Ultrasound-mediated microbubble destruction enhances LMP-1 gene transfection into dendritic cells in vivo / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the transfection efficiency and the optimal conditions of delivering latent membrane protein-1 (LMP-1) gene to dendritic cells (DCs) by ultrasound exposure combined with contrast agent.</p><p><b>METHODS</b>Human DCs were cultured in vivo and transfected with the recombinant plasmid pEGFP-C3-LMP1 under varying conditions including ultrasound intensities, exposure time and microbubble contrast agent concentration. The transfection efficiency was assessed by fluorescent microscopy and flow cytometry, and the cell viability by trypan blue exclusion test.</p><p><b>RESULTS</b>An exposure time of 60 s at MI 1.0 with a microbubble contrast agent concentration of 20% resulted in the optimal effect of delivering the recombinant plasmid pEGFP-C3-LMP1 into the DCs, with a transfection efficiency of (14.37∓2.12)%. Over 90% of the transfected cells were viable after the transfection.</p><p><b>CONCLUSION</b>Microbubble contrast agent combined with ultrasound exposure can enhance the delivery of recombinant plasmid pEGFP-C3-LMP1 into the DCs.</p>

Clinical value of (18)F-FDG positron emission tomography-computed tomography in local liver neoplasm ablation / 南方医科大学学报

<p><b>OBJECTIVE</b>To assess the value of (18)F-fluoro-2-deoxy-D-glucose positron emission tomography-computed tomography ((18)F-FDG PET-CT) in ultrasound-guided local ablation of malignant liver tumors.</p><p><b>METHODS</b>Thirteen patients with 35 local residual tumor foci following previous tumor ablation underwent (18)F-FDG PET-CT and ultrasound-guided local ablation with intratumoral alcohol injection.</p><p><b>RESULTS</b>After the second local ablation guided by (18)F-FDG PET-CT and ultrasound, radioactive defects were detected in the corresponding location in 31 of the 35 residual foci, and after the third local ablation, the other 4 foci also showed radioactive defects.</p><p><b>CONCLUSION</b>(18)F-FDG PET-CT can sensitively and accurately identify tissue necrosis and residual tumors, and serves as an excellent approach for ultrasound-guided local ablation of local residual tumors.</p>

rAAV/BA46-transfected dendritic cells can induce specific cellular immunity / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the feasibility of transfecting breast cancer BA46 gene into dendritic cells (DCs) using adeno-associated virus (AAV) to induce specific cellular immunity.</p><p><b>METHODS</b>Mononuclear cells (DC precursor) were isolated from the peripheral blood of healthy donors by density gradient centrifugation and infected with rAAV/BA46/Neo virus stock (transfection group) or pulsed with 293 cell lysate (control group). In both groups, maturation of the DC precursor was induced by granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor-alpha(TNF-alpha). On day 7, the DCs were collected and mixed with T cells at the ratio of 1 to 20 to induce cytotoxic T lymphocytes (CTL). The capacity of the DCs in stimulating T lymphocyte proliferation was assessed using (3)H-thymidine incorporation assay. The expressions of interferon-gamma (IFN-gamma), IL-4, CD4, CD8, CD25 and CD69 in the CTLs were analyzed with cytometry, and the cytotoxicity of the CTLs was evaluated with (51)Cr-release assay using BA46-positive breast cancer cell line Hs578T as the target.</p><p><b>RESULTS</b>The DCs transfected with BA46 gene exhibited potent capacity to stimulate T lymphocyte proliferation. The CTL population induced by the transfected DCs expressed high levels of CD8, CD69 and IFN-gamma, and showed strong cytotoxicity against BA46-positive breast cancer cell line Hs578T, which was BA46 antigen-specific and MHC-limited.</p><p><b>CONCLUSION</b>The success in BA46 gene transfer in the DCs that induce specific cellular immunity provides the experimental basis for breast cancer immunotherapy using genetically modified cells.</p>

Construction of adenoviral vector encoding soluble human TNFRI-IgGFc cDNA and its expression in human bronchial epithelial cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a recombinant adenovirus vector carrying soluble extracellular region of tumor necrosis factor alpha receptor I-IgGFc (sTNFRI-IgGFc) and express the fusion protein in human bronchial epithelial HBE135-E6E7 cells.</p><p><b>METHODS</b>sTNFRI-IgGFc fusion gene was subcloned into the adenovirus shuttle plasmid pDC316, which was co-transfected with helper plasmid pBHGloxPE1,3Cre into HEK293 cells. The recombinant adenovirus (Ad-sTNFRI-IgGFc) was generated by homologous recombination of the 2 plasmids in HEK293 cells. After identification with PCR, Ad-sTNFRI-IgGFc was amplified and purified, and its titer measured using TCID50 assay. The transcription and expression of sTNFRI-IgGFc gene in the transfected HBE135-E6E7 were detected by RT-PCR and immunohistochemistry.</p><p><b>RESULTS</b>Ad-sTNFRI-IgGFc was successfully constructed with a viral titer of 3 x 10(10) TCID50/ml. The expression of sTNFRI-IgGFc mRNA and protein was confirmed in the transfected HBE135-E6E7 cells.</p><p><b>CONCLUSION</b>The constructed Ad-sTNFRI-IgGFc can effectively infect HBE135-E6E7 cells for efficient expression of sTNFRI-IgGFc protein, which antagonizes the cytolytic effect of TNFalpha in L929 cells, suggesting the potential of adenovirus expressing sTNFRI-IgGFc for local treatment of asthma.</p>

Effects of two different antigen-loading methods on the activity of dendritic cell vaccine for colorectal carcinoma cell inhibition in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the changes in the activity of dendritic cells (DCs) after carcino-embryonic antigen (CEA) gene transfection mediated by recombinant adeno-associated virus type2 (rAAV) and tumor cell lysate.</p><p><b>METHODS</b>Immature DCs isolated from peripheral blood monocytes of HLA-A11-positive healthy volunteers were infected with the rAAV carrying CEA gene or loaded with tumor cell lysate. The surface markers of the DCs such as CD40, CD 1alpha, and CD86 were analyzed by flow cytometry. Interleukin-12 (IL-12) in the supernatants of DCs and interferon-gamma (IFN-gamma) released by the cytotoxic T lymphocytes (CTLs) were determined by ELISA detection kit. The specific killing activity of CTL against LoVo cells was assessed by MTT assay.</p><p><b>RESULTS</b>The DCs following antigen loading with the two methods both highly expressed CD40, CD86 and IL-12, and induced specific CTL that specifically recognized and killed LoVo cells, but the killing effect resulting from rAAV infection of the DCs was much better than that induced by tumor cell lysate loading.</p><p><b>CONCLUSION</b>Both methods of antigen loading can induce mature DCs from peripheral blood monocyte cells, but rAAV infection of the DCs can be more effective than tumor cells lysate loading. DCs infected with rAAV may have the potential to serve as an adjuvant immunotherapy for patients with colorectal carcinoma.</p>