Xiangfeng He

An ocular mucosal administration of nanoparticles containing DNA vaccine pRSC-gD-IL-21 confers protection against mucosal challenge with herpes simplex virus type 1 in mice

Herpes stromal keratitis (HSK) is a chronic inflammatory process caused by the infection of herpes simplex virus type 1 (HSV-1). Development of a HSV-1 vaccine is a priority because these infections are common and cannot be well prevented. It appears that the potential of nanocarriers in DNA vaccination will be required to augment the immune response to DNA vaccines. Therefore, in the study, nanoparticles Fe(3)O(4) coated with glutamic acid, DNA vaccine pRSC-gD-IL-21 and polyethylenimine were prepared and immunized in the mice by ocular mucosal administration. The immune responses and protection efficiency against HSV-1 challenge were also tested. The results showed that the nanoparticles containing DNA vaccine pRSC-gD-IL-21 induced mice to generate higher levels of specific neutralizing antibody, sIgA in tears, and IFN-γ, IL-4 in serum, and to enhance the cytotoxicities of NK cells and splenocytes as well as splenocyte proliferative response to glycoprotein D compared with those of the control mice. More importantly, the mice immunized with the experimental vaccine showed less HSK degree than that of the control mice after HSV-1 challenge of the murine ocular mucosa. In conclusion, an ocular mucosal administration of nanoparticles containing DNA vaccine confers strong specific immune responses and effective inhibition of HSK in a HSV-1 infected murine model.

Using ABCG2-molecule-expressing side population cells to identify cancer stem-like cells in a human ovarian cell line

CSCs (cancer stem cells) are a small subset of cells within a tumour that possesses the characteristics of stem cells and are considered to be responsible for resistance to chemoradiation. Identification of CSCs through stem cell characteristics might have relevant clinical implications. In this study, SP (side population) cells were sorted from a human ovarian cancer cell line by FACS to determine whether cancer stem cell‐like SP cells were present. A very small fraction of SP cells (2.6%) was detected in A2780 cells. SP cells possessed the following characteristics: highly proliferative activity, marked ability for self‐renewal in soft agar and culture medium, high expression of ABCG2, drug resistance to vinblastine in vitro, and strong tumourigenic potential in Balb/c nude mice. It is concluded that there exists in the A2780 cell line a small number of SP cells with high expression of ABCG2. The cells have the characteristics of cancer stem‐like cells, and identification and cloning of such human SP cells can help in improving therapeutic approaches to ovarian cancer in patients.

Identifying tumor stem-like cells in mouse melanoma cell lines by analyzing the characteristics of side population cells

Increasingly more evidence shows that TSCs possess the characteristics of stem‐like cells. However, a link between stem cells and TSCs remains to be shown. We have sorted SP cells and non‐SP cells from the B16F10 cell lines by FACS, and then studied their cellular biological characteristics by using a SFCM culture method, proliferative assay in vitro, clone formation assays in soft agar and normal media, tumorigenic assays in C57BL/6 mice, and resistance to chemotherapy assay in vitro, the quantitative detecting expression of ABCG2 and their CD phenotype analysis by a FCM. We detected 0.96% SP cells in the B16F10 cells and found that they had obvious differences in characteristics from non‐SP cells. They possessed a marked capacity for self‐renewal in soft agar and culture medium, strong tumorigenic potential in C57BL/6 mice, apparent resistance to vinblastin in vitro, upregulated ABCG2 expression, and a high expression of CD44+CD133+CD24+ phenotypes. We conclude that there were a few of SP cells that had the characteristics of tumor stem‐like cells which may provide a useful tool and a readily accessible source for further study when specific TSCs markers are unknown.

Augmenting therapy of ovarian cancer efficacy by secreting IL-21 human umbilical cord blood stem cells in nude mice.

In the present study, CD34+ human umbilical cord blood stem cells (UCBSCs) were engineered to express interleukin-21 (IL-21) and then were transplanted into A2780 ovarian cancer xenograft-bearing Balb/c nude mice. The therapeutic efficacy of this procedure on ovarian cancer was evaluated. The findings from the study indicated that UCBSCs did not form gross or histological teratomas until up to 70 days postinjection. The CD34+ UCBSC-IL-21 therapy showed a consistent effect in the ovarian cancer of the treated mice, delaying the tumor appearance, reducing the tumor sizes, and extending life expectancy. The efficacy was attributable to keeping CD34+ UCBSC-IL-21 in the neoplastic tissues for more than 21 days. The secreted IL-21 not only increased the quantity of CD11a+ and CD56+ NK cells but also increased NK cell cytotoxicities to YAC-1 cells and A2780 cells, respectively. The efficacy was also associated with enhancing the levels of IFN-γ, IL-4, and TNF-α in the mice as well as the high expressions of the NKG2D and MIC A/B molecules in the tumor tissues. This study suggested that transferring CD34+ UCBSC-IL-21 into the nude mice was safe and feasible in ovarian cancer therapy, and that the method would be a promising new strategy for clinical treatment of ovarian cancer.

Effect of downregulation of ZEB1 on vimentin expression, tumour migration and tumourigenicity of melanoma B16F10 cells and CSCs.

Zinc‐finger E‐box binding homeobox 1 (ZEB1) is a master regulator of epithelial‐mesenchymal transition (EMT) and has been implicated in primary epithelial cancer biological processes, such as invasion and metastasis. However, the role of ZEB1 in progression of melanoma and cancer stem cells (CSCs) remains obscure. In this study, the recombinant plasmids of t3 shRNAs targeting mouse ZEB1 were constructed and transfected into melanoma B16F10 cells. The stable transfected cells were selected and the characteristics of ZEB1 downregulated B16F10 cells was assessed. The tumourigenicity of CD44+CD133+CSCs isolated from B16F10 cells stably transfected with the ZEB1‐shRNA2 recombinant was also assessed. ZEB1‐shRNAs B16F10 showed a lower expression of ZEB1 and vimentin, weaker migration, invasiveness, colony forming, and proliferation, and a lower tumourigenicity than the control cells. The tumourigenicity of the ZEB1‐shRNA2 CD44+CD133+CSCs was also inhibited. In conclusion, ZEB1‐shRNA2‐mediated downregulation of ZEB1 expression in B16F10 cells and CSCs is involved in the inhibition of the EMT process. ZEB1 may be a potential target in melanoma targeted.

Human umbilical blood mononuclear cell–derived mesenchymal stem cells serve as interleukin‐21 gene delivery vehicles for epithelial ovarian cancer therapy in nude mice

Ovarian cancer causes more deaths than any other cancer of the female reproductive system, and its overall cure rate remains low. The present study investigated human umbilical blood mononuclear cell (UBMC)–derived mesenchymal stem cells (UBMC‐MSCs) as interleukin‐21 (IL‐21) gene delivery vehicles for ovarian cancer therapy in nude mice. MSCs were isolated from UBMCs and the expanded cells were phenotyped by flow cytometry. Cultured UBMCs were differentiated into osteocytes and adipocytes using appropriate media and then the UBMC‐MSCs were transfected with recombinant pIRES2–IL‐21–enhancement green fluorescent protein. UBMC‐MSCs expressing IL‐21 were named as UBMC‐MSC‐IL‐21. Mice with A2780 ovarian cancer were treated with UBMC‐MSC–IL‐21 intravenously, and the therapeutic efficacy was evaluated by the tumor volume and mouse survival. To address the mechanism of UBMC‐MSC‐IL‐21 against ovarian cancer, the expression of IL‐21, natural killer glucoprotein 2 domain and major histocompatibility complex class I chain‐related molecules A/B were detected in UBMC‐MSC‐IL‐21 and in the tumor sites. Interferon‐γ‐secreting splenocyte numbers and natural killer cytotoxicity were significantly increased in the UBMC‐MSC–IL‐21‐treated mice as compared with the UBMC‐MSCs or the UBMC‐MSC–mock plasmid‐treated mice. Most notably, tumor growth was delayed and survival was prolonged in ovarian‐cancer–bearing mice treated with UBMC‐MSC–IL‐21. Our data provide important evidence that UBMC‐MSCs can serve as vehicles for IL‐21 gene delivery and inhibit the established tumor.

Immunization with DNA Vaccine Expressing Herpes Simplex Virus Type 1 gD and IL-21 Protects against Mouse Herpes Keratitis

The development of novel vaccines to eradicate herpes simplex virus (HSV) is a global public health priority. In this study, we developed a DNA vaccine expressing HSV-1 glycoprotein D (gD) and mouse interleukin-21(IL-21) and intramuscularly inoculated mice 3 times at 2-week intervals with a total of 300 μg/mouse. Two weeks after the last immunization the specific antibody, splenocyte proliferative response to gD, IFN-γ and IL-4 as well as the cytotoxic activities of splenocytes and natural killer (NK) cells were assayed. Immune protection against herpes keratitis was concurrently evaluated in the immunized mice after HSV-1 challenge of the mouse cornea. The results showed that the DNA vaccine pRSC-gD-IL-21 generated higher levels of antibody, IFN-γ and IL-4, and enhanced the splenocyte proliferative response to gD as well as the cytotoxic activity of splenocytes and NK cells to target cells compared with the response in either the pRSC-gD or mock plasmid pRSC immunized mice. Importantly, the pRSC-gD-IL-21 ameliorated herpes keratitis severity and time course after corneal infection with HSV-1. The findings suggest that the DNA vaccine pRSC-gD-IL-21 may induce an immune response that can limit HSV-1 infection and development of herpes keratitis in the immunized mice.

Regulation gene expression of miR200c and ZEB1 positively enhances effect of tumor vaccine B16F10/GPI-IL-21 on inhibition of melanoma growth and metastasis

BackgroundGenetically modified cells have been shown to be one of the most effective tumor vaccine strategies. However, in many cases, such as in melanoma, induction of a potent immune responses against the disease still remains a major challenge. Thus, novel strategies to reinforce tumor vaccine efficacy are needed. Using microRNA (miR) and Zinc-finger E-box binding homeobox (ZEB) have received much attention for potentially regulating tumor progression. To elicit a potent antitumor efficacy against melanoma, we used tumor vaccine in combination with miR200c overexpression or ZEB1 knockdown to assess the efficacy of treatment of murine melanoma.MethodsB16F10 cell vaccine expressing interleukin 21 (IL-21) in the glycosylpho- sphatidylinositol (GPI)-anchored form (B16F10/GPI-IL-21) were developed. The vaccine was immunized into mice challenged by B16F10 cells or B16F10 cells stably transduced with lentiviral-miR200c (B16F10/miR200c) or transfected with the ZEB1-shRNA recombinant (B16F10/shZEB1) or the B16F10/GPI-IL-21 vaccine. The immune responses, tumorigenicity and lung metastasis in mice were evaluated, respectively.ResultsThe vaccination with B16F10/GPI-IL-21 markedly increased the serum cytokine levels of IFN-γ, TNF-α, IL-4 and decreased TGF-β level as well as augmented the cytotoxicity of splenocytes in immunized mice compared with control mice. In addition, the tumor vaccine B16F10/GPI-IL-21 significantly inhibited the tumor growth and reduced counts of lung metastases in mice challenged by B16F10/GPI-IL-21, B16F10/shZEB1 and B16F10/miR200c respectively compared with the control mice challenged by B16F10 cells. The efficacy mechanisms may involve in reinforcing immune responses, increasing expression of miR200c, E-cadherin and SMAD-7 and decreasing expression of TGF-β, ZEB1, Vimentin and N-cadherin in tumor tissues from the immunized mice.ConclusionsThese results indicate that the tumor vaccine B16F10/GPI-IL-21 in combination with miR200c overexpression or ZEB1 knockdown effectively inhibited melanoma growth and metastasis a murine model. Such a strategy may, therefore, be used for the clinical trials.

Reinforcing B16F10/GPI-IL-21 vaccine efficacy against melanoma by injecting mice with shZEB1 plasmid or miR200c agomir.

In this study, we hypothesized that the inhibition of epithelial to mesenchymal transition (EMT) program by knockdown of Zinc-finger E-box binding homeobox 1 (ZEB1) or administration of miR200c agomir would strengthen the B16F10 cells transfected with GPI-anchored IL-21 (B16F10/GPI-IL-21) vaccine efficacy in inhibiting the melanoma metastasis. Our findings from the current study indicated that, when compared with the mice immunized with the B16F10/GPI-IL-21 vaccine alone, the mice immunized with B16F10/GPI-IL-21 vaccine combined with injection of shZEB1 plasmid or miR200c agomir not only meaningfully inhibited EMT of melanoma, reduced the EMT characteristic molecular expression in tumor tissues, but also significantly decreased the Treg cells and TGF-β1, enhanced the cytotoxicities of NK cells and cytotoxic T lymphocytes and the IFN-γ level. Furthermore, the immunotherapeutic combination resulted in inhibiting the melanoma growth and lung metastasis. Our study demonstrated that using the B16F10/GPI-IL-21 vaccine in combination with the down-regulated ZEB1 or miR200c administration effectively elicited anti-tumor immunity and reduced melanoma metastasis by inhibiting the EMT program in the B16F10 melanoma-bearing mice.

Immunotherapy of melanoma by GPI-anchored IL-21 tumour vaccine involves down-regulating regulatory T cells in mouse model

In this study, we developed a tumour cell vaccine expressing a glycosylphosphatidylinositol (GPI)‐anchored IL‐21 to test the effect of immunotherapy of melanoma in mouse model. The results indicated that the tumour vaccine was functional, exhibiting delayed tumour growth and prolonging longevity of tumour bearing mice. The immunotherapeutic effect was associated with decreasing the numbers of CD4+CD25+ Foxp3+Treg (Tregs) cells, increasing IFN‐γ level and promoting lymphocyte‐infiltration in tumour tissues. Overall, our data demonstrate that the GPI‐anchored IL‐21 tumour vaccine regulates immune responses at least in part by down‐regulating Tregs and reveals enhanced efficacy of tumour vaccine therapy of melanoma.