1.Anti-cD20scFv/CD80/CD28/zeta specific T lymphocytes eradicate primary chronic lymphocytic leukemia cells in vitro.
Hong-Lan QIAN ; Kang YU ; Zhi-Jian SHEN ; Bin LIANG ; Sheng LUO ; Chong-Yun XING ; Yong-Xian HU
Chinese Journal of Applied Physiology 2010;26(4):436-439
OBJECTIVETo construct anti-CD20scFv/CD80/CD28/zeta recombinant gene modified T cells, test its effectiveness of eradicating CD20 positive primary chronic lymphocytic leukemia (CLL) cells and provide a promising tool for tumor adoptive immunotherapy.
METHODSThe recombinant vectors were transduced into PA 317 cells and high titer retroviruses were obtained to infect human peripheral blood T lymphocytes. Resistant T cells were obtained by G418 selection for one week. Then transduced T lymphocytes and primary CLL cells were co-cultured. The status of primary chronic lymphocytic leukemia cells were observed by microscope. The level of IL-2 and IFN-gamma in the culture medium were measured.
RESULTSPrimary T cells expressing anti-CD20scFv/IgGFc/CD80/CD28/zeta could be constructed successfully. These T cells were able to lyse CD20+ targets and secrete high levels of IL-2 (1301.00 pg/ml) and IFN-gamma (602.18 pg/ml) in vitro.
CONCLUSION(1) Recombinant gene modified T cells can be constructed successfully. (2) Recombinant gene modified T cells can specially kill CD20 positive primary CLL cells in vitro.
Antigens, CD20 ; genetics ; B7-1 Antigen ; genetics ; CD28 Antigens ; genetics ; Genetic Vectors ; Humans ; Immunotherapy, Adoptive ; Interferon-gamma ; secretion ; Interleukin-2 ; secretion ; Leukemia, Lymphocytic, Chronic, B-Cell ; pathology ; Retroviridae ; genetics ; T-Lymphocytes ; immunology ; secretion ; Transfection ; Tumor Cells, Cultured
2.Enhancement of antigen presenting function of dendritic cells by IL-2 gene modification and its mechanism.
Lifei SUN ; Jiang LIU ; Xuetao CAO ; Minghui ZHANG ; Yihong ZHOU ; Bin LIU ; Hua SHI
Chinese Journal of Hematology 2002;23(5):247-250
OBJECTIVETo investigate the effects of IL-2 gene modification enhancement of the antigen-presenting function of the mouse bone marrow derived dendritic cells and on the activation of CTL induced by MHC class I molecule restricted antigen peptides as well as the related immunological mechanisms.
METHODSDCs were prepared from mouse bone marrow and modified by recombinant IL-2 adenovirus (DC-IL-2). The IL-12 and IFN-gamma levels in culture supernatant of DC and CTL were examined by ELISA, the expression of costimulatory molecules and fluorescent intensity of endocytosis of OVA-FITC in DC by FACS, the capacity of presenting 3LL cell tumor antigen by (3)H-TdR incorporation method, the MHC class I-restricted tumor-antigen-peptide Mut1 of 3LL cells pulsed DC-IL-2 to induce CTL cytotoxicity by (51)Cr 4-hr releasing assay.
RESULTSAfter IL-2 gene modification, DC-IL-2 could produce high level of IL-12 [(78.4 +/- 6.6) pg.(1 x 10(6) cells)(-1).ml(-1)]. The expression of costimulatory molecules on DC-IL-2 was increased, the fluorescent intensity of DC captured OVA-FITC was enhanced, and the proliferation of allo-T cells from 3LL bearing mouse pulsed with Mut1 was also enhanced. Mut1 antigen peptide pulsed DC-IL-2 could induce more potent antigen-specific CTL cytotoxicity and excrete high concentration of IFN-gamma [(1 168 +/- 58.4) pg/ml] in vivo.
CONCLUSIONIL-2 gene modification of DC can activate second signal for DC presenting antigen, and enhance the function for capturing and presenting tumor antigen. DC-IL-2 pulsed with MHC class I restricted tumor-antigen-peptide can induce specific anti-tumor immune response more effectively. Owing to IL-2 gene modification, the functions of IL-12 excretion and T cell activation of DC were promoted, so that the capacity of CTL excreting IFN-gamma was enhanced, which are relevant to the immune mechanism.
Adenoviridae ; genetics ; Animals ; Antigen Presentation ; immunology ; B7-1 Antigen ; genetics ; metabolism ; Dendritic Cells ; cytology ; immunology ; Female ; Interleukin-12 ; secretion ; Interleukin-2 ; genetics ; Lymphocyte Activation ; genetics ; immunology ; Mice ; Mice, Inbred C57BL ; Recombination, Genetic ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; cytology ; immunology
3.Upregulation of Proinflammatory Cytokines in the Fetal Brain of the Gaucher Mouse.
Young Bin HONG ; Eun Young KIM ; Sung Chul JUNG
Journal of Korean Medical Science 2006;21(4):733-738
Gaucher disease is caused by a deficiency of glucocerebrosidase. Patients with Gaucher disease are divided into three major phenotypes: chronic nonneuronopathic, acute neuronopathic, and chronic neuronopathic, based on symptoms of the nervous system, the severity of symptoms, and the age of disease onset. The characteristics of patients with acute neuronopathic- and chronic neuronopathic-type Gaucher disease include oculomotor abnormalities, bulbar signs, limb rigidity, seizures and occasional choreoathetoid movements, and neuronal loss. However, the mechanisms leading to the neurodegeneration of this disorder remain unknown. To investigate brain dysfunction in Gaucher disease, we studied the possible role of inflammation in neurodegeneration during development of Gaucher disease in a mouse model. Elevated levels of the proinflammatory cytokines, IL-1alpha, IL-1beta, IL-6, and TNF-alpha, were detected in the fetal brains of Gaucher mice. Moreover, the levels of secreted nitric oxide and reactive oxygen species in the brains of Gaucher mice were higher than in wild-type mice. Thus, accumulated glucocerebroside or glucosylsphingosine, caused by glucocerebrosidase deficiency, may mediate brain inflammation in the Gaucher mouse via the elevation of proinflammatory cytokines, nitric oxide, and reactive oxygen species.
Up-Regulation/genetics
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Tumor Necrosis Factor-alpha/genetics/secretion
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Reverse Transcriptase Polymerase Chain Reaction
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Reactive Oxygen Species/metabolism
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RNA, Messenger/genetics/metabolism
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Nitric Oxide/metabolism
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Microglia/cytology/metabolism
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Mice, Knockout
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Mice, Inbred ICR
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Mice, Inbred C57BL
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Mice
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Interleukin-6/genetics/secretion
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Interleukin-1/genetics/secretion
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Inflammation/immunology
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Glucosylceramidase/genetics
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Gaucher Disease/*genetics/metabolism/pathology
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Cytokines/*genetics/immunology/secretion
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Cells, Cultured
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Brain/embryology/*metabolism/pathology
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Animals
4.Effects of HIV-1 tat on secretion of TNF-α and IL-1β by U87 cells in AIDS patients with or without AIDS dementia complex.
Li ZHAO ; Shuang Shuang PU ; Wen Hua GAO ; Yuan Yuan CHI ; Hong Ling WEN ; Zhi Yu WANG ; Yan Yan SONG ; Xue Jie YU ;
Biomedical and Environmental Sciences 2014;27(2):111-117
OBJECTIVETo explore the role of HIV-1 tat gene variations in AIDS dementia complex (ADC) pathogenesis.
METHODSHIV-1 tat genes derived from peripheral spleen and central basal ganglia of an AIDS patient with ADC and an AIDS patient without ADC were cloned for sequence analysis. HIV-1 tat gene sequence alignment was performed by using CLUSTAL W and the phylogentic analysis was conducted by using Neighbor-joining with MEGA4 software. All tat genes were used to construct recombinant retroviral expressing vector MSCV-IRES-GFP/tat. The MSCV-IRES-GFP/tat was cotransfected into 293T cells with pCMV-VSV-G and pUMVC vectors to assemble the recombinant retrovirus. After infection of gliomas U87 cells with equal amount of the recombinant retrovirus, TNF-α, and IL-1β concentrations in the supernatant of U87 cells were determined with ELISA.
RESULTSHIV-1 tat genes derived from peripheral spleen and central basal ganglia of the AIDS patient with ADC and the other one without ADC exhibited genetic variations. Tat variations and amino acid mutation sites existed mainly at Tat protein core functional area (38-47aa). All Tat proteins could induce U87 cells to produce TNF-α and IL-1β, but the level of IL-1β production was different among Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen. The level of Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen were obviously higher than that from the non-ADC patient's basal ganglia.
CONCLUSIONTat protein core functional area (38-47aa) may serve as the key area of enhancing the secretion of IL-1β. This may be related with the neurotoxicity of HIV-1 Tat.
AIDS Dementia Complex ; metabolism ; pathology ; virology ; Adult ; Amino Acid Sequence ; Basal Ganglia ; virology ; Cell Line, Tumor ; Gene Expression Regulation, Viral ; Genes, tat ; HIV-1 ; genetics ; pathogenicity ; Humans ; Interleukin-1beta ; biosynthesis ; genetics ; secretion ; Middle Aged ; Molecular Sequence Data ; Neuroglia ; pathology ; secretion ; Spleen ; virology ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics ; secretion ; tat Gene Products, Human Immunodeficiency Virus ; genetics ; physiology
5.Construction of pEgr-TNFalpha plasmid and experimental study on the effect of gene-radiotherapy on mouse melanoma.
Chinese Journal of Oncology 2004;26(3):143-145
OBJECTIVEThe pEgr-TNFalpha plasmid was constructed to investigate the effect of gene-radiotherapy on melanoma and host immune system.
METHODSpEgr-TNFalpha plasmids were constructed and injected into tumor tissue, 36 hours later, the tumors were given 20 Gy X-ray irradiation. Tumor growth at different timepoints was record and immunologic parameters were detected 15 days later.
RESULTSFrom 3 to 15 d after pEgr-TNFalpha gene-radiotherapy the tumor growth was significantly slower than irradiation or genetherapy alone. NK activity, IL-2, TNFalpha and IL-1beta secretion activities of pEgr-TNFalpha gene-radiotherapy group and pEgr-TNFalpha gene group were higher than those of irradiation alone group significantly.
CONCLUSIONThe anti-tumor effect of pEgr-TNFalpha gene-radiotherapy is better than that of either one applied solely, and it can alleviate the lesion caused by radiation therapy.
Animals ; Combined Modality Therapy ; DNA-Binding Proteins ; genetics ; Early Growth Response Protein 1 ; Female ; Genetic Therapy ; Immediate-Early Proteins ; genetics ; Interleukin-2 ; secretion ; Killer Cells, Natural ; immunology ; Melanoma, Experimental ; immunology ; therapy ; Mice ; Plasmids ; Transcription Factors ; genetics ; Tumor Necrosis Factor-alpha ; genetics
6.Preparation of renal cancer vaccine of IL-12-anchored exosomes and its antitumor effect in vitro.
Yao ZHANG ; Xiao-hou WU ; Gang CHEN ; Chun-li LUO ; Jia-mo ZHANG
Chinese Journal of Oncology 2010;32(5):339-343
OBJECTIVETo prepare a vaccine of IL-12-anchored exosomes derived from renal cancer cells and to evaluate its antitumor effect in vitro.
METHODSA mammalian co-expression plasmid of glycolipid-anchor-IL-12 (GPI-IL-12) was constructed by subcloning IL-12A chain gene (P35 subunit) and a fusion gene containing GPI-anchor signal sequence and IL-12B chain gene (P40 subunit) in pBudCE4.1. Confocal laser scanning microscopy and flow cytometry were used to analyze the expression of the fusion proteins. Transmission electron microscopy and Western blot were used to identify the morphology and characteristic molecules of exosomes separated by ultrafiltration and sucrose gradient centrifugation. The function of IL-12-anchored exosomes was determined by IFN-gamma release assay.
RESULTSMammalian co-expression plasmids were successfully constructed. Confocal laser scanning microscopy and flow cytometric analysis of the RC-2-GPI-IL-12 transfectants showed the expression of IL-12 on the cell surface. Exosomes were purified by ultrafiltration and sucrose gradient centrifugation, which were 30-80 nm in diameter, typically saucer-shaped, and expressing HSP70, ICAM-1, G250 and GPI-IL-12. (80.0 +/- 9.6) pg/ml of IL-12 was detected in 10 microg/ml exosomes and it significantly induced the release of IFN-gamma. Stimulation with EXO-IL-12 could efficiently induce antigen-specific cytotoxic T lymphocytes (CTL), resulting in more significant cytotoxic effects in vitro.
CONCLUSIONA vaccine of exosomes-GPI-IL-12 can be obtained from the culture supernatant of renal cancer cells modified to express anchored IL-12. This vaccine expressing IL-12 and tumor associated antigen G250 may become a new strategy for the treatment of renal cancer.
Antigens, Neoplasm ; metabolism ; Cancer Vaccines ; immunology ; Carbonic Anhydrase IX ; Carbonic Anhydrases ; metabolism ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Exosomes ; genetics ; metabolism ; Glycosylphosphatidylinositols ; genetics ; metabolism ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Interferon-gamma ; secretion ; Interleukin-12 ; genetics ; metabolism ; Kidney Neoplasms ; metabolism ; pathology ; Plasmids ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Transfection
7.Effects of traditional tibetan medicine, Fructus Lonicerae microphyllae on phagecytosis and cytokines production of murine macrophages.
Ju-Le WANG ; Yang SUN ; Hui-Ying ZHOU ; Qiang XU ; Zhu DUN
China Journal of Chinese Materia Medica 2006;31(2):145-148
OBJECTIVETo explore the effects of traditional Tibetan medicine, Fructus Lonicerae microphyllae (FLM) on phagecytosis and cytokines production of murine macrophages.
METHODThe phagecytosis of murine macrophages was analyzed by neutral red phagecytosis assay. The activities of IL-1 and TNF-alpha were measured by biological methods. The mRNA of TNF-alpha and INF-gamma expressed by macrophages was detected by RT-PCR.
RESULTThe phagecytosis of murine macrophages was significantly enhanced by FLM at a concentration from 1 microg x mL(-1) to 100 microg x mL(-1) and the secretions of IL-1, and TNF-alpha from macrophages were markedly induced by FLM. Meanwhile, FLM also increased the expression of TNF-alpha mRNA and INF-gamma mRHA from macrophages in vitro.
CONCLUSIONFLM could promote phagecytosis and cytokines production of murine macrophages.
Animals ; Cell Line ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Female ; Fibroblasts ; cytology ; Fruit ; chemistry ; Interferon-gamma ; biosynthesis ; genetics ; Interleukin-1 ; secretion ; Lonicera ; chemistry ; Macrophages, Peritoneal ; metabolism ; physiology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Phagocytosis ; drug effects ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics
8.In vivo ligation of glucocorticoid-induced TNF receptor enhances the T-cell immunity to herpes simplex virus type 1.
Soojin LA ; Eunhwa KIM ; Byungsuk KWON
Experimental & Molecular Medicine 2005;37(3):193-198
GITR (glucocorticoid-induced TNF receptor) is a recently identified member of the TNF receptor superfamily. The receptor is preferentially expressed on CD4+CD25+ regulatory T cells and GITR signals break the suppressive activity of the subset. In this study, we wanted to reveal the in vivo function of GITR in herpes simplex virus type 1 (HSV-1) infection. A single injection of anti-GITR mAb (DTA-1) immediately after viral infection significantly increased the number of CD4+ and CD8+ T cells expressing CD25, an activation surface marker, and secreting IFN-gamma. We confirmed these in vivo observations by showing ex vivo that re-stimulation of CD4+ or CD8+ T cells with a CD4+ or CD8+ T-cell-specific HSV-1 peptide, respectively, induced a significant elevation in cell proliferation and in IFN-gamma secretion. Our results indicate that GITR signals play a critical role in the T-cell immunity to HSV-1.
Animals
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Antibodies, Monoclonal/pharmacology
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CD4-Positive T-Lymphocytes/immunology
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CD8-Positive T-Lymphocytes/immunology
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Cell Proliferation
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Female
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Glucocorticoids/*pharmacology
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Herpes Simplex/*immunology
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Herpesvirus 1, Human/pathogenicity
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*Immunity, Cellular
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Interferon Type II/secretion
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*Lymphocyte Activation
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Mice
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Mice, Inbred BALB C
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Peptide Fragments/metabolism
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Receptors, Interleukin-2/metabolism
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Receptors, Nerve Growth Factor/genetics/immunology/*metabolism
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Receptors, Tumor Necrosis Factor/genetics/immunology/*metabolism
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Research Support, Non-U.S. Gov't
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T-Lymphocytes/*immunology/metabolism/virology