Surface expression of anti-CD3scfv stimulates locoregional immunotherapy against hepatocellular carcinoma depending on the E1A-engineered human umbilical cord mesenchymal stem cells.

Tumor antigens is at the core of cancer immunotherapy, however, the ideal antigen selection is difficult especially in poorly immunogenic tumors. In this study, we designed a strategy to modify hepatocellular carcinoma (HCC) cells by surface expressing anti-CD3scfv within the tumor site strictly, which depended on the E1A-engineered human umbilical cord mesenchymal stem cells (HUMSC.E1A) delivery system. Subsequently, membrane-bound anti-CD3scfv actived the lymphocytes which lysed HCC cells bypassing the expression of antigens or MHC restriction. First, we constructed the anti-CD3scfv gene driven by human α-fetoprotein (AFP) promoter into an adenoviral vector and the E1A gene into the lentiviral vector. Our results showed that anti-CD3scfv could specifically express on the surface of HCC cells and activate the lymphocytes to kill target cells effectively in vitro. HUMSC infected by AdCD3scfv followed by LentiR.E1A could support the adenoviral replication and packaging in vitro 36 h after LentiR.E1A infection. Using a subcutaneous HepG2 xenograft model, we confirmed that AdCD3scfv and LentiR.E1A co-transfected HUMSC could migrate selectively to the tumor site and produce considerable adenoviruses. The new generated AdCD3scfv infected and modified tumor cells successfully. Mice injected with the MSC.E1A.AdCD3scfv and lymphocytes significantly inhibited the tumor growth compared with control groups. Furthermore, 5-fluorouracil (5-FU) could sensitize adenovirus infection at low MOI resulting in improved lymphocytes cytotoxicity in vitro and in vivo. In summary, this study provides a promising strategy for solid tumor immunotherapy.

Stabilization of c-myc G-Quadruplex DNA, inhibition of telomerase activity, disruption of mitochondrial functions and tumor cell apoptosis by platinum(II) complex with 9-amino-oxoisoaporphine.

[Pd(L)(DMSO)Cl2] (1) and [Pt(L)(DMSO)Cl2] (2) with 9-amino-oxoisoaporphine (L), were synthesized and characterized. 1 and 2 are more selectively cytotoxic to Hep-G2 cells versus normal liver cells (HL-7702). Various experiments showed that 2 acted as telomerase inhibitors targeting G4-DNA and triggered cell apoptosis by interacting with c-myc G4-DNA. Furthermore, 2 significantly induced cell cycle arrest at both G2/M and S phase, which leading to the down-regulation of cdc25 A, cyclin D, cyclin B, cyclin A and CDK2 and the up-regulation of p53, p27, p21,chk1 and chk2. In addition, 2 also caused mitochondrial dysfunction. Taken together, we found that 2 exerted its cytotoxic activity mainly via inhibiting telomerase by interaction with c-myc G4-DNA and disruption of mitochondrial function.

High glucose induces inflammatory cytokine through protein kinase C-induced toll-like receptor 2 pathway in gingival fibroblasts.

Toll-like receptors (TLRs) play a key role in innate immune response and inflammation, especially in periodontitis. Meanwhile, hyperglycemia can induce inflammation in diabetes complications. However, the activity of TLRs in periodontitis complicated with hyperglycemia is still unclear. In the present study, high glucose (25 mmol/l) significantly induced TLR2 expression in gingival fibroblasts (p<0.05). Also, high glucose increased nuclear factor kappa B (NF-κB) p65 nuclear activity, tumor necrosis factor-α (TNF-α) and interleukin-lß (IL-1ß) levels. Protein kinase C (PKC)-α and δ knockdown with siRNA significantly decreased TLR2 and NF-κB p65 expression (p<0.05), whereas inhibition of PKC-ß had no effect on TLR2 and NF-κB p65 under high glucose (p<0.05). Additional studies revealed that TLR2 knockdown significantly abrogated high-glucose-induced NF-κB expression and inflammatory cytokine secretion. Collectively, these data suggest that high glucose stimulates TNF-α and IL-1ß secretion via inducing TLR2 through PKC-α and PKC-δ in human gingival fibroblasts.

Intrahepatic transplantation of hepatic oval cells for fulminant hepatic failure in rats.

AIM: To evaluate the effect of intrahepatic trans-plantation of hepatic oval cells (HOC) on fulminant hepatic failure (FHF) in rats. METHODS: HOC obtained from rats were labeled with green fluocescent protein (GFP) or 5, 6-carboxyfluorescein diacetate succinmidyl ester (CFDA-SE). Cell fluorescence was observed under fluorescent microscope at 6, 24, 48 and 72 h after labeling. CFDA-SE labeled HOC (5 x 10(6) cells each rat) were injected into livers of rats with FHF induced by D-galactosamine. Serum albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBil) levels were measured at different time points. Liver function of rats was examined on days 3, 7, 14 and 21 after HOC transplantation. RESULTS: The positive rate of GFP and CFDA-SE labeled HOC was 10% and 90%, respectively, with no significant change in cell viabilities. The survival rate was higher in HOC transplantation group than in control group, especially 48 (9/15 vs 6/15) and 72 h (9/15 vs 4/15) after HOC transplantation. The serum ALT, AST and TBil levels were decreased while the serum Alb level was increased after HOC transplantation. Fluorescence became faded and diffused in liver tissues, suggesting that proliferation and differentiation occur in transplanted HOC. CONCLUSION: CFDA-SE is superior to GFP in labeling HOC, although fluorescence intensity is decreased progressively with cell division. HOC transplantation can improve the liver function and increase the survival rate of recipients.

[Induction of dendritic cells from intra-operative lost blood and their effects on CIK against liver cancer cells in vitro].

OBJECTIVE: To investigate the practical possibility of inducing dendritic cells (DCs) from mononuclear cells in the lost blood during operation of hepatocellular carcinoma (HCC) patients, and attempted to find a new source of precursor cells for the personalized immunotherapy based on DCs. METHODS: Collected lost blood during hepatectomy from 9 HCC patients and human cord blood from 8 cases of healthy donors undergoing caesarean section. Their mononuclear cells were divided into monocytes and nonadherent lymphocytes. RhGM-CSF and rhIL-4 were administered to induce the monocytes differentiation into DCs, and then loaded with different antigens (lysate antigen of autologous liver cancer cells and cell line SMMC-7721 cells). The lymphocytes were induced into cytokine-induced killer cells (CIK) with IL-2, CD3-Ab, gamma-IFN and PHA. MTT assay was performed to detect the proliferation rate of T lymphocytes mediated by DC and the cytotoxicity of CIK to liver cancer cells. RESULTS: DCs induced from monocytes of the intra-operative lost blood possessed typical morphology and phenotypes. Compared with the DCs from cord blood, the DCs from intra-operative lost blood expressed lower level of surface markers, but both could effectively induce proliferation of CIK and enhance the cytotoxicity of activated CIK against liver cancer cells at similar levels. When the DCs from lost blood and their counterpart from cord blood were both loaded with autologous tumor cell antigen, the proliferation rates of CIK were (388.9 +/- 137.3)% and (315.1 +/- 44.5)%, respectively, and the killing rates against tumor cells were (87.1 +/- 8.0)% and (90.0 +/- 5.1)%, respectively. When the two similar DC groups were loaded with lysate antigen of SMMC-7721 cells, the proliferation rates of CIK were (239.9 +/- 48.7)% and (226.3 +/- 32.3)%, respectively, and the killing rates against tumor cells were (76.4 +/- 7.9)% and (81.1 +/- 4.3)%, respectively. There were no significant differences between those two DC groups. The data also showed that the proliferation and cytotoxicity of CIK induced by DCs loaded with autologous antigen were higher than that of DCs loaded with SMMC-7721 antigen. CONCLUSION: Mononuclear cells separated from intra-operative lost blood of HCC patients can be induced into mature DCs, which can effectively activate CIK and significantly increase its killing effect on the liver cancer cells, and may become a new source of DCs to study and develop vaccines for clinical application.