| Hepatitis C virus (HCV) infection is a growing public health problemwith roughly180million of the population infected worldwide, and theprevalence of HCV antibody is3.2%in China. Approximately50%~80%ofthe patients infected with HCV will develop chronic infection. Chronic HCVinfection can progress towards more severe outcomes,10%~15%in the formof cirrhosis after20years and1%~7%in the form of hepatocellular carcinomaannually in patients with cirrhosis. HCV infection is a serious and growingthreat to public health.Interferon (IFN) in combination with ribavirin (RBV) remains aneffective therapy for chronic HCV infection. The treatment duration for HCVgenotype2/3is24weeks and for HCV genotype1is48weeks. However,only55%~60%of chronic hepatitis C (CHC) patients with HCV genotype2/3can achieve sustained virological response (SVR) and the SVR rate is38%~41%in patients with HCV genotype1. So the infection with HCVgenotype1is a cause for the refractory of CHC. Most of patients with CHC inChina are middle-aged and elder, some of them have developed cirrhosis orwith other complications. In addition, because IFN/RBV treatment is relatedto multiple adverse reactions,30~40%of CHC patients can’t tolerate thestandard therapy. Thus, it is important to explore the individualized antiviralstrategy with more efficacy and fewer adverse reactions.Both innate and adaptive immunity have a profound impact on status ofHCV infection and antiviral therapy response. The change of host cellularimmunity before and after treatment may relate to early and sustainedvirological response. Observation the expression changes of immune factorson peripheral monocytes and T cells may contribute to optimizing the antiviral treatment. The related immune factors include Toll-like receptor (TLR)expressing monocytes, programmed death-1(PD-1) expressing T cells,regulatory T cell (Treg) and T cell immunoglobulin and mucin domain-3(Tim-3)/Galectin-9(Gal-9) pathway. Thus, it is important to identify thedynamic changes and modulation mechanisms of these immune mediators forthe forecast of antiviral treatment response and prognosis.In the present study, we aim to explore the individualized antiviralstrategy based on the clinical characters and the immune status of CHCpatients in China, to clarify the role of immune mediators in treatmentresponse by assessing their baseline levels and dynamic changes duringtreatment, and to elucidate the potential antiviral effects of different immuneregulatory manners.Part1Study on the individualized antiviral treatment strategy in patientswith chronic hepatitis CObjective: To explore the individualized antiviral strategy for CHCpatients in China for achieving high rate of virological response withimproved compliance.Methods: This open-label, prospective clinical trial included280CHCpatients recruited from Third Hospital of Hebei Medical University, the FifthHospital of Shijiazhuang City, Bethune International Peace Hospital, XingtaiPeople’s Hospital and Handan Infections Disease Hospital from January2010to December2012. The contaminated ways were investigated. Patientsreceived individualized dosages of IFNα-2b or PegIFNα-2a combined withRBV treatment based on their ages (<65years or≥65years), weights (<60kgor≥60kg), complications (e.g. liver cirrhosis, diabetes, heart disease, thyroiddisorder, etc.) and economic capacity. Treatment duration was determinedbased on the time required to induce virological responses. The rates ofvirological responses and adverse effects among different groups werecompared. HCV genotypes were assessed, and the rates of virologicalresponses and adverse events were compared between patients with differentHCV genotypes. Results: Among280patients,203(72.5%) subjects were contaminatedby transfusion, and30(10.7%) were contaminated by other contaminatedways including previous surgery/injury, stomatologic treatments, acupuncture,endoscopic examination and ear piercing, while47(16.8%) cases werecontaminated in unknown ways. The incidences of rapid virological response(RVR), complete early virological response (cEVR), SVR and adverse eventsin overall cases treated with IFNα-2b (n=171) were52.0%,71.3%,57.9%and7.0%respectively, and the rates were69.7%,89.9%,74.1%and8.3%respectively in patients given PegIFNα-2a (n=109). The incidences of RVRand cEVR were significantly higher in PegIFNα-2a group than those inIFNα-2b group (P <0.01). The RVR, cEVR, SVR and adverse events rates inthe patients given low-dose IFNα-2b (n=68) were44.1%,69.1%,57.1%and10.3%respectively, and they were57.3%,72.8%,60.0%and4.9%respectively in cases treated with routine-dose IFNα-2b (n=103). Theincidences of RVR, cEVR, SVR and adverse events were no significantdifference between the two groups (P>0.05). The RVR, cEVR, SVR andadverse events rates of subjects treated with low-dose PegIFNα-2a (n=48)were66.7%,85.4%,68.8%and12.5%respectively, while they were72.1%,93.4%,81.8%and4.9%respectively in patients received routine-dosePegIFNα-2a (n=61), and those rates were not significantly different betweenthe two groups (P>0.05). The RVR, cEVR, and adverse events rates of CHCpatients with compensated cirrhosis (n=16) were31.3%,81.3%and6.3%respectively, while they were60.6%,78.4%and7.6%respectively in patientswith non-cirrhosis (n=264). The incidences of RVR were significantly higherin non-cirrhosis patients than it in patients with compensated cirrhosis (P <0.05). There were62.3%of146CHC patients infected with HCV genotype1b.The RVR, cEVR and adverse events rates in patients infected with HCVgenotype1were53.2%,71.6%and8.3%respectively, while they were86.5%,83.8%and8.1%respectively in patients with non HCV genotype1, and theRVR rate was significantly higher in non genotype1group (P <0.001).Conclusion: Individualized treatment strategy according to CHC patients’ baseline condition, complications and viral kinetics could achievehigh rate of virological response, improved compliance, prognosis andcost-effectiveness.Part2Dynamic changes of Tregs and PD-1, TLRs expressing cell subsetsand their relationship with antiviral treatment response in patients withHCV infectionObjective: To clarify the dynamic changes of immune mediators ondifferent peripheral subset cells and their relationship with antiviral treatment.Methods: Seventy CHC patients (18~65years old) withoutcomplications were treated either with IFNα-2b plus ribavirin (IFNα-2b/RBV)(n=37) or with PegIFNα-2a plus RBV (PegIFNα-2a/RBV)(n=33) for up to48~72weeks. Twenty age and gender matched healthy subjects were used ascontrols. Frequencies of peripheral CD4+CD25+FoxP3+Tregs, PD-1expressing CD4+T-cells or CD8+T-cells and TLR2, TLR3or TLR4expressing CD14+monocytes were evaluated by flow cytometry at baseline,12,24and48weeks following treatment. The relationship between thedynamic changes and cEVR was analysed.Results: At baseline, frequencies of Tregs (P <0.05), PD-1expressingCD4+T-cells (P <0.05), PD-1expressing CD8+T-cells (P <0.01), TLR2(P<0.05), TLR3(P <0.001) and TLR4(P <0.05) expressing CD14+monocytes were significantly higher in CHC patients compared with thecontrol subjects. When comparing with their baseline levels, Tregs and PD-1expressing CD8+T-cells (P <0.05) were decreased at12weeks in CHCpatients receiving IFNα-2b/RBV or PegIFNα-2a/RBV, and the PD-1expressing CD8+T-cells were further reduced at24weeks (P <0.05), whilethe PD-1expressing CD4+T-cells were significantly decreased at48weeks(P <0.01). TLR2, TLR3or TLR4expressing CD14+monocytes wereincreased (P <0.05) at12weeks in patients receiving IFNα-2b/RBV orPegIFNα-2a/RBV, but returned to baseline levels at24and48weeks. Therate of patients who achieved cEVR in IFNα-2b/RBV group was73.5%(25/34), whilst78.1%(25/32) patients treated with PegIFNα-2a/RBV achieved cEVR. Multivariate analysis identified low baseline TLR3expressing CD14+monocytes as a single predictor of cEVR [odds ratio (OR)=2.11,95%confidence intervals (CI)(1.15-3.88), P <0.05]. Patients withcEVR showed lower Tregs (P <0.01), PD-1expressing CD4+T-cells (P <0.01) and PD-1expressing CD8+T-cells (P <0.05) at12weeks and lowerTLR3(P <0.05), TLR4(P <0.05) expressing CD14+monocytes atbaseline than patient without cEVR.Conclusions: Low peripheral TLR3expressing CD14+monocytes atbaseline could serve as a predictor for cEVR of antiviral therapy in chronicHCV-infected patients. Interferon plus ribavirin could achieve cEVR throughreducing blood CD4+CD25+FoxP3+regulatory T-cells, PD-1expressing CD4+or CD8+T-cells and increasing TLR3or TLR4expressing CD14+monocytes,which provided a new therapeutic target for CHC.Part3Immune modulation role of Tim-3/Gal-9pathway in patients withchronic hepatitis CObjective: To explore the role of Tim-3/Gal-9pathway in chronichepatitis C and the mechanism for its cellular immune modulation function.Method: Forty CHC patients (18~65years old) without complicationswere treated with interferon combined with ribavirin. Twenty age and gendermatched healthy subjects were used as controls. Levels of plasma Gal-9andInterleukin-12(IL-12) were evaluated by ELISA in healthy controls andpatients at baseline,12,24and48weeks following treatment and24weeksafter the end of treatment. The peripheral blood mononuclear cells (PBMC)isolated from whole blood of15patients and15age and gender matchedhealthy subjects were divided into five groups: Control: PBMC were nottreated with any agonist nor antagonist; TLR4agonist group (LPS): PBMCwere treated with LPS; Tim-3antagonist+TLR4agonist group (Anti-Tim-3+LPS): PBMC were treated with anti-Tim-3antibody for30min andsupplemented with LPS; TLR3agonist group [Poly(I:C)]: PBMC were treatedwith Poly(I:C); Tim-3antagonist+TLR3agonist group [Anti-Tim-3+Poly(I:C)]: PBMC were treated with anti-Tim-3antibody for30min and supplemented with Poly(I:C). After incubated for24hours, the levels of IL-12,IFN-α and tumor necrosis factor-alpha (TNF-α) in the supernatant wereevaluated by ELISA and the mRNA expressions of2’-5’ oligoadenylatesynthetase (2’-5’OAS), myxovirus resistance protein A (MxA) and suppressorof cytokine1(SOCS1) extracted from PBMC were detected by quantitativereal-time PCR.Results: Level of plasma Gal-9was significantly higher in CHCpatients compared with the control subjects at each time point (P <0.05).Comparing with its baseline level, plasma Gal-9level was decreased at12,24and48weeks during treatment (P <0.05), but returned to baseline level at24weeks after the end of treatment. There was no significant differenceamong the time points during and after treatment (P>0.05). No significantdifference of the Gal-9level was observed between patients with cEVR andnon-cEVR as well as between SVR and non-SVR at each time point (P>0.05). With respect to the level of plasma IL-12, there was no significantdifference between CHC patients and control subjects (P>0.05), but it wassignificantly higher in CHC patients at12weeks after treatment comparedwith the control subjects (P <0.05) and its baseline level (P <0.01), and itreturned to baseline level at24and48weeks following treatment and24weeks after the end of treatment. No difference of plasma IL-12level wasobserved between patients with cEVR and non-cEVR as well as betweenSVR and non-SVR at each time point (P>0.05). The levels of IL-12inPBMC supernatant of the five groups were low in CHC and healthy subjects,and there was no significant difference among these groups (P>0.05). Theexpression levels of antiviral factors IFN-α,2’-5’OAS and MxA were higherin the four treated groups than those in the Control group (P <0.05). Thelevels of inflammatory cytokine TNF-α and negative immunoregulatorSOCS1were higher in LPS, Poly(I:C) and Anti-Tim-3+LPS groupscompared with Control group (P <0.05). The mRNA expression levels of2’-5’OAS and MxA were higher while the mRNA expression level of SOCS1was lower in Anti-Tim-3+LPS group than LPS group (P <0.05). IFN-α, 2’-5’OAS and MxA expression levels were higher in Anti-Tim-3+Poly(I:C)group than those in Poly(I:C) group (P <0.05) and Anti-Tim-3+LPS group(P <0.05), while SOCS1mRNA expression level was lower inAnti-Tim-3+Poly(I:C) group than Poly(I:C) group (P <0.001). The amountof supernatant TNF-α was lowest in Anti-Tim-3+Poly(I:C) group but it wasnot significantly different compared with other groups (P>0.05). Comparedwith healthy subjects, the levels of IFN-α and2’-5’OAS were lower inAnti-Tim-3+LPS group and the2’-5’OAS mRNA expression level was lowerin Anti-Tim-3+Poly(I:C) group in CHC patients (P <0.05). The levels ofMxA and TNF-α had no significant difference between CHC patients andhealthy subjects in any group (P>0.05), while the SOCS1mRNAexpression level was higher in CHC patients than healthy subjects (P <0.05)in all groups except in Anti-Tim-3+Poly(I:C) group.Conclusions:1The level of plasma negative immunoregulator Gal-9in CHC patientswas significantly elevated. Antiviral treatment could increase the level ofimmune mediator IL-12at early stage. Gal-9expression level was decreasedfollowing the treatment, but increased at24weeks after the end of treatment.2The blocked of Tim-3combined with TLR ligands especially TLR3agonists could induce the expression of antiviral molecules and reduce thelevels of negative immunoregulator, which might provide a possibletherapeutic target for CHC. |
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