Clinical And Experimental Study Of The Relationship Between Virus Infection And Multiple Sclerosis

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 twoResearch of induction of experimental autoimmune encephalomyelitis (EAE) by synthetic viral peptides 1. Selection of IMS-related viral peptides using bioinformatics technologyObjective 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 databasehttp://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 peptidesObjective 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 tocontrol(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 peptidesObjectives 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 mycobacteriumtuberculosis 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 obviousinflammatory 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.