OBJECTIVETo assess the immunotoxicologic effects of genetically modified drought resistant wheat T349 with GmDREB1 gene.

METHODSA total of 250 female BALB/c mice (6-8 week-old, weight 18-22 g) were divided into five large groups (50 mice for each large group) by body weight randomly. In each large group, the mice were divided into five groups (10 mice for each group) by body weight randomly, which were set as negative control group, common wheat group, parental wheat group, genetically modified wheat group and cyclophosphamide positive control group, respectively. Mice in negative control and positive control group were fed with feedstuff AIN-93G, mice in common wheat group, non-genetically modified parental wheat group and genetically modified wheat group were fed with feedstuffs added corresponding wheat (proportion up to 76%) for 30 days, then body weight, organ coefficient of spleen and thymus, peripheral blood lymphocytes phenotyping, serum cytokine, serum immunoglobulin, antibody plaque-forming cell (PFC), serum 50% hemolytic value (HC50), mitogen-induced splenocyte proliferation, delayed-type hypersensitivity (DTH) reaction and phagocytic activities of phagocytes were detected respectively.

RESULTSAfter 30 days raise, among negative control group, common wheat group, non-genetically modified parental wheat group, genetically modified wheat group and cyclophosphamide positive control group, mice body weight were (21.0±0.3), (20.4±0.7), (21.1±1.0), (21.1±1.0), (19.4±1.0) g, respectively (F=7.47, P<0.01); organ coefficient of spleen were (0.407±0.047)%, (0.390±0.028)%, (0.402±0.042)%, (0.421±0.041)%, (0.304±0.048)%, respectively (F=12.41, P<0.01); organ coefficient of thymus were (0.234±0.032)%, (0.246±0.028)%, (0.249±0.040)%, (0.234±0.034)%, (0.185±0.039)%, respectively (F=5.58, P<0.01); the percentage of T cell in peripheral blood were (70.43±4.44)%, (68.33±5.37)%, (73.04±2.68)%, (74.42±2.86)%, (90.42±1.66)%, respectively (F=57.51, P<0.01); the percentage of B cell were (13.89±3.19)%, (15.34±4.84)%, (13.06±4.22)%, (12.93±2.36)%, (3.01±0.96)%, respectively (F=12.79, P<0.01); the percentage of Th cell were (55.87±3.80)%, (55.24±4.60)%, (57.92±3.70)%, (59.57±2.54)%, (77.37±2.31)%, respectively (F=68.58, P<0.01);the Th/Ts ratio were 4.16±0.29, 4.73±0.96, 4.19±0.78, 4.52±0.40, 6.34±0.73, respectively (F=17.57, P<0.01);the serum IgG were (1046.38±210.67), (1065.49±297.22), (1517.73±299.52), (1576.67±241.92), (1155.88±167.05) µg/ml, respectively (F=10.53, P<0.01); the serum IgM were (333.83±18.97), (327.73±27.72), (367.47±27.18), (363.42±46.14), (278.71±24.42) µg/ml, respectively (F=12.11, P<0.01); the serum IgA were (51.69±10.10), (42.40 ± 8.35), (32.11±4.22), (37.12±4.90), (41.45±8.89) µg/ml, respectively (F=8.25, P<0.01); the PFC were (29.2±14.6), (28.0±20.0), (34.8±30.9), (33.2±25.1), (4.8±5.3) per 10(6) splenocyte, respectively (F=3.33, P<0.05); the HC50 were 82.3±6.5, 79.7±4.6, 75.8±4.1, 74.9±3.6, 70.8±2.1, respectively (F=9.99, P<0.01);the LPS-induced splenocyte proliferation were 0.21±0.10, 0.21±0.14, 0.26±0.12, 0.25±0.14, 0.07±0.06, respectively (F=4.18, P<0.05).

CONCLUSIONThe genetically modified drought-resistant wheat T349 was substantially equivalent to parental wheat in the effects on immune organs and immunologic functions of mice, and it didn't show immunotoxicity.

Animals ; Cytotoxicity Tests, Immunologic ; Cytotoxicity, Immunologic ; Droughts ; Female ; Mice ; Mice, Inbred BALB C ; Plants, Genetically Modified ; immunology ; toxicity ; Triticum ; genetics ; immunology ; toxicity

OBJECTIVETo study the expression of B7-H3 mRNA and B7-H3 protein in gastric carcinoma and their clinical significance.

METHODSThe expression of B7-H3 mRNA and B7-H3 protein in gastric carcinoma and the nearby normal tissue of 38 patients was detected by real-time RT-PCR and immunohistochemical assay respectively.

RESULTSB7-H3 mRNA was expressed both in gastric carcinoma and nearby normal tissue, but the expression level in gastric carcinoma was much lower than that in nearby normal tissue. There were no significant differences of B7-H3 mRNA expression among gender, age, histological type, tumor size, lymph node metastasis and invasive depth (all P >0.05). The positive rate of B7-H3 protein expressed in gastric carcinoma was 39.5%. There were no significant differences of B7-H3 protein expression among gender, age, histological type, tumor size, lymph node metastasis and invasive depth (all P >0.05), but there were significant differences among groups of clinical stage (P=0.022) and pathological grade (P=0.039). Kaplan-Meier analysis revealed that disease-free survival or overall survival of the patients with positive B7-H3 expression were significantly longer than those with negative B7-H3 expression (P=0.009 and P=0.010 respectively).

CONCLUSIONDetection of B7-H3 expression in gastric carcinoma will be beneficial to the judgment of the prognosis of gastric carcinoma and the choice of individualized treatment.

Antigens, CD ; genetics ; metabolism ; B7 Antigens ; Biomarkers, Tumor ; genetics ; metabolism ; Cytotoxicity, Immunologic ; Female ; Humans ; Middle Aged ; Neoplasm Staging ; RNA, Messenger ; genetics ; Receptors, Immunologic ; genetics ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology

Cytokines ; secretion ; Cytotoxicity, Immunologic ; Graft vs Leukemia Effect ; Humans ; Immunophenotyping ; Lymphocyte Activation ; Retroviridae ; genetics ; Simplexvirus ; enzymology ; T-Lymphocytes ; immunology ; Thymidine Kinase ; genetics ; Transfection

OBJECTIVETo investigate the influence of hepatitis B virus C protein on the function of natural killer cell.

METHODSRecombinant eukaryotic expression plasmid pHBI-CMV-HBC was constructed and confirmed by double restrictive enzyme digestion and DNA sequencing analysis. Then the recombinant plasmid was transfected into NK-92 cells with lipofectamine encapsuled. The transfected NK-92 cells containing expressive HBV C protein was confirmed by Western Blot analysis. ELISA was employed to determine the IFN-gamma level secreted by NK-92 cells. And finally the cytotoxicities of NK cells were analysed by MTT colorimetry, with the hepatoblastoma cell line (HepG2) as target cell.

RESULTSWestern blotting confirmed the expression of HBV C protein in the NK-92 cells transfected with pHBI-CMV-HBC. NK cytotoxicities and IFN-gamma secretion level of NK-92 cells transfected with recombinant plasmid significantly increased compared to control NK-92 cells transfected with blank plasmid (P < 0.01) and untransfected NK-92 cells(P < 0.01).

CONCLUSIONTransient expression of HBC can increase IFN-gamma secretion and cytotoxicities of NK-92 cells.

Cell Line ; Cytotoxicity, Immunologic ; Hepatitis B ; immunology ; virology ; Hepatitis B Core Antigens ; genetics ; immunology ; Hepatitis B virus ; genetics ; immunology ; Humans ; Interferon-gamma ; immunology ; Killer Cells, Natural ; immunology

OBJECTIVE: To investigate the effect of overexpression of glycosylphosphatidyl-inositol-specific phospholipase D (GPI-PLD) on the biological character of hepatocellular carcinoma cell line HepG2. METHODS: The GPI-PLD gene eukaryon expression vector pcDNA3.1(+)/ GPI-PLD was transiently transfected into HepG2 cell by lipid-media transfection. The untransfected HepG2 and HepG2 transfected with pcDNA3.1(+) were used as controls. After screening with G418, the single clone was obtained. The expression level of GPI-PLD mRNA in HepG2 was identified by reverse transcription polymerase chain reaction (RT-PCR). GPI-PLD activities were analyzed quantitatively by triton-X-114 partition with GPI anchored placental alkaline phosphatase (PLAP) as a substrate. Cell count was used to detect the proliferation of the 3 groups, and complement dependent cytotoxicity (CDC) effects were observed by the staining of trypan blue. Apoptosis cells were analyzed by flow cytometry. Carcinoembryonic antigen (CEA)was detected by enzyme linked immunosorbent assay (ELISA). RESULTS: Compared with HepG2 and pcDNA3.1(+)/HepG2 cell, the levels of GPI-PLD activities and its mRNA from pcDNA3.1(+)/GPI-PLD/HepG2 were increased with almost 2 to 5 times,respectively. The GPI anchored PLAP and CEA released into the medium by GPI-PLD, and the rate of CDC killing on the cells were significantly increased. However, the proliferative capacity was obviously decreased, and the typical apoptosis cells were presented in positive clones and its apoptosis rates were increased significantly. CONCLUSION The stable cell line with overexpression of GPI-PLD has been constructed. The overexpression of GPI-PLD in these cells increases the sensitivity of these cells to CDC killing and impairs the proliferative capacity of cells, and promotes the apoptosis.
Apoptosis ; genetics ; Carcinoma, Hepatocellular ; genetics ; pathology ; Complement Activation ; genetics ; Cytotoxicity, Immunologic ; genetics ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; genetics ; metabolism ; Humans ; Liver Neoplasms ; genetics ; pathology ; Phospholipase D ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Transfection ; Tumor Cells, Cultured ; Up-Regulation

The study was aimed to investigate the expression of HLA class I molecules and MHC class I chain-related molecules A/B (MICA/MICB) in K562 and adriamycin (ADM)-resistant K562 cell lines (K562/AO2) and their effect on cytotoxicity of NK cells. Expression of HLA class I molecules and MICA/MICB on the surface of K562 and K562/AO2 cell lines were analyzed by flow cytometry. Cytotoxicity of NK cells (isolated from 3 healthy persons) against K562 and K562/AO2 cells were detected by LDH releasing assay at different effect-to-target cell ratios (E:T). In blocking experiments, anti-MHC class I monoclonal antibody (McAb) (W6/32, a pan anti-HLA class I antibody) and anti-MHC class I chain-related molecules McAb (BAMO-1, specifically against MICA and MICB) were added to the target cells at E:T of 10:1. The results showed that the expression of MHC class I chain-related molecules on K562 was higher than that on K562/AO2 (P=0.000), and HLA class I molecules were not detectable on both cells. Cytotoxicities of NK cells against K562 and K562/AO2 cells were (29.32 +/- 0.12)%, (45.33 +/- 0.78)%, (58.37 +/- 0.87)%, (72.37 +/- 0.96)% and (12.47 +/- 0.91)%, (24.36 +/- 1.11)%, (33.29 +/- 1.03)%, (53.87 +/- 1.27)% at E:T ratios of 5:1, 10:1, 20:1 and 30:1 respectively (P=0.000), the cytotoxicity of NK cells on K562 cells was significantly higher than that on K562/A02 cells at different E:T ratios. Blocking experiments confirmed that at E:T of 10:1 killing of NK cells against K562 and K562/AO2 cells was efficiently inhibited by BAMO-1, whereas W6/32 had no effect on K562 and K562/AO2 cells. It is concluded that the expression of MHC class I chain-related molecules on K562 and K562/AO2 cells is correlated with NK cell-mediated lysis. NK cells display higher cytotoxicity against parental K562 cells than multi-drug resistant K562/AO2 cells. Down-regulation of MICA/B in multi-drug resistant tumor cell lines leads to reduction of susceptibility to NK lysis.
Cytotoxicity, Immunologic ; immunology ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; immunology ; Genes, MHC Class I ; genetics ; Histocompatibility Antigens Class I ; immunology ; Humans ; K562 Cells ; Killer Cells, Natural ; immunology

OBJECTIVETo investigate the specific cytotoxity of tumor infiltrating lymphocytes (TIL) transfected with chimeric T cell receptor (CTCR) on cells which express KDR.

METHODSA recombinant retroviral plasmid (pMSCVneo-Vhgamma) was constructed by cloning VEGF121-hinger-FcRgamma (Vhgamma) into retroviral vector pMSCVneo. After packaging by PT67, the virus with high titer was used to infect TIL isolated from liver cancer tissues, and then MSCVneo-Vhgamma-TIL was generated. TIL infected with MSCVneo was used as a control. The cytotoxicty of the transgenic TIL on NIH3T3 and HepG2 expressing no KDR and on ECV304 and A375 expressing KDR was detected with MTT colorimetric assay.

RESULTSThe sequences of VEGF121 and hinger-FcRgamma were different from those reported, but the deduced amino sequences were identical to the reported ones. The cytotoxity of TIL infected with MSCVneo on target cell was similar to that of the control TIL; both only had mild cytotoxity on cancer cell line. No significant cytotoxity was found in TIL infected with MSCVneo-cTCR on NIH3T3, but its cytotoxity on ECV304 was significant. The cytotoxity on HepG2 was similar to that of MSCVneo-TIL and uninfected TIL, but cytotoxity on A375 was significantly higher.

CONCLUSIONChimeric T cell receptor permanently grafts TIL cell with predefined new specificity. TIL expressing Vhgamma can selectively recognize and kill vascular endothelial cell and tumor cells which express VEGF receptor KDR.

Animals ; Cytotoxicity, Immunologic ; Humans ; Lymphocytes, Tumor-Infiltrating ; immunology ; Mice ; NIH 3T3 Cells ; Plasmids ; Receptors, Antigen, T-Cell ; physiology ; Retroviridae ; genetics ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; physiology

OBJECTIVE: The efficiency of antitumor immunity induced by dendritic cells (DCs) transfected with total RNA of laryngeal carcinoma cells was explored. METHOD: DCs, induced from human peripheral blood mononuclear cells, were transfected with total RNA of laryngeal carcinoma cells. The specific antitumor immunity of cytotoxic T Lymphocytes (CTLs) that were actived by RNA-transfected DCs were detected by MTT methods in vitro. In vivo, antitumor-specific CTLs were subcutaneously injected into the nude mice previously. After 7 days, the laryngeal carcinoma cells were seeded and the tumor occurrence rate was observed. Tumor-loaded nude mice were treated by specific CTLs once (the treated group) or twice (the retreated group). The growth of the implanted tumor was observed too. RESULT: DCs that transfected with tumor RNA can significantly active CTLs which induced antitumor-specific immune response against laryngeal carcinoma in vitro. In vivo, the tumor occurrence rate of the treatment group was predominantly reduced compared with that of the control (P < 0.01). The implanted tumor size of the treated and retreated groups were both significantly reduced (P < 0.05, P < 0.01) compared with the control too, especially the retreated ones (P < 0.05). CONCLUSION The tumor RNA loaded DCs can significantly active CTLs and the antitumor specific CTLs can both induce antitumor specific immune response against laryngeal carcinoma in vitro and inhibit the growth of the implanted tumor in vivo.
Animals ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Dendritic Cells ; immunology ; Humans ; Immunotherapy, Adoptive ; methods ; Laryngeal Neoplasms ; therapy ; Mice ; Mice, Nude ; RNA, Neoplasm ; genetics ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection

Cytokine-activated T cells (CATs) can be easily expanded and are widely applied to cancer immunotherapy. However, the good efficacy of CATs is rarely reported in clinical applications because CATs have no or very low antigen specificity. The low-efficacy problem can be resolved using T cell antigen receptor-engineered CAT (TCR-CAT). Herein, we demonstrate that NY-ESO-1 HLA-A*02:01-specific high-affinity TCR (HAT)-transduced CATs can specifically kill cancer cells with good efficacy. With low micromolar range dissociation equilibrium constants, HAT-transduced CATs showed good specificity with no off-target killing. Furthermore, the high-affinity TCR-CATs delivered significantly better activation and cytotoxicity than the equivalent TCR-engineered T cells (TCR-Ts) in terms of interferon-γ and granzyme B production and in vitro cancer cell killing ability. TCR-CAT may be a very good alternative to the expensive TCR-T, which is considered an effective personalized cyto-immunotherapy.
Cell Line, Tumor ; Cytokines ; metabolism ; Cytotoxicity, Immunologic ; Genetic Engineering ; HLA-A2 Antigen ; metabolism ; Humans ; Immunotherapy, Adoptive ; methods ; Lymphocyte Activation ; Receptors, Antigen, T-Cell ; genetics ; immunology ; T-Lymphocytes ; immunology

OBJECTIVETo construct anti-CD20scFv/CD80/CD28/zeta recombinant gene modified T cells, test its effectiveness of eradicating CD20+ lymphoma cells and provide a probably new approach to tumor adoptive immunotherapy.

METHODSCD28-zeta cDNA were amplified from vector pBULLET and inserted into pLNCX vector that contained anti-CD20scFv/CD80 gene. The recombinant vectors were transduced into PA317 cells and high titer retroviruses were obtained to infect human peripheral blood T lymphocytes. Resistant T cells were obtained by G418 selection at one week. Then transduced T lymphocytes and lymphoma cell lines Daudi Raji were cocultured. The cytotoxicity and cytokine production of transduced T cells were determined by non-radio-activation cytotoxicity assay and ELISA respectively.

RESULTSThe recombinant eukaryotic vector was constructed successfully as proved by enzyme digestion analysis and sequencing. These T cells were able to lyse CD20+ target cells and secrete high levels of IL-2 and IFN-gamma in vitro.

CONCLUSIONRecombinant gene modified T cells can be constructed successfully. It can specially kill CD20 positive lymphoma cells in vitro.

Antigens, CD20 ; genetics ; immunology ; B7-1 Antigen ; genetics ; immunology ; CD28 Antigens ; genetics ; immunology ; Cell Line ; Cytotoxicity, Immunologic ; Genetic Vectors ; Humans ; Immunotherapy, Adoptive ; Plasmids ; genetics ; T-Lymphocytes ; immunology ; metabolism ; Transfection