The Effect Of Bicyclol Tablets Treatment On Cellular Immune Responses In Patients With Chronic Hepatitis B

BackgroundThe immune responses to HBV and their role in pathogenesis of hepatitis B are incompletely understood.Viral persistence is thought to be related to poor HBV-specific T-cell responses. Helper T cells are found in two distinct cell types, Th1 and Th2, distinguished by the cytokines they produce and respond to and the immune responses they are involved in. Th1 cells produce pro-inflammatory cytokines such as IFN-γ, TNF-β and IL-2, while Th2 cells produce the cytokines IL-4, IL-5, IL-6 , IL-10, and IL-13. The cytokines produced by Th1 cells stimulate the phagocytosis and destruction of microbial pathogens while Th2 cytokines like IL-4 generally stimulate the production of antibodies directed toward large extracellular parasites. Th1 and Th2 are produced by differentiation from a non-antigen exposed precursor cell type, Thp. Exposure of Thp cells to antigen by antigen-presenting cells may result in their differentiation to Th0 cells, not yet committed to become either Th1 or Th2 cells, although the existence of Th0 cells is controversial. Cells committed as either Th1 and Th2 cells are called polarized, whether they are effector cells actively secreting cytokines or are memory cells. The stimulation of Thp cells by exposure to antigen-presenting cells induces the proliferation of undifferentiated cells, and their expression of IL-2 and IL-2 receptor. The differentiation of Th1 cells and Th2 cells depends on the cytokines they are exposed to. IL-12 causes Th1 differentiation and blocks Th2 cell production, while IL-4 causes Th2 differentiationand antagonizes Thl development. IL-18 also induces Thl differentiation. Polarized Thl and Th2 cells also express distinct sets of chemokine receptors that further modify their homing and other cellular responses. Improved studing of Thl and Th2 differentiation will improve our overall understanding of the immune system, its response to infection and aberrant responses that lead to disease. The imbalance of Thl/Th2 type cytokines plays an important role in the pathogenesis of chronic hepatitis B. HBV antigen-specific Th cell activity can also affect the chronicity of hepatitis B. If HBV antigen-specific Thl type cytokines are predominant, the patients may have acute or self-limited infection; if Th2 type cytokines are predominant, the patients tend to have chronic infection. Th2 type cytokines inhibit the cellular immune responses of the host, which may be related with the chronicity of hepatitis B.Correlative clinical studies show that in acute or self-limited hepatitis B, strong T-cell responses to HBV antigens are readily demonstrable in the peripheral blood. These responses involve both major-histocompatibility complex(MHC) class II -restricted, CD4+ helper T cells and MHC class I - restricted, CD8+ cytotoxic T lymphocytes. The antiviral cytotoxic T lymphocytes responses is directed against multiple epitopes within the HBV core, polymerase, and envelope proteins; strong helper T-cell responses to C and P proteins have also been demonstrated in acute infection. By contrast, in chronic hepatitis B, such virus-specific T-cell responses are greatly attenuated, at least as assayed in cells from the peripheral blood. Howerver, antibody responses are vigorous and sustained in both situations(although free antibodies against HBsAg are not detectable in carriers because of the excess of circulating HBsAg). This pattern strongly suggests that T-cell responses, especially the responses of cytotoxic T lymphocytes, play a central role in viral clearance. The mechanisms by which cytotoxic T lymphocytes kill liver cells and cause viral clearance have been incisively investigated in transgenic mice that express viral antigens or contain replication-competent viral genomes in the liver. Because these mice harbor HBV genes in their germ-line DNA, they are largely tolerant HBV proteins, and accordingly, clinically significant liver injury does not develop. However, if antiviral cytotoxic T lymphocytes of syngeneic animals are transferred into such mice, acute liver injury with many of the features of clinical hepatitis Bdevelop.Interferon-a and lamivudine are two effective treatment for hepatitis B. As a direct antiviral effect, Interferon-a also has immunomodulator activity. Interferon-a can induce the expression of IFN-y, and downregulates the levels of IL-4. Although lamivudine is not an immunomodulator, there is strong evidence that successful treatment with lamivudine relies to some extent on an adequate host immune responses.Bicyclol is a synthesized novel anti-hepatitis drug. It has remarkable hepatoprotective and certain anti-hepatitis virus actions. Addition of bicyclol to the cultivated 2.2.15 cell line inhibited secretion of HBeAg and HBsAg as well as HBV-DNA replication, Bicyclol 0.4, 0.6 g/kg given to duck-infected with DHBV significantly reduced serum and liver DHBV-DNA level. The results of mechanistic study on bicyclol indicate that bicyclol is not inhibitor of transaminase, but bicyclol can scavenge free radicals so as to maintain biomembrane stability, moreover, bicyclol can reduce hepatocyte nuclear DNA fracmentation and apoptosis induced by immuno-stimulating compound Con A.ObjectiveTo explore the levels of IL-4^ IL-10 and IFN-y in culture supernatants of peripheral blood mononuclear cells (PBMC) in patients with chronic hepatitis B, and to explore the characteristic of Thl/Th2 type cytokines expression in PBMC in patients with chronic hepatitis B. To study the effect of the levels of IL-4, IL-10 and IFN-y in the supernatants of PBMC cultures in CHB patients treated with Bicyclol, an to investigate the relationship between the expression of Thl/Th2 type cytokines and the effect of Bicyclol therapy. At the same time to explore whether antiviral treatments that is able to inhibit viral replication and to reduce viral load can successfully reconstitute CTL responses in CHB patients.Method1. Thirty patients with chronic hepatitis B at Southern Hospital and Huizhou Municipal Central Hospital between April and July 2004 were included in this study.They were 20 males and 10 females, with the age distribution from 17 to 53 years. All patients were seronegative for markers of hepatitis C, D and E virus. Patients had not received any immunomodulatory treatment in the preceding 3months. All patients were treated with Bicyclol Tablets, 50mg, 3 times one day for six months. In addition, there were seven healthy individuals as controls at the same time.2. PBMCs were separated from fresh, heparinized venous blood by standard density gradient centrifugation with lymphoprep. The interphase layer of cells were washed three times in buffered RPMil 640. The viability of PBMCs were tested with trypan blue exclusion and found to be greater than 95%, and were resuspended at 1 X 106/ml in buffered RPMI 1640, supplemented with 10% heat-inactivated fetal calf serum, and were stimulated with Phytohemagglutinin (PHA) in 24-well plates for 3 days at 37°C in a 100% humidified at mosphere of 5% CO2. IL-4^ IL-10 and IFN- Y production were determined by enzyme-linked immunosorbent assay(ELISA) after 72h cultivation, and to analyse their relation with the presence of HBeAg in serum. Before Bicyclol Tablets therapy, and at 1, 3 and 6 months during Bicyclol Tablets therapy, the production of IL-4> IL-10 and IFN- Y in culture superatants of PBMCs from 30 cases were detected by ELISA as above described.3. During Bicyclol treatment, PBMCs were separated routinely, and stimulated by peptide HBcAgl8-27/rHBcAg/rIL-2, the cytotoxic activity against HBV DNA transfected hepatoma cells(HepG2.2.15) and HepG2 cells were detected by lactate dehydrogenase (LDH) assay after 21 days cultivation.4. All values were expressed as X + S. Data analysis was performed using SPSS 10.0. P values less than 0.05 were considered statistically significant.Results1. The level of IL-4 in the patients with CHB was significantly higher than that in the control (17.18±7.43pg/ml vs 8.24±4.16pg/ml, P<0.05), and the level of IFN-y was significantly lower than that in the control (36.25 ±19.92pg/ml vs 121.68 ± 44.40pg/ml, P<0.01). Compared with the patients with HBeAg negative, the level of IL-4 in the patients with HBeAg positive was significantly higher (20.51 ±9.16pg/ml vs 10.98±5.03pg/ml, P<0.05), however the level of IFN-y was significantly lower (26.03 ± 10.48pg/ml vs 41.94 ±27.42pg/ml, P<0.05). The level of IL-4 was positively correlated with the level of HBV DNA, but the level of IFN-y was negatively correlated with the level of HBV DNA;2. At 6 months during Bicyclol therapy, the level of IFN-y was increased (67.78 ±33.65pg/ml vs 36.25 + 19.92pg/ml, P<0.05) and the level of IL-4 wasobviously decreased (7.42±2.79pg/ml vs 17.18±7.43pg/ml, P<0.01) .The change of the levels of IL-4 and IFN- Y at 3months therapy to pre-therapy were more notable in group with HBeAg postive than those in group with HBeAg negative respectively (7.88+2.78 pg/ml vs 20.51+ 9.16pg/ml, 64.42±36.00pg/ml vs 26.03+ 10.48 pg/ml, respectively, P<0.05) .3. CTL reaction measurements showed a trend to increasing , but there was no significant difference at pre- and post- Bicyclol Tablets treatment.Conclusion1. There was an abnormal cell-mediated immune response in CHB patients, and the imbalance between Thl and Th2 might be correlation with HBV sustained infection, Th2 cell mediated immune response was predominant in CHB, which was associated with the chronicity of HBV infection.2. Bicyclol Tablets treatment not only promoted Thl type cytokine-mediated immune responses but also downregulated Th2 type cytokine-mediated immune responses. Thl/Th2 type cytokines could be a predictor of clinical response during Bicyclol Tablets treatment.3. There was no evidence that Bicyclol Tablets could induce CTL activity.