Adjuvant DNA vaccine pNMM promotes enhanced specific immunity and anti-tumor effects.

DNA vaccines containing only antigenic components have limited efficacy and may fail to induce effective immune responses. Consequently, adjuvant molecules are often added to enhance immunogenicity. In this study, we generated a tumor vaccine using a plasmid encoding NMM (NY-ESO-1/MAGE-A3/MUC1) target antigens and immune-associated molecules. The products of the vaccine were analyzed in 293 T cells by western blotting, flow cytometry, and meso-scale discovery electrochemiluminescence. To assess the immunogenicity obtained, C57BL/6 mice were immunized using the DNA vaccine. The results revealed that following immunization, this DNA vaccine induced cellular immune responses in C57BL/6 mice, as evaluated by the release of IFN-γ, and we also detected increases in the percentages of nonspecific lymphocytes, as well as those of antigen-specific T cells. Furthermore, immunization with the pNMM vaccine was found to significantly inhibit tumor growth and prolonged the survival of mice with B16-NMM+-tumors. Our data revealed that pNMM DNA vaccines not only confer enhanced immunity against tumors but also provide a potentially novel approach for vaccine design. Moreover, our findings provide a basis for further studies on vaccine pharmacodynamics and pharmacology, and lay a solid foundation for clinical application.

Study of the Reliability of Field Test Methods for Physical Fitness in Children Aged 2-3 Years.

Physical fitness measures overall physical health. It is the ability of the body to work effectively and stay healthy during leisure and emergencies. Given the progressive integration of 2-3-year-olds into preschool, physical fitness testing of these children has become increasingly important. We aimed to develop and test the reliability of an appropriate field test method for physical fitness in 2-3-year-olds children. One hundred and three children (44 boys and 59 girls) volunteered for this study. Their height and weight were tested, and the same tester conducted the test twice for handgrip strength, 3 m balance walking, stair climbing, 5 m run, and kicking a ball at one-minute intervals. Pearson correlation coefficient and intraclass correlation coefficient (ICC) were used for reliability testing. The reliability of this field test method for physical fitness was high in the repetitive tests of Chinese 2-3-year-olds for the four items of handgrip strength, 3 m balance walking, stair climbing and 5 m run, and the reliability was moderate for the kicking the ball item. This study indicates that these field-based physical fitness test methods have good reliability and are simple, feasible, safe, and easy to be accepted and understood by 2-3-year-old children; thus, it may be used as a reference for professionals in China and abroad.

Cancerogenic effect of different fragments of the hepatitis C virus core protein.

The hepatitis C virus core protein plays an extremely important role in the hepatocarcinogenesis of hepatitis C virus. Little, however, is known about the oncogenic potency of fragments. Thus, the purpose of the present study is to investigate the cancerogenic effects of the different core protein fragments. Two series of recombinant plasmids containing hepatitis C virus core gene fragments encoding the different-length core protein were constructed using plasmid enhanced green fluorescent protein (pEGFP)-C1 and pcDNA3.1(+), respectively. Human hepatocyte L02 cells transiently transfected with pEGFP-C1-based plasmids were subjected to confocal laser scanning microscopy analysis to determine the localization of the different core protein fragments. The stably transfected L02 cells with the pcDNA3.1(+)-based core protein plasmids were used to investigate the ultrastructural effects of the core protein and the tumorigenicity of L02 cells expressing core protein fragments in athymic nude mice. The full-length core protein and Core130-191 were completely localized in the cytoplasm, while Core1-59 existed exclusively in the nucleus. On the other hand, Core50-140 and Core1-140 were observed in both the nucleus and the cytoplasm. Ultrastructural changes of L02 cells expressing the full-length core protein were comprehensive and included, for example, irregular nuclear, increased nuclear/cytoplasmic ratio and mitochondria swelling. The slight changes were observed in the cells expressing Core50-140 and Core130-191, whereas the ultrastructure of the cells expressing Core1-59 remained normal. All the L02 cells stably expressing different fragments of the core protein, with the exception of the C-terminal truncated fragment Core1-59, could induce the occurrence of tumor in the nude mice. The N-terminal fragment of the core protein, Core1-59, was not oncogenic, while the intermediate and posterior segments of the hepatitis C virus core protein had the cancerogenic potency. In view of the existence of many important immunogenic epitopes in it, the core protein anterior segment might be a safer candidate for the development of hepatitis C virus vaccine.

[Studies on the correlation between titer of antibodies against different function regions of hepatitis C virus and HCV RNA of chronic patients].

OBJECTIVE: To detect humoral immune response against different function regions of hepatitis C virus (HCV) in chronic patients, and further to investigate the correlativity between anti-HCV antibody titers and HCV RNA concentration. METHODS: Using recombinant dominate epitope antigens, e.g. HCV Core, NS3, NS4, NS5 and chimeric HVR1, a set of ELISA test reagents was formulated. Then, titers of antibodies against HCV different regions and the RNA concentration of HCV in chronic patient sera were detected by ELISA and quantitative RT-PCR technique, respectively. RESULTS: Great differences have been noted in antibody titers and positive rate of different HCV function regions in chronic patients. Antibodies against HCV Core and HVR1 have the highest positive rate, then NS3, NS4, and NS5 in sequence. CONCLUSION: The titer of antibodies against different regions of HCV in chronic patients has good correlation with HCV RNA concentration.

[Internal Ribosomal Entry Site-Mediated Expression of Human Multidrug Resistance 1 Gene in Human CD34(+) Cells]

In order to reach the purpose of co-transferring double drug resistance genes into human CD34(+) progenitor cells to broaden the spectrum of drug resistance, the expression efficiency of human multidrug resistance 1 (MDR1) gene mediated by the internal ribosomal entry site (IRES) was investigated. Two retroviral vectors were transferred into packaging cells. One is pSF-DIM containing double drug resistance genes, in which the translation of MDR1 gene was controlled under an IRES from encephalomyocarditis virus. The other is pSF-MDR1 which only contains MDR1 gene controlled under the same promoter of pSF-DIM. The amphotropic retroviral packaging cells PA317/pSF-DIM and PA317/pSF-MDR1 were obtained with titer of 8 x 10(4) and 1.3 x 10(5) cfu/ml respectively. Human cord blood CD34(+) cells were transduced by supernatant infection. Expression of P-gp was detected by flow cytometry. Compared with the untransduced group, the expression of P-gp in pSF-DIM transduced group and pSF-MDR1 transduced group was elevated 10.92% and 28.82% respectively. However, the expression of P-gp in pSF-MDR1 transduced group was higher than that in pSF-DIM transduced group. The result suggests that MDR1 gene can express in the human progenitor cells under control of IRES. It laid the foundation of subsequence research. The reason on the difference in MDR1 gene expression efficiency between pSF-MDR1 transduced group and pSF-DIM transduced group need further research.

[Purification of Human CD40-Ig Fusion Protein and Its Effect on Human Peripheral Blood CFU-T Formation In Virto]

CD40/CD40L interactions play a pivotal role in T cell activation, and take part in many physiologic and pathologic procedures and different levels. In this article, stable CHO transformants secreting human CD40-Ig fusion protein were established through transfection and selection with Lipofectamaine and G418, respectively. In order to obtain great valume of recombinant protein, big batch serum-free cultures of engineered CHO cells were performed in roller-bottle using CHO-II-SFM medium. After cultures, the cell-culture supernatants were harvested, concentrated through ultra-filtration, and finally purified by affinity choromatography with Protein G Sepharose Fast Flow. Human peripheral bloods were collected freshly and seperated with Ficoll, CFU-T was cultured in semi-solid culture system with peripheral blood mononuclear cells (PBMNC). Effect of human CD40-Ig fusion protein on the formation of CFU-T was observed in vitro. The results showed that the yield of human CD40-Ig fusion protein was 30 mg in total 3 liter CHO-II-SFM culture supernatant, and it supposed that the expression level of CD40-Ig in CHO cells was more than 10 micro g/ml. The purity of purified fusion protein is above 95%. Furthermore, compared with human IgG, human CD40-Ig fusion protein significantly inhibited the formation of CFU-T at dose 0.25, 1.0, 4.0, and 10 micro g/ml, it lays a good foundation to evaluate its potential functions in vivo.

[Construction of Expression Vector for Human CD40-Ig Fusion Protein and Its Expression in COS-7 Cells]

CD40/CD40L, besides B7/CD28, is an alternative important costimulation signal transduction pathway. It plays a pivotal role in T cell activation. Moreover, it may play a critical role at many levels of sensitization and effector phases of allograft rejection. In order to get the fusion protein of human CD40 extracelluar region and IgG 1 Fc fragment, and investigate the potential role of blocking CD40/CD40L costimulation pathway in immunotherapy, total RNA was extracted from human lymphoma cell line Daudi, and CD40 gene extracelluar region was amplified by RT-PCR. The PCR products were inserted into pGEM T Easy vector, and the cloning vector pGE40 was obtained. The DNA sequence was analyzed by automatic DNA sequencer. After sequencing, the transient expressing vector was constructed by inserting correct fragment into pIG vector, which contains the genomic human IgG1 Fc (hinge, CH2 and CH3) gene. Hence the recombinant fusion expression vector was constructed successfully, and named after pIG/40 Ig. Then, COS-7 cells were transfected through DEAE-Dextran/chloroquine method. The CD40-Ig fusion protein expressed in COS-7 cell culture supernatant was identified by sandwich ELISA and Western blot. Result showed that the CD40-Ig fusion protein can be detected by sandwich ELISA in the cell culture supernatant. Western blot analysis also showed that it could react with McAbs of mouse anti-human CD40 G28-5 and mouse anti-human Ig gamma chain. There is only one obvious band at the position of relative molecular weight 50 kD, and it is equivalent to the expected value. Above all, the recombinant fusion expression vector pIG/40 Ig was constructed, and CD40-Ig fusion protein gene was expressed in COS-7 cells successfully. It could be laid a foundation to investigate the potential role of CD40/CD40L pathway as the target of GVHD prevention and therapy.