| Part I: The Predictive Value of IL-21 for Recurrence of CHB Patients with HBe Ag-Positive after Discontinuation of EntecavirObjectives: To determine the screening factors in virological relapse and the predictive values of IL-21 and the related Th17 / Th1 / Th2 cytokines for recurrence of chronic hepatitis B(CHB)patients with HBe Ag-positive after discontinuation of entecavir(ETV).Methods: Firstly,we selected 112 CHB patients who meet the withdrawal criteria according to the 2012 Asian-pacific consensus statement on therapy of chronic hepatitis B.We recorded the clinical data within or after ETV treatment,which includes gender,age,genotype,the baseline level of ALT,HBV DNA and HBs Ag,the virological response time(weeks),and the HBs Ag level of HBe Ag seroconversion.Secondly,we detected the serum levels of IL-21 by ELISA and other cytokines of Th1(IL-2?IFN-??TNF-?),Th2(IL-4,IL-6,IL-10),Th17(IL-17A)by cytometric bead array(CBA)in the process of drug withdrawal.We further analyzed the relationship between the serum levels of IL-21 and other detected cytokines with the virological relapse of drug withdrawal.The Cox regression was performed to determine the independent prognostic factors for recurrence and the ROC curve was used to define the best cut-off value.Results:1.After 52 weeks of ETV discontinuation,the recurrence rate was 48.2%(54/112),and the 74%(40/54)recurrence occurred between 12 to 36 weeks after drug withdrawal.2.The age between the relapse group(VR)and the sustained virological response(SVR)was significantly different(P<0.001).The recurrence rates of Age<30,Age of 30-39,Age of 40-49,and Age?50 were 25.0%(8/32),6.7%(11/30),54.2%(13/24)and 84.6%(22/26)respectively,the difference was significantly among Age?50 group and the other three groups.3.The cut-off value of HBs Ag level was 2.5log10IU/ml for HBe Ag seroconversion from 112 CHB patients.AUC was 0.751 according to ROC curve.The recurrence rate of HBs Ag<2.5 patients when HBe Ag seroconvert was 9.5%(2/21)and the HBs Ag levels of 52.4% HBs Ag<2.5 patients were less than 2.0log10IU/ml at the time of drug withdrawal.However,the recurrence rate of HBs Ag?2.5log10 IU /ml patients when HBe Ag seroconvert was 57.1%(52/91).4.At the time of drug withdrawal,the serum level of IL-21 of the sustained virological response(SVR)group was significantly higher than that of recurrence group(VR)(P =0.041).The cut-off value of serum IL-21 levels was 50pg/ml and AUC was 0.811 according to ROC curve.The recurrence rate of patients with serum IL-21 levels ?50pg/ml and <50pg/ml was 25.4%(16/63)and 74.6%(38/49),respectively(P < 0.001).5.The serum levels of IFN-?(Th1)in SVR patients were significantly higher than those in VR patients(P=0.004).And the serum levels of IL-10(Th2)in SVR patients were significantly lower than that in VR patients(P=0.001).The comparison of serum levels of IL-17A(Th17)between the two groups was without significance(P>0.005).6.The prognostic factors of age(P=0.008),serum HBs Ag levels when HBe Ag seroconvert(P=0.019),serum IL-21 levels(P=0.034)and serum HBs Ag levels at the time of drug withdrawal(P=0.045)were closely related to the recurrence after ETV discontinuation according to the Cox regression model.Conclusions:1.Age,serum HBs Ag levels at HBe Ag conversion,serum IL-21 levels and serum HBs Ag levels at the time of drug withdrawal are closely related to the recurrence after ETV discontinuation.2.Patients with Age?50,the serum level of HBs Ag?2.5log10IU/ml at HBe Ag conversion,HBs Ag?2.0log10IU/ml or IL-21<50pg/ml at the time of drug withdrawal have a higher recurrence rate.Part II: The Role of IL-21 in Maintaining Virological Responses after ETV DiscontinuationObjectives: To explore the characteristics underlying IL-21 expression level after ETV discontinuation of CHB patients with HBe Ag-positive and define the role of IL-21 in maintaining virological response after ETV discontinuation.Methods: We conducted a cross-sectional study which included 20 patients with immune tolerance(IT),inactive HBs Ag carriers(IC)and ETV withdrawal(ETV-W).As many as 20 healthy volunteers were selected as controlled group.The protein level of serum IL-21 was detected by ELISA and the m RNA level of PBMC IL-21 by Real-time PCR.Other cytokines of Th1(IL-2?IFN-??TNF-?),Th2(IL-4,IL-6,IL-10),Th17(IL-17A)were examined by cytometric bead array(CBA).We also conducted the longitudinal study which included 78 patients with ETV discontinuation,and we separated them into virology sustained response(SVR)and virological relapse(VR)according to the recurrence in 52 weeks.We detected serum level of IL-21 and Th1/h2/h17 related cytokines during follow-up to further analyze their dynamic changes and expression differences.Results:1.The result of cross-sectional study indicates that serum levels of IL-21 in both groups of IC and ETV-W are higher than the IT group with statistical significance(both P values < 0.001).The result of longitudinal study reveals that serum levels of IL-21 in SVR group stand at a higher level compared to VR group,whose serum level of IL-21 decreased during 52 weeks after ETV discontinuation.2.There is no significant correlation between serum levels of IL-21 and HBs Ag(r=-0.057,P=0.318).However,the correlation in SVR group is significantly negative(r=-0.241,P<0.001).On the contrary,the correlation in VR group is significantly positive(r=0.286,P<0.001).3.The result of cross-sectional study indicates that serum levels of IFN-?(TH1)in both groups of IC and ETV-W are higher than IT group with statistical significance(all P < 0.001).And the serum levels of IL-10 in the groups of ETV-W and IC are lower than that in IT group with significance(All P values < 0.001).4.The result of longitudinal study reveals that serum levels of IFN-? in the group of SVR stand at a higher level compared with VR group during follow-up(P=0.004 and all P < 0.001,respectively)..While,the serum level of IL-10 decreased in SVR group and was lower than the paired IL-10 level in VR group with significance during follow-up.In VR group,the serum level of IFN-? decreased during the first 24 weeks after ETV withdrawal,and after then,the level rose and maintained at baseline level in 52 weeks after ETV discontinuation(P=0.001 and all P < 0.001,respectively)..Conclusions:1.The serum level of IL-21 in SVR group maintained a high level after ETV withdrawal,and negatively correlated with the serum level of HBs Ag which indicated that IL-2l may play an important role in maintaining virological responses in CHB patients of HBe Ag-positive as an immunomodulatory factor.2.The serum level of IFN-?(Th1)in SVR group maintained a high level is conducive to maintaining virological response after ETV withdrawal and high level of IL-10 may be related to virologic relapse.Part III: The Role of CXCR5+CD4+T Cells Secreting IL-21 in Maintaining Virological Responses after ETV DiscontinuationObjectives: To shed light on the correlation between the serum level of IL-21 and the frequency of CXCR5+CD4+T cells and analyze the role of CXCR5+CD4+T cells secreting IL-21 in maintaining virological responses after ETV discontinuation.Methods: We detected the frequency of CXCR5+CD4+T cells of patients with immune tolerance(IT),inactive HBs Ag carriers(IC)and ETV withdrawal(ETV-W)respectively to understand the frequency of CXCR5+CD4+T cells in different periods of HBV infection by means of flow cytometry(FCM).The longitudinal study was conducted to define the correlation between the serum level of IL-21 and the frequency of CXCR5+CD4+T cells after ETV withdrawal.We further performed intracellular cytokine staining after stimulation with PMA/ionomycin and HBV peptides in an effort to determine the frequency of nonspecific and HBV-specific IL-21 producing CXCR5+CD4+T cells.Results:1.The frequency of CXCR5+CD4+T cells is higher in the groups of ETV-W than IT and HC groups with both P values of less than 0.05.2.The frequency of CXCR5+CD4+T cells decreased of 70.3%(26/37)patients in VR group after ETV withdrawal.However,the proportion of patients whose frequency of CXCR5+CD4+T cells decreased was 41.5%(17/41)in SVR group,lower than the proportion in VR group with significance(P=0.013).3.The frequency change of CXCR5+CD4+T cell was consistent with the serum level variation of IL-21,and was positively correlated after ETV discontinuation(r=0.626,P<0.001).4.With the stimulation of PMA/ionomycin,CXCR5+CD4+T cells producing IL-21 was obviously higher than IFN-?,IL-4 and IL-17(all P<0.001).5.With the stimulation of HBV peptides,the frequency of HBV-specific IL-21 producing CXCR5+CD4+T cells in ETV-W group was significantly higher than that in the groups of IT and HC.And the frequency was higher in SVR group compared with VR group.Conclusions: The frequency of CXCR5+CD4+T cell was positively correlated with the serum level of IL-21 after ETV discontinuation and the frequency of HBV-specific IL-21 producing CXCR5+CD4+T cells in SVR group was higher than VR group.These results suggest that CXCR5+CD4+T cells may play an important role through secreting IL-21 in maintaining virological responses after ETV discontinuation. |
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