ANTIGEN ASSOCIATION OF J6-1 CELL MEMBRANE ASSOCIATEDFACTOR RECEPTOR WITH MACROPHAGE COLONYSTIMULATING FACTOR RECEPTOR

Objective: To verify the antigen association of MAF-J6-1 receptor with M-CSFR and to further study the role of M-CSF and its receptor mediated juxtacrine in promoting leukemic cell proliferation. Methods: Monoclonal antibody (McAb) of MAF-J6-1R RE2 and polyclonal antibody (PolyAb) of rhM-CSFR were prepared. The specificity of McAb RE2 to M-CSFR was confirmed by indirect ELISA, cross-neutralizing assay with J6-1 cell colony formation and neutralization test by ELISA. Results: the reactive activity of purified RE2 to M-CSFR was over 1: 16000. The inhibitory activity of M-CSFR and MAF-J6-1R could be blocked by RE2 and anti-M-CSFR antibody. The reactivity of RE2 to M-CSFR could be reduced by M-CSFR. Conclusion: The specificity of RE2 to M-CSFR was confirmed and the antigen association of MAF-J6-1R with M-CSFR was proved. It suggests that M-CSF and its receptor mediated auto-juxtacrine stimulation could be an operative mechanism in either leukemia or nonhematological malignancies.

ENHANCEMENT OF NIH3T3 CELL PROLIFERATION BY EXPRESSING MACROPHAGE COLONY STIMULATING FACTOR IN NUCLEI

Objective: To explore the effects of nuclear M-CSF on the process of tumorigenesis. Methods: Functional part of M-CSF cDNA was inserted into an eukaryotic expression plasmid pCMV/myc/nuc, which can add three NLS to the C-terminal of the expressed protein and direct the protein into the cell nuclei. The constructed plasmid was transferred into NIH3T3 cells and the cell clones were selected by G-418 selection. Cell clones stable expressing target protein were identified by RT-PCR, ABC immunohistochemistry assayand Western blot. Cell growth kinetics analyses throughgrowth curves, cell doubling time, MTT test and anti-sense oligodeoxynucleotide (ASODN) inhibiting cell growth testwere performed to identify cells proliferation potential.Results: The transfected cells showed elevated proliferation potential over the control cells. Conclusion: Abnormalappearance of M-CSF in nucleus could enhance cellproliferation, which suggests that cytokine isoforms within cell nucleus might play transcription factor-like role.

A pilot study on the combined therapy of granulocyte-macrophage colony-stimulating factor and hepatitis B vaccine on chronic hepatitis B virus carrier children

Objective To observe the efficacy of treating intrauterine infected chronic hepatitis B virus (HBV) carrier children with a combination of granulocyte macrophage colony stimulating factor (GM CSF) or hepatitis B immunoglobulin (HBIG) plus recombinant hepatitis B vaccine (rHBvac) Methods A total of 27 chronic HBV infected children, who were born to HBV carrier mothers and received hepatitis B immunoprophylaxis at birth, were randomized into 2 groups: one receiving a combined therapy of 50 μg of GM CSF plus 10 μg of rHBvac injected intramuscularly at the same location (GM CSF group, 14 children) or 200 IU HBIG and 10 μg rHBvac in different muscles (HBIG group, 13 children) on a monthly four dose schedule HBV DNA quantification and other HBV serological markers were tested before and after the four dose therapy Results Twelve children in each group completed the study Of them, 3 children in the GM CSF group and 4 in the HBIG group had elevated serum alanine transaminase (ALT) before the trial, and then 2 in each group became ALT normal after the treatment Before the therapy, hepatitis B e antigen (HBeAg) positivity was found in nine children in the GM CSF group and 10 in the HBIG group One from each group had an HBeAg/anti HBe seroconversion after the treatment The quantity of HBV DNA was significantly lower after the treatment ( P =0 023) in GM CSF group, but was not significantly reduced in HBIG group No subjects were found to be negative for hepatitis B surface antigen (HBsAg) after the treatment, and no serious adverse events occurred in either group Conclusion Combined GM CSF and rHBvac therapy inhibit HBV replication in carrier children who were not protected after treatment with immunoprophylaxis

Immune Responses of Dendritic Cells Loaded with Antigens from Apoptotic Cholangiocarcinoma Cells Caused by Y-Irradation

Objective To investigate the induction cytotoxic T cells (CTLs) with antitumor activity and therapeutic efficacy after dendritic cells(DCs) acquired antigen from apoptotic cholangiocarcinoma cells caused by y-irradiation.Methods DCs from peripheral blood mononuclear cells (PBMC) that maintain the antigen capturing and processing capacity charateristicof immature cells have been established in vitro, using granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Then, in cholangiocarcinoma cells apoptosis was induced by J-irradiation. The experimental groups were as follows: (1) coculture ofDCs and apoptotic cancer cells and T cells; (2) coculture of DCs and necrotic cancer cells and T cells; (3) coculture of DCs, culturedcancer cell and T cells. They are cocultured for 7 days. DCs and T cells were riched, isolated and their antitumor response was tested.Results The cells had typical dendritic morphology, expressed high levels of GDI a and B7, acquired antigen from apoptotic cells causedby y-irradiation and induced an increased T cell stimulatory capacity in mixed lymphocyte reactions (MLR) .Conclusion DCs obtained from PBMCs using GM-CSF and IL-4 can efficiently present antigen derived from apoptotic cells caused by y-irradiation and efficiently induce T cells. This strategy, therefore, may present an effective approach to transduce DCs with antigen.

CONSTRUCTION AND EXPRESSION OF THE REPLICATION-DEFI-CIENT ADENONIRUS VECTOR OF HUMAN GM-CSF

The full-length cDNA encoding human Granulocyte macrophage colony stimulating factor (GM CSF) was cloned by RT PCR, placed under the control of CMV promoter, and inserted into adenovirus vector of E1 substitution type, pAx1cw. Subsequently, the cassette cosmid was cotransfected into 293 cells together with EcoT22I digested Ad5 TPC, and the replication deficient recombinant adenoviruses(Ad) of human GM CSF were generated efficiently by homologous recombination, with the titers of 1.51×10 9pfu/ml. 48 hours after infection with prepared human GM CSF recombinant adenoviruses in vitro, HeLa cells and primary human skin fibroblasts expressed high levels of human GM CSF (80～400ng/ 10 6cells/24hr). These suggest that the recombinant Ad of human GM CSF prepared by COS/TPC method is effective in mediating GM CSF gene transfer and might be used in cancer gene therapy.

IP10, KC and M-CSF Are Remarkably Increased in the Brains from the Various Strains of Experimental Mice Infected with Different Scrapie Agents

Activation of inflammatory cells and upregulations of a number of cytokines in the central nervous system(CNS)of patients with prion diseases are frequently observed.To evaluate the potential changes of some brain cytokines that were rarely addressed during prion infection,the levels of 17 different cytokines in the brain homogenates of mice infected with different scrapie mouse-adapted agents were firstly screened with Luminex assay.Significant upregulations of interferon gamma-induced protein 10(IP10),keratinocyte chemoattractant(KC)and macrophage colony stimulating factor(M-CSF)were frequently detected in the brain lysates of many strains of scrapie infected mice.The upregulations of those three cytokines in the brains of scrapie infected mice were further validated by the individual specific ELISA and immunohistochemical assay.Increased specific mRNAs of IP10,M-CSF and KC in the brains of scrapie infected mice were also detected by the individual specific qRT-PCRs and IP10-specific digital PCR.Dynamic analyses of the brain samples collected at different time points post infection revealed the time-dependent increases of those three cytokines,particularly IP10 during the incubation period of scrapie infection.In addition,we also found that the levels of IP10 in cerebral spinalfluid(CSF)of 45 sporadic Creutzfeldt–Jakob disease(sCJD)patients were slightly but significantly higher than those of the cases who were excluded the diagnosis of prion diseases.These data give us a better understanding of inflammatory reaction during prion infection and progression of prion disease.

Characteristics of ovarian cancer cells transduced by the bicistronic retroviral vector containing GM-CSF and HSV-TK genes

Objectives To explore whether HSV-TK (herpes simplex virus thymidine kinase) and GM-CSF (granulocyte-macrophage colony-stimulating factor) genes could be linked by internal ribosome entry site (IRES) in one retroviral vector and expressed by ovarian cancer cells following transfection, and to observe the characteristics of the transduced cells.Methods Retroviral vector pLGM-I-TK was constructed by linking the HSV-TK gene and GM-CSF gene with the IRES sequence. By using the “ping-pong' technique, pLGM-I-TK was transfected into the packaging cell line, PA317, to produce a PA317/TK-GM cell line. Using the resulting viral supernatant to infect the ovarian cancer cell line SKOV3, PCR and RT-PCR were used to explore the integration and transcription of HSV-TK and GM-CSF genes. The cytotoxicity of GCV (gancyclovir) on SKOV3/TK-GM was determined by MTT assay and the bystander effect of the HSV-TK/GCV system was also assessed. ELISA was used to measure the expression of GM-CSF by the transgene cells.Results The bicistronic retroviral vector constructed could be successfully transduced into PA317 and the titer of the retroviral vector was about 8.6×105?cfu/ml. PCR and RT-PCR demonstrated the successful integration and transcription of HSV-TK and GM-CSF genes transduced into the SKOV3 cell. SKOV3/TK-GM cells could be killed by GCV, and the IC50 was 0.7?μg/ml. The bystander effect was demonstrated. The expression level of GM-CSF in SKOV3/TK-GM was 60.4?ng*ml-1*106 cells-1*2 days-1.Conclusion The IRES sequence can be used to construct retroviral vectors to facilitate co-transfection of two genes. SKOV3/TK-GM cells can simultaneously express the HSV-TK and GM-CSF genes with biological activities which could be useful for enhancing the function of immune cells on the basis of suicide gene therapy.