Objective To construct an eukaryotic expression plasmid containing gene coding for the hemagglutinin-neuraminidase(HN)of newcastle disease virus(NDV)oncolytic strain Italien,and then to express the protein in eukaryotic cell.Method The HN cDNA was synthesized from viral RNA by RT-PCR,and the eukaryotic expression vector of HN gene(named pcDNA3.1-HN)was constructed.The vector pcDNA3.1-HN was transfected into CHO-K1 cell by liposome,and G418 was used to select stable clones expressing HN gene.The expression of HN protein was visualized by Western blot and Immunofluorescence microscopy.Results Restriction analysis and DNA sequencing proved that HN gene was correctly cloned into expression vector.Western blot analysis and immunofluorescence showed that the HN was expressed in CHO-K1 cells.Conclusion The HN cDNA of NDV was successfully cloned into eukaryotic vector which showed good expression of HN protein in CHO-K1 cells.

Objective To evaluate the teaching effectiveness of applying article structure analysis to improve medical graduate students' ability to read research articles.Methods 48 medical graduate students from basic medicine school of the Fourth Military Medical University were randomly and equally divided into the experimental group and control group.In the reading ability training of scientific research papers,the experimental group used the teaching of the structure analysis,while the control group used the collective self study.Before and after the training,the two groups of students were implemented a unified reading ability test and self reading ability evaluation survey,and after the training,the teaching satisfaction survey was conducted among the experimental group only.SPSS 16 was used to analyze the correlation data and Wilcoxon for signed-rank t est.Results After training the reading ability test results showed that reading ability score (P=0.013),consumed reading time score (P=0.003) and reading efficiency (P=0.004) of the experimental group were significantly higher than those of control group.The two groups of students' self-evaluation of the reading ability showed that after training,the scores of the students in the experimental group were higher than those in the control group,and the differences were statistically significant (P values were less than 0.05).The experimental group students' teaching satisfaction survey to article structure analysis showed students' score in 6 survey contents were greater than 3,namely degree of evaluation was more than general,among which,the score of four survey contents was more than 4,that is to achieve satisfied or very satisfied.Conclttsion Applying article structure analysis can significantly improve medical graduate students' ability to read research articles.

Glioblastoma(GBM)is a lethal cancer with limited therapeutic options.Dendritic cell(DC)-based cancer vaccines provide a promising approach for GBM treatment.Clinical studies suggest that other immu-notherapeutic agents may be combined with DC vaccines to further enhance antitumor activity.Here,we report a GBM case with combination immunotherapy consisting of DC vaccines,anti-programmed death-1(anti-PD-1)and poly I:C as well as the chemotherapeutic agent cyclophosphamide that was integrated with standard chemoradiation therapy,and the patient remained disease-free for 69 months.The patient received DC vaccines loaded with multiple forms of tumor antigens,including mRNA-tumor associated antigens(TAA),mRNA-neoantigens,and hypochlorous acid(HOCl)-oxidized tumor lysates.Furthermore,mRNA-TAAAs were modified with a novel TriVac technology that fuses TAAs with a destabilization domain and inserts TAAs into full-length lysosomal associated membrane protein-1 to enhance major histo-compatibility complex(MHC)class Ⅰ and Ⅱ antigen presentation.The treatment consisted of 42 DC cancer vaccine infusions,26 anti-PD-1 antibody nivolumab administrations and 126 poly I:C injections for DC infusions.The patient also received 28 doses of cyclophosphamide for depletion of regulatory T cells.No immunotherapy-related adverse events were observed during the treatment.Robust antitumor CD4+and CD8+T-cell responses were detected.The patient remains free of disease progression.This is the first case report on the combination of the above three agents to treat glioblastoma patients.Our results suggest that integrated combination immunotherapy is safe and feasible for long-term treatment in this patient.A large-scale trial to validate these findings is warranted.

Lung cancer is the most common incident cancer and the leading cause of cancer death. In recent years, the development of tumor immunotherapy especially chimeric antigen receptor T (CAR-T) cell has shown a promising future. Epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific mutation expressed in various types of tumors and has been detected in non-small cell lung cancer with a mutation rate of 10%. Thus, EGFRvIII is a potential antigen for targeted lung cancer therapy. In this study, CAR vectors were constructed and transfected into virus-packaging cells. Then, activated T cells were infected with retrovirus harvested from stable virus-producing single clone cell lines. CAR expression on the surfaces of the T cells was detected by flow cytometry and Western blot. The function of CAR-T targeting EGFRvIII was then evaluated. The EGFRvIII-CAR vector was successfully constructed and confirmed by DNA sequencing. A stable virus-producing cell line was produced from a single clone by limited dilution. The culture conditions for the cell line, including cell density, temperature, and culture medium were optimized. After infection with retrovirus, CAR was expressed on more than 90% of the T cells. The proliferation of CAR-T cells were induced by cytokine and specific antigen in vitro. More importantly, EGFRvIII-CART specifically and efficiently recognized and killed A549-EGFRvIII cells with an effector/target ratio of 10:1 by expressing and releasing cytokines, including perforin, granzyme B, IFN-γ, and TNF-α. The in vivo study indicated that the metastasis of A549-EGFRvIII cells in mice were inhibited by EGFRvIII-CART cells, and the survival of the mice was significantly prolonged with no serious side effects. EGFRvIII-CART showed significantly efficient antitumor activity against lung cancer cells expressing EGFRvIII in vivo and in vitro. Therefore, CAR-T targeting EGFRvIII is a potential therapeutic strategy in preventing recurrence and metastasis of lung cancer after surgery.
Animals ; Carcinoma, Non-Small-Cell Lung ; immunology ; therapy ; Cell Line, Tumor ; ErbB Receptors ; immunology ; metabolism ; Female ; Humans ; Immunotherapy, Adoptive ; methods ; Lung Neoplasms ; immunology ; therapy ; Mice ; Mice, Inbred NOD ; Receptors, Chimeric Antigen ; immunology ; T-Lymphocytes ; immunology ; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. This malignancy is associated with poor prognosis and high mortality. Novel approaches for prolonging the overall survival of patients with advanced HCC are urgently needed. The antitumor activities of adoptive cell transfer therapy (ACT), such as strategies based on tumor-infiltrating lymphocytes and cytokine-induced killer cells, are more effective than those of traditional strategies. Currently, chimeric antigen receptor T-cell (CAR-T) immunotherapy has achieved numerous breakthroughs in the treatment of hematological malignancies, including relapsed or refractory lymphoblastic leukemia and refractory large B-cell lymphoma. Nevertheless, this approach only provides a modest benefit in the treatment of solid tumors. The clinical results of CAR-T immunotherapy for HCC that could be obtained at present are limited. Some published studies have demonstrated that CAR-T could inhibit tumor growth and cause severe side effects. In this review, we summarized the current application of ACT, the challenges encountered by CAR-T technology in HCC treatment, and some possible strategies for the future direction of immunotherapeutic research.
Adoptive Transfer ; methods ; Carcinoma, Hepatocellular ; immunology ; therapy ; Humans ; Immunotherapy, Adoptive ; methods ; Liver Neoplasms ; immunology ; therapy ; Lymphocytes, Tumor-Infiltrating ; cytology ; Randomized Controlled Trials as Topic ; Receptors, Chimeric Antigen ; T-Lymphocytes ; cytology