| Part one:The study on peripheral immune regulation of PD-1in primary intractable epilepsy in patientObjective: Primary intractable epilepsy etiology is unknown.With the pathogenesis of continuous exploration,a lot of evidence that the occurrence of epilepsy may be related to non-neurological changes.Immune avoidance factors play an important role in recurrent epilepsy.CD4+CD25high regulatory T cells(T regulatory cells,Treg)and programmed cell death protein 1(PD-1)have now become key participants in immunomodulation.So we study on PD-1 pathway with CD4 + CD25 low and CD4 + CD25 high Treg cells in pathogenesis of intractable epilepsy and strive to find the pathways in etiology and treatment in intractable epilepsy.This study investigated differences in the number of expression of Treg cells and CD4+CD25high and CD4+CD25low T lymphocytes of PD-1,differences in cytokine concentration in peripheral blood and in patients with partial seizures,intractable status epilepticus and healthy volunteers.To explore their possible role in the development and progression of primary intractable epilepsy.Method:In this study,41 patients with primary intractable epilepsy were randomly selected from November 2015 to March 2017 in patients with epilepsy in the department of epilepsy patients established in our department and recent 26 inpatients of primary intractable status epilepticus as the experimental group,25 cases healthy volunteers.Collected peripheral blood from them.Isolated peripheral blood mononuclear cells(PBMC).Fluorescent antibody labeling and flow cytometry were used to compare the differences in the number of expression CD4+ T cells,CD4 +CD25high and CD4+CD25low Treg cells,and the difference of PD-1 expression between CD4+CD25high and CD4+CD25low Treg lymphocytes,ELISA was used to determine the difference of 1L-10 concentration in patients with partial seizures,intractable status epilepticus and healthy volunteers.Clear the age,sex,degree of attack and the course of the impact of the above factors.To determine the difference in the percentage of regulatory T cells,PD-1 in different Treg cells and concentration of IL-10 of patients with primary intractable epilepsy.Result: 1.Sex,age,course of disease,frequency of onset and percentage of CD4+CD25low T cells in peripheral blood were not significantly different between the patients with partial seizures,intractable status epilepticus and healthy volunteers(P>0.05).2.The percentage of PD-1+CD4+CD25low and PD1+CD4+CD25high Treg lymphocytes in peripheral blood in patients with partial seizures,intractable status epilepticus was significantly higher than that of the control group,the difference was statistically significant(P<0.05).3.The percentage of lymphocytes,CD4+ T lymphocyte percentage,percentage of CD4+CD25high Treg cells in peripheral blood in patients with partial seizures,intractable status epilepticus was significantly higher than that of the control group,the difference was statistically significant(P<0.05).4.The percentages of T lymphocyte,CD4+ T lymphocyte and CD4+CD25high Treg cells in patients with intractable status epilepticus were significantly lower than those of partial seizures.The percentages of PD1+CD4+CD25high and PD-1+CD4+CD25low Treg cells was significantly higher than that of partial seizures group,the difference was statistically significant(P<0.05).5.The expression of IL-10 in serum of epilepsy group was significantly higher than that of healthy control group,of that in patients with intractable status epilepticus group significantly higher than that of partial seizures group.The difference was statistically significant(P<0.05).6.Incidence of sex,age,duration and frequency of occurrence associated with each outcome(spearman correlation)analysis,the percentages of PD-1+CD4+CD25low Treg cells and CD4+ T lymphocyte were decreased with age,and it was correlation(P<0.05).There was no statistically significant correlation between the age of the results in healthy control group and patients with intractable status epilepticus group.7.The correlation between PD1+CD4+CD25high Treg/CD4+CD25high Treg ratio was statistically analyzed.The ratio was significantly higher in epilepsy patients than in control group.And the rate was increased with the degree of seizures.The difference was statistically significant(P<0.05).Conclusion: Our study found that the increased expression of PD-1 on CD4+CD25high and CD4+CD25low Treg cells in patients with primary intractable epilepsy,imbalance of CD4+ T cell population,changes of PD-1+CD4+CD25high Treg/CD4+CD25high Treg ratio,and the difference of IL-10.Our results suggest that increased expression of PD-1 is likely to be associated with an immunosuppressive process in patients with primary primary intractable epilepsy.This pathway participates in immunocompromised regulation of primary intractable epilepsy.So we speculate that the changes of PD-1 immune pathway lead to systemic immune status and inflammatory environment changes and T cell phenotype and inflammatory factor secretion levels which causing autoimmune tolerance,eventually leading to the development of primary intractable epilepsy and the occurrence of drug resistance.Part two:The study on central immune regulation of PD-1in primary intractable epilepsy in patientObjective: Cerebrospinal fluid and brain tissue extracellular fluid are directly connected.Different parts of the brain tissue metabolites can be secreted into the cerebrospinal fluid.At the same time neuronal loss,injury and synaptic damage,blood-brain barrier damage will cause cerebrospinal fluid protein and cytokines and other specific changes.The cellular immune response of the central nervous system is often reflected in the cerebrospinal fluid,so cerebrospinal fluid has important clinical research value.So the analysis of the components of cerebrospinal fluid immune cells has become an important way to evaluate the immune function of the central nervous system.We measured the percentage of lymphocytes,CD4 + T lymphocytes,PD-1 in Treg lymphocytes and the concentration of IL-10 in the cerebrospinal fluid of patients with primary intractable epilepsy.To compare the expression of IL-10 and PD-1 in CD4+CD25high and CD4+CD25low T lymphocytes in cerebrospinal fluid of patients with primary intractable epilepsy group and healthy control group,and to explore their roles in central immunization.Method: In this study,recent 26 inpatients of primary intractable status epilepticus as the experimental group were selected from November 2015 to March 2017 in patients,25 cases healthy volunteers.Collected cerebrospinal fluid from them.Isolated peripheral blood mononuclear cells(PBMC).T lymphocyte proliferation in cultured RPMI1640.Fluorescent antibody labeling and flow cytometry were used to compare the differences in the number of expression CD4+ T cells,CD4+CD25high and CD4+CD25low Treg cells,and the difference of PD-1 expression between CD4+CD25high and CD4+CD25low Treg lymphocytes,ELISA was used to determine the difference of 1L-10 concentration in patients with intractable status epilepticus and healthy volunteers.Clear the age,sex,degree of attack and the course of the impact of the above factors.To determine the difference in the percentage of regulatory T cells,PD-1 in different Treg cells and concentration of IL-10 of patients with primary status epilepticus.To determine and compare the immune relationship between PD-1,Treg cells and IL-10 in the cerebrospinal fluid of patients with primary intractable status epilepticus and healthy contral group.Result: 1.Sex,age,course of disease,frequency of seizure,percentages of CD4+CD25low T cells and CD4+ T cells in cerebrospinal fluid were not significantly different between the patients with intractable status epilepticus and healthy volunteers(P>0.05).2.The percentage of PD-1+CD4+CD25low and PD1+CD4+CD25high Treg lymphocytes in cerebrospinal fluid in patients with intractable status epilepticus was significantly lower than that of the control group,the difference was statistically significant(P<0.05).3.The percentage of lymphocytes,PD-1+CD4+CD25low Treg cells in cerebrospinal fluid in patients with intractable status epilepticus was significantly higher than that of the control group,the difference was statistically significant(P<0.05).4.The concentration of IL-10 in cerebrospinal fluid of intractable status epilepticus group was significantly higher than that of healthy control group.The difference was statistically significant(P<0.05).5The correlation between PD1+CD4+CD25high Treg/CD4+CD25high Treg ratio was statistically analyzed.The difference of the ratio was no statistically significant(P>0.05).Conclusion: The percentage of PD-1+CD4+CD25high Treg in cerebrospinal fluid was significantly higher in intractable status epilepticus group than in controls.We suggest that PD-1 expression is abnormal in patients with ntractable status epilepticus due to CD4CD25 T cells for the reason of that on the one hand that was inhibited the normal proliferation of T cells and thereby reducd the number of CD4CD25 high Treg.On the other hand that was inhibited the immunosuppressive function of CD4+CD25+ Treg cells.Because of the decrease in the number and function of CD4+CD25+ Treg,the inhibitory ability of T cells is not enough,and thus enhance the activity of T cells,induce immune balance and increase the immune pathological damage,leading to the occurrence and development of the disease.In summary,the decrease in the number of CD4+CD25high Tregs and the up-regulation of its expression of PD-1 resulted in further reduction of the immunosuppressive activity of Tregs,which may play an important role in the central immune mechanism of primary intractable epilepsy persistence.Part three:The effect of valproic acid on immune regulation of PD-1 in patients with primary intractable epilepsyObjective:Epilepsy recurrent and chronic course of disease have led to changes in immune status,Immune-mediated seizure pathogenesis may lead to the possibility of intractable epilepsy resistance.Valproic acid may mediate different T lymphocyte responses by regulating the ability of dendritic cells to modulate inflammation,and they may provide complementary immunomodulatory effects for the treatment of inflammatory-related diseases.Therefore,we determined the difference of PD-1 expression in CD4+CD25high and CD4+CD25low T lymphocytes and IL-10 in peripheral blood of patients before and after(d0)(d3)the treatment with valproic acid of intractable status epilepticus,and to compare the relationship between the concentration of valproic acid and the clinical effect of epilepsy control.To explore its immune function in the treatment of intractable status epilepticus.Method: In this study,recent 26 inpatients of primary intractable status epilepticus as the experimental group were selected from November 2015 to March 2017 in patients,25 cases healthy volunteers.We used the valproic acid as a single factor in treatment of intractable status epilepticus.The peripheral blood and cerebrospinal fluid were collected at different time points before and after epilepticus control.We determinated the concentration of sodium valproate in peripheral blood and cerebrospinal fluid by HPLC.Fluorescent antibody labeling and flow cytometry were used to compare the differences in the number of expression CD4+ T cells,CD4+CD25high and CD4+CD25low Treg cells,and the difference of PD-1 expression between CD4+CD25high and CD4+CD25low Treg lymphocytes,ELISA was used to determine the difference of 1L-10 concentration in patients with intractable status epilepticus and healthy volunteers.The changes of the above indexes before and after treatment with valproic acid and the correlation between valproic acid concentration and clinical epilepsy treatment,the influence of age,sex and course of disease on the above factors were comparied.Result: 1 The percentages of PD-1+CD4+CD25low,PD-1+CD4+CD25high Treg lymphocytes,and CD4+ lymphocytes in the patients with primary intractable status epilepticus treated with valproic acid(d3)were significantly lower than befor the treatment(d0)(P <0.05).while the percentage of CD4+CD25low and CD4+CD25high,the percentage of lymphocytes and CD4+ T lymphocytes were significantly higher than those of d0 group.The differences was statistically significant(P <0.05).2 There was no correlation between the drugs concentration of plasma or cerebrospinal fluid of valproic acid and the epilepsy control start time.And no correlation between plasma concentration and drug concentration in cerebrospinal fluid.But the concentration of drug in cerebrospinal fluid was significantly lower than the plasma concentration.There was no significant difference in the concentration of valproic acid at different time(d0 and d3)during the treatment(P >0.05).3 Valproic acid can control the primary refractory status epilepticus peripheral blood IL-10,d3 significantly lower than d0(P <0.05).4 The percentages of CD4+CD25high + PD1/CD4+CD25high T cells in normal group and d0,d0 and d3 group were significantly different(P <0.05).There was no significant difference between the normal group and the d3 group(P>0.05).Conclusion: The percentage of CD4+CD25high and CD4+CD25low Treg and concentration of IL-10 in peripheral blood of patients with intractable status epilepticus was significantly lower after treatment with valproic acid.And after treatment,the percentage of CD4+CD25+ T cells was significantly higher,but still lower than the healthy control group.We hypothesized that the normal proliferation of T cells in patients with primary intractable status epilepticus was widely inhibited.CD4+CD25+T cells played immunosuppressive and immunosuppressive effects of PD-1 after active antiepileptic persistence.The numbers and function of CD4+CD25+ Treg were increased,thus which inhibited the body immune balance disorders and reduced the immune pathological damage.In summary,antiepileptic status treatment can increase the number of CD4+ CD25 high Treg,and reduce the PD-1 molecule negative regulation of Treg immunosuppressive activity.Immunomodulation plays an important role in the treatment of intractable epilepsy. |
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