Hepatitis B virus replication is upregulated in proliferated peripheral blood lymphocytes.

Increasing evidence indicates that the hepatitis B virus (HBV) replicates in peripheral blood mononuclear cells (PBMCs), but at a low level. The present study aimed to establish a reliable and sensitive method that effectively detects HBV viral products for monitoring antiviral therapy, organ transplantation screening, and diagnosing occult HBV infection. In the present study, PBMCs (obtained from six healthy volunteers) were inoculated with HBV, and cultured with phytohemagglutinin (PHA) and interleukin­2 (IL­2) to stimulate cell proliferation. PBMCs were harvested, and quantitative detection of HBV DNA in cell suspension and intracellular hepatitis B surface antigen (HBsAg) was conducted on days 0, 1, 6 and 12, respectively. In situ hybridization, immunohistochemistry and reverse transcription­polymerase chain reaction (RT­PCR) were performed to analyze the HBV infection. The results demonstrated that HBV DNA increased concurrently with proliferation of PBMCs isolated from three of six healthy volunteers, and the mean number of PBMCs on day 12 was 13.61 times higher than the initially seeded cell number (P<0.01). The mean copies of HBV DNA at day 12 were 2.98 times higher compared with initial levels (P<0.05). Furthermore, intracellular HBsAg levels increased concurrently with proliferation of PBMCs in one group of cultured PBMCs, which was accompanied by increased HBV DNA levels. In addition, HBV nucleic acids were detected in PBMCs using in situ hybridization. Intracellular HBsAg was observed in PBMCs and HBV RNA was also detected by RT­PCR. The present study demonstrated that HBV replicates in proliferating PBMCs, which were induced by PHA and IL­2. This method offers a novel investigative tool to detect HBV infection in PBMCs and to monitor the course of HBV infection.

A DNA vaccine against chimeric AFP enhanced by HSP70 suppresses growth of hepatocellular carcinoma.

Alpha-fetoprotein (AFP) is produced principally in fetal liver, gastrointestinal tract and the yolk sac which is temporarily present during embryonic development. AFP is overexpressed in the majority of hepatocellular carcinoma (HCC) and thus offers an attractive target for immunotherapy against this neoplasm. Here, we report that anti-HCC effects were achieved in a therapeutic setting with a DNA vaccine encoding mouse AFP and co-expressing heat shock protein 70 (HSP70) gene. We also demonstrated that this vaccine elicited a marked and highly effective AFP specific CTL response against AFP-positive target cells. This vaccine also induced the prolongation of life span in mice bearing the tumor and the eradication of HCC. It is anticipated that vaccine strategies such as this may contribute to the effective future treatment of hepatocellular carcinoma.

[Immunotherapy with a chimeric AFP and HSP70 gene DNA vaccine targeting on a murine hepatocellular carcinoma].

OBJECTIVES: To investigate immune responses and the anti-tumor effect of a constructed Chimeric AFP-Mt.HSP70 DNA vaccine in mice. METHODS: Chimeric AFP-Mt.HSP70 was constructed by molecular clone techniques. Spleen cells derived from mice immunized twice were induced to secrete IFN gamma and were assayed using ELISA. The activity of the cytotoxic lymphocytes (CTL) derived from spleen cells was assayed using lactate dehydrogenase (LDH). 4 x 10(6) Hepa1-6 cells/200 microl were injected subcutaneously into the right axilla of each mouse bearing the tumor. The anti-tumor effect of the recombinant DNA vaccine was evaluated by measuring tumor sizes of the mice. RESULTS: AFP-specific CTL reaction was induced by our chimeric DNA vaccine and Mt.HSP70 enhanced this effect (P < 0.05). The CTL activity was about 32% at E/T=50:1. The IFN gamma secreted by spleen cells of mice immunized with chimeric plasmids was about 200 pg/ml. It was higher than those in the other groups; Tumor sizes of mice immunized with fused plasmids were smaller than those in the other groups. Survival times of mice immunized with the fused plasmids were prolonged. CONCLUSION: Chimeric DNA vaccine can induce AFP-specific CTL reaction and has an anti-tumor effect on transplanted tumors in our murine experiment.

[The role of dendritic cell and macrophage in hepatoma antigen-presenting].

OBJECTIVE: To study the role of dendritic cells (DCs) and macrophages, differentiated from the same individual peripheral blood monocytes, in tumor antigen- presenting. METHODS: DCs and macrophages were differentiated from human peripheral blood monocytes by adding both Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) or GM-CSF only. Then they were loaded with tumor antigen at different concentrations and cocultured with autologous T cells in 96-well flat-bottomed microtiter plates for five days at 37 degrees C, 5% CO(2). (3)H-thymine was added before the culture terminated, and twelve hours later, the cells were gathered to test the cpm value. RESULTS: Both DCs and macrophages chased with tumor antigen could strongly stimulate the proliferation of autologous T cells, especially DCs. The stimulation effect with 20 microl/ml antigen was the most remarkable and the cmp values were 11,950.3 +/-1621.8, 8,708.5 +/-176.1, 402.5+/-43.1 in DCs group, Macrophages group, and lymphocytes group, respectively. CONCLUSION: The antigen presenting role of DCs is stronger than that of macrophages from the same individual.

[Transient expression of fusion protein with a chimeric HBsAg-HSP70 construct in HepG2 cells].

OBJECTIVE: To investigate transient expression of fusion protein with a chimeric HBsAg-HSP70 construct in HepG2 cells. METHODS: Enkaryotic expression plasmids inserted HBsAg gene or chimeric HBsAg-HSP70 gene were prepared and transfected into HepG2 cells by means of cationic liposome. mRNA were detected by RT-PCR and proteins expressed in the cells were detected by immunocytochemistry 48 hours later. HBsAg in cultured supernatants and cell lysates were assayed by ELISA. RESULTS: Fusion protein (HBsAg-HSP70) transient expression in HepG2 cells were confirmed by RT-PCR, immunocytochemistry or ELISA, but fusion protein was not assayed in cell cultured supernatants by ELISA. CONCLUSIONS: Transfection of HepG2 cells with a chimeric HBsAg-HSP70 construct leads to express fusion protein, but it does not secrete into cell cultured supernatants.