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Background & Aims Liver cancer has a very dismal prognosis due to lack of effective therapy. Here, we studied the therapeutic effects of hyper-interleukin15 (hyper-IL-15), which is composed of IL-15 and the sushi domain of the IL-15 receptor α chain, on metastatic and autochthonous liver cancers. Methods Liver metastatic tumour models were established by intraportally injecting syngeneic mice with murine CT26 colon carcinoma cells or B16-OVA melanoma cells. Primary hepatocellular carcinoma (HCC) was induced by diethylnitrosamine (DEN). A hydrodynamics-based gene delivery method was used to achieve sustained hyper-IL-15 expression in the liver. Results Liver gene delivery of hyper-IL-15 robustly expanded CD8 + T and NK cells, leading to a long-term (more than 40days) accumulation of CD8 + T cells in vivo , especially in the liver. Hyper-IL-15 treatment exerted remarkable therapeutic effects on well-established liver metastatic tumours and even on DEN-induced autochthonous HCC, and these effects were abolished by depletion of CD8 + T cells but not NK cells. Hyper-IL-15 triggered IL-12 and interferon-γ production and reduced the expression of co-inhibitory molecules on dendritic cells in the liver. Adoptive transfer of T cell receptor (TCR) transgenic OT-1 cells showed that hyper-IL-15 preferentially expanded tumour-specific CD8 + T cells and promoted their interferon-γ synthesis and cytotoxicity. Conclusions Liver delivery of hyper-IL-15 provides an effective therapy against well-established metastatic and autochthonous liver cancers in mouse models by preferentially expanding tumour-specific CD8 + T cells and promoting their anti-tumour effects.

Berberine is a well‑known component of the Chinese herbal medicine Huanglian (Coptis chinensis), and is capable of inhibiting the proliferation of multiple cancer cell lines. However, information available regarding the effect of berberine on prostate cancer cell growth is limited. In the present study, LnCaP and PC‑3 human prostate cancer cell lines were selected as in vitro models in order to assess the efficacy of berberine as an anticancer agent. A cell proliferation assay demonstrated that berberine inhibited cell growth in a dose‑and time‑dependent manner. Further investigation revealed berberine significantly accumulated inside cells that were in the G1 phase of the cell cycle and enhanced apoptosis. Western blot analysis demonstrated that berberine inhibited the expression of prostate‑specific antigen and the activation of epidermal growth factor receptor (EGFR), and it attenuated EGFR activation following EGF treatment in vitro. In conclusion, the results indicate that berberine inhibits the proliferation of prostate cancer cells through apoptosis and/or cell cycle arrest by inactivation of the EGFR signaling pathway.

Graft-versus-host disease (GVHD) is a prevalent and potential complication of hematopoietic stem cell transplantation. An animal model, xenogeneic GVHD (X-GVHD), that mimics accurately the clinical presentation of GVHD would provide a tool for investigating the mechanism involved in disease pathogenesis. Murine models indicated that inhibiting IL-21 signaling was a good therapy to reduce GVHD by impairing T cell functions. We sought to investigate the effect of exogenous human IL-21 on the process of X-GVHD. In this study, human IL-21 was expressed by hydrodynamic gene delivery in BALB/c-Rag2⁻/⁻ IL-2RΓc⁻/⁻ (BRG) immunodeficient mice which were intravenously transplanted human peripheral blood mononuclear cells (hPBMCs). We found that human IL-21 exacerbated X-GVHD and resulted in rapid fatality. As early as 6 days after hPBMCs transplanted to BRG mice, a marked expansion of human CD19⁺ B cells, but not T cells, was observed in spleen of IL-21-treated mice. Compared with control group, IL-21 induced robust immunoglobulin secretion, which was accompanied by increased accumulation of CD19⁺ CD38(high) plasma cells in spleen. In addition, we demonstrated that B-cell depletion was able to ameliorate X-GVHD. These results are the first to find in vivo expansion and differentiation of human B cells in response to IL-21, and reveal a correlation between the expansion of B cells and the exacerbation of xenogeneic GVHD. Our findings show evidence of the involvement of B cells in X-GVHD and may have implications in the treatment of the disease.

Results of recent investigations have demonstrated the plasticity of mesenchymal stem cells (MSC) can differentiate into neural lineages. In this study, we explored the experimental condition of differentiation into neuron-like cells or rhodopsin (RHOS)-positive cells induced by epidermal growth factor (EGF) and taurine in vitro and to investigate their biological characteristics. MSC were obtained from umbilical cord blood (UCB) of term deliveries. Cultured cells were treated with Dulbecco's modified Eagle's medium/F12 (pH 7.0-7.2) supplemented with 30 ng/ml EGF. After the third cell passage, the cells were trysinized and analyzed with a flow cytometer using the following monocloned antibodies: CD90, CD29, CD34, CD44, and CD45. Taking another MSC of the third passage, its basal medium was replaced with alpha minimum essential medium supplemented with taurine (50 micromol/L). Cells were cultured for an additional 8-10 d, fixed, and then immunocytochemically analyzed. Primary antibodies included the following: neuron-specific enolase (NSE), RHOS, and nestin. In our study, we isolated a cell population derived from UCB, which possesses morphological characteristics similar to those of MSC isolated from bone marrow. In the cytometric analysis, MSC did not present labeling for the hematopoietic line (CD34 and CD45) and were positive for CD29, CD44, and CD90. After induction by taurine, 80.5 +/- 16.2% of the cell population expressed NSE, 36.8 +/- 9.6% expressed RHOS, and 29.6 +/- 9.3% expressed Nestin, while only 7.9 +/- 3.5% expressed NSE in the control group. This study demonstrates that partial MSC induced by taurine and EGF can differentiate into neuron-like cells or RHOS-positive cells in vitro, which may provide a promising therapeutic strategy for the treatment of some forms of retinal degeneration.

Increasing evidence indicates the immunosuppressive nature of the local environment in tumor. The present study was focused on analyzing the immune status within hepatocellular carcinoma. In contrast to the increasing number of CD4(+) T cells, CD8(+), CD3(-)CD56(+), CD3(+)CD56(+), and gammadeltaT cells were all found to be under-represented in tumor infiltrating lymphocytes. Notably, the relative abundance of CD3(+)CD56(+) cells appeared to be correlated with patient survival. Functional analysis demonstrated that CD4(+) cells in the tumor tended to produce more IL-10 but less IFN-gamma, whereas CD8(+) cells showed impaired capacity for the production of both IFN-gamma and perforin. Consistent with previous reports, we observed a significant increase of Foxp3(+) cells in the tumor tissue. Intriguingly, although over 90% of CD4(+)CD25(high) cells were found to be Foxp3(+), the majority of Foxp3(+) cells were identified in the CD4(+)CD25(medium) and CD4(+)CD25(-) subsets. In support of its role as a negative regulator, CD4(+)CD25(high) cells suppressed the proliferation of CD4(+)CD25(-) cells isolated from the same tissues in an APC dependent manner. In conclusion, the tumor microenvironment of hepatocellular carcinoma is featured by the presence of multiple immunosuppressive factors.

To explore the effects of siRNA-mediated downregulation of integrin-linked kinase (ILK) gene expression on the proliferation and apoptosis in retinoblastoma cells.Experiment study. Human retinoblastoma cells, HXO-Rb(44) cells, were divided into four groups: ILK siRNA intervention group, control siRNA intervention group, empty liposome group and blank control group. ILK siRNA was transfected into HXO-Rb(44) cells by lipofection. The expression of ILK mRNA and protein was detected at 48 hours after transfection by RT-PCR and Western blot, respectively. Cell proliferation inhibition was measured by Cell Counting Kit-8 assay. The apoptosis was detected by Annexin/PI double immunofluorescence and flow cytometry. Statistical method adopted one-way ANOVA between each group overall comparison.The HXO-Rb(44) cells were transfected by ILK siRNA successfully with lipofection. RT-PCR analysis showed that the expression of ILK mRNA in the ILK siRNA intervention group was significantly decreased as compared to the control siRNA intervention group, empty liposome group and blank control group (0.12 ± 0.02 vs. 0.45 ± 0.04, 0.42 ± 0.05 and 0.40 ± 0.04, F = 12.781, P = 0.000). Similar results were obtained for protein expression as revealed by Western blot analysis (0.10 ± 0.03 vs. 0.38 ± 0.07, 0.40 ± 0.05 and 0.43 ± 0.03, F = 18.647, P = 0.000). The proliferation inhibition rate in the ILK siRNA intervention group (35.0%) was higher than that in the blank control group (0%), control siRNA intervention group (2.1%) and empty liposome group (1.8%) (F = 23.573, P = 0.000). The results of immunofluorescence and flow cytometry showed that ratio of Annexin positive cells was highest in the ILK siRNA intervention group (26.5%), which was 5.0%, 4.6%, 5.3% in the empty liposome group, blank control group, control siRNA intervention group, respectively (F = 65.217, P = 0.000).ILK siRNA can downregulate the expression of ILK gene in HXO-Rb(44) cells inhibited the cell proliferation and induced their apoptosis.

Background Agonist antibodies against CD137 (4–1BB) on T lymphocytes are used to increase host anti-tumor immunity, but often leading to severe liver injury in treated mice or in patients during clinical trials. Interleukin-6 (IL-6) has been reported to protect hepatocyte death, but the role of IL-6 in protecting chronic T cell-induced liver diseases is not clearly defined due to lack of relevant animal models. We aimed to define the role of IL-6 in CD8+ T cell-mediated liver injury induced by a CD137 agonistic mAb (clone 2A) in mice. Methods/Principal Findings We expressed IL-6 in the liver by hydrodynamic gene delivery in mice treated with 2A or control mAb and studied how IL-6 treatment affected host immunity and T cell-mediated liver injury. We found that ectopic IL-6 expression in the liver elevated intrahepatic leukocyte infiltration but prevented CD8+ T cell-mediated liver injury. In IL-6 treated mice, CD8+ T cells proliferation and IFN-γ expression were inhibited in the liver. We discovered that IL-6 increased accumulation of Gr-1+CD11b+ myeloid derived suppressor cells (MDSCs) in the liver and spleen. These MDSCs had the ability to inhibit T cells proliferation and activation. Finally, we showed that the MDSCs were sufficient and essential for IL-6-mediated protection of anti-CD137 mAb-induced liver injury. Conclusions/Significance We concluded that IL-6 induced Gr-1+CD11b+ MDSCs in the liver to inhibit T cell-mediated liver injury. The findings have defined a novel mechanism of IL-6 in protecting liver from CD8+ T cell-mediated injury.

To evaluate the role of different concentration of all-trans retinoic acid (ATRA) on the morphology, proliferation and apoptosis in inducing umbilical cord mesenchymal stem cells (MSC) into neuron-like cells in vitro, and screen the optimal concentration of ATRA.It was an experimental study. The third passage of MSC was placed in 24-well cell culture plates at a density of 1 × 10(4)/well. After the adherent of cells, the medium was changed to DMEM/F-12 containing different concentration of ATRA (0.25 µmol/L, 0.5 µmol/L, 1.0 µmol/L, 2.0 µmol/L, 4.0 µmol/L) for 24 h respectively. The cells cultured without ATRA were taken as the control group. After another 24 h, the morphologic changes of induced cells were observed by inverted microscope and cell proliferation, apoptosis of ATRA was analyzed using the MTT colorimetric assay. We take another control group and ATRA groups to detect the apoptotic and positive stained percentage of induced cells by Annexin V-FITC/PI combining flow cytometry. The optimal concentration of ATRA was determined by all the above-mentioned index. According to the nature of the material, analysis of variance (ANOVA) was employed for absorption value and apoptosis rate in different concentration of ATRA for 24 h, t test for further comparison between two groups. T-test were also used between the positive expression of induced neuron-like cells and the control group.Compared to the control group, ATRA at the concentration of 0.25 µmol/L did not inhibit the proliferation of umbilical cord MSC obviously (t = 0.72, 1.32, P0.05). Part of MSC were floating instantly at the moment of adding ATRA of 4.0 µmol/L and no adherent cells were observed after 24 h' culture. Exposed to ATRA at the concentration of ≥ 1.0 µmol/L for 24 h, the proliferation of MSC were significantly inhibited, showing a dose-dependent manner (t = 8.8, 18.9, 22.1; P0.01). 0.5 µmol/L of ATRA did not affect the proliferation of cells and its morphology remained normal; 1.0 µmol/L of ATRA affected very few cells; but 2.0 µmol/L of ATRA cultured for 24 h inhibited the proliferation of cells obviously than 1 h, and the cells increased in size and became flattened. Flow cytometry showed that the rate of apoptosis between the control group and ≥ 1.0 µmol/L groups were significantly different (t = 9.88, 19.95, 31.61; P0.01).In the process of inducing umbilical cord MSC into neuron-like cells, 0.5 µmol/L ATRA was the optical concentration. ≥ 1.0 µmol/L ATRA can inhibit the cell proliferation, increase the apoptosis of cells significantly and caused obvious damages.

Proliferation and migration of retinal pigment epithelial (RPE) cells play a crucial role in proliferative vitreoretinopathy (PVR)-related pathology. Cytokines, including EGF, can result in RPE cell activation and cause PVR. In this study, integrin-alpha(5) expression was first studied in PVR membranes by immunofluorescence. Then the effect of EGF on integrin-alpha(5) expression was determined by flow cytometry, Western blot analysis and the reverse-transcription polymerase chain reaction (RT-PCR) in the ARPE-19 cell line. Proliferation and migration of ARPE-19 cells were measured by the methylthiazolyldiphenyl-tetrazolium bromide and Boyden chamber assays. We found that a higher level integrin-alpha(5) was present at the RPE cell surface in PVR compared with normal retina. EGF could dose dependently increase integrin-alpha(5) mRNA and protein levels in vitro. EGF promoted ARPE-19 cell proliferation and migration. Neutralizing integrin-alpha(5) by specific anti-integrin-alpha(5) antibody abolished most of the effects of EGF. The study provided evidence that EGF might influence PVR by promoting integrin-alpha(5) expression and subsequent proliferation and migration of RPE.