Multiple Sclerosis Pathogenesis

[Introduction]Multiple sclerosis (MS) is a primary demyelinating disease of the central nervous system (CNS) with preponderance in young females. Paralysis, sensory disturbances, incoordination, and visual impairment are common features. Strong evidence showed that MS is sustained by blood-derived T cells specific for CNS antigens, such as myelin. However, since the first report of MS patient about 160 years ago, the etiology and pathogenesis of MS remain unclear and no effective therapeutic approach has been developed so far. "Research on MS" has become one of the hot spots for researchers in the field of neuroimmunology. At home, systemic studies on MS are still a long way to go.[Objective]Part one: To determine a dose response of T cell recognition of different myelin antigens we would use, to establish a more effectiv procedure for T lymphocyte proliferation assay, and examine the effects of myelin lipids to T lymphocyte proliferation.Part two: To compare the proliferative responses of T cell lines (TCL)stimulated by delipidated or intact myelin to 11 kinds of myelincomponents in MS patients and in normal controls, and to identify therole of delipidated process in first onset MS patients.Part three: To identify whether T cells from MS patients would bespecifically activated by self myelin antigen in vivo and to determine theimmunodominant and encephalitogenic epitope .Part four: To distinguish the characters of chronic relapsing phase of MSand of remitting and to investigate how MS is perpetuated after onset.[Methods]27 MS patients with relapsing-remitting phase were enrolled as test groupand 12 volunteers as normal controls (NC), which all were verified to beHLA-DR15+. Mononuclear cells (MNC) were isolated from heparinizedwhole blood on Histopaque gradients. Cells would be cultured forvarious purposes.Part one: The optimal concentration of each kind of myelin antigens wasfound using 1 X 105 MNC per well in a 7 day proliferation assay. Enrichedadherent cell proliferation assay was developed by removal of non-adherent cells from the antigen presenting cells (APC). And then the assay was used to investigate the effects of galactocerebroside or sphingosine to T lymphocyte proliferation. Part two: Human TCLs, established from mononuclear cells of MSpatients or controls, by stimulating with intact myelin or delipidatedmyelin for short term culture in vitro. These TCLS were further tested ifthey could recognized 11 antigens, including mylin basic protein (MBP),proteolipid protein (PLP) and their synthetic peptides. Theimmunodominant epitope would be then distinguished in both myelinTCLs, and contrast between MS group and controls.Part three: Proliferating cells are more likely to undergo geneticmutation, so the HPRT ( hypoxanthine-guanine phosphoribosyltransferase) gene mutant T cells were isolated by culturing MNC in thepresence of 10 uM 6-thioguanine and then tested for their ability torecognize 19 myelin antigens.Part four: MNCs were isolated from individual MS patients in bothexacerbation and remit phases, and were paired for finding theirdistinctions of repertoire and intensity of T cell recognition to myelinantigen by T cell cloning method. Serial MNC samples (more thanthree times at least 6 months interval) were used in enriched adherent cellproliferation assay, and HPRT gene mutant T cells method. Theinteraction of T lymphocyte betweem serial time-points with intactmyelin and mylin components was contrasted.[Results]All data were listed in 22 tables and 20 figures in detail.Part one: The optimal concentration of myelin or delipidated myelin is50-150ug/ml, PLP or MBP is 25-100ug/ml. Each synthetic peptide is used as 20-50ug/ml. Enriched adherent cell proliferation assay showed a higher degree of respose than mixed cell population. The effect of lipids on T cell...