Susceptibility Genes, Cognitive Impairment And Cellular Mechanism Of Immunological Treatment In MS

Multiple sclerosis (MS) is a T cell mediated autoimmune disease with the involvement of genetic and environmental factors. Natural killer (NK) cells are critical regulators of innate and adaptive immunity. NK cells from MS patients decreased in number and were defective in cytokine production. As many diseases were associated with NK cell defection, it was largely unknown whether the alteration of NK cells in number and function preceded the development of MS or resulted from MS. Given MS is a genetic susceptible disease, we explored KIR (killer immunoglobulin-like receptor) and their HLA-C (human leukocyte antigen) ligand genes in the first part of our experiment. RT-PCR was used to detect the genotype of KIR and HLA-C in MS patients and controls. Odds Ratio risk test was applied to evaluate the risk of KIR/HLA-C genes in MS. We found that MS patients with higher frequency of inhibitory KIR2DL2 had increased risk of MS. The sole existence of HLA-C1 and HLA-C2 ligand gene did not increase the risk of MS. Carriers of HLA-C1 ligand gene had a higher risk for MS than noncarriers among individuals also carrying KIR2DL2 gene, which suggested KIR2DL2 execute its functions mainly through combination with HLA-C1 ligand.MS manifested variable symptoms and signs according to the disparate lesions.40%-70% MS patients acquired cognitive impairment, it could appeare early in the course of MS. However, the cognitive insult was always ignored because of the prominent motor disability, early detection and treatment of cognitive impairment would improve the quality of life in MS patients. In the second part of our experiment, Digit Symbol Modality Test (DSMT), Semantic Verbal Fluency Test (SVFT) and Digit Span Test (DST) were used to evaluate the cognitive functions in RRMS patients. We found impairment in information processing speed, memory, sustained attention and executive function in MS patients. Corpus calllosum (CC) is the largest white matter bundles connecting both hemispheres and plays a pivotal role in cognition and information integration. The possibility of DTT reflecting the cognitive functions was determined by the correlation analysis of fibers through CC with cognitive functions. By analyzing the neural fibers through CC between MS patients and healthy controls, the volume, FA and DR of fibers through genu, splenium and whole portion of CC were moderately correlated with the cognitive function and could partly reflect the cognitive function. As to the DTT study in fibers through lesion and NAWM, structural damage of the neural fibers through the lesion was found in MS patients, while only FA values decreased in the fibers through the NAWM opposite the lesion, which suggested FA value be an early merit evaluating white matter damage.The treatment of MS was tough, especially for the severe types. Mitoxantrone (MX) was the first cytotoxic drug licensed in the treatment of vicious relapsing-remitting multiple sclerosis (RRM), secondary progressive multiple sclerosis (SPMS) and progressive relapsing multiple sclerosis (PRMS). Microglias are the macrophages and antigen presenting cells residing in the CNS, besides processing antigens to the T cells infiltrating the CNS and reactivating the T cells, they can secrete many profiles of cytokines to regulate types of T cells and promote the progressing of MS. As MX was a small molecule, it could penetrate the damaged blood brain barrier (BBB) and affect the CNS cells directly. So in the third part of our experiment, the therapeutic mechanisms of MX on CNS microglias were studied. LPS was used to stimulate the BV2 microglial cells,0, 0.02,0.2,2,20 and 200 ng/ml MX were selected to treat the cells. MTT and flow cytometric analysis showed that MX induced cell death in a dose-dependent manner predominantly from late apoptosis and necrosis above concentration of 2 ng/ml. So 0.02 and 0.2 ng/ml MX were further studied to explore its immunoregulatory effects on BV2 cells. Elisa method was used to detect the production of IL-10 and IL-23p19. We found that LPS alone increased the production of pro-inflammatory IL-23p19, but did not affect the expression of anti-inflammatory IL-10. MX with 0.02 and 0.2 ng/ml down-regulated the expression of IL-23p19 and increased the production of IL-10. The third part of our experiment suggested that besides cytotoxic effects, MX also exerted immunoregulatory function in microglias. The study on the therapeutic effects of MX may provide clues to develop new drugs.In conclusion, KIR genes were found to be involved in the susceptibility of MS; the RRMS patients had an early cognitive impairment in information processing speed, memory, sustained attention and executive functions, the fibers through CC could partly reflect the cognitive functions; while there was visible structural damage of neural fibers, only FA values decreased in the fibers through NAWM and could be used as an early merit of white matter alteration; Besides cytotoxic roles, MX also exerted immunoregulatory functions, it seemed that the balance of pro-inflammatory and anti-inflammatory cytokines played pivotal roles in the treatment of MS.