CD19 chimeric antigen receptor-redirected T cells combined with epidermal growth factor receptor pathway substrate 8 peptide-derived dendritic cell vaccine in leukemia.

BACKGROUND: Chimeric antigen receptor (CAR)-T cell therapy opens a new era for cancer treatment. However, in prolonged follow-up, relapse has emerged as one of the major obstacles. Dendritic cell (DC) vaccination is a promising treatment to eradicate tumor cells and prevent relapse. The epidermal growth factor receptor (EGFR) pathway substrate 8 (Eps8) gene is involved in regulating cancer progression and is considered an attractive target for specific cancer immunotherapy. The purpose of this study was to explore a combinatorial therapy using CAR-T cells and a DC vaccine such as Eps8-DCs to increase leukemia treatment efficacy. METHODS: We pulsed DCs with Eps8-derived peptides to generate Eps8-DCs, engineered T cells to express a second-generation CAR specific for CD19, and analyzed the effects of the Eps8-DCs on the in vitro expansion, phenotype and effector functions of the CD19 CAR-T cells. RESULTS: The Eps8-DCs significantly reduced the activation-induced cell death and enhanced the proliferative potential of CAR-T cells during in vitro expansion. In addition, the expanded T cells co-cultured with the Eps8-DCs exhibited an increased percentage of central memory T cells (Tcms) and a decreased percentage of effector memory T cells (Tems). The Eps8-DCs enhanced CD19 CAR-T cell immune functions, including cytokine production, CD107a degranulation activity and cytotoxicity. DISCUSSION: This study demonstrates that Eps8-DCs exert synergistic effect on CD19 targeting CAR-T cells and paves the way for clinical trials using the combination of DC vaccination and engineered T cells in relapsed leukemia.

[Effect of Silencing Eps8 Gene Expression on the Biology Activity of Human Leukemia K562 Cells and Its Molecular Mechanism].

OBJECTIVE: The study was to investigate the effect of silencing Eps8 gene expression on proliferation and apoptosis of human leukemia K562 cells and its molecular mechanism. METHODS: The expetriments were divided into 3 groups, including blank control group(K562 cells without treatment), K562-shRNA group(K562 cells transfected by specific Eps8-shRNA lenticiral vector) and K562-NC group(K562 cells transfected by negtive control lenticiral vector). K562 cells with stably-silenced Eps8 gene were constucted by lentibirus-mediated RNA technology. The efficacy of transfection was observed by fluorescence microscopy and the changes of Eps8 mRNA and protein level were detected by RT-PCR and Western blot respectively. Cell proliferation was confirmed by typan blue exclusion and MTT method. The apoptosis rate of cells was analysed by the flow cytometry, and colony forming was detected by methylcellulose colony forming assay. The protein level change of phosphrylated-AKT were detected by Western blot. RESULTS: Stably-silenced Eps8 gene K562 cells and the negative control cells were successfully constructed. Compared with the blank control group and the K562-NC group, the proliferation of K562-shRNA cells were siginificantly inhibited(P<0.05); the apoptosis of K562-shRNA cells increased(P<0.05). In addition, the methylcellulose colony forming assay showed that the colony forming was dramatically suppressed in K562-shRNA cells (P<0.05). Furthermore, knocking down Eps8 gene reduced the protein level of AKT phosphrylation at both residue Ser437 and Thr308(P<0.05), while there was no obvious change in the level of total-AKT(P>0.05). Knocking down Eps8 gene reduced the protein level of m-TOR phosphrylation and PRAS40 phosphrylation (P<0.05), while there was no obvious change in the level of total-mTOR and PRAS40 (P>0.05). CONCLUSION: Silencing Eps8 gene through lentvirus can inhibit the cell proliferation and promote the apoptosis of human leukemia K562 cells, which possibly relates with the inhibition of AKT/mTOR activation.

[A Feasibility Study of closing the small bowel with high-frequency welding device].

This study aimed to evaluate the feasibility and effectiveness of closing the small bowel in an ex vivo porcine model with high-frequency welding device. A total of 100 porcine small bowels were divided into two groups, and then were closed with two different methods. The fifty small bowels in experimental group were closed by the high-frequency welding device, and the other fifty small bowels in comparison group were hand-sutured. All the small bowels were subjected to leak pressure testing later on. The speed of closure and bursting pressure were compared. The 50 porcine small bowels closed by the high-frequency welding device showed a success rate of 100%. Compared with the hand-sutured group, the bursting pressures of the former were significantly lower (P<0.01) and the closing process was significantly shorter (P<0.01). The pathological changes of the closed ends mainly presented as acute thermal and pressure induced injury. Experimental results show that the high-frequency welding device has higher feasibility in closing the small bowel.