on T-helper 1 Cell and T-helper 2 Cell Cytokine Synthesis in Patients with Hepatitis B Virus e Antigenpositive Chronic Hepatitis B

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1 The Journal of International Medical Research 21; 38: Effect of Thymosin-α 1 on T-helper 1 Cell and T-helper 2 Cell Cytokine Synthesis in Patients with Hepatitis B Virus e Antigenpositive Chronic Hepatitis B Y-F JIANG 1, Z-H MA 1, P-W ZHAO 1, Y PAN 1, Y-Y LIU 2, J-Y FENG 1 AND J-Q NIU 1 1Department of Hepatology, and 2 Department of the Digestive System, First Hospital, Jilin University, Changchun, China Thymosin-a 1 ) has been shown to be effective treatment for chronic hepatitis B virus (HBV) infection. This study investigated the immune response after TA 1 monotherapy in 25 HBV e antigen (HBeAg)-positive patients randomized to receive either 1.6 mg active TA 1 (group A), 1.6 mg recombinant TA 1 (group B) or 3.2 mg recombinant TA 1 (group C) monotherapy for 52 weeks. The percentages of T-helper 1 ( 1) cytokineproducing T-cells (interleukin-2 [IL-2], interferon-g [IFN-g], tumour necrosis factor-a) and 2 cytokine-producing T- cells (IL-4) were analysed using flow cytometry. In all patients treated with TA 1, cytokine levels and the proportion of peripheral blood mononuclear cells producing these cytokines were significantly increased, compared with baseline and healthy controls. The proportions of each cytokine-producing cell increased gradually over time and were restored to normal levels, and proportions of IFN-g and IL-4-producing cells reached higher levels than in normal (healthy) controls. The results showed that treatment with TA 1 increased cytokine production, especially IFN-g, and higherdose TA 1 exhibited better efficacy against HBV, compared with other treatments studied. KEY WORDS: THYMOSIN-a 1 ; IMMUNOMODULATORY DRUGS; CHRONIC HEPATITIS B; T-HELPER 1 CELLS (T H 1); T-HELPER 2 CELLS (T H 2); CYTOKINES Introduction Chronic hepatitis B virus (HBV) infection is one of the most common infectious diseases and affects 4 million people. 1 The infection leads to complications such as liver cirrhosis and hepatocellular carcinoma and, as a result, contributes to the deaths of millions of people per year worldwide. 2 China is among the countries that are highly endemic for HBV, with approximately 8% of the population being chronically infected with the virus. 2 Despite the introduction of universal vaccination against hepatitis B in China, persistent HBV infection is still a serious problem. 2,3 Chronic HBV infection is a dynamic state involving an interaction between the virus, hepatocytes and the immune system of the 253

2 host. The use of antiviral agents that have different mechanisms of action may help to minimize toxicity and the development of drug-resistant viral mutants. 4,5 There is a general consensus that the magnitude of the cytotoxic T-cell response to HBV is the principal determinant of viral clearance and/or suppression. 6 From an immunological point of view, the aim of treatment should be to restore effective T-cell control. It has been shown that a vigorous, polyclonal and multispecific cytotoxic and T-helper ( ) cell response to HBV is readily detectable in the peripheral blood of patients with self-limited hepatitis B, but this response is weak, antigenically restricted or undetectable in patients with chronic infection. 7 Thus, the T- cell response is believed to play a pivotal role in the outcome of HBV infection. Human CD4 + T-cells consist of two distinct cell subsets: 1 cells producing interferon-γ (IFNγ) and interleukin-2 (IL-2); and 2 cells releasing IL-4 and IL-1. 8 Thymosin-α 1 ) is an immune-modifier peptide hormone secreted by the thymus that can trigger maturational events in lymphocytes, augment T-cell function and promote reconstitution of immune defects. 9 It plays a very important role in the cellular immune response by triggering T-cell maturation and modulating the immune function. 1 Clinical trials of thymosin treatment for immunodeficiency or cancer indicate that this agent is non-toxic, enhances immune responsiveness and augments specific lymphocyte functions including lymphoproliferative responses to mitogens, T- cell maturation, antibody production and T- cell-mediated cytotoxicity. 11,12 The aims of the present study were: (i) to evaluate the impact of TA 1 treatment on levels of 1 (IL-2, IFN-γ and tumour necrosis factor-α [TNF-α]) and 2 (IL-4) cytokineproducing cells in the peripheral blood of patients with HBV e antigen (HBeAg)- positive chronic hepatitis B infection; and (ii) to assess the immune mechanism of the anti- HBV effect of TA 1 and its correlation with clinical indicators. Patients and methods STUDY DESIGN This was a randomized, controlled clinical trial. The study was approved by the Ethical Committee of the First Hospital of Jilin University (Changchun, China) according to the 1964 Declaration of Helsinki. All patients provided written informed consent prior to study enrolment. INCLUSION/EXCLUSION CRITERIA Patient inclusion criteria were: age 2 58 years; positive for HBV markers (HBV surface antigen [HBsAg] and HBeAg); alanine transaminase (ALT) levels above the upper limit of normal during the previous 2 weeks; and HBV DNA 15 copies/ml. Patients with hepatocellular carcinoma, decompensated liver disease, autoimmune-type liver disease, those positive for antihepatitis C virus, anti- HIV and antihepatitis D virus antibodies, and patients using lamivudine or other antiviral drugs in the 3 months before commencing the present study were excluded. Healthy subjects with normal liver function, no HBV markers and no history of therapy with glucocorticosteroids or other drugs that could have changed the immune function within the previous 3 months were included as controls. TREATMENT SCHEDULES Eligible patients were randomly assigned by a computer-generated randomization scheme to one of three groups: group A, 1.6 mg active TA 1 (Zadaxin ; Patheon Italia SpA, Ferentino, Italy) subcutaneously twice a week; group B, 1.6 mg TA 1 (recombinant TA 1, 254

3 Genescience Pharmaceutical Co., Ltd, Changchun, China) subcutaneously twice a week; or group C, 3.2 mg TA 1 (recombinant TA 1, Genescience Pharmaceuticals Co., Ltd) subcutaneously twice a week. Patients were injected subcutaneously with TA 1 every Tuesday and Friday for 52 weeks. Active TA 1 (Zadaxin ) is a chemically synthesized drug and additional by-products are produced during synthesis. Thus, in the present study, recombinant TA 1, which is obtained by genetic engineering methods and extracted from Escherichia coli, was used to avoid such by-products. SEROLOGICAL ANALYSES Blood samples were collected and serum was used to measure aspartate transaminase (AST), ALT, bilirubin, albumin, complete blood count and markers of HBV replication (cytokine levels, HBV DNA and biochemical indicators of liver function) on enrolment (before treatment) and at 13, 21, 37 and 52 weeks after treatment. Serological analyses were performed in healthy controls at the time of enrolment. Standard biochemical techniques were used to analyse serological samples, unless specific techniques are described below (i.e., for cytokine staining and virological assessment). FLOW CYTOMETRY ANALYSIS OF INTRACELLULAR CYTOKINE STAINING Blood cells were stimulated for cytokine secretion and analysed by flow cytometry, according to a previously reported protocol. 13 For the analysis of intracellular cytokine production, 1 µl of blood was diluted with Iscove s modified Dulbecco s medium (1:1 volume). The diluted blood was stimulated with 5 ng/ml phorbol 12- myristate 13-acetate (PMA; Sigma-Aldrich, St Louis, MO, USA) plus 2 µg/ml ionomycin for 6 h; 1 µg/ml brefeldin A was added during the last 2 h. The cytokine-producing cells of interest in this study were CD3 + CD4 + cells; however, as PMA stimulation can induce downregulation of CD4 from the cell surface, stimulated cells were stained using anti-cd3- peridinin chlorophyll protein complex (PerCP), anti-cd8-fluorescein isothiocyanate (FITC) or anti-cd8-phycoerythrin (PE) antibodies to stain CD3 + CD8 + cytotoxic T- cells, and a CD3 + CD8 T-cell inverter gate was used to select for CD4 + cells. After the cells were permeabilized and fixed using fixing reagent (Caltag Laboratories Inc., Burlinghame, CA, USA), anti-il-2 FITC, anti-tnf-α FITC, anti-il-4 PE, anti-ifn-γ PE or isotope-matched control antibodies were added for 3 min, and cells underwent flow cytometry (FACSCalibur TM ; Becton, Dickinson and Co., Franklin Lakes, NJ, USA) and were analysed using FlowJo flow cytometry analysis software, version (FlowJo, Ashland, OR, USA) according to the manufacturer s instructions. Isotypematched antibodies were used as controls for all the samples. VIROLOGICAL ASSESSMENT Levels of HBV DNA were measured using a luciferase quantitation detection kit (Roche Diagnostics, Mannheim, Germany). HBsAg, anti-hbs, HBeAg and anti-hbe were determined using commercial Murex Mikrotiterplatten enzyme immunoassay kits (Abbott Laboratories, Abbott Park, IL, USA). STATISTICAL ANALYSES All data were analysed using SAS software, version 8. (SAS Institute Inc., Cary, NC, USA) and are expressed as median (range). Student s t-test and the χ 2 -test were used to compare demographic data in the active treatment and control groups. The Wilcoxon 255

4 signed rank test, Kruskal Wallis test and Spearman s correlation were used for analysis of immune and antiviral results from the TA 1 - treated groups. Levels of cytokine-producing cells were analysed by mutation status with the Kruskal Wallis test, the χ 2 -test and Fisher s exact test. A P-value <.5 was considered to be statistically significant. Results DEMOGRAPHIC BACKGROUND A total of 25 Chinese patients with chronic HBV infection (17 males and eight females) were included in this randomized controlled trial; 2 unrandomized healthy control subjects were also included. The mean ± SD age of the chronic HBV-infected patients was 42.7 ± 2.8 years and the mean ± SD disease duration was 6.37 ± 4.62 years. All of the 25 chronic HBV-infected patients completed 52 weeks of treatment. Baseline data for the patients and controls (Table 1) revealed no statistically significant between-group differences. On initial screening, HBsAg, HBeAb and HBV DNA were detected in all patients and they had an HBV DNA load in the range of log 1 copies/ml. Median serum ALT and AST levels were in the range and U/l, respectively, across all patients. No betweengroup differences were noted for other serological parameters analysed (e.g. bilirubin, albumin, complete blood counts), which were normal unless otherwise stated. TABLE 1: Baseline demographic and biochemical data for patients with chronic hepatitis B virus (HBV) infection treated with thymosin-a 1 ) and healthy controls Active TA a 1 a Recombinant TA mg 1.6 mg 3.2 mg (group A) (group B) (group C) Controls b Parameter n = 9 n = 8 n = 8 (n = 2) Age, years Mean ± SD 46. ± ± ± ± 11.8 Median Range Sex, n (%) Male 7 (77.8) 7 (88.9) 6 (75) 14 (7) Female 2 (22.2) 1 (11.1) 2 (25) 6 (3) HBV DNA, log 1 copies/ml Mean NA Range Alanine transaminase, U/l Mean Range Aspartate transaminase, U/l Mean Range a Active or recombinant TA 1 was administered subcutaneously twice a week for 52 weeks. b Control group consisted of healthy subjects with normal liver function and no HBV markers; they received no treatment and were sampled only at enrolment. No statistically significant differences were found between any of the groups in terms of baseline demographic and biochemical data (P >.5). NA, not analysed. 256

5 PROFILE OF CYTOKINE-PRODUCING T H 1/T H 2 CELLS AT BASELINE The percentages of cytokine-producing CD3 + CD4 + T-cells at baseline in patients with chronic HBV were compared with those in control subjects (Table 2). The percentages of 1 and 2 cells producing the cytokines IL- 2, IFN-γ, TNF-α and IL-4 were significantly lower in patients with chronic HBV compared with healthy controls (P <.1). CHANGES IN CYTOKINE- PRODUCING T H 1/T H 2 CELL PROFILES AFTER TA 1 TREATMENT The baseline and post-treatment percentages of cytokine-producing CD3 + CD4 + T-cells across all the TA 1 treatment groups is shown in Table 3. Continuous treatment with TA 1 for up to 52 weeks gradually increased levels of 1 and 2 cytokine-producing CD3 + CD4 + T-cells. At week 13, the levels of each cytokine-producing cell type were not enhanced significantly compared with baseline levels. Over time, the levels continued to increase significantly (P <.5) and became restored to normal levels. In addition, the levels of IFN-γ- and IL-4- producing cells were higher than those for normal controls (Table 2). At 52 weeks of treatment with TA 1 there were significantly increased percentages of 1 and 2 CD3 + CD4 + cells producing IL-2, IFN-γ, TNF-α and IL-4 compared with baseline in all three groups (Fig. 1). BIOCHEMICAL AND VIROLOGICAL RESPONSES TO TA 1 TREATMENT In groups B and C, ALT levels started to decline soon after treatment and normalized between weeks 13 and 37, remaining normal to week 52 (Fig. 2A). In the group receiving active TA 1 (group A), a similar trend was seen for ALT, except for the week 21 data (Fig. 2A). Serum HBeAg levels showed a steady decline in group C, but not in groups A and B over the treatment period (Fig. 2B). Serum HBsAg showed no change following active drug or TA 1 treatment (Fig. 2C). The baseline serum HBV DNA load was high in all patients, but decreased only in groups A and C following TA 1 treatment (Fig. 2D). ASSOCIATION BETWEEN CYTOKINE- PRODUCING T H 1/T H 2 CELLS AND CLINICAL PARAMETERS No correlation was observed between the viral load, ALT, HBsAg, HBeAg, and levels of cytokine-producing 2 cells in chronic HBV patients. A significant correlation was observed between AST and IFN-γ (r =.461, P =.23). TABLE 2: Baseline percentages of CD3 + CD4 + T-cells producing the four intracellular cytokines in patients with chronic hepatitis B infection prior to thymosin-a 1 treatment, compared with healthy controls All patients Control Cytokines (n = 25) (n = 2) Interleukin-4.6 (.1 8.7) a 5.69 ( ) Interferon-γ.3 (. 2.8) a 5.73 ( ) Interleukin-2.6 (.1 6.2) a ( ) Tumour necrosis factor-α 1. ( ) a ( ) Data expressed as median percentage (range). a P <.1 for all patients versus the control group (Kruskal Wallis test). 257

6 TABLE 3: Percentages of CD3 + CD4 + T-cells producing intracellular cytokines following various periods of thymosin-a 1 ) treatment Treatment period (weeks) Cytokines (baseline) Interleukin-2.6 (.1 6.2) 2.5 ( ) 9.65 a (.3 16.) 2.65 a ( ) 7.8 a ( ) Interferon-γ.3 (. 2.8) 2.4 ( ) 5.5 a ( ) 15.7 a ( ) 16.2 a ( ) Tumour necrosis factor-α 1. ( ) 1.1 ( ) 19.6 a ( ) 29.7 a ( ) 7.9 a ( ) Interleukin-4.6 (.1 8.7) 5.4 ( ) 3. ( ) 15.1 a ( ) 22.9 a ( ) Data expressed as median percentage (range). Patients received active TA 1 (1.6 mg, n = 9) or recombinant TA 1 (1.6 mg or 3.2 mg, n = 8 per group) a P <.5 for all three treatment groups versus baseline (Kruskal Wallis test). Discussion As 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. 14 Over the past 2 years, many antiviral or immunomodulatory agents have been used in the treatment of chronic HBV infection. The immune modifier, TA 1, can trigger lymphocyte maturation, augment T-cell function and promote reconstitution of immune defects. 1 The magnitude of the cytotoxic T-cell response to HBV is thought to be the principal determinant of viral clearance and/or suppression, 6 and the aim of treatment should be to restore effective T- cell control. 15 In recent years, research has focused on the pathogenic processes involved in chronic HBV infection and it has been found that cytokines may play complex roles in these processes. 4,6 T-helper cells, which are divided into 1 and 2 cells, are one of the major cell populations to secrete cytokines. 7,8 A balance between 1 and 2 cells is required to maintain normal immune function but, in HBV-infected patients, the immune response to HBV is defective, which is an important contributing factor to the development of chronic HBV infection. 16 An imbalance between the 2 responses may be responsible for the dysregulated immune status seen in HBV-infected patients. In the present study, levels of cytokine-producing CD3 + CD4 + T-cells at baseline were significantly lower in patients with chronic HBV-infection compared with healthy controls, suggesting that both 1 and 2 cells were functionally impaired in chronic HBV-infected patients. Developing and updating antiviral drugs, and confirming their antiviral effects, enables the influence of antiviral therapy on 258

7 A 5 B mg active TA 1 (group A) 1.6 mg recombinant TA 1 (group B) 3.2 mg recombinant TA 1 (group C) IL-2 + CD3 + CD4 + (%) IFN-γ + CD3 + CD4 + (%) C TNF-α + CD3 + CD4 + (%) (baseline) (baseline) D (baseline) (baseline) IL-4 + CD3 + CD4 + (%) 5 4 FIGURE 1: Analysis of T-helper ( ) cell ( 2) cytokine-producing CD3 + CD4 + cells in chronic hepatitis B virus (HBV) patients after (baseline), 13, 21, 37 and 52 weeks of treatment with 1.6 mg active thymosin-α 1 ) (group A), 1.6 mg recombinant TA 1 (group B) or 3.2 mg recombinant TA 1 (group C) administered subcutaneously twice weekly for: (A) interleukin-2 (IL-2)-producing cells, (B) interferon-γ (IFN-γ)- producing cells, (C) tumour necrosis factor-α (TNF-α)-producing cells and (D) interleukin-4 (IL-4)-producing cells (P <.5 versus baseline; Kruskal Wallis test, χ 2 - test and Fisher s exact test) immune status to be evaluated, rather than directly studying the impaired immune status induced by HBV in chronically infected patients. 15 Studies have elucidated the physiological and pathological roles of 2 and provided theoretical evidence for 2-mediated immune diseases. 9,17 Thymosin-α 1 may sequentially trigger T- cell maturation, modulate the host s immune balance, and enhance the immune response of mature T-cells against antigens and other stimuli. 18 As a superordinary immunomodulator, TA 1 has significant antiviral and antineoplastic effects, and shows a good therapeutic effect in adjunctive therapy against viral diseases and malignant tumours. 19 It can also promote bone marrow stem cells to develop into T- cells, resulting in the enhancement of cellular immune function. 2 TA 1 is an amino acid peptide that affects immunoregulatory T-cell function, promotes TNF-α, IFN-γ, IL-2 259

8 A 6 5 B mg active TA 1 (group A) 1.6 mg recombinant TA 1 (group B) 3.2 mg recombinant TA 1 (group C) ALT (U/l) HBeAg (IU/ml) C (baseline) (baseline) D 12 HBsAg (IU/ml) HBV DNA (log 1 copies/ml) (baseline) (baseline) FIGURE 2: Analysis of the biochemical and virological responses in chronic hepatitis B virus (HBV)-infected patients after (baseline), 13, 21, 37 and 52 weeks of treatment with 1.6 mg active thymosin-α 1 ) (group A), 1.6 mg recombinant TA 1 (group B) or 3.2 mg recombinant TA 1 (group C) administered subcutaneously twice weekly for serum levels of: (A) alanine transaminase (ALT), (B) HBV e antigen (HBeAg), (C) HBV surface antigen (HBsAg) and (D) HBV DNA (P <.5 versus baseline; Wilcoxon signed rank test, Kruskal Wallis test and Spearman s correlation) and IL-3 production by normal human lymphocytes, and increases lymphocyte IL-2 receptor expression. 15,16,21 The efficacy of TA 1 in the treatment of patients with chronic HBV infection has been studied in several trials. 12,21,22 The present study showed that cytokine production by peripheral blood mononuclear cells taken from chronic HBV patients was increased by TA 1 treatment compared with baseline: our data showed that TA 1 treatment stimulated the immune response, and that higher doses of TA 1 further induced a 1 response. TA 1 alone is able to increase antiviral protein synthesis in HBeAg-negative HBV-infected patients while, in association with IFN, it stimulates IL-2 synthesis and inhibits the production of IFNinduced IL The present study also demonstrated that TA 1 alone could increase cytokine production in HBeAg-positive HBV- 26

9 infected patients, and that higher doses of TA 1 exhibited better efficacy against HBV. Despite the limited number of patients, the present study demonstrated that TA 1 could enhance IFN-γ production compared with healthy controls. This finding simply reflects the immune system in healthy subjects, rather than a 1 polarization in HBV patients, as previously reported. 23 At present, the immunomodulators for treating chronic HBV infection include thymic peptide, TA 1, levamisole, immune ribonucleic acid, and traditional Chinese medicines. 15 Immunomodulators could enhance the human immune response, specifically the immune response against HBV, and recognize the target cells infected by HBV for elimination. 16 Chronic HBVinfected patients usually have a poor immune response and develop immune tolerance against HBV, which results in a failure to eliminate HBV from the body. 9 Immunomodulatory agents could activate the human immune system and interrupt the development of immune tolerance, ultimately helping to eliminate HBV. 6,9,15 In the present study, the levels of cytokineproducing 1 and 2 T-cells increased over the course of long-term TA 1 treatment. The increase in the level of cytokines at various stages of treatment was also significantly increased compared with the baseline levels. The enhanced 2 immune response, especially of IFN-γ, related to TA 1 treatment that was observed in the present study suggests that the antiviral effect of this drug may have an effect on immune regulation. The higher dose of TA 1 (3.2 mg) exhibited better efficacy compared with the other treatments studied. Furthermore, the altered cytokine-producing T- cell profiles in chronic HBV-infected patients, in particular those secreting IFN-γ, may provide additional biomarkers for disease diagnosis and prognosis. Further research is needed to understand the underlying mechanism involved in the potential immunoregulatory function of TA 1. Acknowledgements This study was supported by grants from the Eleventh 5-year Plan for AIDS and Viral Hepatitis (No. 28ZX12-4), Ministry of Health (No ), National Natural Science Foundation of China (No and ), and Jilin Province Science and Technology Agency (No ). Conflicts of interest The authors had no conflicts of interest to declare in relation to this article. Received for publication 7 June 21 Accepted subject to revision 12 June 21 Revised accepted 11 October 21 Copyright 21 Field House Publishing LLP References 1 Mahboobi N, Agha-Hosseini F, Mahboobi N, et al: Hepatitis B virus infection in dentistry: a forgotten topic. J Viral Hepat 21; 17: World Health Organization (WHO): Hepatitis B. WHO Fact Sheet, No. 24, revised October 2 (available at: mediacentre/factsheets/fs24/en/). 3 Li DD, Ding L, Wang J, et al: Prevalence of hepatitis B virus genotypes and their relationship to clinical laboratory outcomes in Tibetan and Han Chinese. J Int Med Res 21; 38: Lavanchy D: Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J Viral Hepat 24; 11: Maddrey WC: Hepatitis B: an important public health issue. J Med Virol 2; 61: Bertoletti A, Gehring AJ: The immune response during hepatitis B virus infection. J Gen Virol 26; 87: Mosmann TR, Sad S: The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today 1996; 17:

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