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The purpose of the experiment malaria infection is one of the main challenges of today's threat to human health, more than one million children die from malaria each year. Cerebral malaria (cerebral malaria, CM), referred to as cerebral malaria, one of the most serious complications of malaria, the leading cause of death and malaria infection. CM seriousness determinants are not entirely clear, but it may come from both the host and parasite. Ultimately, parasites and immune response interactions decided host infection finale. Therefore, the basic research about the the CM occurred immune mechanism is a prerequisite for development of effective anti-malaria vaccines and drugs. Plasmodium berghei murine malaria (Plasmodium berghei ANKA, P.bANKA) human diseases have many common features, is the best model of the existing CM. Studies suggest that CM is infected erythrocytes (parasitized red blood cells, pRBC) adhesion and cytokine effector cell-mediated strong host inflammatory response to the combined effect of cerebrovascular diseases caused. Related studies have shown that activation of T cell-mediated immune response involved in disease induction and CD4 T cells and CD8 T cells may promote brain lesions. In fact, Thl response play a key role in the control of parasitaemia also promote CM occurred. CD4 CD25 regulatory T cells (regulatory T cells, Tregs) is different from Thl and Th2 immunomodulatory effects of T-cell subsets. Tregs activation preliminary tests have proved one of the mechanisms of the parasite to evade the host immune Tregs activated with approximately Plasmodium (Plasmodium yoelii 17XL, P.yoelii 17XL) related to the susceptibility of the infected mice, Tregs by dendritic cells (Dendritic cells, DCs) response induced IL-10 production and Th1 response of CD4 T-cell apoptosis regulation. To this end the study dynamic observation of normal infected mice and Tregs eliminate pro-inflammatory cytokines in the mice, the anti-inflammatory cytokine expression levels and Tregs proliferation changes, in order to clarify the role of Tregs in the occurrence of cerebral malaria and its associated mechanisms. Experimental methods, experimental animal models to build and CM lesion assessment 6-8w, female C57BL / 6 intraperitoneally infected with 1 × 106 P.berghei ANKA parasitized erythrocytes (pRBC) build of CM susceptible Model, infected BALB / c and DBA / 2 mouse build the CM resistance model. Experimental group (6 / group) and control group (6 / group) the 1d and infection of mice before infection 1d by intraperitoneal injection 1mganti-CD25mAb (7D4, rat IgM; Bio Express) and an equal volume of phosphate-buffered saline (PBS) to build Tregs to eliminate mouse model. Daily monitoring mouse twice in six days after infection to evaluate the clinical experimental cerebral malaria (Experimental cerebral malaria, ECM) symptoms, these symptoms to determine ECM score: fur ruffled, trembling, paralysis of limbs, convulsions, coma. Each a symptom appears to give a cumulative score ≥ 4 points will be considered for heavy malaria. Flow cytometry spleen cell suspension in CD4 T cell populations within the percentage of CD4 CD25 Foxp3 Tregs prior to infection and 3 after infection, 5d 8d conventional aseptic remove the spleen of mice using 0.17mol / adjusting spleen cells LNH4C1 lysis erythrocytes, in RPMI1640 containing 10% FCS (Fetal calf serum, FCS) is a final concentration of 1 × 107/ml. Each sample with the FITC-conjugated anti-mouse CD4/L3T4 (GK1.5; BD Pharmingen), APC-conjugated anti-mouse CD25/IL-2 receptor alpha (PC61; BD Pharmingen) and PE-CONJUGATED anti-of Foxp3 (the clone FJK16s; eBioscience) tri-color analysis, another negative control tube. Spleen cell suspension for flow cytometry staining tube was added a freshly prepared 1 × 107/ml 0.1ml, and used to Fcγ III / Ⅱ incubated in blocking non-specific staining of the fluorescent antibody blocking antibody, then add an anti-CD4-FITC and anti- CD25-APC mAb surface staining, after the permeable membrane is fixed, using anti-Foxp3-PE mAb intracytoplasmic staining and eluted with 1? S in PBS and washed twice, and suspended in 500μlPBS, flow cytometer (Becton Dickinson, USA ) were detected. And lateral scattering angle before determining lymphocyte population and CD4 T cells painted door, analysis and calculation of CD4 CD25 Foxp3 Tregs percentage of CD4 T cell populations. 3, using a double antibody sandwich ELISA assay spleen cell culture supernatant of IFN-γ and TNF-α, IL-6, IL-17, IL-10 and Griess method detects NO2-content (1) Preparation of spleen cell samples were infected with 3 before and after infection, 5d, 8d conventional aseptic remove the spleen of mice with 0.17mol / L NH4Cl lysis erythrocyte. Containing 10? The S the RPMI1640 adjustment spleen cells at a final concentration of 1 × 107/ml, the 500μl of spleen cells in 24-well plates, each well was added to the suspension at 37 ° C, 5% CO2 for 48h. Note that the of natural spleen cells and purified by the NA / LE Hamster Anti-Mouse CD3E (145-2C11, bd by Pharmingen, 0.56μl/well) and purified Na / LE Hamster Anti-mouse CD28 (37.51, the BD Pharmingen, 0.112μl/well ) at 37 ° C, 5% CO2 in co-cultured for 48h to stimulate IL-17, and then the culture supernatant, -80 ℃ until cytokines and NO detection. (2) double-antibody sandwich ELISA kits were used to detect the spleen cell culture supernatant of IFN-γ, TNF-α, IL-6, IL-10, IL-17 secretion level microplate reader at 450nm OD value was measured. Results to the standard kit standard curve analysis applications the SoftMax Pro 4.3.1Ls software, and calculate the content of cytokines (pg / ml). (3) Griess method detection spleen cell culture supernatant of NO2-content 100μl supernatant and Griess reaction solution 100μl at room temperature the reaction 10min, with NaNO2 as standards, OD value microplate detection at 550nm. Experimental results, the infection rate, survival rate and disease assessment of cerebral malaria to C57BL / 6 mice were injected the toxicity P.bANKA blood stage parasites induced, C57BL / 6 mice at 8-11 days after infection, died of paralysis coma, parasitaemia 10-20%. In contrast, the resistant BALB / c and DBA / 2 mice experienced a similar parasite growth but does not occur brain lesions, anemia and excessive the parasitaemia (gt; 60%) 3-4w died after infection. From the clinical score of cerebral malaria symptoms, C57BL / 6 mice score higher than the BALB / c and DBA / 2 mice. 2 during susceptible and resistance in mice infected spleen cell culture supernatant of pro-inflammatory cytokines, anti-inflammatory cytokines and NO levels the dynamic detection CM susceptible C57BL / 6 mice and resistance to BALB / c and DBA / 2 mouse spleen cell culture supernatant proinflammatory cytokines IFN-γ, TNF-α, IL-6, IL-17 and NO in infections began to increase after 5 days after infection, reached the peak decreased 8 days after infection (C57BL / 6 mice showed a slight decline). Reached the peak five days after the three mouse spleen cell culture supernatant of anti-inflammatory cytokine IL-10 in infection in C57BL / 6 mice 8 days after infection, IL-10 levels decreased significantly, to resist the BALB / c and DBA / 2 mice a slight decline in the level of IL-10. Susceptible mouse spleen cell culture supernatant of proinflammatory cytokines and NO levels significantly higher resistance to murine IL-10 levels were significantly lower than the resistance rat. 3 splenic CD4 T cells at different time points after infection of susceptible and resistant mice Tregs percentage and absolute number of CM susceptible mice and resistant mouse spleen CD4 T cells in Tregs percentage and absolute number began to increase in the infection, infection three days, reached the peak, five days to eight days after infection gradually decreased. During infection, Tregs percentage and absolute number of splenic CD4 T cells in C57BL / 6 mice was significantly lower than the BALB / c and DBA / 2 mice. 4, after the elimination of in vivo CD25 Tregs effect analysis CD25 eliminated C57BL / 6, BALB / c and DBA / 2 mice spleen CD4 T cells, Tregs percentage was significantly lower than the corresponding control rats. Compared with normal mice infected the Tregs were elimination rate of C57BL / 6 mice survived significantly increased after infection 5,6,7 days infection rate decreased significantly, CM symptoms of clinical score is also a corresponding reduction, CD25 eliminate the C57BL / 6 mice does not occur Cerebral malaria symptoms, died at 3-4 weeks after infection anemia and excessive parasitaemia. CD25 elimination of BALB / c and DBA / 2 mice and normal mice infected with the infection process, but in the early stage of infection parasitaemia temporary control, eight days after CD25 eliminate the Tregs' fast recovery inhibit T cell activation can not control parasitaemia 3-4 weeks after infection, these mice died of excessive parasitaemia and severe anemia. CD25 eliminate obvious reversal of IFN-γ and TNF-α, IL-6, IL-17, IL-10 and the production of NO. CD25 eliminate the DBA / 2 mice and CD25 eliminate the BALB / c mice infection process and cytokine production in a similar procedure. Different time Tregs with IFN-γ, TNF-α, NO and IL-10 and parasitaemia correlation analysis of cerebral malaria susceptible mice susceptible and resistance mice infected and resistance during murine infection Tregs quantity with IFN-γ , TNF-α, NO produce a negative correlation, IL-10 generates a positive correlation with the level of parasitemia. Conclusion 1, the establishment of balance between the pro-inflammatory response and anti-inflammatory response occurs on the control of heavy-duty malaria is essential, Tregs is important to maintain this balance adjustment factor. 2, Tregs regulate the balance between the inflammatory response and anti-inflammatory response mediated CM occurrence and infection outcome by modification of a pro-inflammatory response.
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