| Objective1. To evaluate the clinical efficacy of treating the CHB patients by Nourishing Kidney and Eliminating Toxicity and Nucleotide Analogue. In order to explore the advantage of therapy of Traditonal Chinese Medicine.2. To compare the differences on the function of monocyte-derived dendritic cells between healthy donors and CHB patients, and explore the mechanism of HBV persistent infection.3. To evaluate the changes on the antigen presentation of dendritic cells in the patients who received the therapy of Nourishing Kidney and Eliminating Toxicity from the perspectives of cellular immunology methods, and to find out the possible methods to break the status of HBV persistence infection.4. We can interpretthe relationship between the immunosecpae of constant HBV infection and the Traditional Chinese Medicine's latent-evil theory-"pestilence incubated in the liver blood due to the kidney deficiency", and can finally try to discover the essential pathogenesis of latent evil of CHB, and provide a theoretical basis ofr treatment of Traditional Chinese Medicine in CHB.Methods1. To gather 49 CHB patients, and divid into two groups stochastically, and treat them with the therapy of Nourishing Kidney and Eliminating Toxicity,Nucleotide Analogue. To examinate the changes of serum and virologic marks before,3 months and 6 months after being treated. 2. Generation of monocyte-derived DCs in peripheral blood from control group and patients with CHB:monocytes were islated from blood samples containing heparin obtained using Ficoll density gradient centrifugation. After 2 hours of culturing in 6-well plates the adherent cells were cultured in RPMI supplemented with 10%fetal bovine serum. Recombine human granulocyte macrophage-colony stimulating factor and IL-4 were added to DCs cultures. In the day 9, Recombinant human TNF-a and IL-1βwere added in the culture. Collecting the mature DCs. The presence of DCs surface markers were evaluated by flow cytometry using a panel of phycoerythrin or fluorescein isothiocyanate-conjugated monoclonal antibodies specific for CD14, CD80, CD83, CD86 and HLA-DR. The autologous mixed leukocyte reaction (AMLR) was used to test the capacity of DCs to initiate a primary immune response.3. Evaluate the special cytokines of Th cells stimulated by DCs:In the day 9 of generation of monocyte-derived DCs, HBsAg was added, then measured the level of IL-12,IFN-γ,IL-10 by ELISA.4. To gather the moncytes which were islated from blood samples containing heparin obtained using Ficoll density gradient centrifugation. Flow cytometry was used to evaluate the PD-1 expression in CD8+T cells. In order to know the changes of immunosuppressive cytokines of cytoimmunity.5. The effect of CTL response induced by HBcAg-pulsed dendritic cell: HBcAg-pulsed dendritic cells were stimulus which was cultured with DCs. ELISPOT was an effective method to evaluate the CTL activity, which mainly reflected by the level of IFN-γscreted from actived CTL. Therefore, we can consider if there was something to do with the improved clinical index and the celluar immunity.Results1. Measured HBV-DNA and ALT of the two groups, we found that there was no difference before the treatments.3 months later, the level of HBV-DNA and ALT decreased observably, P<0.05 in Nucleotide Analogue groups. HBV-DNA and ALT of the Nourishing Kidney and Eliminating Toxicity group also decreased, but not obviously, while the level of the two marks decreased observably after 6 months of treatment.2. We successfully induced the monocyte-derived dendritic cells from healthy donors and CHB patients. The dendritic cells we generated displayed typical characteristics on morphology. There was significant difference in the expression of dendritic cells'surface markers such as CD14, CD80, CD83,CD86 and HLA-DR from healthy donors and CHB patients. However, the increased level of the surface markers of the TCM group is more than the Nucleotide Analogue groups after 6 months of treatment. We found that the stimulate index(SI) detected at the mix ratio 1:40(DC:autologous T cells) in healthy donors was higher than the patients, which had significant difference statistically.3. The stimulate index (SI) detected at the most appropriate ratio in healthy donors was higher than before therapy group, after therapy group and HBcAg-pulsed group, which had difference between groups statistically. We found that the level of IL-12 which secreted by dendritic cells in healthy group and patients were (705.9±126.1) VS (583.5±135.8) respectively, which were statistical significance. After 6 months treatment, the level of IL-12 and IFN-γin TCM therapy group were higher than the Nucleotide Analogue groups.4. The virus antigenic specific CTL of CHB patients was (50.58±20.93) VS (20.26±13.85) respectively. The level of TCM therapy group is higher than the Nucleotide Analogue groups. And PD-1 which expressed on the surface of CD8+T cells has decreased in both two groups, but not statistical significance in compare. However after 6 months treatment the level of PD-1 is lower than that before therapy's in TCM group. HBcAg-pulsed DC could active more cytotoxic T lymphocytes, which secreted high level IFN-γ, compared to the DC without antigen loaded.Conclusions1. HBV has been eliminated, liver function of CHB has recovered and the clinical sumptoms has been made better, which can be achieved through both methods. And the Nucleotide Analogue groups is better than the TCM therapy group on eliminating HBV, but not on restoring liver function in a short time. But the TCM therapy would show the advantage on eliminating HBV and restoring liver function for a long time. We found that the therapy of Nourishing Kidney and Eliminating Toxicity would had a positive effect on immunity of CHB patients. 2. The ability of peripheral blood dendritic cells from patients with chronic hepatitis B infection to stimulate reproduction of lymphocytes was down regulation. Considering the comparison research of DC s surface marker, we believed that the down regulation was related to the lower expression level of adhesive molecules of DC for the low level expression of adhesive molecules may lead to down regulation of antigen presentation ability of DC and hence affect its stimulative ability. Probably, an enhanced antigen presentation ability of DC is beneficial to the clearance of virus in HBV infected patients.3. There is no obvious difference of expression of CHB patient's DC's surface marker between pre-therapy group and post-therapy group that treated by Nourishing Kidney and Eliminating Toxicity, but the level of expression is up regulated. This phenomenon suggested that Nourishing Kidney and Eliminating Toxicity can up regulate the expression of adhesive molecules of DC. The up regulation of CD83 expression level of DC after stimulated by HbcAg may relate to the degree of maturity and the antigen presentation ability. The results of surface marker expression level of Dc and AMLR suggested that HBcAg charge could up regulate the antigen presentation ability of DC and enhance humoral and cellular immunological response induced by DC.4. PD-1's expression on CD8+T cells decreased, while the co-stimulatory molecule CD137 up regulated in Nourishing Kidney and Eliminating Toxicity group. It is indicated that the Triditional Chinese medical therapy can promise the cellular immune function.The Nourishing Kidney and Eliminating Toxicity was a effective remedy in the therapy of CHB and it could regulate the expression of a series of cell factors. Our work suggested that C could up regulate the function of cellular immunity of patients which probablly relates to the enhancement of antigen presentation ability of DC. The prospect of using antigen-charge DC as an immune adjuvant in the immune therapy of CHB was promising.
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