Objective To investigate the levels of peripheral blood CD8+ CD28- regulatory lymphocytes and their clinical values in the patients with multiple sclerosis (MS). Method From October 2005 to August 2008, 51 patients with active rehpsing-remitting MS were enrrolled from Department of Neurology of the First Affil-iated Hospital of Wenzliou Medical College. The diagnostic criteria for MS were the 2005 revisions to the "McDon-ald criteria". All the admitted patients received 1 g of methylprednisoione per day intravenously for 5 days, fol-lowed by 60 mg prednisone per day orally for 12 days,and tapered in 6 weeks. Fourteen patients were reevaluated after corticosteroid therapy. Twenty healthy individuals ,as normal controls,matched for age and sex with the MS patients were also enrolled in this study. The percentages of peripheral blood T cells (CD8+ CD28-, CD8+CD28+, CD8+, CD4+ CD8-) were measured by flow cytometric analysis. Parametric statistical analysis were per-formed using standard methods, and linear regression analysis was conducted using Pearson correlation test. Re-sults (1)Compared with controls,the patients with active MS had significantly lower percentage of CD8+ CD28-T cells [(18.48±9.89)% vs. (24.48±4.86)%, P <0.01], and higher percentage of CD8+ CD28+ T cells [(12.23±4.31) % vs. (8.55±3.49) %, P <0.01]. (2)The percentage of CD8+ CD28- T cells was negative-ly correlated with that of CD4+ CDS- T cells (r = -0.488, P < 0.01). (3) After corticosteroid therapy, the per-eentage of peripheral blood CD8+ CD28- / CD8+ CD28+ T cells didn' t significantly decrease or increase in 14 ac-tive MS patients (P > 0.05). Conclusions The decrease of peripheral blood CD8+ CD28- regulatory T cells might be associated with the pathogenesis of MS, and CD8+ CD28- regulatory T cells perhaps played their roles through CD4+ T cells. Corticosteroid therapy could not reverse the levels of CD8+ CD28- T cells.

Objective To induce experimental allergic encephalomyelitis (EAE) in female C57BL/6 mice with the extracellular domain of myelin oligedendroglia glycoprotein(MOG~(Igd)). Percentages of CD4~+ CD25~+ T cell (Tr) were tested , and also normalized expressions of Foxp3. Methods Molecular cloning technology was used to produce MOG~(Igd) fusion protein. The MOG~(Igd)-TrxA fusion protein and TrxA protein were purified by metal chelate affinity chromatography (MCAC). Mice were injected s. c. in the flank with 300 μg MOG~(Igd) in complete Frcund's adjuvant (CFA) supplemented with 4 μg/μl Mycobacterium tuberculosis. H37Rv. Mice received 0.4 ml emulsion of spinal cord homogenate of guinea pigs (GPSCH) in positive control group, and the same volume emulsiom of TrxA in negative control group, while mice served as normal control received only saline/adjuvant. Mice were monitored two times a day for continuously 30 days by double bind. Clinical scores and histopathology were evaluated. Then, mice were sacrificed. The spinal cord and brain were removed and fixed in buffered formalin. Horizontal sections taken from the central nervous system(CNS) were stained with haematoxylin and eosin (HE), and Kluver-Barrera staining. Also, immunohistochemistry was performed. Percentages of CD4~+ CD25~+ T cells were tested through flow cytometric analysis, and real-time PCR was performed to test normalized expressions of Foxp3 mRNA. Then, correlations between the two were performanced. Results Mice in both MOG group and GPSCH group shew chronic non-remitting course. The onset of disease, time when the most severe clinical symptoms happened and the clinical score between the two groups shew no significant differnces (P>0.05). However, neither in TrxA treated group nor in normal control group did animals exhibit clinical signs of EAE. Histologic sections of the brain and spinal cord taken from affected animals shew perivascular infiltration of mononuclear cells, gliosis, and multifocal demyelination. Lesions scattered throughout the CNS including brainstem, spinal cord, cerebellum, and penventricular white matter. There were significant differences between MOG group and TrxA group in the level of lesion-ceutric AQP-4 expression showing up by immunohistochemistry (P<0.05). Percentages of CD4~+ CD25~+ T cells in MOG group and GPSCH group were (4.71±1.61) % and (1.44±0.65) %, respectively, both of which were significantly lower than those in the normal control group or TrxA treated group (P<0.01). And the difference between MOG group and GPSCH group also reached statistics meaning (P<0.01). Normalized expression of Foxp3 mRNA in MOG group was 2.26± 1.97, and was not significantly higher than the 1.44±1.20 level in GPSCH group (P>0.05). However, they beth were statistically lower than that in the negative control group, namely 8.58±3.34 (P<0.01). Percentages of CD4~+ CD25~+ T cells was statistically correlated with expressions of Foxp3 mRNA (P< 0.05). Conclusion EAE induced in C57BL/6 mice with MOG~(Igd) is reproduceable. It shares the similar clinial signs and pathologic features with human multiple sclerosis(MS). Thus, we find a good way to further study the immune mechanisms of MS and also to search for the effective treatments.