thymosin-alpha1 and interferon-alpha on Th1 and Th2 cytokine synthesis in chronic hepatitis B

In vitro effect of thymosin-alpha1and interferon-alpha on Th1 and Th2cytokine synthesis in patients with eAg-negative chronic hepatitis BE.Loggi,1A.Gramenzi,1M.Margotti,1C.Cursaro,1S.Galli,2G.Vitale,1E.Grandini,1A.Scuteri,1R.Vukotic,1P.Andreone1and M.Bernardi11Department of Internal Medicine,Cardioangiology and Hepatology and2Microbiology Section,Department of Clinical and Experimental Medicine,University of Bologna,Bologna,ItalyReceived April2007;accepted for publication October2007SUMMARY.Thymosin alpha-1(T a1)has been shown to be effective in chronic hepatitis B treatment.This study inves-tigated the effect of T a1and interferon-alpha(IFN a)on cytokine production by peripheral blood mononuclear cells (PBMCs)of12patients with eAg-negative chronic hepatitis B(HBV).We evaluated the effect of incubation with T a1, IFN a or both on the synthesis of T-helper1(Th1)cytokines [interleukin-2(IL-2),IFN c]and Th2cytokines(IL-4,IL-10) and of antiviral protein2¢,5¢-oligoadenylate synthetase (2¢,5¢-OAS)in patients and in a group of10healthy controls. Concerning Th1profile,controls showed lower IL-2syn-thesis than HBV patients.In HBV setting,IFN a/T a1combi-nation was able to increase IL-2production significantly, when compared with baseline condition.About the Th2-cytokines,controls showed statistically lower synthesis of IL-4and higher production of IL-10,than HBV patients.In these latter,IFN a increased the synthesis of IL-10compared with baseline.Interestingly,both T a1alone and the IFN a/ T a1combination reversed this effect.Finally,compared with baseline,the synthesis of2¢,5¢-OAS was significantly higher in the presence of T a1and IFN a alone,and in the presence of IFN a/T a1association,while no differences were found be-tween controls and HBV patients.In conclusion,in PBMCs from eAg-negative HBV patients,T a1alone was able to in-crease the antiviral protein synthesis,while in association with IFN a,it stimulated the IL-2synthesis and inhibited the IFN-induced IL-10production.These results need further investigations,but reinforce the idea of an immunothera-peutic approach for chronic hepatitis B.Keywords:hepatitis B,immune response,thymosin alpha-1, T-helper1,T-helper2.INTRODUCTIONHepatitis B virus(HBV)is one of major causes of chronic liver disease worldwide[1].Chronic hepatitis B can range from a persistent asymptomatic carrier state to a more or less progressive chronic infection,potentially evolving to cir-rhosis,hepatocellular carcinoma and liver failure[2].HBV is not a cytopathic virus and liver injury is mainly mediated by the host immune response against virus-infected liver cells and by the production of cytokines.However,the pathoge-netic mechanisms responsible for both viral persistence and variable course of infection have only partly been clarified.It has been shown that a vigorous,polyclonal and multi-specific cytotoxic and T-helper(Th)cell response to HBV is readily detectable in the peripheral blood of patients with self-limited hepatitis B,but is weak,antigenically restricted or undetectable in patients with chronic infection[3].Thus, this T-cell response is believed to play a pivotal role in the outcome of HBV.Disturbed functions of CD4Th lymphocytes in the course of chronic hepatitis B have been well documented[4]. Human CD4T cells are known to be functionally heteroge-neous,containing two distinct Th subsets:Th1-producing interferon-gamma(IFN c)and interleukin(IL)-2,and Th2-releasing IL-4,IL-5and IL-10[5,6].In immunopathogenesis of chronic hepatitis B,it has been suggested that a prefer-ential shift towards a Th1-polarized phenotype could be associated with a spontaneous viral clearance or with a successful control of infection[7],while an early commit-ment towards a Th2synthesis plays a significant role in disease progression[8].On the other hand,some authors also showed that the Th1cytokine profile correlates withAbbreviations:IFN,interferon;IL,interleukin;2¢,5¢-OAS,2¢,5¢-oli-goadenylate synthetase;HBV,hepatitis B virus;PBMCs,peripheralblood mononuclear cells.Correspondence:Pietro Andreone MD,Servizio di SemeioticaMedica,Department of Internal Medicine,Cardioangiology andHepatology,University of Bologna,Via Massarenti,9,Bologna,Italy.E-mail:andreone@med.unibo.itJournal of Viral Hepatitis,2008,15,442–448doi:10.1111/j.1365-2893.2007.00960.xhepatic inflammatory activity and liver damage progression [9].Thymosin alpha-1(T a1)is a synthetic polypeptide of thymic origin that stimulates the maturation of thymocytes, the differentiation into active T cells and restores T-cell function by T-cell-mediated antibody production[10–13]. T a1concentration has been found to be low in patients with chronic HBV[14],and clinical trials with T a1indicates that it could be at least as effective as IFN a[15,16].The aim of the present study was to evaluate theÔin vitroÕeffect of IFN a,T a1,and of their association on the produc-tion of Th1(IL-2,IFN c)and Th2cytokines(IL-4,IL-10),as well as the synthesis of an antiviral protein,the2¢,5¢-oli-goadenylate synthetase(2¢,5¢-OAS),by peripheral blood mononuclear cells(PBMCs)of patients with eAg-negative chronic HBV infection.PATIENTS AND METHODSPatientsTwelve patients[male/female:9/3;median age(years): 44.5,range:19–62]with HBeAg-negative chronic hepa-titis B were enrolled in this study.All patients were hepatitis B surface antigen-positive and hepatitis B e antigen-negative for at least1year and had detectable serum HBV-DNA within the last3months before starting the study.Chronic hepatitis B was confirmed by histo-logical evaluation in all cases.No patient had decompen-sated liver disease,detectable antibodies against hepatitis C,D or human immunodeficiency viruses;none had evi-dence of hepatocellular carcinoma or had received any anti-viral drug.Ten healthy subjects[male/female:6/4;median age (years):38,range:28–52],seronegative for any HBV sero-logical markers,without a clinical history of hepatitis and without symptoms or signs of liver disease were studied as controls.Their demographic characteristics were not signif-icantly different from those of patients.All subjects gave written informed consent to the study that conformed to the Helsinki declaration.MethodsVenous blood samples were collected into heparinized tubes and immediately processed.PBMCs were obtained by centrifugation of blood over Ficoll solution(Sigma-Aldrich,Milan Italy)and washed three times in RPMI1640medium containing10%heat inactivated fetal calf serum(Gibco Invitrogen,Milan,Italy), 2m M L-glutamine,50U/mL penicillin,50l g/mL strepto-mycin and10m m HEPES(Gibco Invitrogen).The cells were resuspended at the concentration of1·106PBMC/mL and were then subjected to four different experimental condi-tions,all in the presence of Concanvalin A(ConA)at 10mg/mL(Sigma);as previously reported[17,18],ConA was used as a polyclonal stimulus for triggering cytokine production.The incubation conditions were as follows:1culture medium alone(baseline condition);2culture medium containing500U/mL of IFN a(Wellfer-on,Glaxo Wellcome;Verona,Italy);3culture medium containing10l g/mL of T a1(SciClone Pharmaceuticals,San Mateo,CA,USA);4culture medium containing IFN a and T a1together.Peripheral blood mononuclear cells of healthy controls were cultured only in the presence of culture medium alone without addition of IFN a and/or T a1.The cells were cultured for24h at37°C in atmosphere containing5%CO2,and then harvested for cytokine analysis:after centrifugation at 500·g,the supernatants were collected and stored at–80°C for subsequent test.The cytokine production was assessed by enzyme-linked immunoadsorbent assay(ELISA)according to manufac-turerÕs instructions(R&D Systems,Minneapolis,MN,USA), using the supernatants harvested from cell cultures.Optical density was determined by plate photometer at450nm,and corrected by subtraction of readings at540nm.The values of patients were obtained by interpolation with the standard curve.Both the patients and the standard samples were as-sessed in duplicate.The activity of2¢,5¢-OAS was determined in the super-natants of the cell cultures incubated in the absence of ConA,using a radioimmunological assay(Eiken Chemical, Tokyo,Japan).The sensitivity of the method was 10–810pmol/dL.Serum HBV markers were tested by Chemioluminescent Microparticles Immunoassay(CMIA; ArchitectÒAbbott Laboratories,Aprilia,Italy).Serum HBV-DNA was measured quantitatively by real-time PCR assay(AffigeneÒHBV trender;Alfa Wassermann,Bologna, Italy).The limit of detection for this test was50IU/mL. Nucleotide polymorphisms at nucleotide(nt)1762and nt1764in the basal core promoter region and at codon28in the precore region were identified by line probe assay Inno-Lipa HBV PreCore(Innogenetics Srl,Pomezia,Italy).Viral genotype was assessed by TrugeneÒHBV Genotyping Kit (Bayer Diagnostic,Tarrytown,NY,USA).Statistical analysisThe data are expressed as median(range).The statistical analysis was performed by non-parametric test;in partic-ular,comparison between the different experimental conditions was analysed by WilcoxonÕs test for paired data and correlations between variables were evaluated by SpearmanÕs correlation coefficient.A value of P<0.05was considered statistically significant.Data processing was carried out with the SPSS statistical package for Windows.T a1and IFN a and cytokine synthesis in HBV443RESULTSThe main biochemical and virological characteristics of pa-tients are listed in Table 1.Th1-cytokinesAt baseline,the concentration of IL-2of HBV-infected pa-tients was significantly higher when compared with healthy controls [435(75–1828)vs 86(25–139)pg/mL,P =0.000](Fig.1a).Concerning the HBV patients,com-pared with the baseline condition,the addition of IFN a or T a 1to the culture medium did not significantly modify the IL-2production (Fig.1b).Interestingly,the association IFN a /T a 1induced a significant increase in IL-2when com-pared both with baseline [1081(239–2930)vs 435(75–1828)pg/mL,P =0.02]and with T a 1alone [1081(239–2930)vs 652.5(100–2195)pg/mL,P =0.01].The IFN c concentrations (Fig.2a)did not show any sig-nificant differences between patients and controls at the baseline conditions [2877(1567–6738)vs 2957(170–8650)pg/mL,P =0.6].In HBV patients,the IFN c level in the different stimulate conditions was not significantly dif-ferent with respect to baseline.However,the association IFN a /T a 1was able to increase IFN c synthesis significantly compared with T a 1alone [3320(1165–9130)vs 3000(315–7780)pg/mL,P =0.03](Fig.2b).No correlation was found between HBV-DNA serum levels and ALT levels and Th1-cytokines both in baseline and in stimulated cultures.Th2-cytokinesAt baseline,compared with controls,the IL-4levels were significantly higher in patients [0(0–83)vs 41(0–207)pg/mL,P =0.01](Fig.3a),while the IL-10levels were signifi-cantly lower [1051(295–3150)vs 2666(439–4118)pg/mL,P =0.003](Fig.4a).As far as HBV patients are con-cerned,IL-4production (Fig.3b)in the presence of IFN a washigher when compared with all the other experimental conditions,even if the increase reached statistical signifi-cance only when compared with the association IFN a /T a 1[67(0–238)vs 15(0–132)pg/mL;P =0.03].Regarding IL-10production (Fig.4b),the addition of IFN a alone resulted in a significant increase in comparison with all the other experimental conditions:baseline [1705(570–4530)vsTable 1Biochemical and virological characteristics of patientsCase no.Sex Age GPT (U/L)HBV-DNA (IU/mL)Nucleotide at 1762/1764Nucleotide at 1896Genotype 1M 47941831823T/AA D 2F 5417787320A/A &A/G G D 3M 56323375A/G G/A D 4M 242562259125T/A A D 5F 49100207525T/A A D 6M 477839310A/G A D 7F 6225100T/A A D 8M 3934390A/G A C 9M 261401907690A/G A D 10M 1922283260T/A G D 11M 42580361350T/A G D 12M36409960A/GAD444 E.Loggi et al.1051.5(295–3150)pg/mL,P=0.003],T a1alone[1705(570–4530)vs656.5(280–2060)pg/mL,P=0.003],and IFN a/T a1together[1705(570–4530)vs982.5(458–23205)pg/mL,P=0.003].Finally,the IL-10concentra-tion in the presence of T a1alone was found significantly lower than baseline condition[656.5(280–2060)vs1051.5 (295–3150)pg/mL,P=0.02].No correlation was found between HBV-DNA serum levels and alanine aminotransferase(ALT)levels and Th2-cyto-kines both in baseline and in stimulated cultures.2¢,5¢-OASAt baseline,no difference was found between2¢,5¢-OAS levels of patients and controls[51[0–14930)vs40(21–139)pmol/mL,P=0.7](Fig.5a).In HBV patients(Fig.5b), the2¢,5¢-OAS level at baseline was significantly lower com-pared with all the other experimental conditions.In fact,the addition of either IFN a[118(0–25690)vs51(0–14930) pmol/mL,P=0.03]or T a1[168(0–22620)vs51(0–14930)pmol/mL,P=0.03]induced a significant increase in2¢,5¢-OAS production,when compared with baseline condition.The IFN a/T a1association produced a further in-crease in2¢,5¢-OAS concentration,but it reached statistical significance only when compared with baseline[255(44–29200)vs51(0–14930)pmol/mL,P=0.01].No corre-lation was found between HBV-DNA serum levels and ALT levels and2¢,5¢-OAS level both in baseline and in stimulated cultures.DISCUSSIONAs HBV is a non-cytopathic virus,the immune response against virus-infected liver cells and the production of inflammatory cytokines are thought to be responsible for both liver disease and viral clearance[19].It has been shown that individuals with self-limited HBV infection mount a vigorous polyclonal Th and cytotoxic(CTL)re-sponse to multiple viral epitopes;these responses can be persistently found following viral clearance[20–23].On the contrary,these responses are usually weak or absent in patients who fail to clear the virus and become chronically infected[3,4,24].Furthermore,a consistent lymphocyte infiltrate with Th-1profile has been found in the liver of patients chronically infected by HBV,suggesting a patho-genetic role in liver injury[25].Although these data are still controversial,there is general agreement about the concept that hyporesponsiveness of HBV-specific T cells is an important determinant of virus persistence in chronic HBV infection.Thus,therapeutic strategies should aim to correct this deficiency[26].T a1and IFN a and cytokine synthesis in HBV445Thymosin-a1is an immunoregulatory agent capable of triggering maturational events in lymphocytes,to augment T-cell function,to promote reconstitution of immune defects, to amplify the expression of class I HLA and to increase natural killer cell activity[11–13,27].In PBMCs of patients with chronic hepatitis C,T a1has been shown to enhance the Th1immune response[17].Data of trials with T a1in chronic hepatitis B indicate that it may be at least as effective as IFN a[15,16].Interestingly,T a1 is better tolerated than IFN a and seems to induce a gradual and sustained increase in virological response overtime after discontinuation of treatment[15,28,29].We performed this study to evaluate the effect of T a1,alone or in association with IFN a on cytokine and antiviral protein2¢,5¢-OAS synthesis from PBMCs of patients with chronic hepatitis B.Despite the limited number of patients,this study dem-onstrated that PBMCs of patients with chronic HBV infection produce more IL-2than those of healthy controls.On the contrary,IFN c production was not different between patients and controls.Thisfinding could simply reflect the absence of stimulation of the immune system in healthy subjects,rather than a Th1polarization in HBV patients,as previously reported[30].A predominant shift of the immune response towards a Th1profile has been suggested to be crucial for the eradication of HBV,while a prevalent Th2response seems to favour viral persistence[21,31].Therefore,it is possible to speculate that the use of immunomodulatory agent capable of reinforcing the Th1response could be advantageous to control HBV infection.Our in vitro data show that T a1alone,as well as IFN a alone are not able to modify either IL-2or IFN c production compared with base-line condition.However,in association,they are able to in-duce a significant increase in IL-2not only compared to baseline but also compared to T a1alone.As we have not studied the local cytokine production in HBV-infected liver samples,this IL-2increased production needs further investigations.IL-2is a pleiotropic cytokine with several antiviral and immunomodulatory proprieties that have been demonstrated to downregulate hepatocellular HBV gene expression by an indirect,noncytopathic process[32].Thus, we can hypothesize that the association between IFN a and T a1could have a role in controlling HBV chronic infection. As far as the Th2cytokines are concerned,at baseline,the behaviours in patients and controls were completely opposed and more difficult to explain.In particular,the higher value of IL-4concentration in patients is not unexpected as it446 E.Loggi et al.reflects the Th2polarization in chronic infection,as previ-ously described[9,33].On the other hand,ourfinding of lower levels of IL-10in HBV patients is in contrast to pre-vious data[30].These discrepancies could be partially be-cause of different experimental design and different study population.IL-10is a potent immunosuppressor that has been recently shown to play a key role in T-cell exhaustion and viral persistence in animal models of viral infection[34]. In HBV patients,Hyodo et al.[30]found a large number of IL-10-secreting cells after stimulation of PBMCs with HBcAg, while no specific stimulation was performed in our study. Furthermore,it should be pointed out that our study sample included only patients with precore mutant infection (HBeAg-negative,HBV-DNA-positive).It is well known that HBeAg is responsible for immunotolerance and T cell anergy [35].We can speculate that in our patients the precore mutation could have some influence in IL-10synthesis,even though further studies are needed in this setting.Our results show that IL-4production by PBMCs of HBV patients was not modified by the different experimental condition compared to baseline.Nevertheless,IFN a alone significantly increased IL-10synthesis,while T a1induced a significant decrease with respect to baseline.Incubation of T a1in combination with IFN a was able to reverse the IFN-induced increase of IL-10.Finally,we confirmed that T a1amplifies the synthesis of IFN-induced antiviral protein 2,5-OAS[17].In summary,T a1alone and IFN a alone were found to reduce and increase respectively only the synthesis of IL-10 by PBMCs of patients with HBeAg-negative chronic HBV infection.Furthermore,both drugs alone or in association significantly increased the antiviral protein2,5-OAS pro-duction.On the other hand,the association T a1plus IFN a induced a significant increase in IL-2and blocked the IL-10 increase induced by IFN a.If this profile could be effectively advantageous in the treatment of chronic hepatitis B,more information is needed about intrahepatic environment.It is well known that nucleoside/nucleotide analogues,widely used for treatment of chronic HBV,are able to achieve the suppression of viral replication,but at the same time,they are inadequate to induce recovery of the immune system [36].In this view,compounds able to modulate the host immune response,should be further investigated to boost or increase HBV-immune response.ACKNOWLEDGEMENTSThis study was in part supported by Associazione per la Ricerca sulle Malattie Epatiche(ARME).REFERENCES1Lok AS,Mc Mahon BJ.Practice Guidelines Committee, American Association for the Study of Liver Diseases,2001.Chronic hepatitis B.Hepatology2001;34:1225–1241.2Villeneuve JP.The natural history of chronic hepatitis B virus infection.J Clin Virol2005;34(Suppl.1):S139–142. 3Bertoletti A,Gehring AJ.The immune response during hepatitis B virus infection.J Gen Virol2006;87:1439–1449.4Chisari FV,Ferrari C.Hepatitis B virus immunopathogene-sis.Annu Rev Immunol1995;13:29–60.5Mosmann TR,Sad S.The expanding universe of T-cell subsets:Th1,Th2and more.Immunol Today1996;17: 138–146.6OÕGarra A.Cytokines induce the development of function-ally heterogeneous T helper cell subsets.Immunity1998;8: 275–283.7Ferrari C,Missale G,Boni C,Urbani S.Immunopathogenesis of hepatitis B.J Hepatol2003;39(Suppl.1):S36–42.8Akpolat N,Yahsi S,Godekmerdan A,Demirbag K,Yalniz M.Relationship between serum cytokine levels and histopath-ological changes of liver in patients with hepatitis B.World J Gastroenterol2005;11:3260–3263.9Jiang R,Feng X,Guo Y et al.T helper cells in patients with chronic hepatitis B virus infection.Chin Med J(Engl)2002;115:422–424.10Li CL,Zhang T,Saibara T et al.Thymosin alpha1acceler-ates restoration of T cell-mediated neutralizing antibody response in immunocompromised hosts.Int Immunophar-macol2002;2:39–46.11Chien RN,Liaw YF.Thymalfasin for the treatment of chronic hepatitis B.Expert Rev Anti Infect Ther2004;2:9–16.12Knutsen AP,Freeman JJ,Mueller KR,Roodman ST,Bou-hasin JD.Thymosin-alpha1stimulates maturation of CD34+stem cells into CD3+4+cells in an in vitro thymic epithelia organ coculture model.Int J Immunophar-macol1999;21:15–26.13Giuliani C,Napolitano G,Mastino A et al.Thymosin-alpha1 regulates MHC class I expression in FRTL-5cells at tran-scriptional level.Eur J Immunol2000;30:778–786.14Sherman KE,Jones CC,Goldstein AL,Naylor PH.Low thy-mosin alpha-1concentrations in patients chronically in-fected with the hepatitis B virus.Viral Immunol1991;4: 195–199.15Andreone P,Cursaro C,Gramenzi A et al.A randomized controlled trial of thymosin-alpha1versus interferon alfa treatment in patients with hepatitis B e antigen antibody and hepatitis B virus DNA positive chronic hepatitis B.Hepatology1996;24:774–777.16Chien RN,Liaw YF,Chen TC,Yeh CT,Sheen IS.Efficacy of thymosin alpha1in patients with chronic hepatitis B:a ran-domized,controlled trial.Hepatology1998;27:383–387. 17Andreone P,Cursaro C,Gramenzi A et al.In vitro effect of thymosin-alpha1and interferon-alpha on Th1and Th2 cytokine synthesis in patients with chronic hepatitis C.J Viral Hepat2001;8:194–201.18Andreone P,Gramenzi A,Loggi E et al.In vitro effect of indomethacin and interferon-alpha on Th1and Th2cyto-kine synthesis in patients with chronic hepatitis C.Cytokine 2004;26:95–101.19Thomas HC,Montano L,Goodall A et al.Immunological mechanism in chronic hepatitis B virus infection.Hepatology 1982;2:116S–121S.T a1and IFN a and cytokine synthesis in HBV44720Penna A,Artini M,Cavalli A et al.Long-lasting memory T cell responses following self-limited acute hepatitis B.J Clin Invest1996;98:1185–1194.21Penna A,Del Prete G,Cavalli A et al.Predominant T-helper 1cytokine profile of hepatitis B virus nucleocapsid-specific T cells in acute self-limited hepatitis B.Hepatology1997;25: 1022–1027.22Rehermann B,Pasquinelli C,Mosier SM,Chisari FV.Hepa-titis B virus(HBV)sequence variation of cytotoxic T lym-phocyte epitopes is not common in patients with chronic HBV infection.J Clin Invest1995;96:1527–1534.23Rehermann B,Ferrari C,Pasquinelli C,Chisari FV.The hepatitis B virus persists for decades after patientsÕrecovery from acute viral hepatitis despite active maintenance of a cytotoxic T-lymphocyte response.Nat Med1996;2:1104–1108.24Webster GJ,Reignat S,Brown D et al.Longitudinal analysis of CD8+T cells specific for structural and nonstructural hepatitis B virus proteins in patients with chronic hepatitis B:implications for immunotherapy.J Virol2004;78:5707–5719.25Barnaba V,Franco A,Paroli M et al.Selective expansion of cytotoxic T lymphocytes with a CD4+CD56+surface phenotype and a T helper type1profile of cytokine secretion in the liver of patients chronically infected with Hepatitis B virus.J Immunol1994;152:3074–3087.26Bertoletti A,Naoumov NV.Translation of immunological knowledge into better treatments of chronic hepatitis B.J Hepatol2003;39:115–124.27Serrate SA,Schulof RS,Leondaridis L,Goldstein AL,Sztein MB.Modulation of human natural killer cell cytotoxic activity,lymphokine production,and interleukin2receptor expression by thymic hormones.J Immunol1987;139: 2338–2343.28Chan HL,Tang JL,Tam W,Sung JJ.The efficacy of thymosin in the treatment of chronic hepatitis B virus infection:a meta-analysis.Aliment Pharmacol Ther2001;15:1899–1905.29Lau e of immunomodulatory therapy(other than interferon)for the treatment of chronic hepatitis B virus infection.J Gastroenterol Hepatol2000;15(Suppl.):E46–E52.30Hyodo N,Tajimi M,Ugajin T,Nakamura I,Imawari M.Frequencies of interferon-gamma and interleukin-10 secreting cells in peripheral blood mononuclear cells and liver infiltrating lymphocytes in chronic hepatitis B virus infection.Hepatol Res2003;27:109–116.31Bertoletti A,DÕElios MM,Boni C et al.Different cytokine profiles of intraphepatic T cells in chronic hepatitis B and hepatitis C virus infections.Gastroenterology1997;12:193–199.32Guilhot S,Guidotti LG,Chisari FV.Interleukin-2downre-gulates hepatitis B virus gene expression in transgenic mice by a posttranscriptional mechanism.J Virol1993;67: 7444–7449.33Kobayashi K,Ishii M,Igarashi T et al.Profiles of cytokines produced by CD4-positive T lymphocytes stimulated by anti-CD3antibody in patients with chronic hepatitis C.J Gas-troenterol1998;33:500–507.34Blackburn SD,Wherry EJ.IL-10,T cell exhaustion and viral persistence.Trends Microbiol2007;15:143–146.35Milich DR,Chen MK,Hughes JL,Jones JE.The secreted precore antigen can modulate the immune response to the nucleocapsid:a mechanism for persistence.J Immunol1998;160:2013–2021.36Boni C,Penna A,Bertoletti A et al.Transient restoration of anti-viral T cell responses induced by lamivudine therapy in chronic hepatitis B.J Hepatol2003;39:595–6.448 E.Loggi et al.。