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Objective: To investigate the curative effect of local application of CpG-oligodeoxynucleotide (CpG-ODN) combined with 4-1BB monoclonal antibody in hepatoma-bearing mice, and to evaluate the effect of 4-1BB monoclonal antibody on CpG-ODN immunotherapy. Methods: H22 single cell suspension was injected subcutaneously into the axilla and four limbs of the BALB/c male mice to establish a tumor-bearing mice model. After 7 days, 30 mice with corresponding tumor-bearing volume were screened and randomly divided into model control group, CpG group and CpG+4-1BB group, and the drug was injected into the tumors of left lower extremity. The same batch of normal mice was selected as normal control group. Survival of mice was recorded. Tumor-bearing volume and organ index were calculated. Serum levels of interleukin (IL) - 12 and interferon (IFN) gamma and spleen CD8(+)T lymphocyte ratio were measured. The measurement data were analyzed by analysis of variance. The survival rate of each group of mice was analyzed by log-rank test. Results: Mice in the model control group with tumor-bearing volume had a sustained growth before the execution. CpG group and the CpG+4-1BB group [(976.08 ± 29.55) mm(3), (47.25 ± 0.93) mm(3))] tumor-bearing volume was decreased than model group [(1 336.52 ± 39.40) mm3] ( F = 5 329.273, P < 0.05). CpG+4-1BB group distant tumor-bearing volume [(611.83 ± 113.02) mm3] was decreased than model group and CpG group [(1 406.62 ± 51.09) mm(3), (1 380.01 ± 51.44) mm3] ( F = 247.160, P < 0.05), but there was no significant difference between the CpG group and the model group ( P > 0.05). Serum IL-12 concentration (23.90 ± 2.33 pg/ml), IFN-γ concentration (103.02 ± 6.10 pg/ml) and spleen CD8(+)T cell ratio (4.54 ± 0.62%) in the model group were lower than those in the normal group ( P < 0.05). Serum IL-12 concentration in CpG group and CpG+4-1BB group (29.21 ± 2.23 pg/ml, 37.04 ± 1.49 pg/ml), IFN-γ concentration (116.12 ± 4.08 pg/ml, 138.65 ± 1.72 pg/ml), CD8(+)T cell ratio (6.65 ± 0.64%, 12.73 ± 0.88%) were higher than the model group, while CpG+4-1BB group was higher than the CpG group ( P < 0.05). The survival rate of CpG+4-1BB group was higher than that of model group and CpG group ( χ (2) = 25.544, P < 0.05), but there was no significant difference between CpG group and model group ( P > 0.05). There was no significant difference in organ index between the four groups ( P > 0.05). Conclusion: 4-1BB monoclonal antibody combined with CpG-ODN therapy can shrink hepatoma-bearing capacity, inhibit the growth of distant tumors and significantly prolong the survival time of mice.

Aptamers are capable of binding a wide range of biomolecular targets with high affinity and specificity. It has been widely developed for diagnostic and therapeutic purposes. Because of unique three dimensional structures and cell-membrane penetration, aptamers inhibit virus infection not only through binding specific target, such as the viral envelope, genomic site, enzyme, or other viral components, but also can be connected to each other or with siRNA jointly achieve antiviral activity. Taking human immunodeficiency virus and hepatitis C virus as examples, this paper reviewed the effects and mechanisms of aptamers on disturbing viral infection and replication steps. It may provide an insight to the development of aptamer-based new antiviral drugs.

Objective: To investigate the efficacy of cryoablation combined with CpG ODN in the treatment of murine transplanted colon carcinoma., Methods: Colon carcinoma model was established by subcutaneously inoculating CT26 cells into the right flank in BALB/c mice. The tumor-bearing mice were randomly divided into 4 groups:the group of PBS injected in peritumoral area, the group of cryoablation, the group of cryoablation combined with CpG ODN, the group of CpG ODN injected in peritumoral area. The tumor size changes were measured. Serum levels of interleukin (IL)-12 and interferon-gamma(IFN-gamma) were assayed by ELISA. The rates of CD3(+)CD4(+)T, CD3(+)CD8(+)T lymphocytes in serum were counted with flow cytometry. Mice in the cryoablation group and the combined group with tumor regression were re-challenged with CT26 cells., Results: The survival time of cryoablation group and combined therapy group were (80.3 + or - 5.4) days and (83.8 + or - 5.5) days, respectively, longer than (53.7 + or - 3.7) days in PBS group and (51.5 + or - 6.8) days in CpG ODN group(all P<0.05). The suppress rates of tumor cells in cryoablation group and combined therapy group were 83.8% and 86.2% respectively. After 20 days following treatment, CD3(+)CD4(+)T/CD3(+)CD8(+)T ratio and the concentrations of IL-12 and IFN-gamma in mice serum of cryoablation group and combined therapy group were higher than those in PBS group and CpG ODN group(all P<0.05). No significant difference was found in CD3(+)CD4(+)T/CD3(+)CD8(+)T ratio between cryoablation group and combined therapy group(P>0.05). However, the concentrations of IL-12 and IFN-gamma in combined therapy group were higher than those of cryoablation group(all P<0.05). After re-challenging, tumor formation rate in the cryoablation combined with CpG ODN group was 16.7%, significantly lower than that in the cryoablation group(83.8%)(P<0.05)., Conclusion: Cryoablation combined with CpG ODN can increase antitumor immune response in mice, and therefore can decrease the tumor formation when re-challenged with CT26 cells.

Objective: To study the protective effects on the infection of Schistosoma japonicum in C57BL/6 mice induced by dendritic cells DCs pulsed with GST in combination with CpG oligodeoxynucleotide., Methods: GST was purified and used to stimulate DC2.4 cell line. Antigen loading was analyzed by immunofluorescence method. Thirty-five C57BL/6 mice were divided into seven groups(5 mice per group). Mice in groups A, B, C, D and E were immunized subcutaneously with DCs, DCs treated with PSA, DCs pulsed with GST, DCs stimulated with GST+CpG ODN, DCs stimulated with CpG ODN, respectively. For the above five groups, each mouse received 100 microl cell suspension at the density of 10(7)/ml subcutaneously for three times at 2-week intervals. Each mouse of group F was immunized subcutaneously with 50 microg GST formulated in complete Freund's adjuvant first, and 50 microg, 10 microg GST respectively in incomplete Freund's adjuvant for the last two doses. Group G received PBS and served as control. Serum samples were collected 10 days after the final immunization, and were analyzed for specific antibodies by ELISA. At two weeks after the final immunization, each mouse were challenged by 30 +/- 1 cercariae of S. japonicum. Six weeks after infection the mice were sacrificed, and number of worms was counted., Results: Light green fluorescence was observed in dendritic cells under the fluoroscope after pulsing with GST which indicated the protein loaded dendritic cells. The IgG level in groups C, D and F was 0.555 2 +/- 0.078 9, 0.715 0 +/- 0.052 3, and 2.127 0 +/- 0.411 5, respectively, all higher than that of group G (P < 0.05). The worm reduction rate of group D was 53.3%, followed by group F (24.0%) and group C (21.3%). There was no significantly difference in the worm reduction rate between group D and groups F and C (P > 0.05)., Conclusion: Dendritic cells pulsed with GST in combination with CpG oligodeoxynucleotide induce significant immunoprotection against the infection of Schistosoma japonicum.

Objective: To evaluate the therapeutic effect of intranasal oligodeoxynucleotides with CpG motifs (CpG ODN) in prevention of allergic rhinitis in juvenile guinea pigs., Methods: Juvenile guinea pigs aged from 7 to 10 weeks were administrated with CpG ODN alone or combined with OVA at single dose concentration intranasally (on day 0, 5, 10, 15 in sequence) while control and blank group were administrated with saline. Both experimental and control animals were again sensitized by OVA (on day 18, 25), and 14 days after second sensitization animals were challenged by OVA intranasally (on days 39 and 46). Two hours after challenge, the animals were sacrificed. Then Hemotoxin and Eosin stain were carried out to analyze local eosinophilic reactions and nasal lesions. Local and systemic cytokines interleukin IL-5 and IFN-γ levels were examined by ELISA. Immunofluorescence was carried out with ICAM-1 antibody. Statistical analysis was performed using a SPSS 11.0 software., Results: In CpG ODN-administration or CpG ODN with OVA-administration group allergic rhinitis symptoms were not as severe as model control group (P < 0.05). Compared with the model control group, CpG ODN-administration did not increase production of OVA-specific Th1 cytokine IFN-γ but decreased productions of ovalbumin-specific Th2 cytokines IL-5 both in serum and nasal specimen (q value were 3.890 and 4.019, P < 0.05). Moreover, nasal lesions with infiltration of mean (x ± s) eosinophils (20.0 ± 9.6) in CpG group animal were prominently reduced by the CpG ODN-treatment compared with the control animals (53.5 ± 19.8) and CpG+OVA group (9.5 ± 5.7) were lower than CpG-M+OVA group (49.2 ± 18.9), the differences were significant (q value were 3.785 and 4.576, P < 0.05). Immunofluorescence results showed lower ICAM-1 expression in nasal specimen of CpG group compared with model group and CpG plus OVA group animal to CpG mimics plus OVA group (Z value were 3.697 and 3.765, P < 0.05)., Conclusions: Intranasal administration of CpG oligodeoxynucleotides with or without allergen may be an effective way to prevent the development of allergic rhinitis.

Objective: To investigate the therapeutic and immunological effects of microwave ablation (MA) combined with CpG ODN in mice bearing transplanted colon carcinoma., Methods: A mouse model bearing colon carcinoma was established by subcutaneously inoculating CT26 cells into the right flank of Balb/c mice. The tumor-bearing mice were randomized into control group with PBS injection in the peritumoral area, MA group, MA combinated with CpG ODN group, and CpG ODN group with CpG ODN injection in the peritumoral area. The tumor volume changes were observed, and serum CD3(+)CD4(+) and CD3(+)CD8(+) T lmyphocytes were analyzed by flow cytometry after the treatments. Serum levels of interleukin (IL)-2, IL-12 and IFN-gamma were detected by ELISA. The mice in the MA group and the combined treatment group showing tumor regression were rechallenged with CT26 cells., Results: No significant difference was found in the number of serum CD3(+), CD3(+)CD4(+), or CD3(+)CD8(+) T lymphocytes between the 4 groups. The ratio of CD3(+)CD4(+)/CD3(+)CD8(+) T lymphocytes in the combined treatment group and MA group were 1.58-/+0.10 and 1.53-/+0.13, respectively, significantly higher than that in PBS group and CpG ODN group (P<0.05). The serum concentration of IL-2, IL-12 and IFN-gamma in the combined treatment group were 64.6-/+7.4 pg/ml, 314.1-/+26.9 pg/ml and 61.9-/+7.3 pg/ml, respectively, significantly higher than those in the other 3 groups (P<0.05). The tumor formation rate in the combined treatment group was significantly lower than that in MA group (25.0% vs 75.0%, P<0.05)., Conclusion: CpG ODN can enhance the immunity and decrease the tumor formation rate following a rechallenge with CT26 cells in mice treated with MA.

Aim: To investigate the potential effect of a self-designed C-type CpG ODN (D-SL01) on coxsackie virus B3 (CVB3) infection., Methods: HeLa cells infected by CVB3 were used as cell a model and BALB/c mice inoculated with CVB3 were used as an animal model for evaluating the anti-viral effect of D-SL01., Results: The supernatants of D-SL01-treated PBMC protected HeLa cells from CVB3 infection and Vero E6 cells from VSV infection. However, in vivo study showed that D-SL01 reduced the body weight of CVB3-infected mice and aggravated the pathological changes related to myocarditis in mice when they were intraperitonealy injected for 6 days in succession., Conclusion: When used for the treatment of viral-infected diseases in vivo, CpG ODN may function in a complicated way, which indicates its administration time, route and dosage, should be taken into full consideration.

To investigate the anti-tumor and side effect of CpG ODN 1826 and CpG ODN2006 as an adjuvants on leukemic tumor in mouse-models, an acute lymphocytic leukemic tumor in mouse model was established, then inoculated inactivated L1210 cells alone or with different vaccine adjuvants were injected subcutaneously into each DBA/2 model mouse at different times. The activities of mice, the tumor formation rate and the growth status of leukemic tumor were observed. The tumor was examined by pathologic section. The results showed that the vaccine of inactivated L1210 cells and CpG ODN 1826 could decrease the leukemic tumor formation rate, slow down the growth of leukemic tumor mass in mice and obviously cause necrosis of tumor cells, but it could not prolong the life spans of the tumor-burden mice; while CpG ODN2006 could not only decrease the tumor formation rate, slow down the growth of tumor mass in mice and result in obvious necrosis of tumor cells, but also could eliminate the existing tumor mass in mice, and prolong the life spans of the tumor-burden mice. It is concluded that using CpG ODN2006 as an adjuvant enhances the anti-tumor effect against the leukemic tumor, prolong the life span of tumor-burden mice without obvious side effect.

Objective: To investigate whether CpG ODN can affect the antitumor responses of DC-tumor cell vaccine against Lewis lung cancer., Methods: CpG oligonucleotides 1826 (ODN 1826) were used to promote maturation of DCs in vitro. By fusing DCs with Lewis lung carcinoma L3-8 cells, DC-based tumor cell vaccines were developed. To determine the immune responses to the vaccines, T cell proliferation and cytotoxicity were done in vitro. Therapeutic and prophylactic immunization with DC vaccines were performed in C57BL/6 mice bearing Lewis lung carcinoma., Results: Bone marrow cells cultured in the presence of GM-CSF and IL-4 plus additional ODN 1862 appeared typical morphology of DCs. FACS analyses showed that the mean fluorescence index (MFI) of CD40 expression of DCs stimulated with and without CpG ODN was 24 and 11, respectively, and that of CD86 expression was 75 and 33, respectively. IL-12 secreted by DCs cultured with ODN 1826 was 10-fold as high as that without ODN 1826. Significant T-cell proliferation and T cell-mediated cytotoxicity against L3-8 was induced in vitro. Marked inhibition of tumor growth in L3-8 bearing mice was observed upon prophylactic and therapeutic immunizations with the vaccine., Conclusion: CpG ODN can enhance the antitumor responses of DC vaccine by promoting DC maturation.

Objective: To induce stronger humoral and cell mediated immune response against HBsAg and seek more effective methods to treat hepatitis B virus infection., Methods: 21-mer phosphorothioate oligodeoxynucleotide (ODN) and its control ODN were synthesized and used as an adjuvant of HBsAg and commercial hepatitis B vaccine to enhance their immune responses against HBsAg. Four groups of 33 mice received 2 doses of the mixtures (1.67 microg HBsAg: 16.5 microg CpG ODN) at an interval of 15 days. Group A: HBsAg only, 7 mice; group B: HBsAg + CpG ODN, 10 mice; group C: commercial hepatitis B vaccine, 6 mice; group D: commercial hepatitis B vaccine + CpG ODN, 10 mice. Serum anti-HBs levels and cytotoxic T lymphocyte response (CTL) of splenocytes were assayed 15 days after the last vaccination., Results: The mean absorbent values of blood anti-HBs in the 4 groups were 0.109, 0.435, 0.422 and 0.575, respectively. The rates of CTL response were 8.5%, 37.0%, 1.5%, and 28.0%, respectively., Conclusions: (1) CpG ODN markedly improves humoral (anti-HBs) and cellular immune response (CTL) induced by HBsAg immunization. (2) There is a synergistic interaction between aluminum and CpG ODN in enhancing the humoral and cellular immune responses against HBsAg. Therefore, CpG ODN might be used to treat chronic hepatitis B virus infection after its safety and effectiveness have been confirmed.