| Hepatitis C virus(HCV) infection remains a big threatening to public health. According to the statistics by world health organization(WHO), approximately 185 million people infected HCV worldwide, and thrity-five thousand people infected annually. The sum of HCV-related death is nearly 350 thousand, for the fifth of all infectious diseases caused death. The rates of HCV infection increase with years in our country, and 220 thousand new cases were reported in 2013. The virus usually persists, and among 55%~85% of the patients with HCV develop chronic hepatitis C(CHC), that is closely associated with cirrhosis and hepatocellular carcinoma(HCC). So timely and effective treatment is benefit for preventing progression of the disease and improving the quality of life of the patients.Recently, the European and American countries have successfully developed many categories of directing antiviral agents(DAAs), and satisfactory results were obtained after clinical application. But the DAAs are expensive, and their long-term effectiveness need follow-up study, make the interferon(IFN) and ribavirin(RBV) dual therapy remain the standard of care for CHC in China. According to many researches, the sustained virological reponse(SVR) rates of full-dose and overall-period of treatment were up to 78%~93.5%. But the side-effects and bad tolerance of the combination therapy, makes optimization of treatment strategy essential for improving patients compliance and SVR rates for Chinese HCV-infected patients.Both innate and adaptive immunity have close relationship with the prognosis of HCV infection and the response to antiviral therapy. Especially the weak status of HCV-specific cellular immune response, may serve as the main factor for chronic infection. T helper 17(Th17) cells which produce interleukin-17(IL-17) as its main effector play an important role in immune-mediated inflammatory diseases. IL-23, the upstream factor of Th17 cells, which modulation is related to the survival and population expansion of Th17 cells. Many reports have demonstrated that IL-23 is involved in immune and inflammatory response in chronic liver diseases. But the effect of IL-23 modulation on the expressions of Th17 cells and their related antiviral molecules in chronic HCV infection remains unclear. Therefore, the studies of baseline plasma IL-23 levels, Th17 cells function in peripheral blood mononuclear cells(PBMCs) as well as the modulation of IL-23/IL-17 axis and their relationship with antiviral response in HCV-infected patients, are very important for the optimization of treatment strategy and determination of prognosis. Part 1 Study on the optimization of treatment strategy in patients withchronic hepatitis CObjective: To select the individual antiviral strategy for chronic HCV-infected patients in China for improving the cure rate of IFN or peg-IFN α and the compliance of patients.Methods: This open-label, prospective clinical study recruited 652 HCV-infected patients from Third Hospital of Hebei Medical University, Xingtai People’s Hospital, Handan Infections Disease Hospital, Bethune International Peace Hospital, Cangzhou Infections Disease Hospital, First Hospital of Hebei Medical University, the Fifth Hospital of Shijiazhuang City, Weichang County Hospital from January 2010 through March 2015. Patients received different treatment strategies based on their ages(< 65 years or ≥ 65 years), weights(< 60 kg or ≥ 60kg), complications(e.g. liver cirrhosis, diabetes, heart disease, thyroid disorder, etc.) and economic capacity. Routine-dose group: patients received IFNα-2b 500 MIU by subcutaneous injection every other day or pegylated interferon(Peg IFN) α-2a 180μg, Peg IFNα-2b 80μg by subcutaneous injection once a week respectively plus RBV 1200mg/d; low-dose group: patients received IFNα-2b 100 MIU ~300MIU by subcutaneous injection every other day or Peg IFNα-2a 67.5~135μg, Peg IFNα-2b 50μg by subcutaneous injection once a week respectively plus RBV 900mg/d. Total periods of treatment were made according to the time virological responses occurred, that is to say HCV RNA load detected below lowest detection limit should continue treatment for 44 weeks and keep follow-up for 24 weeks after the end of treatment. Blood routine, blood biochemicals, oral glucose tolerance test(OGTT), HCV RNA load, erythrocyte sedimentation rate, three factors associated with rheumatism, autoantibodies, thyroid functions, functions of T lymphocytes subsets, four fibrosis indexes, alpha-fetoprotein and abdominal ultrasound examination should be monitored before and during treatment. The rates of virological responses in different treatment groups, included rapid virological response(RVR), complete early virological response(c EVR) and sustained virological response(SVR) were analyzed; related factors included host factors, viral factors(viral load, genotypes), the time of treatment response and adverse effects, included influenza-like illness, evanescent myelosuppression, Thyroid dysfunction, mental disorder, tetter and so on were also assessed.Results:1 General characteristics of the patients Among the 652 patients, 482 subjects(73.93%) were contaminated by transfusion, 73(11.20%) were contaminated by previous surgery/injury, and others were 97 subjects(14.87%). A total of 364 HCV-infected patients underwent HCV genotype assay, of which genotype 1b was 260(71.43%) and genotype 2a was 76(20.88%). A total of 240 patients underwent IFNα-2b treatment, of which low-dose group was 109 subjects, included 50 male and 59 female, the average age was 49.56±12.45 years old, the average Body mass index(BMI) was 23.57±3.32 kg/m2; the routine-dose group was 131 subjects, included 53 male and 78 female, the average age was 45.92±10.23 years old, the average BMI was 24.14±3.22 kg/m2. A total of 357 patients underwent Peg IFNα-2a treatment, of which low-dose group was 92 subjects, included 33 male and 59 female, the average age was 50.83±13.41 years old, the average BMI was 23.72±3.46 kg/m2; the routine-dose group was 265 subjects, included 120 male and 145 female, the average age was 46.73±11.36 years old, the average BMI was 23.69±3.04 kg/m2. A total of 55 patients underwent Peg IFNα-2b treatment, of which low-dose group was 29 subjects, included 16 male and 13 female, the average age was 51.19±13.70 years old, the average BMI was 22.83±2.78 kg/m2; the routine-dose group was 26 subjects, included 12 male and 14 female, the average age was 48.20±13.44 years old, the average BMI was 24.32±2.35 kg/m2, there was no significant difference in sex, age, BMI, baseline ALT, HCVRNA load or HCV genotype between the routine-dose and low-dose groups above(P > 0.05).2 Treatment responses The RVR, c EVR and SVR rates in the patients received routine-dose and low-dose of IFNα-2b were 64.12%, 90.08%, 80.68% and 41.28%, 68.81%, 71.43% respectively. The incidences of RVR, c EVR were significantly higher in routine-dose group than those in low-dose group(P all < 0.001). The RVR, c EVR and SVR rates of patients treated with routine-dose and low-dose of Peg IFNα-2a were 74.34%, 94.34%, 88.84% and 63.04%, 83.70%, 83.10% respectively. The incidences of RVR, c EVR were significantly higher in routine-dose group than those in low-dose group(P < 0.05, P < 0.01, respectively). The RVR, c EVR and SVR rates of subjects received routine-dose and low-dose of Peg IFNα-2b were 73.08%, 100%, 100% and 34.48%, 79.31%, 64.71% respectively. The incidences of RVR, c EVR and SVR were significantly higher in routine-dose group than those in low-dose group(P < 0.01, P < 0.05, P < 0.05, respectively). The RVR, c EVR and SVR rates in overall cases treated with IFNα-2b were 53.75%, 80.42% and 76.82% respectively, were 71.43%, 91.60% and 87.58% respectively in patients given Peg IFNα-2a, and were 52.73%, 89.09% and 80.00% respectively in patients received Peg IFNα-2b. The incidences of RVR, c EVR and SVR were significantly different among three types of IFNs(P < 0.001, P < 0.001, P < 0.05, respectively). The RVR rate of Peg IFNα-2a group was significantly higher than that of IFNα-2b group and Peg IFNα-2b group(P < 0.001, P < 0.01, respectively), while the c EVR and SVR rates of Peg IFNα-2a group were markedly higher than those in IFNα-2b group(P < 0.001, P < 0.05, respectively). The RVR, c EVR and SVR rates of HCV patients with compensated cirrhosis(n=38) and non-cirrhosis(n=614) were 34.21%, 57.89%, 66.67% and 65.15%, 89.09%, 84.87% respectively. The incidences of RVR, c EVR and SVR were significantly higher in non-cirrhosis patients than those in patients with compensated cirrhosis(P < 0.001, P < 0.001, P < 0.05, respectively). The RVR, c EVR, SVR rates in patients infected with HCV genotype 1(n=282) and with non HCV genotype 1(n=82) were 56.38%, 84.75%, 81.40% and 79.27%, 92.68%, 85.94% respectively, the RVR rate was significantly higher in non-genotype-1 group(P < 0.001). The RVR, c EVR and SVR rates in genotype-1 patients(n=94) and non-genotype-1 patients(n=33) treated with IFNα-2b were 43.62%, 72.34%, 70.91% and 72.73%, 93.94%, 86.36% respectively, the RVR and c EVR rates were significantly higher in non-genotype-1 group(P < 0.01, P < 0.05, respectively). The RVR, c EVR and SVR rates in genotype-1 patients(n=163) and non-genotype-1 patients(n=39) treated with Peg IFNα-2a were 65.64%, 92.02%, 86.99% and 87.18%, 92.31%, 86.49% respectively, the RVR rate was significantly higher in non-genotype-1 group(P < 0.01). The RVR, c EVR and SVR rates in genotype-1 patients(n=25) and non-genotype-1 patients(n=10) treated with Peg IFNα-2b were 44.00%, 84.00%, 64.29% and 70.00%, 90.00%, 80.00% respectively, the RVR, c EVR and SVR rates were not significantly different between genotype-1 group and non-genotype-1 group(P > 0.05).3 Adverse events The adverse events rates in the patients received IFNα-2b, Peg IFNα-2a and Peg IFNα-2b were 5.42%, 9.24% and 3.64% respectively; the adverse events rates of HCV patients with compensated cirrhosis and non-cirrhosis were 2.63% and 6.65% respectively; the adverse events rates in the patients infected with HCV genotype 1 and with non HCV genotype 1 were 5.03% and 7.41% respectively;the adverse events rates in the patients received low-dose and routine-dose of IFNα-2b were 7.34% and 3.82% respectively, of Peg IFNα-2a were 12.12% and 5.66% respectively, of Peg IFNα-2b were 3.45% and 3.48% respectively, there was not any significant difference between or among the groups above(P > 0.05).Conclusion: Optimization of antiviral treatment strategy based on the age, weight, complications and economic capability of patients, and modulation of the period of treatment based on the RVR could improve SVR rate, toleration, compliance and prognosis. Part 2 The role of Th17 cells in chronic hepatitis C and their relationship with IFN/peg IFN+RBV treatment responseObjective: To clarify the dynamic changes of Th17 subsets and Th17 immune-associated cytokines, and determine the correlations with antiviral treatment response.Methods: A total of sixty-six patients with chronic HCV infection were treated with IFNα-2b/RBV(n=13) or Peg IFNα-2a/RBV(n=53) for 48~72 weeks. Twenty age- and sex-matched healthy people were selected as the controls. Frequencies of IL-17 A, IFN-γ and IL-21-producing PBMCs were determined by Flow Cytometry at baseline, 12, 24 and 48 weeks following treatment, and their relationships with treatment responses were evaluated.Results: The frequencies of IL-17A(median was 4.16%) and IFN-γ-producing PBMCs(8.17%) were dramatically higher while IL-21-producing PBMCs(2.15%) were lower in the HCV patients at baseline than those in the healthy controls(1.18%, 1.38%, 4.51%, respectively), P all < 0.001. After IFNα-2b/RBV or Peg IFNα-2a/RBV administration, 54.5% achieved RVR(39/66) and 89.4% achieved c EVR(59/66). The frequencies of IL-17A(1.00%) and IFN-γ-producing PBMCs(3.26%) were both markedly decreased by 12 weeks after treatment(P < 0.001, P < 0.01, respectively). There was not significant difference between 24(1.57%) or 48 weeks(0.90%) with 12 weeks in the frequencies of IL-17A-producing PBMCs(P > 0.05). Slight reduction was seen in IFN-γ-producing PBMCs at 24 weeks(1.83%) of treatment(P > 0.05), and at 48 weeks(1.51%)showed marked difference as compared with 12 weeks(P < 0.001). IL-21-producing PBMCs increased in 12 weeks of treatment(4.01%, P < 0.05), and decreased to near baseline levels by 24 weeks(1.91%, P < 0.001), and further reduced at 48 weeks of treatment compared with baseline(1.44%, P < 0.05). The frequencies of IL-21-producing PBMCs were obviously higher in patients with RVR(2.18%) than without RVR(1.88%) at baseline(P < 0.05).Conclusions: Interferon in combination with ribavirin could achieve virological response through reducing IL-17 A and IFN-γ-producing PBMCs and increasing IL-21-producing PBMCs together with their antiviral activity, which might be a new approach for chronic hepatitis C treatment. Part 3 Immune modulation role of IL-23 on Th17 cells and the expression of antiviral molecules in chronic hepatitis CObjective: To clarify the plasma IL-23 levels of chronic HCV-infected patients and the effect of IL-23 modulation on Th17 activity, to develop the targeting immunotherapy for paitents with chronic HCV infection.Method: The plasma IL-23 levels of 55 HCV-infected patients and 20 age- and sex-matched healthy subjects were assessed by enzyme-linked immunosorbent assay(ELISA). PBMCs were isolated from fresh blood samples of 24 HCV patients and 15 healthy subjects using Ficoll density gradient centrifugation. Then PBMCs were divided into 3 groups: Control group, not treated with any agonist or antagonist; IL-23 agonist group, treated with human IL-23 recombinant protein; IL-23 antagonist group, treated with anti-human IL-23 p19 functional grade purified. All of these groups were incubated in 5% CO2 at 37℃ for 48 hours. The levels of IL-23 in the supernatant of the control groups were assessed by ELISA. PBMC m RNA expressions of IL-17 A, IL-21, IL-22, IFN-γ, janus kinase 1(JAK1), signal transducer and activator of transcription 1(STAT1), STAT3, interferon regulatory factor 9(IRF9), myxovirus resistance protein A(Mx A), suppressor of cytokine signaling 3(SOCS3), T-box expressed in T cells(T-bet), GATA binding protein 3(GATA3) and forkhead box P3(Fox P3) in all groups were evaluated by quantitative real-time polymerase chain reaction(q RT-PCR).Results: The IL-23 levels in plasma and supernatant of PBMCs were dramatically higher in HCV patients(median was 107.17 pg/ml, 23.18 pg/ml, respectively) than those in the healthy controls(69.03 pg/ml, 6.06 pg/ml, respectively and P < 0.05, P < 0.001, respectively). There was no obvious difference between patients with RVR(103.65 pg/ml) and non-RVR(121.07 pg/ml) in baseline plasma IL-23 levels(P > 0.05). PBMC m RNA levels of IL-17A(P < 0.05, P < 0.05, P < 0.01, respectively), IL-22(P < 0.001, P < 0.001, P < 0.01, respectively), IFN-γ(P < 0.01, P < 0.01, P < 0.05, respectively) and GATA3(P < 0.01, P < 0.05, P < 0.05, respectively) in control group, IL-23 agonist group and IL-23 antagonist group of the HCV patients were all obviously higher than those of healthy controls. The m RNA levels of IL-21(P both < 0.05) and STAT3(P both < 0.001) of HCV patients were markedly higher in IL-23 agonist group and IL-23 antagonist group. Higher negative-feedback regulator SOCS3 m RNA levels were found in control group and IL-23 agonist group of HCV patients than those of healthy controls(P < 0.01, P < 0.05, respectively). The transcription factor Fox P3 m RNA levels were higher in control group and IL-23 antagonist group of the patients(P < 0.05, P < 0.001, respectively). Higher m RNA levels of signaling molecule STAT1 and antiviral protein Mx A were only observed in control group(P < 0.01, P < 0.001, respectively). While there was no significant difference in m RNA levels of transcription factor T-bet, signaling molecules JAK1 and IRF9 among three groups(P > 0.05). In groups of healthy controls, PBMC m RNA expressions of Mx A and SOCS3 were obviously increased in IL-23 agonist group(P both < 0.01). The m RNA expressions of JAK1, STAT3, IRF9, IL-22 and IFN-γ were apparently decreased in IL-23 antagonist group(P < 0.05, P < 0.01, P < 0.01, P < 0.01, P < 0.05, respectively). The m RNA expressions of STAT1 were obviously increased in IL-23 agonist group(P < 0.01) and markedly decreased in IL-23 antagonist group(P < 0.01). Fox P3 m RNA expressions were up-regulated in IL-23 antagonist group compared with those in IL-23 agonist group(P < 0.01). There was not any significant difference in m RNA levels of IL-17 A, IL-21, T-bet and GATA3 among the three groups(P > 0.05). In HCV patients, PBMC m RNA expressions of IL-21 and IFN-γ were elevated in IL-23 agonist group(P < 0.01, P < 0.05, respectively). And the m RNA expressions of IL-22, STAT1 and IRF9 were markedly decreased in IL-23 antagonist group(P < 0.01, P < 0.001, P < 0.01, respectively). The m RNA expressions of STAT3, Mx A and SOCS3 were apparently increased in IL-23 agonist group(P < 0.001, P < 0.01, P < 0.01, respectively) and markedly decreased in IL-23 antagonist group(P < 0.05, P < 0.05, P < 0.01, respectively). Fox P3 m RNA expressions slightly decreased in IL-23 agonist group(P > 0.05) and inversely up-regulated in IL-23 antagonist group(P < 0.01). There was no marked difference in m RNA levels of IL-17 A, JAK1, GATA3 and T-bet among the three groups(P > 0.05).Conclusions: The plasma and PBMC expressed IL-23 levels were elevated in chronic HCV patients; IL-23 modulation could increase the expressions of Th17 cells and their immune cytokines IL-17 A, IL-21, IL-22, IFN-γ as well as antiviral protein Mx A, suppress the expressions of transcription factors Fox P3 and GATA3, and promote anti-HCV immune response, which suggested that IL-23 and its downstream Th17 cells could serve as important immune targets for the treatment of patients with chronic HCV infection. |
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