Enhanced anti-tumor immunity ex vivo induced by GM-CSF gene transducted dendritic cell vaccine

Objective： The aim of the study was to investigate whether dendritic cell （DC） precursors, recruited by injection of chemokine ligand 3 （CCL3）, induce enhanced anti-tumor immunity after granulocyte-macrophage colony stimulating factor （GM-CSF） transfection in mice ex vivo. Methods： The 615 mice were injected with CCL3 via the tail vein. Freshly isolated B220–CD11c＋ cells were cultured with cytokines. For adenoviral （Ad）-mediated gene transduction, DCs were transferred AdGM-CSF gene at different ratios of multiplicity of infection （MOI） to determine the optimal gene transfection conditions, and detecting the expression of GM-CSF after transfection. The variation of GM-CSF gene-modified DCs were analyzed by morphological observation, phenotype analysis, and mixed lymphocyte reaction （MLR）. DCs were loaded with gastric cancer antigen obtained by frozen and thawed method. The stimulated DCs vaccination induced T lymphocytes, and the killing effect of T cells to gastric cancer cells was assayed by MTT. INF-γ production was determined with the INF-γ ELISA kit. Results： B220–CD11c＋ cells numbers increased after CCL3 injection. ELISA results showed that after GM-CSF gene modification, DC could produce high level of GM-CSF. When DCs were transferred AdGM-CSF gene at MOI equal to 1：100, GM-CSF level in culture supernatants reached saturation [（130.00 ± 12.61） pg/mL]. After GM-CSF gene-modification, DCs tended to more maturated through morphological observation and were phenotypically identical to typical DC and gained the capacity to stimulate allogeneic T cells. T lymphocytes stimulated with DC transduced with GM-CSF gene showed the specific killing effect on gastric carcinoma cells and produced high level of INF-γ [（1245.00 ± 13.75） pg/mL]. Conclusion： CCL3-recruited DCs modified by adenovirus-transducted GM-CSF could produce high level of GM-CSF, which tended to more maturated, and the capacity of activating allogeneic T lymphocytes proliferation was enhanced greatly. Moreover, they could stimulate specific cytotoxic T lymphocyte （CTL） to gastric cancer ex vivo.

The molecular mechanisms of Yang Wei Kang Liu powder on anticancer and reducing chemotherapy side-effect in combination with chemotherapy

Objective:We studied the molecular mechanisms of Yang Wei Kang Liu Power(YWKL,traditional Chinese medicine for nourishing stomach and anticancer) on anticancer and reducing chemotherapy side-effect in combination with chemotherapy.Methods:615 pre-cancer mouse model of YWKL for 10 days and CTX 1 time,semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR) to detect bone marrow granulocyte-macrophage colony-stimulating factor(GM-CSF) gene and cancer proto-oncogene Bcl-2,c-myc expression.Results:YWKL in combination with chemotherapy could obviously promoted the expression of GM-CSF gene and inhibited the expression of Bcl-2 and c-myc oncogenes of FC 615 mice.Conclusion:The molecular mechanisms of anticancer and reducing chemotherapy side-effect of YWKL in combination with chemotherapy are to promote the expression of GM-CSF gene and inhibit the expression of Bcl-2 and c-myc oncogenes.

Analysis of the GM-CSF and GM-CSF/IL-3/IL-5 receptor common beta chain in a patient with pulmonary alveolar proteinosis

Objective To investigate the expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and GM-CSF/IL-3/IL-5 receptor common beta chain (βc receptor) in an adult patient with idiopathic pulmonary alveolar proteinosis (PAP), so as to demonstrate the possible association of the GM-CSF and βc receptor with the pathogenesis of human PAP.Methods The GM-CSF levels were measured with a commercial ELISA kit (sensitivity 5?pg/ml) and the βc receptor expression on the cell surface was detected by flow cytometry analysis. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was employed to detect the expression of the GM-CSF mRNA and the βc receptor mRNA in peripheral blood mononuclear cells and alveolar macrophages. The entire coding regions of the GM-CSF cDNA and the βc receptor cDNA were sequenced by the Sanger dideoxy-mediated chain termination method to detect possible mutations.Results The patient with PAP failed to release the GM-CSF protein either from circulating mononuclear cells or from alveolar macrophages. The expression of the GM-CSF mRNA was normal after the stimulation of lipopolysaccharide, whereas a point mutation at position 382 of the GM-CSF cDNA from 'T' to 'C' was revealed by cDNA sequencing, which caused a change in amino acid 117 of the protein from isoleucine to threonine. The βc receptor expression on the cell surface was normal, and the βc receptor mRNA expression and the sequence of the entire coding region of the βc receptor were also normal.Conclusions The decreased GM-CSF production is associated with the pathogenesis of human PAP. A point mutation of the GM-CSF cDNA may contribute to the decreased GM-CSF production in our adult PAP patient. The mutation of the βc receptor in some of paediatric patients with PAP may not be a common problem in adult patients.