Development of prophylactic recombinant HPV58-attenuated Shigella live vector vaccine and evaluation of its protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model.

To develop a prophylactic recombinant HPV58L1-attenuated Shigella live vector vaccine and evaluate its protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model, the HPV58L1 gene was cloned into vector pUCmt, and then subcloned into the suicide vector pCVD442. The recombinant plasmid pCVD442-HPV58L1 was introduced into attenuated Shigella (sf301:deltavirG) with the helper plasmid PRK2013 by filter mating. The positive colonies were harvested and confirmed by polymerase chain reaction. The expression of the HPV58L1 protein with a molecular weight of 60 kDa was confirmed by western blot. The ability of the interested protein to self-assemble into virus-like particles was identified by transmission electron microscope, and murine erythrocyte hemagglutination assay. The guinea pig keratoconjunctivitis model was used to evaluate the protective efficacy and immunogenicity of the vaccine. Animal experiments showed that there was no keratoconjunctivitis occurred in the immunized group (HPV58-attenuated Shigella), and the serum levels of anti-HPV58L1-IgG and -IgA were obviously increased (P < 0.05), but the anti-sf301 LPS-IgG just slightly increased (P > 0.05). Enzymelinked immunosorbent spot assay showed that HPV58L1-specific IgA-antibody-secreting cells (ASC) and IgG-ASC of spleen and lymph nodes were also obviously increased (P < 0.01). In this study, a recombinant HPV58L1-attenuated Shigella live vector vaccine was successfully constructed, and it could induce strong humoral immune responses in the immunized animals, and induce protective antibody production.

HPV-16E6 can induce multiple site phosphorylation of p53.

Modulation of the activity of tumor suppressor p53 is a key event in the replication of many viruses. They could manipulate p53 function through modification of phosphorylation for their own purpose. However, there are scarce data on the relationship between high risk human papillomavirus (HPV) E6 protein and p53 phosphorylation status. Therefore, we used a mammalian green fluorescence protein (GFP) expression system to express HPV-16E6 with GFP fusion proteins in wild-type p53 cell lines, 293T, MCF-7, and SMMC-7721 to trace the traffic and subcellular location of E6 protein. By immunoblotting, we determined the positive phosphorylated sites of p53 in the context of HPV-16E6. Using immunofluorescence techniques, we observed the distribution of phosphorylated p53 in all the cells we used. In conclusion, HPV-16E6 was predominantly located in nuclei of wild-type p53 cells, and it was able to induce phosphorylation of p53 at multiple sites, such as Ser15, Ser20, and Ser392. The level and time of these phosphorylated sites of p53 were different in HPV-16E6 expressing cells. Furthermore, the phosphorylated p53 was localized in the nuclei together with HPV-16E6.

Two different HPV-11E6 fusion proteins trap p53 in the cytoplasm and induce apoptosis.

Mucosal high risk human papillomaviruses (HPVs) have been shown to be the major cause of cervical cancer. However, the reason why the low risk HPVs only cause proliferative but non-invasive lesions of infected epithelia remains elusive. Because p53 interacts with high risk HPVs E6 and plays a very important role in carcinogenesis, it is assumed that low risk HPVs E6 might interact with p53 in a different pattern. We used mammalian green fluorescent protein (GFP) tagged and polyhistidine (His) tagged proteins expression systems to express HPV-11E6 fusion proteins in wild-type (wt)p53 cell lines, such as 293T and MCF-7 cells to trace the traffic and location of E6s and p53. We showed that: (1) Following transfection, HPV-11E6 was predominantly expressed in the cytoplasm; (2)Using immunocytochemistry and Western blotting, endogenous wt p53 was shown to be trapped in cytoplasm by HPV-11E6 expression. (3) Apoptosis was increased in HPV-11E6 expressed cells. In conclusion, the entrapment of endogenous wt p53 in cytoplasm by the low risk HPV-11E6 may be one of the reasons why low risk HPV is not able to induce malignant transformation.

In GFP with high risk HPV-18E6 fusion protein expressed 293T and MCF-7 cells, the endogenous wild-type p53 could be transiently phosphorylated at multiple sites.

BACKGROUND: Infected cells recognize viral replication as a DNA damage stress and elicit the host surveillance mechanism to anti-virus infection. Modulation of the activity of tumor suppressor p53 is a key event in the replication of many viruses. They could manipulate p53 function through phosphorylation modification for their own purpose. But there is rarely research about p53 phosphorylation status in the context of HPV-E6. Therefore, we investigated whether p53 could be phosphorylated by HPV-E6. METHODS: We used a mammalian green fluorescence protein (GFP) expression system to express HPV-18E6 with GFP fusion proteins (GFP-18E6) in wild-type (wt) p53 cell lines, such as 293T and MCF-7 cells to trace the traffic and subcellular location of E6 protein. By immunofluorescence technique and immunoblotting, we determined the positive phosphorylated sites of p53 and observed the distribution of phosphorylated p53 in the context of GFP-18E6. RESULTS: GFP-18E6 was predominantly located in nuclei of wt p53 cell lines, and it could induce transient phosphorylation of p53 at multiple sites, such as Ser15, Ser20, and Ser392. All the three sites of phosphorylated p53s were localized in nuclei together with GFP-18E6. CONCLUSION: In GFP with high risk HPV-18E6 fusion protein expressed 293T and MCF-7 cells, the endogenous wt p53 could be transiently phosphorylated at multiple sites.

Proteomic analysis of chromium cytotoxicity in cultured rat lung epithelial cells.

Chromium (Cr) has been widely used in industry for more than one century. Exposure to hexavalent Cr compounds is strongly associated with increasing risk of lung cancer. Extensive researches at DNA level indicated that generation of ROS from the reduction of Cr(VI) leading to DNA damage is the major cause of the toxicity and carcinogenicity of Cr(VI). The present study in cellular and protein levels confirmed that Cr(VI) induced apoptosis of lung epithelial cells (LEC) via ROS generation. To view the differentially expressed proteins in the process of Cr(VI) reduction, subcellular proteomics was applied and allowed the identification of more than 30 proteins with expression alteration. Most of those proteins are correlated with ROS-elicited responses, which were further validated by Western blotting analysis, induction of p53 pathway and antioxidative treatment. The current findings provided additional evidence in protein level to support the claim that ROS generated during the process of Cr(VI) reduction are involved in the Cr(VI)-induced toxicity and carcinogenesis.

[Preparation and activity detection of chicken egg yolk IgY antibody against human papillomavirus 16 type L1 main capsid protein].

OBJECTIVE: To prepare highly specific chicken egg yolk IgY antibody against human papillomavirus 16 type L1 main capsid protein (HPV16L1) for detection of HPV16L1. METHODS: Purified HPV16L1 protein was used to immunize the hens, from which the eggs were collected since one week after the first immunization. The egg yolk was separated and the IgY antibody purified by PEG-6000 method. The bioactivity of the antibody was tested using enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was performed to detect the HPV16L1 in the CHO cells transfected with the recombinant pcDNA-EGFP-HPV16L1 plasmid (containing EGFP-HPV16L1 fusion gene) for assessing the specific affinity of IgY to HPV16L1. RESULTS: After 3 immunizations of the hens, the titer of the purified IgY antibody against HPV16L1 from the egg yolk reached 1:10240. The IgY bound specifically to the EGFP-HPV16L1 protein expressed in the transfected CHO cells. CONCLUSION: High titer IgY can be prepared by immunization of the hens with HPV16L1 protein, and the prepared IgY can be used for HPV16L1 detection at the cellular level.

Heparin chromatography to deplete high-abundance proteins for serum proteomics.

BACKGROUND: Serum is a very informative sample for disease diagnosis. However, a few of the high-abundance proteins existing in serum make the identification of disease-specific serum biomarkers extremely challenging using currently available technologies. A highly promising first step for most analytical approaches of serum is to deplete as many of the high-abundance proteins as possible. METHODS: We introduced the traditional method of heparin chromatography coupled with protein G sepharose to deplete the high-abundance proteins for serum proteomics. RESULTS: Compared with the multiple affinity removal system (MARS) column (a commercial version to deplete 6 major proteins in serum), heparin chromatography can deplete more high-abundance proteins in a single step, especially many high molecular-weight proteins. Using this simple and inexpensive method to pretreat serum for 2-DE analysis, more protein spots can be visualized. IgGs depletion by protein G sepharose can further enhance the resolution of the resulting serum proteome. CONCLUSIONS: Heparin chromatography coupled with protein G appears to be an efficient and economical strategy to pretreat serum for serum proteomics.

[Construction of prophylactic recombinant HPV58-attenuated Shigella vector live vaccine and evaluation of its protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model].

AIM: To construct the prophylactic recombinant HPV58-attenuated Shigella vector live vaccine and evaluate its protective efficacy and immunogenicity in the Guinea pig Keratoconjunctivitis model. METHODS: The HPV58 L1 gene was cloned into PUCMT, and the recombinant plasmid HPV58 L1-pCVD442 was constructed and introduced into attenuated Shigella (Sf301: deltavirG) with helper plasmid PRK2013 by filter mating. After homologous recombination, the positive colonies in Sf301: deltavirG strains contained HPV58L1 gene were selected and verified by PCR. The expressed HPV58L1 protein was harvested and analyzed by SDS-PAGE and Western blot. The bio-activity of the interested protein was identified by mouse erythrocyte hemagglutination assay, and the VLP formation was proved with transmission electron microscope. Guinea pig Keratoconjunctivitis model was used to evaluate protective efficacy and immunogenicity of the vaccine. 20 days after immunization, serum HPV58L1-IgG, IgA level and serum sf301 LPS- IgG, IgA level were measured by ELISA, and HPV58 L1-specific IgA-ASC and IgG-ASC of spleen and lymph nodes (SVCLN, MDLN, MSLN and pp) were measured by ELISPOT assay. RESULTS: HPV58 L1 protein with MW 60 kDa was confirmed by Western blot. The ability of the interested protein to self-assemble into VLPs was identified by transmission electron microscope, and the result of murine erythrocyte hemagglutination assay indicated that the given proteins expressed by the recombinant bacillus had similar characteristics as the natural HPV58L1 protein.In the Guinea pig Keratoconjunctivitis Model, animal immune results showed that there were no Keratoconjunctivitis occurred in the immune group(HPV58-attenuated Shigella), and after Sf301 attacking, 80% eyes had no Keratoconjunctivitis occurrence. 20 days after immunization, serum HPV58L1-IgG, IgA level were obviously increased; but serum sf301 LPS-IgG position was just slightly increased; ELISPOT results showed that HPV58 L1-specific IgA-ASC and IgG-ASC of spleen and lymph nodes were also obviously increased. CONCLUSION: Recombinant HPV58L1-attenuated Shigella vector live vaccine could induce strong humoral immune responses in the immunized animals.

Anti-CD3 scFv-B7.1 fusion protein expressed on the surface of HeLa cells provokes potent T-lymphocyte activation and cytotoxicity.

The targeting of tumor cells by cytotoxic T lymphocytes is a promising strategy for biotherapy, but T cells require 2 signals via the T-cell receptor - CD3 complex and CD28 molecules for activation. To bridge the gap between cytotoxic T lymphocytes and tumor cells, our objective in this study was to describe the construction and the cell surface-anchored expression of a fusion protein, anti-CD3 scFv-B7.1, derived from inserting a fusion gene encoding anti-CD3 scFv and the extra-cellular domain of B7.1 fused by the splicing by overlap extension method into a mammalian expression vector, pDisplay. Transfection of the recombinant vector by electroporation into HeLa cells resulted in the production of protein migrating at approximately 57 kDa under reducing conditions. The expressed fusion protein could bind to T lymphocytes and induce strong T-cell activation. Meanwhile, a potent cytotoxicity was induced in the mixed culture of T-cell-modified tumor cells in a 96 h methyl-thiazolyl-diphenyl tetrazolium bromide assay. Our results indicate that this bifunctional protein, through activating T lymphocytes to lyse homologous human carcinomas, may be of potential value for T-cell-based immunotherapeutical treatment protocols in vivo.

[Identification of metastasis-associated proteins of ovarian cancer by proteomics].

OBJECTIVE: To investigate the differenal protein expression profiles of ovarian tumor cell lines with distinct metastatic abilities. METHODS: The ovarian cancer cell line HO8910 and HO8910pm, derived from same parental cells but exhibited different metastatic ability, were investigated by two-dimensional gel electrophoresis (2-DE)-MALDI-TOF-MS proteomic approach. RESULTS: Thirty-nine proteins were detected by 2-DE to have expression disparity levels over 2 folds between two cell lines. Eighteen of them were identified by MALDI-TOF-MS. The proteins are involved in apoptosis, extra cellular matrix (ECM), cytoskeleton, growth factor, glycolysis, protein metabolism and immune system. CONCLUSION: The data are valuable for the identification of differentially expressed proteins involved in the biological behavior of human ovarian cancer.

[In situ analysis of distribution of immunocompetent cells in tumor's local draining lymph nodes].

AIM: To observe the distribution of immunocompetent cells (ICCs) in tumor's local draining lymph nodes (LDLN) at different stages of disease including non-metastasis, micro-metastasis and late metastasis. METHODS: 71 LDLN from 22 breast cancer, 28 LDLN from 7 gastric carcinoma were analysed by using catalyzed signal amplification (CSA) immunohistochemical staining. Monoclonal antibodies (mAbs) to perforin, granzyme B, CD8, CD56, CD68, S-100, CD134 and CD25 were used to detect the amount and functional change of ICCs. RESULTS: Paracortical hyperplasia and sinus histocytosis was mainly observed in tumor's LDLN. As it was reported, the density of S100(+) dendritic cells (DCs) was decreased as the draining lymph nodes became tumor-containing from the status of tumor free (P<0.05), and the morphology of DCs turned to be inactivated. As the lymph nodes were invaded by tumors, the densities of CD134(+) lymphocytes and CD25(+) cells were significantly increased (P<0.01). Furthermore, there was a significant trend of decreasing in the number of activated cytotoxic T lymphocytes (granzyme B(+) cells) in LDLN as tumor progressed from non/micro-metastasis, early metastasis to advanced stage metastasis (P<0.01). There was no obvious difference in distribution of ICC between non/micro-metastasis sentinel lymph nodes and non-sentinel lymph nodes. CONCLUSION: The dynamic change of the ICCs in LDLN implied that immune response of tumor bearing host tends to be inhibited with the progress of tumors.

[Immunopotentiation of novel adjuvant SWZY on the poorly immunogenic murine melanoma vaccine].

AIM: To evaluate the immunopotentiation of novel adjuvant SWZY on the cancer vaccine of poorly immunogenic melanoma. METHODS: C57BL/6 mice immunized with inactivated D5 melanoma cells were divided into 6 groups: the control without adjuvants, with FCA, FCA+IL-2+GM-CSF, FIA+IL-2+GM-CSF, FIA+SWZY, and FIA+SWZY+IL-2+GM-CSF. Three days after completion of immunization, DTH response, specific killing activity of splenocytes and the level of IFN-gamma and IL-10 in serum and splenocytes' culture supernatant were assayed in half of the mice in each group. The rest were subject to live D5 tumor cell challenge. Three weeks after tumor inoculation, the same immunological parameters were measured. RESULTS: DTH response, splenocytes' killing activity of all the experimental groups were markedly higher than those in the control group (P<0.05), but decreased as the tumor grew larger. The level of IFN-gamma in serum or splenocyte culture supernatant of each experimental group was significantly higher than that of the control (P<0.05) before tumor formation, whereas IL-10 level was lower than that of the control (P<0.05). After tumor formation, the level of IFN-gamma in all groups decreased while that of IL-10 increased in general, but the levels of IFN-gamma and IL-10 in group FCA and FIA+SWZY didn't change very much. CONCLUSION: All kinds of adjuvants can enhance the cell-mediated immune response against poorly immunogenic tumor to some extent. The novel adjuvant SWZY can strengthen immunoresponses similar to FCA. With few harmful side-effect, SWZY might be a promising adjuvant for cancer vaccine.

[Inhibition of the expression of p42MAPK in HeLa cell line by RNA interference].

OBJECTIVE: To screen for siRNAs that inhibit the expression of p42(MAPK) in HeLa cell line. METHODS: Three p42(MAPK) siRNAs and one random siRNA were synthesized using Silencer siRNA Construction Kit, and labeled with Cy-3 for measurement of transfection effect. SiRNAs were transfected into HeLa cells by Lipofectamin 2000. The expression of p42(MAPK) was analyzed by Western blot. The biological effect of siRNAs on HeLa cell growth was monitored by MTT and flow cytometry. RESULTS: Two siRNAs (siRNA-2 and siRNA-3) among three tested were identified to be able to downregulate the p42(MAPK) expression. A concurrent growth retardation of HeLa cell line was observed in comparison with the control. CONCLUSION: Inhibition of p42(MAPK) expression with siRNA technique can inhibit the proliferation of HeLa cells.

Predicting the nuclear localization signals of 107 types of HPV L1 proteins by bioinformatic analysis.

In this study, 107 types of human papillomavirus (HPV) L1 protein sequences were obtained from available databases, and the nuclear localization signals (NLSs) of these HPV L1 proteins were analyzed and predicted by bioinformatic analysis. Out of the 107 types, the NLSs of 39 types were predicted by PredictNLS software (35 types of bipartite NLSs and 4 types of monopartite NLSs). The NLSs of the remaining HPV types were predicted according to the characteristics and the homology of the already predicted NLSs as well as the general rule of NLSs. According to the result, the NLSs of 107 types of HPV L1 proteins were classified into 15 categories. The different types of HPV L1 proteins in the same NLS category could share the similar or the same nucleocytoplasmic transport pathway. They might be used as the same target to prevent and treat different types of HPV infection. The results also showed that bioinformatic technology could be used to analyze and predict NLSs of proteins.

Proteomics-based analysis of a pair of glioma cell lines with different tumor forming characteristics.

Glioma is the most common malignant disease in the brain, and recurrence is the main cause of death from this disease. Tumor recurrence involves multiple steps, and requires the accumulation of the altered expression of many different proteins. Identification of the recurrence associated protein profile in glioma cell lines will be helpful in clarifying the molecular mechanisms underlying glioma recurrence. In this report, two glioma cell lines with distinct tumor forming ability in vitro and in vivo were chosen and the different protein expression patterns were analyzed by proteomics method. To confirm the utility of this method, we validated the differential expression of one protein, cathepsin D, by immunohistochemistry analysis. Forty-six proteins appeared differently between two cell lines and 18 of them were identified. These 18 are involved in cell proliferation, DNA replication, protein synthesis, invasion, angiogenesis and neurotrophic factor. All of these molecules are important in tumor growth, and a subset of them may be related to glioma recurrence. These findings may contribute to the discovery of new diagnostic markers and therapeutic targets of glioma.

[Small interfering RNA-mediated MAPK p42 silencing induces apoptosis of HeLa cells].

OBJECTIVE: To observe the effect of small interfering RNA (siRNA)-induced MAPK p42 silencing on the survival of HeLa cells. METHODS: Two siRNAs targeting at the MAPK p42 gene and one random siRNA were synthesized respectively by Silencer siRNA Construction Kit and transfected into HeLa cells by Lipofectamin 2000. The expression of p42(MAPK) in the transfected HeLa cells was analyzed by Western blotting and immunohistochemistry, and the morphology of cells were observed with electron microscope. TUNEL assay and Annexin V/PI staining were employed for detecting the cell apoptosis. RESULTS: The expression of p42(MAPK) in the HeLa cells was remarkably suppressed after transfection with the two siRNAs, reduced by about 2.5 and 3.2 folds respectively in comparison with the negative control. Chromatin margination in the cell nuclei were observed in the transfected cells, and TUNEL assay and Annexin V/PI staining further confirmed the occurrence of cell apoptosis. CONCLUSION: In vitro MAPK p42 siRNA-1 and siRNA-2 transfection can specifically silence the gene expression and induce apoptosis of HeLa cells.

[The in situ analysis of cytokine mRNA expression in immunological microenvironment about non-small cell lung cancer].

AIM: Using Non-small cell lung cancer (NSCLC) as subject, to explore the characteristics of immune response in immunological microenvironment at the tumor site and its effect on anti-tumor immunity. METHODS: Using in situ hybridization (ISH), the expressions of IL-2, INF-gamma, IL-12 (p40), IL-18, IL-4, IL-10, TGF-beta1, IL-1, IL-3, IL-8, GM-CSF, TNF-alpha and TGF-alpha mRNAs in lymphocytes and tumor cells from five fresh pleural effusion samples and 18 tumor tissue samples of NSCLC patients were detected by in situ hybridization with digoxin end-labeled oligonucleotide probe, using 5 tuberculous pleurisy patients as control. RESULTS: In pleural effusion and tumor tissue of NSCLC patients, the expression levels of IL-4, IL-10, TGF-alpha, and TGF-beta1 mRNAs were significantly higher than those of IL-2, IL-12, IL-18 and INF-gamma mRNAs. In contrast, the analysis of tuberculous pleural effusion samples revealed lower levels of above-mentioned cytokine mRNA. There was no significant difference among expression levels of these cytokine mRNAs. CONCLUSION: The expressions of type II cytokine mRNAs and immunosuppressive cytokine mRNAs in pleural effusion and tumor tissue of NSCLC patients occupied a dominant position, suggesting that the immunological microenvironment about tumor be in an immunosuppressive state. The present study has contributed to a better understanding the mechanisms of tumor escape and provides important experimental basis for working out the scheme for an effective immunomodulatory treatment of NSCLC patients.