were sacrificed at day 15 post immunization. Frozen sectons of lumbar spinal cord were obtained and lymph node cells' suspension were prepared. The lymph node cells of 7 groups were stimulated in vitro with guinea pig MBP 68-86 and PBS (as control), respectively. The lymphcytes proliferation assay with or without MBP 68-86 presence were determined by 3H-TdR method. ELISA (sandwich method) were applied to measure cytokine IFN-γ and IL-10 production by lymphcytes of each group with or without MBP 68-86 stimulation. ELISA (indirect method) were used to examine the cross-reactivities between the antibodies induced by MBP 68-86/MBP and four synthetic peptides. Hematoxylin-erosin-stained (HE) spinal cord sections of each rat were examined for inflammatory cell infiltration. Immunohistochemistry methods were used to classify the infiltration cells. ELISA (indirect method) were applied to detect the antibodies to four synthetic viral peptides in 18 MS patients and 8 NND patients.
Results 1) EAE induction: 4 rats in group B and 2 rats in group E developed clinical symptoms of EAE around day 13-15 post immunization. 3 rats in group B were scored 3 and 1 rat was scored 1. 1 rat in group E were scored 1 and the other was scored 0.5. The mean body weight of rats in group B and E reduced significantly compared to CFA group (P<0.05). 2) The T cell proliferation assay revealed that lymph node cells of rats in group B and E responded vigorously to MBP 68-86 with statistically significance(P<0.05). 3) Lymph node cells of the rats immunized with MBP 68-86 and JC virus peptide could produce high levels of IFN- γ upon MBP 68-86 stimulation, with statistically significance(P<0.05) compared to CFA group. 4) There were not much differences in IL-10 production with or without MBP 68-86 presence. 5) There were not obvious cross-reactivities among sera antibodies of the rats immunized with MBP 68-86 and four viral peptides. 6) There were no obvious
inflammatory cells in HE stained spinal cord sections of rats of group A, C, D, F and G. Extensive inflammatory cells in parenchyma and perivascular cuffing were found in rats with clinical EAE symptoms of group B and E. 7) Immunohistochemistry findings indicated there were no obvious T cell and macrophage infiltration in spinal cord sections of group A, C, D, F and G. And microglial cells were not activated in these groups. While large amounts of T cells and macrophages were found, with microglial cells' activation in spinal cord sections of EAE rats in group B and E. 8) There were no statistically differences of CSF antibodies to four synthetic viral peptides between 18 MS patients' group and 8 NND patients' group. Conclusions 1) EAE model were successfully induced using JC virus peptide which shared similar amino acid sequence with MBP according to animal behavioral, immunological and pathological evidences. JC virus peptide was encephalitogenic. But compared with guinea pig 68-86 immunized group, the incidence of EAE was lower and disease severity were milder in JC virus immunized group. 2) JC virus peptide had weak cross-reactivity with the antibody induced by guinea pig MBP 68-86, which inferred JC virus peptide might have similar T cell epitopes with guinea pig MBP 68-86, but not B cell epitopes. This is consistant with the results from above part. 3) Our research provided some evidence for molocular mimicry hypothesis in pathogenesis of MS.
引文
1. Johnson RT. The virology of demyelinating diseases. Ann Neurol, 1994,36:S54-S60.
2. Noseworthy J, Lucchinetti C, Rodriguez M, et al. Multiple sclerosis. N Eng J Med, 2000,343:938-952.
3. Ault GS, Stoner GL. Two major types of JC virus defined in progressivemultifocal leukoencephalopathy brain by early and late coding region DNA sequences. J Gen Virol, 73:2669-2678.
4. Oleszak EL, Chang JR, Friedman H, et al. Theiler's virus infection: a model for multiple sclerosis. Clinical Microbiology Reviews, 2004, 174-207.
5. Haring J, Perlman S. Mouse hepatitis virus. Current Opinion in Microbiology, 2001, 4:462-466.
6. Moore FGA, Wolfson C. Human herpes virus 6 and multiple sclerosis. Acta Neurol Scand, 2002, 106:63-83.
7. Munch M, Riisom K, Christensen T, et al. The significance of Epstein-Barr virus seropositivity in multiple sclerosis patients. Acta Neurol Scand, 1998,97:171-174.
8. Ferrante P, Omodeo-Zorini E, Caldarelli-Stefano R, et al. Detection of JC virus DNA in cerebrospinal fluid from multiple sclerosis patients. Mult-Scler, 1998, 4(2):49-54.
9．吴笃初，吕传真，汪无级等．多发性硬化与乙肝病毒感染．中国神经精神疾病杂志，1992，18(4)：222-224．
10. Wekerle H, Hohlfeld R. Molecular mimicry in multiple sclerosis. N Eng J Med, 2003,349:185-186.
11. Lang HLE, Jacobsen H, Ikemizu S, et al. A functional and structural basis for TCR cross-reactivity in multiple sclerosis. Nat Immunol, 2002,3:940-943.
12. Link H, Xiao BG. Rat models as tool to develop new immunotherapies. Immunological Reviews, 2001,184:117-128.
13. Poser CM. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. AnnNeurol, 1983, 13:227-231.
14. Wandinger KP, Jabs W, Siekhaus A, et al. Association between clinical disease activity and Epstein-Barr virus reactivation in MS. Neurology, 2000,55:178-184.
15．林友才，杜英．20例乙肝病毒慢性感染者脑脊液乙肝病毒标志物检测情况分析．赣南医学院学报，1996，16(2)：155-156．
16. Lane TE, Buchmeier MJ. Murine coronavirus infection: a paradigm for virus-induced demyelinating disease. Trends in Microbiology, 1997, 5(1):9-13.
17. Fujinami RS, Oldstone MBA. Amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity. Science, 1985,230:1043-1045.
18. Munch M, Hvas T, Christensen A, et al. The implications of Epstein-Barr virus in multiple sclerosis- a review. Acta Neurol Scand,1997, 169:59-64.
19. Wagner HJ, Hennig H, Jabs WJ, et al. Altered prevalence and reactivity of anti-Epstein-Barr virus antibodies in patients with multiple sclerosis. Viral Immunology, 2000,13(4):497-502.
20. Ascherio A, Munger KL, Lennette ET, et al. Epstein-Barr virus antibodies and risk of multiple sclerosis. JAMA, 2001,286(24):3083-3088.
21. Levin LI, Munger KL, Rubertone MV, et al. Multiple sclerosis and Epstein-Barr virus. JAMA, 2003,289(12):1533-1536.
22. Myhr KM, Riise T, Barrett-Connor E, et al. Altered antibody pattern to Epstein-Barr virus but not to other herpesviruses in multiple sclerosis: a population based case-control study from western Norway. J Neurol Neurosurg Psychiatry, 1998,64:539-542.
23. Bray PF, Luka J. Antibodies against Epstein-Barr nuclear antigen (EBNA) in multiple sclerosis CSF, and two pentapeptide sequence identities between EBNA and myelin basic protein. Neurology, 1992, 42:1798-1804.
24. Novoa LJ, Nagra RM, Nakawatase T, et al. Fulminant demyelinating encephalomyelitis associated with productive HHV-6 infection in an immunocompetent adult. J Med Virol, 1997,52:301-308.
25. Mackenzie IRA, Carrigan DR, Wiley CA. Chronic myelopathy associated with human herpesvirus-6. Neurology, 1995,45:2015-2017.
26. Challoner PB, Smith KT, Parker JD, et al. Plaque-associated expression of human herpesvirus 6 in multiple sclerosis. Proc Natl Acad Sci USA 1995,92:7440-7444.
27. Braun DK, Dominguez G, Pellett PE. Human herpes virus 6. Clin Micr Rev 1997,10:521-567.
28. Taus C, Pucci E, Cartechini E, et al. Absence of HHV-6 and HHV-7 in cerebrospinal fluid in relapsing-remitting multiple sclerosis. Acta Neurol Scand, 2000,101:224-228.
29. Nielsen L,Moller-Larsen A, Munk M, et al. Human herpesvirus-6 immunoglobulin G antibodies in patients with multiple sclerosis. Acta Neurol Scand, 1997,169:76-78.
30. Ablashi DV, Eastman HB, Owen CB, et al. Frequent HHV-6 reactivation in multiple sclerosis(MS) and chronic fatigue syndrome (CFS) patients.Journal of clinical virology, 2000,16:179-191.
31. Ongradi J, Rajda C, Marodi CL, et al. A pilot study on the antibodies to HHV-6 variants and HHV-7 in CSF of MS patients. J Neurovirol 1999;5:529-532.
32. Mirandola P, Stefan A, Brambilla E, et al. Absence of human herpesvirus 6 and 7 from spinal fluid and serum of multiple sclerosis patients. Neurology 1999;53: 1367-1368.
33. Ferrante P, Mancuso R, Pagani E, et al. Molecular evidence for a role of HSV-1 in multiple sclerosis clinical acute attack. J-Neurovirol, 2000, 6 Suppl 2:S109-114.
34. Sanders VJ, Felisan S, Waddell A, et al. Detection of herpesviridae in multiple sclerosis brain tissue and controls by polymerase chain reaction. J Neurovirol, 1996;2(4):249-258.
35. Nicoll JA, Kinrade E, Love S. PCR-mediated search for herpes simplex virus DNA in sections of brain from patients with multiple sclerosis and other neurological disorders. J-Neurol-Sci, 1992, 113(2): 144-151.
36. Heinzlef O, Roullet E, Johanet C, et al. Prevalence of serum antibodiesto hepatitis C virus is not increased in patients with multiple sclerosis. J Neurol Neurosurg Psychiatry, 1996,61 (6):655-656.
37．Andreas D，Baxevanis，Francis Ouellette BF著，李衍达，孙之荣等译．生物信息学：基因和蛋白质分析的实用指南．北京：清华大学出版社，2000．
38．张成岗，贺福初编著．生物信息学方法与实践．科学出版社．2002．
39. Deibler GE, Martensson RE, Kies MV. Large scale preparation of myelin basic protein from central nervous tissue of several animal species. Prep Biochem, 1972, 1:139-165.
40. St.Louis J, Chan EL, Singh B, et al. Suppression of experimental allergic encephalomyelitis in the Lewis rat, by administration of an acylated synthetic peptide of myelin basic protein. J of Neuroimmunol, 1997, 73:90-100.
41. Link H, Xiao BG. Rat models as tool to develop new immunotherapies Immunological Reviews, 2001,184:117-128.
42. Gran B, Hemmer B, Vergelli M, et al. Molecular mimicry and multiple sclerosis: degenerate T-cell recognition and the induction of autoimmunity. Ann Neurol, 1999,45:559-567.
43. Tselios T, Apostolopoulos V, Dalian I, et al. Antagonistic effects of human cyclic MBP87-99 altered peptide ligands in experimental allergic encephalomyelitis and human T-cell proliferation. J Med Chem, 2002, 45:275-283.
44. Wucherfennig KW, Strominger JL. Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell, 1995, 80:695-705.
45. Boerman RH, Bax JJ, Beekhuis-Brussee JA, et al. JC virus and multiple sclerosis:a refutation? Acta-Neurol-Scand, 1993,87(5):353-355.
46. Stoner GL, Agostini HT, Ryschkewitsch CF, et al. JC virus excreted by multiple sclerosis patients and paired controls from Hungary. Mult-Scler, 1998, 4(2):45-48.
47. Ferrante P, Omodeo-Zorini E, Caldarelli-Stefano R , et al. Detection of JC virus DNA in cerebrospinal fluid from multiple sclerosis patients. Mult-Scler, 1998,4(2):49-54.
48. Bogdanovic G, Priftakis P, Hammarin AL, et al. Detection of JC virus in cerebrospinal fluid (CSF) samples from patients with progressive multifocal leukoencephalopathy but not in CSF samples from patients with herpes simplex encephalitis, enteroviral meningitis, or multiple sclerosis. J-Clin-Microbiol, 1998,36(4):1137-1138.
49. Rose NR, Mackay IR. Molecular mimicry: a critical look at exemplary instances in human diseases. Cell-Mol-Life-Sci, 2000,57(4):542-551.
50. Albert LJ, Inman RD. Molecular mimicry and autoimmunity. The New England Journal of Medicine, 1999, 341:2068-2074.
51. Gautam AM, Liblau R, Chelvanayagam G , et al. A viral peptide with limited homology to a self peptide can induce clinical signs of experimental autoimmue encephalomyelitis. Journal of Immunology, 1998, 161:60-64.
52. Lenz DC, Lu L, Conant SB, et al. A chlamydia pneumoniae-specific peptide induces experimental autoimmune encephalomyelitis in rats. Jounal of Immunolgoy, 2001,167:1803-1808.
53. Lang HLE, Jacobsen H, Ikemizu S. et al. A functional and structural basis for TCR cross-reactivity in multiple sclerosis. Nat Immunol 2002;3:940-943.
1. Braun DK, Dominguez G, Pellett PE. Human herpes virus 6. Clin Micr Rev, 1997,10:521-567.
2. Novoa LJ, Nagra RM, Nakawatase T , et al. Fulminant demyelinating encephalomyelitis associated with productive HHV-6 infection in an immunocompetent adult. J Med Virol, 1997,52:301-308.
3. Mackenzie IRA, Carrigan DR, Wiley CA. Chronic myelopathy associated with human herpesvirus-6. Neurology, 1995,45:2015-2017.
4. Sola P, Merelli E, Marasca R,et al. Human Herpesvirus 6 and multiple sclerosis: survey of anti-HHV-6 antibodies by immunotfluorescence analysis and of viral sequences by polymerase chain reaction J Neurol Neurosurg Psychiatry 1993,56:917-919.
5. Challoner PB, Smith KT, Parker JD, et al. Plaque-associated expression of human herpesvirus 6 in multiple sclerosis. Proc Natl Acad Sci USA 1995,92:7440-7444.
6. Fogdell HA, Soldan SS, Jacobson. Association of chronic progressive neurological disease and ubiquitous viral agents: lessons from human herpesvirus 6 and multiple sclerosis. Mol Psychiatry, 2002,7 Suppl 2: S29-31.
7. Liedtke W, Malessa R, Faustmann PM, et al. Human herpesvirus 6 polymerase chain reaction findings in human immunodeficiency virus associated neurological disease and multiple sclerosis. J Neurovirol, 1995,1:253-258.
8. Taus C, Pucci E, Fie A, et al. Absence of HHV-6 and HHV-7 in cerebrospinal fluid in relapsing-remitting multiple sclerosis. Acta Neurol Scand, 2000,101:224-228.
9. Ablashi DV Lapps W, Kaplan M, et al. Human herpesvirus 6 infection in multiple sclerosis: a preliminary report. Multiple Sclerosis, 1998,4:490-496.
10. Ongradi J, Rajda C, Marodi CL, et al. A pilot study on the antibodies to HHV-6 variants and HHV-7 in CSF of MS patients. J Neurovirol, 1999,5:529-532.
11. Soldan SS, Berti R, Salem N ,et al. Association of human herpes virus 6 (HHV-6) with multiple sclerosis: increased IgM response to HHV-6 early antigen and detection of serum HHV-6 DNA. Nat Med, 1997,3: 1294-1397.
12. Alvarez LR Martin EC. Heras V. et al. Active human herpesvirus 6 infection in patients with multiple sclerosis. Arch Neurol, 2002,59(6):929-933.
13. Ablashi DV, Eastman HB, Owen CB, et al. Frequent HHV-6 reactivation in multiple sclerosis and chronic fatigue syndrome patients. J Clin Virol, 2000,16:179-191.
14. Mirandola P, Stefan A, Brambilla E, et al. Absence of human herpesvirus 6 and 7 from spinal fluid and serum of multiple sclerosis patients. Neurology, 1999,53: 1367-1368.
15. Soldan SS, Leist TP, Juhng KN, et al. Increased lymphoproliferative response to human Herpesvirus type 6A variant in multiple sclerosis patients. Ann Neurol, 2000,47:306-313.
16. Enbom M, Wang FZ, Fredrikson S, et al. Similar humoral and cellular immunological reactivities to human herpesvirus 6 in patients with multiple sclerosis and controls. Clin Diagn Lab Immunol, 1999,6:545-549.
17. Knox KK, Brewer JH, Henry JM ,et al. Human herpes virus 6 and multiple sclerosis: Systemic active infections in patients with early disease. Clin Inf Dis, 2000,31:1321-1322.
18. Blumberg BM, Mock DJ, Powers JM, et al. The HHV-6 paradox: ubiquitous commensal or insidious pathogen? A two-step in situ PCR approach. J Clin Virol, 2000,16:159-178.
19. Enbom M. Human herpesvirus 6 in the pathogenesis of multiple sclerosis. APMIS, 2001,109(6):401-411.
20. Ascherio A, Munch M. Epstein-Barr virus and multiple sclerosis. Epidemiology, 2000,11(2):220-224.
21. Ascherio A, Munaer KL, Lennette ET, et al. Epstein-Barr virus antibodies and risk of multiple sclerosis. JAMA, 2001,286:3083-3088.
22. Wagner HJ, Hennig H, Jabs WJ, et al. Altered prevalence and reactivitv of anti-Epstein-Barr virus antibodies in patients with multiple sclerosis. Viral immunology, 2000,13(4):497-502.
23. Myhr KM, Riise T, Barrett-Connor E, et al. Altered antibody pattern to Epstein-Barr virus but not to other herpes viruses in multiple sclerosis: a population based case-control study from western Norway. J Neurol Neurosurg Psychiatry, 1998,64:539-542.
24. Rand KH, Houck H, Denslow, et al. Epstein-Barr virus nuclear antigen-1(EBNA-1) associated oligoclonal bands in patients with multiple sclerosis. J Neurol Sci, 2000,173 (1) :32-39.
25. Haahr S, Munch M. The association between multiple sclerosis and infection with epstein-Barr virus and retrovirus. J Neurovirol, 2000,6 Suppl 2: S76-79.
26. Herman MA, Zhang SM, Lipworth L, et al. Multiple sclerosis and age at infection with common viruses. Epidemiology, 2001,12 (3):301-306.
27. Levin LI, Munger KL, Rubertone MV, et al. Multiple sclerosis and infection with Epstein-Barr virus. JAMA, 2003,289:1533-1536.
28. Lily O. Epstein-Barr virus and risk of multiple sclerosis. JAMA, 2003,290:192.
29. Fujinami RS, Oldstone MBA. Amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity. Science, 1985, 230:1043-1045
30．吴笃初，吕传真，汪无级等．多发性硬化与乙肝病毒感染．中国神经精神疾病杂志，1992，18(4)：222-224．
31．能友生，卢洁，苏美芳等．多发性硬化112例临床分析．江西医学院学报，1995．35(1)：51．
32. Heinzlef O, Roullet E, Johanet C, et al. Prevalence of serum antibodies to hepatitis C virus is not increased in patients with multiple sclerosis. J Neurol Neurosurg Psychiatry, 1996,61 (6):655-656.
33. Sauleda S, Rio J, Montalban X, et al. Lack of association between hepatitis G virus and multiple sclerosis. J Neurol Neurosurg Psychiatry, 1998,64(2):283.
34. Bock M, Stoye JP Endogenous retroviruses and the human germline. Curr Opin Genet Dev, 2000,10:651-655.
35. Rasmussen HB, Lucotte G, Clausen J. Endogenous retrovirus and multiple sclerosis. J Neurovirol, 2000,6: S80-84.
36. Perron H, Gratacap B, Lalande B, et al. In vitro transmission and antigenicity of a retrovirus isolated from a multiple sclerosis patient. Res Virol, 1992,143:337-350.
37. Christensen T, Dissing SP, Riemann H, et al. Molecular characterization of HERV-H variants associated with multiple sclerosis. Acta Neurol Scand, 2000,101:229-238.
38. Johnston JB, Silva C, Holden J, et al. Monocyte activation and differentiation augment human endogenous retrovirus expression: implications for inflammatory brain diseases. Ann Neurol, 2001,50:434-442.
39. Kolson DL, Gonzalez SF. Endogenous retroviruses and multiple sclerosis. Ann Neruol, 2001,50(4):429-430.
40. Sanders VJ, Felisan S, Waddell A, et al. Detection of herpesviridae in multiple sclerosis brain tissue and controls by polymerase chain reaction. J Neurovirol, 1996,2(4):249-258.
41. Boerman RH, Bax JJ, Beekhuis-Brussee JA, et al. JC virus and multiple sclerosis:a refutation? Acta-Neurol-Scand, 1993,87(5):353-355.
42. Stoner GL, Agostini HT, Ryschkewitsch CF, et al. JC virus excreted by multiple sclerosis patients and paired controls from Hungary. Mult-Scler, 1998, 4(2):45-48.
43. Ferrante P, Omodeo-Zorini E, Caldarelli-Stefano R , et al. Detection of JC virus DNA in cerebrospinal fluid from multiple sclerosis patients. Mult-Scler, 1998,4(2):49-54.
44. Bogdanovic G, Priftakis P, Hammarin AL, et al. Detection of JC virus in cerebrospinal fluid(CSF) samples from patients with progressive multifocal leukoencephalopathy but not in CSF samples from patients with herpes simplex encephalitis, enteroviral meningitis, or multiple sclerosis. J Clin Microbiol, 1998,36(4):1137-1138.
45. Ohara Y. Multiple sclerosis and measles virus. Jpn J Infect Dis, 1999;52(5): 198-200.
46. Lang HLE, Jacobsen H, Ikemizu S, et al. A functional and structural basis for TCR cross-reactivity in multiple sclerosis. Nat Immunol, 2002,3:940-943.
47. Wekerle H, Hohlfeld R. Molecular mimicry in multiple sclerosis. N Eng J Med, 2003,349:185-186.
48. Kim BS,Palma JP, Inoue A,et al. Pathogenic immunity in Theiler's virus-induced demyelinating disease:a viral model for multiple sclerosis. Arch Immunol Ther Exp, 2000,48:373-379.
49. Liu MT,Keirstead HS,Lane TE.Neutralization of the chemokine CXCL10 reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis. J Immunol,2001,167:4091 -4097.
50. Murray PD, Krivacic K, Chernosky A,et al. Biphasic and regionally-restricted chemokine expression in the central nervous system in the Theiler's virus model of multiple sclerosis. Curr Neurol Neurosci Rep, 2003,3:265-271.
51. Sharief MK, Matthews H, Noori MA. Expression ratios of the Bcl-2 family proteins and disease activity in multiple sclerosis. J Neuroimmunol, 2003,134(1-2): 158-165.
52. Sakai T, Inoue A, Koh CS, et al. Serum levels of apoptosis-related molecules in patients with multiple sclerosis and human T-lymphotropic virus Type I-associated myelopathy. J interferon Cytokine Res, 1999,19(9):999-1004.
53. Scarisbrick IA, Rodriguez M. Hit-Hit and Hit-Run: viruses in the playing field of multiple sclerosis. Curr Neurol Neurosci Rep, 2003,3:265-271.
1. Kurtzke JF. MS epidemiology worldwide: one view of current status. Acta Neurol Scand, 1995,161 (Suppl):23-33.
2. Pozzilli C, Romano S, Cannoni S. Epidemiology and current treatment of multiple sclerosis in Europe today. J Rehabil Res Dev, 2002,39(2): 175-185.
3. Pugliatti M, Sotgiu S, Rosati G. The world prevalence of multiple sclerosis. Clin Neurol Neurosurg, 2002,104(3): 182-191.
4. Rosati G. The prevalence of multiple sclerosis in the world: an update. Neruol Sci, 2001,22:117-139.
5. Gilmore M, Grennan E. A pilot study of the relationship between multiple sclerosis and the physical environment in northwest Ireland. Environ-Geochem-health, 2003,25(1): 157-163.
6. Sotgiu S, Pugliatti M, Sanna A, et al. Multiple sclerosis complexity in selected populationstthe challenge of Sardinia, insular Italy. Eur J Neurol, 2002,9(4):329-341.
7. Ranzato F, Perini P,Tzintzeva E, et al. Increasing frequency of multiple sclerosis in Padova, Italy: a 30 Year epidemiological survery. Multi Scler, 2003,9(4):387-392.
8. Kaufman MD, Johnson SK, Moyer D, et al. Multiple sclerosis: severity and progression rate in African Americans compared with whites. Am J Phys Med Rehabil, 2003,82(8):582-590.
9. Kira J. Multiple sclerosis in the Japanese population. Lancet Neurol,2003,2(2): 117-127.
10. Lau KK, Wong LKS, Li LSW, et al. Epidemiological study of multiple sclerosis in Hong Kong Chinese: questionnaire survery.Hong Kong Med J, 2002,8(2):77-80.
11. Barnett MH, Williams DB, Day S, et al. Progressive increase in incidence and prevalence of multiple sclerosis in Newcastle, Australia: a 35-year study, J Neurol Sci,203,213(1-2):1-6.
12. Voskuhl RR. Gender issues and multiple sclerosis. Curr Neruol Neruosci Rep, 2002, 2(3):277-286.
13. Levin LA, Lessell S. Optic neuritis and multiple sclerosis. Arch Ophthalmol, 2003,121(7):944-949.
14. Noseworthy J, Lucchinetti C, Rodriguez M, et al. Multiple sclerosis. N Eng J Med, 2003,343: 938-952.
15. Health-related quality of life and depression in an Italian sample of multiple sclerosis patients. J Neurol Sci, 2003,211(1-2):55-62.
16. Giordano M, Dalfonso S, Momigliano R. Genetics of multiple sclerosis: linkage and association studies. Am J Pharmacogenomics, 2002,2(1):37-58.
17. Sawcer S, Maranian M, Setakis E, et al. A whole genome screen for linkage disequilibrium in multiple sclerosis confirms disease associations with regions previously linked to susceptibility. Brain, 2002,125(6):1337-1347.
2. Noseworthy J, Lucchinetti C, Rodriguez M, et al. Multiple sclerosis. N Eng J Med, 2000,343:938-952.
3. Ault GS, Stoner GL. Two major types of JC virus defined in progressivemultifocal leukoencephalopathy brain by early and late coding region DNA sequences. J Gen Virol, 73:2669-2678.
4. Oleszak EL, Chang JR, Friedman H, et al. Theiler's virus infection: a model for multiple sclerosis. Clinical Microbiology Reviews, 2004, 174-207.
5. Haring J, Perlman S. Mouse hepatitis virus. Current Opinion in Microbiology, 2001, 4:462-466.
6. Moore FGA, Wolfson C. Human herpes virus 6 and multiple sclerosis. Acta Neurol Scand, 2002, 106:63-83.
7. Munch M, Riisom K, Christensen T, et al. The significance of Epstein-Barr virus seropositivity in multiple sclerosis patients. Acta Neurol Scand, 1998,97:171-174.
8. Ferrante P, Omodeo-Zorini E, Caldarelli-Stefano R, et al. Detection of JC virus DNA in cerebrospinal fluid from multiple sclerosis patients. Mult-Scler, 1998, 4(2):49-54.
9．吴笃初，吕传真，汪无级等．多发性硬化与乙肝病毒感染．中国神经精神疾病杂志，1992，18(4)：222-224．
10. Wekerle H, Hohlfeld R. Molecular mimicry in multiple sclerosis. N Eng J Med, 2003,349:185-186.
11. Lang HLE, Jacobsen H, Ikemizu S, et al. A functional and structural basis for TCR cross-reactivity in multiple sclerosis. Nat Immunol, 2002,3:940-943.
12. Link H, Xiao BG. Rat models as tool to develop new immunotherapies. Immunological Reviews, 2001,184:117-128.
13. Poser CM. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. AnnNeurol, 1983, 13:227-231.
14. Wandinger KP, Jabs W, Siekhaus A, et al. Association between clinical disease activity and Epstein-Barr virus reactivation in MS. Neurology, 2000,55:178-184.
15．林友才，杜英．20例乙肝病毒慢性感染者脑脊液乙肝病毒标志物检测情况分析．赣南医学院学报，1996，16(2)：155-156．
16. Lane TE, Buchmeier MJ. Murine coronavirus infection: a paradigm for virus-induced demyelinating disease. Trends in Microbiology, 1997, 5(1):9-13.
17. Fujinami RS, Oldstone MBA. Amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity. Science, 1985,230:1043-1045.
18. Munch M, Hvas T, Christensen A, et al. The implications of Epstein-Barr virus in multiple sclerosis- a review. Acta Neurol Scand,1997, 169:59-64.
19. Wagner HJ, Hennig H, Jabs WJ, et al. Altered prevalence and reactivity of anti-Epstein-Barr virus antibodies in patients with multiple sclerosis. Viral Immunology, 2000,13(4):497-502.
20. Ascherio A, Munger KL, Lennette ET, et al. Epstein-Barr virus antibodies and risk of multiple sclerosis. JAMA, 2001,286(24):3083-3088.
21. Levin LI, Munger KL, Rubertone MV, et al. Multiple sclerosis and Epstein-Barr virus. JAMA, 2003,289(12):1533-1536.
22. Myhr KM, Riise T, Barrett-Connor E, et al. Altered antibody pattern to Epstein-Barr virus but not to other herpesviruses in multiple sclerosis: a population based case-control study from western Norway. J Neurol Neurosurg Psychiatry, 1998,64:539-542.
23. Bray PF, Luka J. Antibodies against Epstein-Barr nuclear antigen (EBNA) in multiple sclerosis CSF, and two pentapeptide sequence identities between EBNA and myelin basic protein. Neurology, 1992, 42:1798-1804.
24. Novoa LJ, Nagra RM, Nakawatase T, et al. Fulminant demyelinating encephalomyelitis associated with productive HHV-6 infection in an immunocompetent adult. J Med Virol, 1997,52:301-308.
25. Mackenzie IRA, Carrigan DR, Wiley CA. Chronic myelopathy associated with human herpesvirus-6. Neurology, 1995,45:2015-2017.
26. Challoner PB, Smith KT, Parker JD, et al. Plaque-associated expression of human herpesvirus 6 in multiple sclerosis. Proc Natl Acad Sci USA 1995,92:7440-7444.
27. Braun DK, Dominguez G, Pellett PE. Human herpes virus 6. Clin Micr Rev 1997,10:521-567.
28. Taus C, Pucci E, Cartechini E, et al. Absence of HHV-6 and HHV-7 in cerebrospinal fluid in relapsing-remitting multiple sclerosis. Acta Neurol Scand, 2000,101:224-228.
29. Nielsen L,Moller-Larsen A, Munk M, et al. Human herpesvirus-6 immunoglobulin G antibodies in patients with multiple sclerosis. Acta Neurol Scand, 1997,169:76-78.
30. Ablashi DV, Eastman HB, Owen CB, et al. Frequent HHV-6 reactivation in multiple sclerosis(MS) and chronic fatigue syndrome (CFS) patients.Journal of clinical virology, 2000,16:179-191.
31. Ongradi J, Rajda C, Marodi CL, et al. A pilot study on the antibodies to HHV-6 variants and HHV-7 in CSF of MS patients. J Neurovirol 1999;5:529-532.
32. Mirandola P, Stefan A, Brambilla E, et al. Absence of human herpesvirus 6 and 7 from spinal fluid and serum of multiple sclerosis patients. Neurology 1999;53: 1367-1368.
33. Ferrante P, Mancuso R, Pagani E, et al. Molecular evidence for a role of HSV-1 in multiple sclerosis clinical acute attack. J-Neurovirol, 2000, 6 Suppl 2:S109-114.
34. Sanders VJ, Felisan S, Waddell A, et al. Detection of herpesviridae in multiple sclerosis brain tissue and controls by polymerase chain reaction. J Neurovirol, 1996;2(4):249-258.
35. Nicoll JA, Kinrade E, Love S. PCR-mediated search for herpes simplex virus DNA in sections of brain from patients with multiple sclerosis and other neurological disorders. J-Neurol-Sci, 1992, 113(2): 144-151.
36. Heinzlef O, Roullet E, Johanet C, et al. Prevalence of serum antibodiesto hepatitis C virus is not increased in patients with multiple sclerosis. J Neurol Neurosurg Psychiatry, 1996,61 (6):655-656.
37．Andreas D，Baxevanis，Francis Ouellette BF著，李衍达，孙之荣等译．生物信息学：基因和蛋白质分析的实用指南．北京：清华大学出版社，2000．
38．张成岗，贺福初编著．生物信息学方法与实践．科学出版社．2002．
39. Deibler GE, Martensson RE, Kies MV. Large scale preparation of myelin basic protein from central nervous tissue of several animal species. Prep Biochem, 1972, 1:139-165.
40. St.Louis J, Chan EL, Singh B, et al. Suppression of experimental allergic encephalomyelitis in the Lewis rat, by administration of an acylated synthetic peptide of myelin basic protein. J of Neuroimmunol, 1997, 73:90-100.
41. Link H, Xiao BG. Rat models as tool to develop new immunotherapies Immunological Reviews, 2001,184:117-128.
42. Gran B, Hemmer B, Vergelli M, et al. Molecular mimicry and multiple sclerosis: degenerate T-cell recognition and the induction of autoimmunity. Ann Neurol, 1999,45:559-567.
43. Tselios T, Apostolopoulos V, Dalian I, et al. Antagonistic effects of human cyclic MBP87-99 altered peptide ligands in experimental allergic encephalomyelitis and human T-cell proliferation. J Med Chem, 2002, 45:275-283.
44. Wucherfennig KW, Strominger JL. Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell, 1995, 80:695-705.
45. Boerman RH, Bax JJ, Beekhuis-Brussee JA, et al. JC virus and multiple sclerosis:a refutation? Acta-Neurol-Scand, 1993,87(5):353-355.
46. Stoner GL, Agostini HT, Ryschkewitsch CF, et al. JC virus excreted by multiple sclerosis patients and paired controls from Hungary. Mult-Scler, 1998, 4(2):45-48.
47. Ferrante P, Omodeo-Zorini E, Caldarelli-Stefano R , et al. Detection of JC virus DNA in cerebrospinal fluid from multiple sclerosis patients. Mult-Scler, 1998,4(2):49-54.
48. Bogdanovic G, Priftakis P, Hammarin AL, et al. Detection of JC virus in cerebrospinal fluid (CSF) samples from patients with progressive multifocal leukoencephalopathy but not in CSF samples from patients with herpes simplex encephalitis, enteroviral meningitis, or multiple sclerosis. J-Clin-Microbiol, 1998,36(4):1137-1138.
49. Rose NR, Mackay IR. Molecular mimicry: a critical look at exemplary instances in human diseases. Cell-Mol-Life-Sci, 2000,57(4):542-551.
50. Albert LJ, Inman RD. Molecular mimicry and autoimmunity. The New England Journal of Medicine, 1999, 341:2068-2074.
51. Gautam AM, Liblau R, Chelvanayagam G , et al. A viral peptide with limited homology to a self peptide can induce clinical signs of experimental autoimmue encephalomyelitis. Journal of Immunology, 1998, 161:60-64.
52. Lenz DC, Lu L, Conant SB, et al. A chlamydia pneumoniae-specific peptide induces experimental autoimmune encephalomyelitis in rats. Jounal of Immunolgoy, 2001,167:1803-1808.
53. Lang HLE, Jacobsen H, Ikemizu S. et al. A functional and structural basis for TCR cross-reactivity in multiple sclerosis. Nat Immunol 2002;3:940-943.
1. Braun DK, Dominguez G, Pellett PE. Human herpes virus 6. Clin Micr Rev, 1997,10:521-567.
2. Novoa LJ, Nagra RM, Nakawatase T , et al. Fulminant demyelinating encephalomyelitis associated with productive HHV-6 infection in an immunocompetent adult. J Med Virol, 1997,52:301-308.
3. Mackenzie IRA, Carrigan DR, Wiley CA. Chronic myelopathy associated with human herpesvirus-6. Neurology, 1995,45:2015-2017.
4. Sola P, Merelli E, Marasca R,et al. Human Herpesvirus 6 and multiple sclerosis: survey of anti-HHV-6 antibodies by immunotfluorescence analysis and of viral sequences by polymerase chain reaction J Neurol Neurosurg Psychiatry 1993,56:917-919.
5. Challoner PB, Smith KT, Parker JD, et al. Plaque-associated expression of human herpesvirus 6 in multiple sclerosis. Proc Natl Acad Sci USA 1995,92:7440-7444.
6. Fogdell HA, Soldan SS, Jacobson. Association of chronic progressive neurological disease and ubiquitous viral agents: lessons from human herpesvirus 6 and multiple sclerosis. Mol Psychiatry, 2002,7 Suppl 2: S29-31.
7. Liedtke W, Malessa R, Faustmann PM, et al. Human herpesvirus 6 polymerase chain reaction findings in human immunodeficiency virus associated neurological disease and multiple sclerosis. J Neurovirol, 1995,1:253-258.
8. Taus C, Pucci E, Fie A, et al. Absence of HHV-6 and HHV-7 in cerebrospinal fluid in relapsing-remitting multiple sclerosis. Acta Neurol Scand, 2000,101:224-228.
9. Ablashi DV Lapps W, Kaplan M, et al. Human herpesvirus 6 infection in multiple sclerosis: a preliminary report. Multiple Sclerosis, 1998,4:490-496.
10. Ongradi J, Rajda C, Marodi CL, et al. A pilot study on the antibodies to HHV-6 variants and HHV-7 in CSF of MS patients. J Neurovirol, 1999,5:529-532.
11. Soldan SS, Berti R, Salem N ,et al. Association of human herpes virus 6 (HHV-6) with multiple sclerosis: increased IgM response to HHV-6 early antigen and detection of serum HHV-6 DNA. Nat Med, 1997,3: 1294-1397.
12. Alvarez LR Martin EC. Heras V. et al. Active human herpesvirus 6 infection in patients with multiple sclerosis. Arch Neurol, 2002,59(6):929-933.
13. Ablashi DV, Eastman HB, Owen CB, et al. Frequent HHV-6 reactivation in multiple sclerosis and chronic fatigue syndrome patients. J Clin Virol, 2000,16:179-191.
14. Mirandola P, Stefan A, Brambilla E, et al. Absence of human herpesvirus 6 and 7 from spinal fluid and serum of multiple sclerosis patients. Neurology, 1999,53: 1367-1368.
15. Soldan SS, Leist TP, Juhng KN, et al. Increased lymphoproliferative response to human Herpesvirus type 6A variant in multiple sclerosis patients. Ann Neurol, 2000,47:306-313.
16. Enbom M, Wang FZ, Fredrikson S, et al. Similar humoral and cellular immunological reactivities to human herpesvirus 6 in patients with multiple sclerosis and controls. Clin Diagn Lab Immunol, 1999,6:545-549.
17. Knox KK, Brewer JH, Henry JM ,et al. Human herpes virus 6 and multiple sclerosis: Systemic active infections in patients with early disease. Clin Inf Dis, 2000,31:1321-1322.
18. Blumberg BM, Mock DJ, Powers JM, et al. The HHV-6 paradox: ubiquitous commensal or insidious pathogen? A two-step in situ PCR approach. J Clin Virol, 2000,16:159-178.
19. Enbom M. Human herpesvirus 6 in the pathogenesis of multiple sclerosis. APMIS, 2001,109(6):401-411.
20. Ascherio A, Munch M. Epstein-Barr virus and multiple sclerosis. Epidemiology, 2000,11(2):220-224.
21. Ascherio A, Munaer KL, Lennette ET, et al. Epstein-Barr virus antibodies and risk of multiple sclerosis. JAMA, 2001,286:3083-3088.
22. Wagner HJ, Hennig H, Jabs WJ, et al. Altered prevalence and reactivitv of anti-Epstein-Barr virus antibodies in patients with multiple sclerosis. Viral immunology, 2000,13(4):497-502.
23. Myhr KM, Riise T, Barrett-Connor E, et al. Altered antibody pattern to Epstein-Barr virus but not to other herpes viruses in multiple sclerosis: a population based case-control study from western Norway. J Neurol Neurosurg Psychiatry, 1998,64:539-542.
24. Rand KH, Houck H, Denslow, et al. Epstein-Barr virus nuclear antigen-1(EBNA-1) associated oligoclonal bands in patients with multiple sclerosis. J Neurol Sci, 2000,173 (1) :32-39.
25. Haahr S, Munch M. The association between multiple sclerosis and infection with epstein-Barr virus and retrovirus. J Neurovirol, 2000,6 Suppl 2: S76-79.
26. Herman MA, Zhang SM, Lipworth L, et al. Multiple sclerosis and age at infection with common viruses. Epidemiology, 2001,12 (3):301-306.
27. Levin LI, Munger KL, Rubertone MV, et al. Multiple sclerosis and infection with Epstein-Barr virus. JAMA, 2003,289:1533-1536.
28. Lily O. Epstein-Barr virus and risk of multiple sclerosis. JAMA, 2003,290:192.
29. Fujinami RS, Oldstone MBA. Amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity. Science, 1985, 230:1043-1045
30．吴笃初，吕传真，汪无级等．多发性硬化与乙肝病毒感染．中国神经精神疾病杂志，1992，18(4)：222-224．
31．能友生，卢洁，苏美芳等．多发性硬化112例临床分析．江西医学院学报，1995．35(1)：51．
32. Heinzlef O, Roullet E, Johanet C, et al. Prevalence of serum antibodies to hepatitis C virus is not increased in patients with multiple sclerosis. J Neurol Neurosurg Psychiatry, 1996,61 (6):655-656.
33. Sauleda S, Rio J, Montalban X, et al. Lack of association between hepatitis G virus and multiple sclerosis. J Neurol Neurosurg Psychiatry, 1998,64(2):283.
34. Bock M, Stoye JP Endogenous retroviruses and the human germline. Curr Opin Genet Dev, 2000,10:651-655.
35. Rasmussen HB, Lucotte G, Clausen J. Endogenous retrovirus and multiple sclerosis. J Neurovirol, 2000,6: S80-84.
36. Perron H, Gratacap B, Lalande B, et al. In vitro transmission and antigenicity of a retrovirus isolated from a multiple sclerosis patient. Res Virol, 1992,143:337-350.
37. Christensen T, Dissing SP, Riemann H, et al. Molecular characterization of HERV-H variants associated with multiple sclerosis. Acta Neurol Scand, 2000,101:229-238.
38. Johnston JB, Silva C, Holden J, et al. Monocyte activation and differentiation augment human endogenous retrovirus expression: implications for inflammatory brain diseases. Ann Neurol, 2001,50:434-442.
39. Kolson DL, Gonzalez SF. Endogenous retroviruses and multiple sclerosis. Ann Neruol, 2001,50(4):429-430.
40. Sanders VJ, Felisan S, Waddell A, et al. Detection of herpesviridae in multiple sclerosis brain tissue and controls by polymerase chain reaction. J Neurovirol, 1996,2(4):249-258.
41. Boerman RH, Bax JJ, Beekhuis-Brussee JA, et al. JC virus and multiple sclerosis:a refutation? Acta-Neurol-Scand, 1993,87(5):353-355.
42. Stoner GL, Agostini HT, Ryschkewitsch CF, et al. JC virus excreted by multiple sclerosis patients and paired controls from Hungary. Mult-Scler, 1998, 4(2):45-48.
43. Ferrante P, Omodeo-Zorini E, Caldarelli-Stefano R , et al. Detection of JC virus DNA in cerebrospinal fluid from multiple sclerosis patients. Mult-Scler, 1998,4(2):49-54.
44. Bogdanovic G, Priftakis P, Hammarin AL, et al. Detection of JC virus in cerebrospinal fluid(CSF) samples from patients with progressive multifocal leukoencephalopathy but not in CSF samples from patients with herpes simplex encephalitis, enteroviral meningitis, or multiple sclerosis. J Clin Microbiol, 1998,36(4):1137-1138.
45. Ohara Y. Multiple sclerosis and measles virus. Jpn J Infect Dis, 1999;52(5): 198-200.
46. Lang HLE, Jacobsen H, Ikemizu S, et al. A functional and structural basis for TCR cross-reactivity in multiple sclerosis. Nat Immunol, 2002,3:940-943.
47. Wekerle H, Hohlfeld R. Molecular mimicry in multiple sclerosis. N Eng J Med, 2003,349:185-186.
48. Kim BS,Palma JP, Inoue A,et al. Pathogenic immunity in Theiler's virus-induced demyelinating disease:a viral model for multiple sclerosis. Arch Immunol Ther Exp, 2000,48:373-379.
49. Liu MT,Keirstead HS,Lane TE.Neutralization of the chemokine CXCL10 reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis. J Immunol,2001,167:4091 -4097.
50. Murray PD, Krivacic K, Chernosky A,et al. Biphasic and regionally-restricted chemokine expression in the central nervous system in the Theiler's virus model of multiple sclerosis. Curr Neurol Neurosci Rep, 2003,3:265-271.
51. Sharief MK, Matthews H, Noori MA. Expression ratios of the Bcl-2 family proteins and disease activity in multiple sclerosis. J Neuroimmunol, 2003,134(1-2): 158-165.
52. Sakai T, Inoue A, Koh CS, et al. Serum levels of apoptosis-related molecules in patients with multiple sclerosis and human T-lymphotropic virus Type I-associated myelopathy. J interferon Cytokine Res, 1999,19(9):999-1004.
53. Scarisbrick IA, Rodriguez M. Hit-Hit and Hit-Run: viruses in the playing field of multiple sclerosis. Curr Neurol Neurosci Rep, 2003,3:265-271.
1. Kurtzke JF. MS epidemiology worldwide: one view of current status. Acta Neurol Scand, 1995,161 (Suppl):23-33.
2. Pozzilli C, Romano S, Cannoni S. Epidemiology and current treatment of multiple sclerosis in Europe today. J Rehabil Res Dev, 2002,39(2): 175-185.
3. Pugliatti M, Sotgiu S, Rosati G. The world prevalence of multiple sclerosis. Clin Neurol Neurosurg, 2002,104(3): 182-191.
4. Rosati G. The prevalence of multiple sclerosis in the world: an update. Neruol Sci, 2001,22:117-139.
5. Gilmore M, Grennan E. A pilot study of the relationship between multiple sclerosis and the physical environment in northwest Ireland. Environ-Geochem-health, 2003,25(1): 157-163.
6. Sotgiu S, Pugliatti M, Sanna A, et al. Multiple sclerosis complexity in selected populationstthe challenge of Sardinia, insular Italy. Eur J Neurol, 2002,9(4):329-341.
7. Ranzato F, Perini P,Tzintzeva E, et al. Increasing frequency of multiple sclerosis in Padova, Italy: a 30 Year epidemiological survery. Multi Scler, 2003,9(4):387-392.
8. Kaufman MD, Johnson SK, Moyer D, et al. Multiple sclerosis: severity and progression rate in African Americans compared with whites. Am J Phys Med Rehabil, 2003,82(8):582-590.
9. Kira J. Multiple sclerosis in the Japanese population. Lancet Neurol,2003,2(2): 117-127.
10. Lau KK, Wong LKS, Li LSW, et al. Epidemiological study of multiple sclerosis in Hong Kong Chinese: questionnaire survery.Hong Kong Med J, 2002,8(2):77-80.
11. Barnett MH, Williams DB, Day S, et al. Progressive increase in incidence and prevalence of multiple sclerosis in Newcastle, Australia: a 35-year study, J Neurol Sci,203,213(1-2):1-6.
12. Voskuhl RR. Gender issues and multiple sclerosis. Curr Neruol Neruosci Rep, 2002, 2(3):277-286.
13. Levin LA, Lessell S. Optic neuritis and multiple sclerosis. Arch Ophthalmol, 2003,121(7):944-949.
14. Noseworthy J, Lucchinetti C, Rodriguez M, et al. Multiple sclerosis. N Eng J Med, 2003,343: 938-952.
15. Health-related quality of life and depression in an Italian sample of multiple sclerosis patients. J Neurol Sci, 2003,211(1-2):55-62.
16. Giordano M, Dalfonso S, Momigliano R. Genetics of multiple sclerosis: linkage and association studies. Am J Pharmacogenomics, 2002,2(1):37-58.
17. Sawcer S, Maranian M, Setakis E, et al. A whole genome screen for linkage disequilibrium in multiple sclerosis confirms disease associations with regions previously linked to susceptibility. Brain, 2002,125(6):1337-1347.