| PART ONE RESEARCH OF IMMUNE FUNCTION OF CD4+T CELLS IN SEVERE HAND,FOOT,AND MOUTH DISEASEObjective:To evaluate immune function of CD4+T cells and investigate the potential role in T cell exhaustion in severe hand,foot,and mouth disease.To investigate the correlation between the expression of proinflammatory and anti-inflammatory cytokines associated with CD4+T cells and severity of disease and prognosis in severe hand,foot,and mouth disease to find out predict factors for further immunotherapy.Methods:From January 2014 to January 2016,clinical data were collected of total of 433 children of HFMD and 50 healthy controls,who were divided into five groups by clinical charateristics:the death group,the survival group,the severe group,the mild group,the recessive infection group.Absolute quantity statistics total number of peripheral white blood cells,lymphocytes,neutrophils,CRP and percentage of CD3+T cells,CD3+CD4+T cells,CD3+CD8+T cells were collected.IgM and IgG of Coxsackie virus type 16 group A and enterovirus 71 were measured by ELISA.Peripheral blood of children with hand,foot and mouth disease and normal control group were collected and cultivated,IFN-γ levels of supernatantand after PHA stimulation were measured by ELISA.The serum of 433 cases of children with HFMD and divided into five groups:death group of 30 cases of death,survival group of 58 cases,severe group of 167 cases,mild group of 118 cases,recessive infection group of 60 cases and 50 cases of surgical inpatients without infection as control group were collected,IFN-α、IFN-β、IFN-y、IL-2、IL-4、IL-6、IL-8、IL-10、IL-12、IL-18、IL-21、TNF-α、IL-1β、GM-CSF、TGF-β were measured by quantitative cytokines antibody microarray.Statistics was performed by using SPSS 19.0 software.Data of normal distribution were presented as Average±Standard Deviation and data of skewed distribution with Median(Min,Max).One-way analysis of variance and Kraskal-Wallis Rank-Sum test were selected respectively for different comparisons.Results:1.The results showed that death group of white blood cell count,neutrophil count,CRP median,blood sugar were 19.00± 10.79×109/L,14.52±1.52×109/L,9.18mg/L,12.74±9.99mmol/L,much higer than the survival group,severe group,the mild group(P<0.05).Lymphocyte count was 2.51 ± 1.85×109/L in survival group lower than in severe group(P<0.05).2.Peripheral blood percentage of CD3+T cells and CD4+T cells,CD8+T cells of death group were 51.19± 13.05、23.68± 12.47、22.26±5.24,survival group was 49.07± 10.79、23.92±6.57、20.25±6.52,CD3+T cells of survival group are always lower than mild group and normal control group(P<0.05).The death and survival group and CD4+T cells decreased compared with normal control group(P<0.05).3.Serum IFN-gamma,interferons alpha,IFN-beta in children with death group significantly increased compare with severe group,the mild group,the recessive infection group increased compare with normal control group(P<0.05).Proinflammatory factor TNF alpha,beta,IL-6,IL IL-1-2,IL-12 levels increased significantly both in death group and survival group compare with severe group,the mild group and normal control group(P<0.05).IL-18 and IL-8 levels significantly increased of the deathgroup(P<0.05).Inrecessive infection group,IL-1beta,IL-6,IL-8 levels higher than normal control group(P<0.05).In the death group,anti-inflammatory factors of IL-10,TGF-beta levels were high than survival group,severe group,mild group,the recessive infection group and normal control group(P<0.05).IL-4 levels increased in mild,recessive infection group and normal control group(P<0.05),but there was no significant difference between death group,survival group and severe group.The levels of GM-CSF,IL-21 of death groupincreased significantly compare with the severe group,the mild group,the recessive infection group and normal control group(P<0.05).4.IGRA test showed that death group and survival group’s T cells function of IFN-gamma release reduce compared with the mild group.Conclusion:1.The proinflammatory factor,anti-inflammation factor levels are increased by severity of disease,but the death group had higher anti-inflammation factor IL-10 which reveal that CARS exist in severe hand,foot,and mouth disease.2.IL-8,IL-10,IL-18 may be used as a promising predictor of worsening condition of the patient in severe HFMD.3.Increased levels of IL-1 β,IL-6 and IL-8 in early response phase may be a protective factor.4.T cell exhaustion occurs in severe HFMD.PART TWO ASSOCIATION OF NEGATIVE CO-STIMULATORY MOLECULE TIM-3,PD-1,LAG-3,CTLA-4 EXPRESSION,GENE POLYMORPHISM AND SEVERE HAND,FOOT,AND MOUTH DISEASEObjective:To explore the expression of negative co-stimulator molecular Tim-3,PD-1,LAG-3,CTLA-4 mRNA in PBMCs and the levels of serum soluble molecular in severe hand,foot,and mouth disease,and determine whether there is correlation between negative co-stimulator molecular and clinical status of HFMD.Furthermore,the other objective of this study is to investigate the relationship of single nucleotide polymorphisms in Tim-3,LAG-3,PD-1,CTLA-4 gene and severity of Enterovirus 71 infectious HFMD,and to evaluate susceptibility to Enterovirus 71 infectious HFMD.Methods:1.From January 2014 to January 2016,ACD-anticoagulated venous blood of 200 cases with hand,foot and mouth disease and healthy controls were collected,Tim-3,PD-1,LAG-3,CTLA-4 mRNA expression in peripheral blood mononuclear cells(peripheral blood mononuclear cell,PBMC)were analyzed by quantitative real-time PCR(qRT-PCR).2.The serum of 411 cases of children with HFMD were collected and divided into six groups:30 cases of the death group,58 cases of the survival group,145 cases of the severe group,68 cases of the mild group,60 cases of the recessive infection group and 50 cases of surgical inpatients without infection as the normal control group.The level of sTim-3,sPD-1,sLAG-3,sCTLA-4 were explored by quantitative antibody microarray.The difference between all groups were compared,and the relationship between serum levels of negative co-stimulator molecular and disease severity was analyzed.3.In this study,145 patients with HFMD and 104 cases with recessive infection of EV71,89 health controls were recruited.The polymorphisms were detected by imLDRTM.The alleles and genotypes frequency between groups were analyzed by apposite statistical methods.Results 1.Tim-3 mRNA expression of the death group and the survival group increased comparing with the mild group and the normal control group(P<0.05).2.PD-1 mRNA of the death group increased significantly comparing with the normal control group(P<0.05),the survival group increased significantly comparing with the severe group,the mild group and normal control group(P<0.05).LAG-3 mRNA of the death group increased significantly comparing with the recessive infection group and normal control group(P<0.05).CTLA-4 mRNA of the death group and the survival group increased significantly comparing with the severe group,the mild group and normal control group.There was no difference between the death group and the survival group.2.The level of sTim-3 in the death group was 28.8 pg/ml higher than survival group,severe group,mild group,the recessive infection group and control group(P<0.05),while sTim-3 of the recessive infection group increased significantly than normal control group(P<0.05).The levels of sPD-1 in death group and survival group were 665.40 pg/ml and 519.45 pg/ml,obviously higher than the mild group and normal control group(P<0.05),sPD-1 of the recessive infection group was 605.00 pg/ml higher than normal control group(P<0.05).The level of sLAG-3 in death group was 70.50 pg/ml increased significantly compare with the severe group,the mild group,the recessive infection group and the normal control group(P<0.05),sLAG-3 levels of the recessive infection group was 27.80 pg/ml higher than normal control group(P<0.05).The level of sCTLA-4 in the death group was 9.80 pg/ml higher than the severe group and the recessive infection group(P<0.05).The frequencies of genotypes TT and allele T in Tim-3 rs13170556 polymorphism were significantly higher in cases of HFMD and the recessive infection group than the control group(P<0.05).The frequencies of genotypes TT in PD-1 rs10204525 polymorphism were significantly higher in the recessive infection group than the control group(P<0.05).LAG-3 gene polymorphisms were distributed similarly between the HFMD group and recessive infection group comparing with the control group.The frequencies of allele G in CTLA-4 rs231775 polymorphism were significantly higher in cases of the control group than the recessive infection group(P<0.05).The frequencies of allele G in CTLA-4 rs231775 polymorphism were significantly higher in cases of the survival group than the death group(P<0.05).Conclusion:Negative co-stimulator molecular Tim-3,CTLA-4,PD-1,LAG-3 may play an important role in T cell exhaustion to impair the immunity of host in hand,foot and mouth disease.Genotypes TT and allele T in Tim-3 rs13170556 polymorphism is associated with susceptibility to EV71 infected HFMD in Chinese patients.The allele G of CTLA-4 rs231775 polymorphism may have negative effects on prognosis of HFMD by modifying the expression and functions of CTLA-4. |
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