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The potential application of dendritic cells (DC) sensitized with cytosine-phosphoric acid-guanine (CpG) oligodeoxynucleotide (ODN) and tumor antigen as a vaccine against murine melanoma was investigated with freshly isolated mouse bone marrow-derived dendritic cells. For the DC vaccine preparation, DC were sensitized with the B16 tumor antigen and CpG ODN was used to promote further maturation of the DC. The immunogenic activity of the vaccine was evaluated in vitro by determining the proliferation of T lymphocytes and the killing effect of cytotoxic T lymphocytes (CTL) on B16 tumor cells. The DC vaccine was injected intraperitoneally and tumor inhibition in mice bearing B16 xenografts was examined. All mice were cared for under an approved SIMM Institutional Animal Care and Use Committee (IACUC) protocol. In vitro, this DC vaccine promoted the proliferation of T lymphocytes and showed a potent killing effect on the target B16 cells. In vivo experiments showed that after treatment or pre-immunization both the tumor volume and weight were significantly decreased. The DC vaccine with CpG ODN and tumor antigen exhibited an inhibitory effect against melanoma, providing a potential method for melanoma cancer treatment.
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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) mm3, (47.25 ± 0.93) mm3)] 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) mm3, (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 (χ2 = 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.
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@#Objective: To investigate the role of CpG ODN (CpG oligodeoxynucleotide) adjuvant in enhancing the anti-bladder cancer response induced by MAGE-3 (melanoma antigen gene -3) antigen and its molecular mechanism. Methods: Mononuclear cells were isolated from HLA-A2 type peripheral blood of healthy donors by Ficoll method to prepare mature DC by conventional means. DC surface markers were detected by flow cytometry. MTT assay was used to detect the promotion effect of DCs sensitized by different means (MAGE-3, CpGODN, MAGE-3+CpG ODN, irrelevant control antigen) on the proliferation of T lymphocytes and the killing effect of CTL on BIU-87 tumor cells. The tumor mass of nude mice bearing BIU-87 bladder cell xenograft were examined on Day 7 and 11 after CpG ODN+MAGE-3 sensitized DC treatment. The expression of Bcl-2/Bax protein was detected by Western blotting while the proliferation level of xenograft cells was detected by MTT assay. Results: DCs sensitized by CpG ODN combined with MAGE-3 antigenic peptides could promote the proliferation of T lymphocytes and significantly enhance the killing effect of CTLon target BIU-87 cells (P< 0.05). Compared with other sensitized DCs, in vivo experiments showed that 7 and 11 days after treatment, both the tumor volume and weight were significantly reduced (all P<0.05), and the proliferation ability of xenograft tumor was decreased (P<0.05). Compared with other sensitization means, CpG ODN+MAGE-3 especially exhibited obvious inhibitive effect on tumor growth on Day 11, and significantly promoted the proliferation of splenic monocytes of tumor bearing mice (P<0.01); moreover, Bcl-2 expression in xenograft tissues significantly decreased（P<0.01）while Bax expression significantly increased（P<0.05 or P<0.01）on Day 3 after treatment. Conclusion: CpG ODN can promote the inhibitory effect of MAGE-3 sensitized DC on bladder cancer BIU-87 cells, which will provide experimental basis for clinical application of DC vaccine in bladder cancer treatment.
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Objective To study the treatment effects of newborn mice murine Cytomegalovirus (MCMV) hepatitis by using CpG Oligodeoxynucleotide 2395 (CpG ODN2395).Methods Specific pathogen-free BALB/c newborn mice were divided into 3 groups according to the broods randomly:control group,virus group and treatment group.In control group 9 g/L sodium chloride 20 × 10-6 L was intraperitoneally injected every other day.In virus group MCMV (TCID50 =108.31/L) 20 × 10-6 L was intraperitoneally injected once and 9 g/L sodium chloride solution 20 × 10-6 L was intraperitoneally injected every other day.In treatment group murine MCMV(TCID50 =10S31/L) 20 × 10-6 L was intraperitoneally injected once and from the first day CpG ODN2395 20 mg/kg was intraperitoneally injected every other day.Growths and development of mice were observed.Murine body weights were measured.Pathology of livers was observed by means of hematoxylin and eosin stain.The levels of serum ALT and IFN-α were measured by adopting enzyme linked immunosorbent assay.MCMV loads in liver were measured by way of polymerase chain reaction.The expression levels of IFN-α mRNA in liver were detected by using reverse transcription polymerase chain reaction.The expression levels of Toll-like receptor 9 (TLR9) and myeloid cell differentiation factor 88 (MyD88) in liver were detected by adopting Western blot.The results were analyzed by using SPSS 16.0 statistics software.Results 1.Compared with control group and treatment group,growth and development of virus group mice fell behind and on day 7,14 body weights were lowest(F =18.919,25.543,all P ＜ 0.05).Growth and development of treatment group mice were between control group and virus group.Body weights of treatment group mice were lower than those of control group,and there was statistical difference on day 7 (t =3.187,P ＜ 0.05).2.Compared with control group and treatment group,the levels of ALT in virus group was highest.ALT levels of treatment group were higher than those of control group and treatment group(F =11.407,11.791,154.656,all P ＜ 0.05).3.The pathologic change of liver tissue:the HAI of virus group reached the peak on day 3,then decreased gradually.The HAI of treatment group also reached the peak on day 3,but liver damage was obviously less than those of virus group.The liver damage also relieved gradually and the mean of HAI was obviously lower than that of virus group on day 7 and 14.4.MCMV DNA in liver was negative at control group.The magnitude of viral loading in livers of virus group was higher than that of treatment group.5.The levels of IFN-α in treatment group and virus group reached a peak on day 3 and declined gradually on day 7,14.The levels of IFN-α on treatment group were higher than that of virus group and control group.The levels of IFN-α virus group were higher than those of control group,but had no statistical difference.6.The mRNA expression of IFN-α in livers of treatment group and virus group began to increase on day 3 and reached a peak on day 7,and declined on day 14.The mRNA expression of IFN-α was higher than that of virus group and control group.The mRNA expression of virus group was higher than that of control group.7.The expressions of TLR9 and MyD88 in livers of treatment group were higher than that of virus group and control group.The expressions of TLR9 and MyD88 of virus group were higher than those of control group.Conclusions CpG ODN2395 can obviously improved liver function and histopathological lesions and reduce MCMV DNA load within liver tissues as well.CpG ODN2395 can improve the expression levels of TLR9 in liver and activate secretion interferon alpha by MyD88-dependent pathway,which were likely to play an important role in its treatment of murine CMV infection.
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CpG oligodeoxynucleotides (ODN) have potent immunostimulatory effects and can enhance the anti-cancer activity of cancer treatments. CpG ODN directly induced the activation and maturation of plasmacytoid dendritic cells, stimulated the secretion of Th1-type cytokines, and enhanced the differentiation of B cells into antibody-secreting plasma cells. CpG oligodeoxynucleotides as vaccine adjuvants can enhance both the humoral and cellular responses to antigens in some clinical trails. CpG ODN was applied in several clinical trials as an adjuvant of tumor vaccine. CpG ODN alone had anti-tumor activity and had synergistic effects with other anti-tumor therapies including monoclonal antibodies, chemotherapy, radiotherapy, cytokines, etc. Compared with standard regimen, in the phase Ⅲ clinical trial CpG ODN did not prolong the median survival time and induced severe adverse reaction in the treatment of ⅢB-Ⅳ stage non-small cell lung cancer. But CpG ODN had definite anti-cancer activity in other clinical trials. The safety and efficacy of CpG ODN in anti-tumor therapy needs to be further verified in clinic.
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Objective To investigate whether a novel mucosal adjuvant (DNA containing six base pair motifs consisting of an unmethylated CpG dinucleotide flanked by two 5′ purines and two 3′ pyrimidines, CpG Oligodeoxynucleotide, CpG ODN),which has not been shown to have significant toxicity,could be an ideal mucosal adjuvant for the development of a H. pylori vaccine in mice model. Methods C57BL/6 mice were orally or intranasally immunized with H. pylori whole cell sonicate(WCS) / cholera toxin (CT) or WCS /CpG ODN, and the corresponding control groups were set. Mice were dosed once a week for four weeks. One week after the last immunization, all animals were challenged by live H. pylori (5?10 8) three times in a five day duration. Two and 8 weeks after the last challenge, all animals were sacrificed to examine infection of H. pylori. Sera, saliva, gastric juice were collected to measure the concentrations of IgG, IgG1, IgG2a and IgA by ELISA. Results The protecting rates against H. pylori infection were 75%(9/12), 70% (7/10) and 0 (0/10) in the group of WCS/CT orally, WCS/CpG ODN intranasally and WCS/CpG ODN orally, respectively. Significantly higher levels of serum IgG2a antibody was found in the group immunized with WCS plus CpG ODN than those found in the sham immunized controls ( P
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Objective To identify the putative CpG-N ODNs in adenovisus 2 DNA (Adv2 DNA) and Adv5 DNA by comparing the sequence difference among Adv2, 5, 12 DNA and E.coli (EC) DNA. Methods Sequences of Adv2, 5, 12 DNA and EC DNA were obtained from the Entrez Nucleotides database at NCBI. The specific CpG motifs of Adv2 DNA and Adv5 DNA were identified after above sequences were analyzed and compared by softwares such as DNATools, BioEdit, and so on. All the 12-ODNs with specific CpG motif core were searched from Adv2 DNA and Adv5 DNA. Results 19 specific CpG motifs were ascertained and 504 12-ODNs were detected in Adv2 DNA and Adv5 DNA. Conclusion 504 12-ODNs were putative CpG-N ODNs in Adv2 DNA and Adv5 DNA.
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0.05); serum HBsAb levels of pregnant mice and neonatal mice in group with CpG-1826 (20 ?g)+hepatitis B vaccine significantly higher than those in group with CpG-1826 (10 ?g, 40 ?g)+ hepatitis B vaccine,hepatitis B vaccine and control respectively(P0.05).Conclusions Combination injection of CpG-1826 20 ?g and hepatitis B vaccine can markedly increase serum antibody levels of pregnant mice and neonatal mice, but don′t affect the survival quantity, the growth and development of neonatal mice.CpG-1826 is an ideal immune adjuvant for neonates with immature immune system during pregnancy.
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Objective To obtain a potent bacterial DNA antagonizing CpG oligonucleotides (CpG-N ODN) from the structures of Adv2, 5 DNA. Methods Ten putative CpG-N ODNs were synthesized and investigated. Their abilities to inhibit the TNF-? release from hPBMC were observed. Based on the above results, the putative CpG-N ODN was redesigned according to the relationship between the structure and free energy using RNA structure software (version 3.71). Eleven putative CpG-N ODNs were synthesized and screened. Results Out of the ten initial CpG ODNs, ODN101 was the only CpG-N ODN with weak activity to inhibit TNF-? release from hPBMC induced by CpG-N ODN. After redesign, five CpG-N ODNs with strong activity were confirmed. Conclusion Six CpG-N ODNs were obtained with activity to inhibit TNF-? release from hPBMC induced by CpG-N ODN.