Fabrication of functionalized magnetic microspheres based on monodispersed polystyrene for quantitation of allyl-benzodioxoles coupled with gas chromatography and mass spectrometry.

Monodispersed magnetic microspheres were synthesized by the magnetization of the aminized polystyrene cores and the subsequent polymerization of allyl glycidyl ether, divinylbenzene and N-vinyl-2-pyrrolidone, which exhibited excellent monodispersity in aqueous solution and high efficiency for allyl-benzodioxoles extraction. Various techniques, including scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR) and vibrating sample magnetometry (VSM) were employed to characterize the composites. Coupled with gas chromatography-mass spectrometry (GC-MS), a sensitive and simple magnetic solid phase extraction (MSPE) procedure based on the prepared microspheres was established for determination of allyl-benzodioxoles in cola-flavoured drinks. The factors affecting the extraction procedure, such as pH value, adsorbent amount, adsorption time, desorption solution and desorption time were optimized. The developed method was characterized by a high recovery (spiked at 5 µg L-1, 25 µg L-1 and 250 µg L-1), a low detection limit (0.05 µg L-1 for safrole and 0.08 µg L-1 for myristicin), a good linearity (correlation coefficients higher than 0.9990 for both) and a satisfactory repeatability (relative standard deviation below 3.7% for both, n = 3). The approach proposed here was confirmed to be fast and reliable for quantitative analysis of allyl-benzodioxoles in cola samples, especially at a trace level.

Type-specific and cross-reactive antibodies induced by human papillomavirus 31 L1/L2 virus-like particles.

The aim of this study was to determine whether antibodies induced by human papillomavirus (HPV) type 31 L1/L2 virus-like particles (VLPs) could cross-react with VLPs of the closely related HPV-16 and distantly related HPV-11, and to investigate the potential role of the L2 protein in L1/L2 VLPs in inducing cross-neutralizing antibodies. Antisera were prepared from rabbits immunized with intact or denatured HPV-31 L1/L2 VLPs. Cross-reaction and cross-neutralization were analysed by Western blotting and ELISA, and by haemagglutination inhibition, respectively. Western blotting results showed that H31 L1/L2 (D) antiserum (antiserum from a rabbit immunized with denatured HPV-31 L1/L2 VLPs) could cross-react with the L1 protein of HPV-11 and -16. HPV-31 L1/L2 VLP antiserum showed strong cross-reaction with and cross-neutralization of HPV-16 VLPs, but this was significantly less with HPV-11 VLPs. In addition, the cross-neutralizing activity against HPV-16 L1/L2 VLPs was slightly higher than that against HPV-16 L1 VLPs, although the difference was not statistically significant. Epitope-blocking ELISA showed that mAb H16.V5 could partially inhibit the cross-reaction of HPV-31 L1/L2 VLP antiserum with HPV-16 L1/L2 VLPs. These results suggested that (i) H31 L1/L2 (D) antiserum could cross-react with L1 protein from both closely related and distantly related HPV types, but HPV-31 L1/L2 VLP antiserum could only cross-neutralize closely related HPV types, (ii) surface-exposed epitopes of the L2 protein in L1/L2 VLPs may induce only low titres of cross-neutralizing antibodies and (iii) certain epitopes that cross-reacted with HPV-31 L1/L2 VLP antiserum are located close to the epitopes recognized by mAb H16.V5. These findings may provide suggestions for the design of multivalent VLP vaccines.

Encapsidating artificial human papillomavirus-16 mE7 protein in human papillomavirus-6b L1/L2 virus like particles..

BACKGROUND: Human papillomaviruses (HPVs) can infect squamous or mucosal epithelia and cause cervical cancer or genital warts. Coinfection with multiple HPV types is a common finding of many epidemiological studies. Therefore, it is necessary to develop a vaccine, which can eradicate established HPV infections and prevent other HPV infections. In this study, we generated chimeric virus like particles (cVLPs) composed of HPV-6b L1, HPV-6b L2 and one artificial HPV-16 mE7 proteins. METHODS: The artificial HPV-16 mE7 gene was designed by codon modification, point mutation and gene shuffling then chemically synthesized and subcloned behind HPV-6b L2. HPV-6b L1 and L2-mE7 were expressed in insect cells by using Bac-to-Bac system. The generated cVLPs were purified by CsCl gradient ultracentrifuge and analyzed by immunoblot, electron microscope and haemagglutination assay. RESULTS: The HPV-6b L1 and L2-mE7 proteins were well expressed in insect cells and could selfassemble into cVLPs, whose diameter was about 55 nm and similar to that of HPV-6b L1/L2 VLPs. Intact cVLPs could be recognized by H6.M48 neutralizing monoclonal antibody and HPV-6b L2 polyclonal antibody, while the denatured cVLPs, but not the intact cVLPs, were reactive to HPV-16 E7 polyclonal antibody. HPV-6b L1/L2-mE7 cVLPs haemagglutinated mouse erythrocytes as efficiently as HPV-6b L1/L2 VLPs did. CONCLUSIONS: The insertion of the 158 amino acid HPV-16 mE7 protein behind L2 did not disrupt the correct assembling of cVLPs. The morphological characteristics and haemagglutinating activity of cVLPs were similar to those of HPV-6b L1/L2 VLPs. The cVLPs retained conformational B cell epitopes of HPV-6 VLPs and HPV-16 mE7 protein had an internal location in the cVLPs. Therefore, large modified E7 protein with higher immunogenicity could be incorporated into cVLPs by fusing to the C-terminus of L2, which would help to improve the therapeutic effects of L1/L2-E7 cVLPs.

[Enhancement of herpes simplex virus-1 glycoprotein-D DNA vaccine induced specific immune responses by coimmunization with interleukin-2 genetic adjuvant].

OBJECTIVE: To investigate the immune responses and protection from virus challenge, induced by the coinjection of IL-2cDNA with herpes simplex virus type 1 (HSV-1) glycoprotein-D (gD) DNA vaccine. METHODS: Two DNA vaccines (pgD and pIL-2) were constructed by inserting the gD gene and IL-2 cDNA into the eukaryotic expression vector pcDNA3.1, respectively. The BALB/c mice were inoculated intramuscularly three times at 2-week intervals. Two weeks after the final immunization, mice were bled for antibody assay and spleen cells were separated for Th cell proliferation and cytokine assays. Delayed type hypersensitivity (DTH) response was detected by the pinna-swelling test. Corneal protection under HSV-1 virus challenge was continuously observed with slit-lamp microscope. RESULTS: IL-2 cDNA coinjection remarkably enhanced the specific IgG2a level when compared with gD plasmid vaccination alone. Th cell proliferation and secretion of cytokines (IL-2 and IFN-gamma) were significantly increased by IL-2 cDNA coinjection. However, the production of IL-10 was inhibited. The DTH response was also enhanced by IL-2 coinjection. When the mice were challenged with HSV-1, the cornea epithelial lesions were significantly alleviated by IL-2 coinjection as compared with gD vaccination alone. CONCLUSION: IL-2 cDNA can enhance both the humoral and cellular immune responses, and thus increase the vaccine potency.

The role of CD40-CD154 interaction in cell immunoregulation.

CD40, a member of the nerve growth factor/tumor necrosis factor receptor superfamily, and its ligand, CD154, play essential roles in cell immune responses. The results of many studies have indicated that CD40-CD154 interaction can upregulate costimulatory molecules, activate antigen-presenting cells (APCs), influence T-cell priming and T-cell-mediated effector functions as well as participate in the pathogenic processing of chronic inflammatory diseases, such as autoimmune diabetes, graft rejection, atherosclerosis, and cancer. Ligation of CD40 on cancer cells was also found to produce a direct growth-inhibitory effect through cell cycle blockage and/or apoptosis with no overt side effects on normal cells and treatment with CD154 can heighten tumor rejection immune response as well. However, systemic treatment with CD154 has some potential risks. Therefore, searching for efficient and safe strategies of CD154-based cancer therapy has been a hot topic in human cancer research. This review focuses on the latest discovered functions of CD40-CD154 interaction in cell immune responses and on the new findings of CD154-based human cancer therapy.

[Enhancement of human papillomavirus type 16E6E7 vaccine-induced specific immune response by coimmunization with B7-1 co-stimulatory gene].

OBJECTIVE: To develop a therapeutic vaccine against human tumors associated with human papillomavirus type 16E6E7 (HPV16E6E7) which is modified from a Chinese patient of the cervical cancer which possessing the antigenicity and no transforming activity, and explore more active vaccine for inducing cellular immunity with mouse co-stimulatory molecular B7-1 gene. METHODS: The modified E6E7 gene expression plasmid pVR1012-fmE6E7 was constructed and transfected Cos-7 cells, and the E7 protein specific expression was testified by immunofluorescence assay. C57BL/6 mice were immunized intramuscularly with pVR1012-fmE6E7 alone or in combination with B7-1 gene expression plasmid (pcDNA3.1-B7-1). The activity of cytotoxic T lymphocytes (CTLs) was analyzed with 51Cr specific release assay and the specific antibody in sera was analyzed by indirect ELISA. HPV16 positive C57BL/6 tumor cells C3 were inoculated subcutaneously in the vaccinated mice to assay the growth of transplanted tumors. RESULTS: The specific CTLs and antibody from immunized mice were induced efficaciously by the E6E7 gene immunization, and co-administration of B7-1 gene could significantly enhanced the CTLs immune responses of fmE6E7, and protected 33% immunized mice against C3 tumor cells challenge. In contrast, all the mice immunized only with fmE6E7 gene developed transplanted tumors after C3 cells challenge. There was no difference in E7 specific antibody responses between mice immunized with the E6E7 gene only and co-administration with B7-1 gene. CONCLUSIONS: The modified E6E7 gene can be used as target gene for developing DNA vaccine, and B7-1 gene may represent an attractive adjuvant for enhancement of the specific cellular immune responses.

Enhancement of DNA vaccine potency against herpes simplex virus 1 by co-administration of an interleukin-18 expression plasmid as a genetic adjuvant.

In this study, the immune-modulatory and vaccine effects of using an interleukin (IL)-18 expression plasmid as a genetic adjuvant to enhance DNA vaccine-induced immune responses were investigated in a mouse herpes simplex virus 1 (HSV-1) challenge model. BALB/c mice were immunized by three intramuscular inoculations of HSV-1 glycoprotein D (gD) DNA vaccine alone or in combination with a plasmid expressing mature IL-18 peptide. Both the serum IgG2a/IgG1 ratio and T helper 1-type (Th1) cytokines [IL-2 and interferon (IFN)-gamma] were increased significantly by the co-injection of the IL-18 plasmid compared with the injection of gD DNA alone. However, the production of IL-10 was inhibited by IL-18 plasmid co-injection. Furthermore, IL-18 plasmid co-injection efficiently enhanced antigen-specific lymphocyte proliferation and the delayed-type hypersensitivity response. When mice were challenged with HSV-1 at the cornea, co-injection of IL-18 plasmid with gD DNA vaccine showed significantly better protection, manifested as lower corneal lesion scores and faster recovery. These experiments indicate that co-injection of an IL-18 plasmid with gD DNA vaccine efficiently induces Th1-dominant immune responses and improves the protective effect against HSV-1 infection.

Expression of human papillomavirus type 6 L1 and L2 isolated in China and self assembly of virus-like particles by the products.

To study variations of genome late region of human papillomavirus type 6 (HPV-6) isolated in China and assembling capabilities of the encoded capsid proteins, HPV-6 L1 and L2 sequences were cloned and used for expression in Bac-to-Bac baculovirus expression systems (Gibco BRL). Based upon L1 and L2 overlapping sequence two sequences (GenBank accession number AY015006, AY015008) of HPV-6 late region (2869 bp long) were assembled and classified into HPV-6b by phylogenetic analysis. Compared with prototype sequence, nine point mutations were found, including four missense mutations. L1, instead of L2, could self-assemble into virus-like particles (VLPs) in Sf9 nucleus. VLPs self-assembled by L1 alone (L1-VLPs) and by L1 plus L2 (L1+L2-VLPs) were purified and further characterized. Both types of VLPs were spherical particles with a diameter of approximately 50 nm. L1+L2 VLPs comprising L1 and L2 in the molar ratio of about 4:1 possessed the HPV-6 L1 VLP surface and conformational epitopes. In co-expression assay with a series of MOI combination of L1 and L2 recombinant baculoviruses (total MOI=10), existence of L2 of certain level enhanced L1 production by 0.8 fold and VLP production by 3 - 4 folds under experimental conditions. In conclusion, variation rate of HPV-6 genome late region is less than 0.28% and the substitutions A to G at position 7081 and G to A at 7099 may represent region characteristics. The cloned HPV-6 L1 and L2 sequences can be expressed efficiently in Sf9 cells, and the expressed products (L1 or L1+L2) can self-assemble into VLPs that resemble naturally occurring virions.

[Determination of chemical constituents of essential oil from the fruit of Eucalyptus globulus by GC-MS].

OBJECTIVE: The chemical components of essential oil from fruit of Eucalyptus globulus were analyzed by GC-MS. METHOD: The essential oil were extracted by steam distillation, then separated by capillary gas chromatography. The amount of the component from essential oil were determined by normalization methods. Chromatographic conditions were: capillary column DB-WAX (0.32 mm x 30 m, 0.25 microm) was used, column temperature: initial temperature at 40 degrees C for 3 min,ramping 5 C x min(-1) to 250 degrees C (holding for 10 min) the detector MS. RESULT: 31 component from the fruit of E. globulus were identified, which accounted for over 93.7% of total volatile oil. CONCLUSION: The methods is reliable, stabilize and can be applied to identify the volatile oil from the fruit of E. globulus.

[Construction of herpes simplex virus type I glycoprotein D DNA vaccine and its preliminary study].

OBJECTIVE: The goal of this study was to construct a eukaryotic expression plasmid containing the gene encoding herpes simplex virus type I glycoprotein D (HSV-1, gD) and evaluate its utility for DNA immunization in mice. METHODS: The gD gene was amplified from viral DNA using PCR with EcoR I and BamH I restriction sites encoded on 5' and 3' ends, respectively. The PCR fragment was inserted into the transfer vector pGEM-T Easy. gD was then cut from this vector and inserted into the EcoR I and BamH I sites in the pcDNA3.1 at the multiple cloning sites (MCS). The recombinant plasmid, pcDNA3.1-gD1, was transfected into COS-7 cells using Lipofectamine according to the manufacture's instructions. The expression of the glycoprotein D was analyzed by immunoblotting of the cell lysates. 4-6 weeks old BALB/C mice were given two injections at tibia anterialis muscle, each containing 100 micrograms of plasmid DNA, on days 0 and 15. pcDNA3.1 was used as negative control. Blood samples were taken from all mice at weeks 0, 2, 4, and 6 after the first inoculation. Standard indirect ELISA was employed to evaluate the levels of specific total Ig in serum. RESULTS: The recombinant plasmid was confirmed with restriction digestion and sequencing to contain target gene segment and expressed in COS-7 cells in vitro shown by Western blotting. The pcDNA3.1-gD1 immunized group induced specific antibody response as compared to the negative control, and the titer was about 1:2000. CONCLUSIONS: The recombinant plasmid pcDNA3.1-gD1 is potential to be used as a candidate vaccine, for the treatment of HSV-1 infection.