Morphological observation of nuclear sub-localization of human angiogenin in HUVECs.

Angiogenin, a potent angiogenic factor, was cloned and expressed by Escherichia coli and then purified with gel filtration chromatography. Approximately 90% pure angiogenin was obtained to generate a monoclonal antibody. Using western immunoblotting and ELISA, we confirmed that monoclonal antibody C46 secreted from hybridoma cells stably and specifically binds to angiogenin. The fused protein angiogenin-EGF was then expressed in HUVECs, and the subcellular localization of the recombinant protein was determined by confocal microscopy and TEM assay. Recombinant angiogenin was found to mainly concentrate in the pars granulosa of the nucleus, where the protein accumulates to form ribonucleoprotein particles.

[Preparation and purification of polyclonal antibody against chymopapain].

OBJECTIVE: To prepare and purify polyclonal antibody against chymopapain, and to make a foundation for establishing an immunossay for chymopapain. METHODS: New Zealand rabbit was immunized with chymopapain. Antiserum was purified by Protein A and analyzed by ELISA. RESULTS: The titer of the antiserum obtained in this experiment by ELISA was up to 1:380000 and the purity was proved to be high by SDS-PAGE.

[Construction and identification of anti-chymopapain scFv phage display library].

AIM: To construct phage display library of anti-chymopapain scFv. METHODS: V(H) and V(L) gene repertoires were amplified from splenocyte mRNA by RT-PCR and joined by a (Gly(4)ser)3 linker to obtain scFv genes. The scFv genes were then cloned into phagemid pFAB5C to construct phage display library. Affinity selection and ELISA were used to identify specific phage antibody to chymopapain. RESULTS: After 4 rounds of panning, high affinity scFv was obtained. CONCLUSION: Phage display library of anti-chymopapain scFv was successfully constructed, and scFv with binding ability to chymopapain was obtained.

[Rapid serum-free culture of dendritic cells from human peripheral blood monocytes and their intracellular signal transduction].

OBJECTIVE: To explore the methods for rapid in vitro culture of the dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) under serum-free conditions and ascertain whether intracellular signal transduction pathway differs between calcium ionophore (CI) and tumor necrosis factor (TNF)- alpha during their induction of dendritic cell differentiation. METHODS: PBMCs isolated from healthy donors were plated in serum-free medium supplemented with 50 ng/ml rhGM-CSF. Cells cultured overnight were induced to differentiate with 100 ng/ml A23187 or 50 ng/ml TNF-alpha, given before or 30 min after pre-treatment with 0.5 mug/ml cyclosporine A (CsA). After culture for 40 h, the cell morphology was observed under phase-contrast microscope, and the surface markers on treated PBMCs were analyzed by flow cytometry. MTT colorimetry was employed to assess the proliferation of the allogeneic T cells. RESULTS: PBMCs of healthy donors treated with 50 ng/ml rhGM-CSF in combination with 100 ng/ml CI or 50 ng/ml TNF-alpha for 40 h exhibited typical morphology of DCs with rapidly decreased CD14 expression and increased expressions of CD83 and co-stimulatory molecules (CD80 and CD86), showing also enhanced ability of stimulating allogeneic T cell proliferation. Calcineurin antagonist CsA inhibited the differentiation induced by CI, but not that induced by TNF-alpha. CONCLUSIONS: Under serum-free conditions, both CI and TNF-alpha are capable of inducing rapid DC differentiation from human PBMCs, but the intracellular signal transduction of CI-induced differentiation is different from that induced by TNF-alpha.

[Signal transduction in the differentiation of human peripheral blood mononucleocytes towards dendritic cells induced by calcium ionophore].

AIM: To explore the intracellular signal transduction pathway in the differentiation of human peripheral blood mononucleocytes (PBMCs) towards dendritic cells (DCs) induced by calcium ionophore (CI). METHODS: PBMCs isolated from a healthy donor were cultured with A23187 plus rhGM-CSF, 100 microg/L each. In some experiments, PBMCs were cultured for 30 minutes with W-7 (10 micromol/L), CsA(0.5 mg/L) or KT5926(1 micromol/L) before addition of rhGM-CSF and A23187. After culture for 40 hours, morphological change of the cells were observed under phase contrast microscope; surface markers on treated PBMCs were analyzed by flow cytometry; the proliferation of allogeneic human T cells stimulated by the treated PBMCs was detected by MTT colorimetry. RESULTS: PBMCs of the healthy donor cocultured with rhGM-CSF plus CI for 40 hours had the typical morphology of DCs, with decreased CD14 expression, and increased CD83, CD80 and CD86 expressions. The proliferation of allogeneic T cells stimulated by PBMCs treated with A23187 plus rhGM-CSF was strengthened. But the morphological changes, surface marker expressions and the ability to enhancing proliferation of allogeneic T cells were inhibited to different degrees by W-7, CsA or KT5926. CONCLUSION: The differentiation of PBMCs towards DCs by CI may be modulated by Ca (2+)/calmodulin and multiple signal transduction pathways downstream.

[The induction of differentiation into dendritic cells from HL-60 cells by calcium ionophore].

AIM: To explore whether the promyelocytic leukemia cell line HL-60 may differentiate into activated dendritic cells (DCs) by A23187, a calcium ionophore. METHODS: The HL-60 cells were cultured in common medium alone or with various concentrations of A23187 (25-1 600 microg/L) and rhGM-CSF (100 microg/L). After culture for 24-96 hours, the cellular morphological change was observed under light microscope and electron microscope. Surface makers on treated HL-60 cells were analyzed by flow cytometry. The proliferation of allogeneic human T cells was detected by MTT colorimetry. RESULTS: Under the condition of a suitable dose (200 microg/L) of A23187 treatment of HL-60 cells for 24 hours, the expression of CD83 molecule, a characteristic marker on DCs, was highest. The typical dendritic outgrowth appeared at a time when HL-60 cells were treated with A23187 for 72 hours. However, when HL-60 cells were treated with A23187 for 96 hours, the expressions of CD80 (B7.1), CD86 (B7.2), MHC-class II molecule and CD54 on HL-60 cells reached peak, and marked activation of allogeneic T cells occurred. CONCLUSION: Calcium ionophore A23187 can induce the HL-60 cells to differentiate into activated DCs-like cells.

[Construction of recombinant vector pEGFP-ANG and expression of angiogenin gene in HUVECs].

AIM: To express the recombinant angiogenin protein in mammalian cells. METHODS: Human ANG full-length cDNA was obtained by chemical synthesis. The target gene ANG was inserted into eukaryotic expression vector pEGFP-C2. The recombinant plasmid was transfected into the human umbilical vein endothelial cells (HUVECs) via Lipofectin transfection. The expression of ANG gene in transfected HUVECs was detected by fluorescence microscopy and immunohistochemical staining. RESULTS: DNA sequencing showed the sequence of the synthetic ANG was correct and amino acid sequence of ANG was right. The green fluorescence could be seen in transfected HUVECs under fluorescence microscope. Immunohistochemical staining detection showed that ANG expressed in transfected cells. CONCLUSION: Human ANG full-length cDNA has been obtained. The ANG protein was expressed in mammalian cells successfully.

[Development of toxin targeting to VEGF-KDR].

OBJECTIVE: To develop toxin targeting vascular endothelial growth factor receptor II (VEGF-II/KDR) fused with a KDR-binling peptide screened from peptide library. METHODS: By affinity to KDR molecular which expressed specifically by new born vascular endothelial cell, peptides to KDR were screened from C7 peptide library by phage display. Among them, a peptide binding to KDR with high affinity termed as P5 was selected and fused to the N-terminal of Shiga toxin subunit A (StxA). The protein (P5-StxA) was expressed in E. coli. RESULTS: ELISA and Western blot were applied to characterize the binding interaction between the fusion protein, P5-StxA and KDR. Cytotoxicity assay showed that P5-StxA maintained similar toxicity to cell as StxA. In the model of angiogenesis, P5-StxA inhibited selectively VEGF-induced growth of preexisting vessels of the chick chorioallantoic membrane. CONCLUSION: These studies demonstrate the small peptide, P5, maybe be used as carrier of toxin targeting to KDR.

[The effect of calcium ionophore on dendritic cells derived from peripheral blood mononuclear cells].

AIM: To explore the effect of calcium ionophore (CI) on dendritic cells (DC) derived from peripheral blood monocytes. METHODS: Peripheral blood monocytes from healthy donors were treated with 100 microg/L rhGM-CSF, 100 microg/L rhGM-CSF plus (10 microg/L) CI, and 100 microg/L rhGM-CSF plus (100 microg/L) respectively. After cultivation of 40 hours, cellular morphology was observed under light microscope and electron microscope. Surface markers on treated PBMCs were analyzed by flow cytometry. MTT colorimetry was used to detect proliferation of autologous T cells. RESULTS: Peripheral blood monocytes treated with 100 microg/L rhGM-CSF plus 100 microg/L CI for 40 hours showed typical morphology of DCs. Simultaneously there was a decrease in CD14 expression and increase in HLA-DR, CD40, CD83 and CD86 expressions on these cells. In addition, PBMCs treated with 100 microg/L rhGM-CSF pass 100 microg/L CI for 40 hrs. Could evidently stimulate proliferation of autologous T cells. CONCLUSION: CI can markedly enhance transformation of peripheral blood monocytes induced by GM-CSF into DCs.

[Dendritic cells transfected with carcinoembryonic antigen-vaccinia recombinant virus induces CEA-specific immunity mediated by cytotoxic T lymphocytes in vitro].

OBJECTIVE: To observe whether dendritic cells (DCs) transfected with carcinoembryonic antigen (CEA)-vaccinia recombinant virus (rV-CEA) induces cytotoxic T lymphocyte-mediated CEA-specific immunity in vitro. METHODS: DCs derived from peripheral blood monocytes were transfected with rV-CEA and then cocultured with autologous T cells. The proliferation of the induced T cells and their cytotoxic activity against CEA-secreting tumor cells were assessed and compared with those of T cells induced by DCs without rV-CEA transfection. RESULTS: T cells induced by DCs transfected with rV-CEA showed specific cytotoxicity against CEA-secreting tumor cells. CONCLUSION: DCs transfected with rV-CEA can elicit activation of CEA-specific T cells.