[Toll-like receptor 4 expression of human peripheral blood mononuclear cells treated by recombinant hIFN-α-2b-BCG and its role of immune activation].

OBJECTIVE: To examine the toll-like receptor 4 (TLR4) expression of human peripheral blood mononuclear cells (hPBMC) treated with recombinant bacillus Calmette-Guérin (rBCG) and its role of immune activation. METHODS: hPBMC was treated with recombinant human interferon (hIFN)-α-2b-BCG (rBCG) or wild BCG (wBCG) in vitro and the TLR4 expression detected by flow cytometry. The TLR4 functional blocking antibodies were applied for intervening the TLR4 signaling pathway of hPBMC. Then rBCG, wBCG, hIFN-α-2b and phosphate-buffered solution (PBS) were used to stimulate the hPBMC of blocking and non-blocking groups. The changes of human tumor necrosis factor-alpha (hTNF-α), human interleukin-12 (hIL-12) and hIFN-γ between the blocking and non-blocking groups by ELISA. RESULTS: The expression of TLR4 in hPBMC treated with rBCG or wBCG were stronger than that treat with PBS (all P < 0.05). In TLR4 non-blocking groups the expressions of hTNF-α and hIFN-γ were rBCG group > wBCG group > hIFN-α-2b group > PBS group, the expressions of hIL-12 was hIFN-α-2b group > PBS group > rBCG group > wBCG group (all P < 0.05). Application of TLR4 functional blocking antibodies to intervene hPBMC 48 h, comparing the changes in rBCG group, wBCG group, hIFN-α-2b group and PBS group, the expressions of hIFN-γ in TLR4 blocking groups (27.3 ± 1.2, 20.6 ± 0.9, 20.3 ± 0.8, 18.4 ± 0.7)were significantly inhibited than those in non-blocking groups (84.6 ± 1.3, 34.0 ± 1.0, 24.9 ± 0.9, 22.9 ± 0.7) (all P < 0.05). The expressions of hTNF-α in TLR4 blocking groups (1431 ± 28, 1032 ± 21, 104 ± 6, 109 ± 4) were significantly inhibited than those in non-blocking groups (1553 ± 28, 1065 ± 31, 343 ± 6, 299 ± 4) (all P < 0.05). The expressions of hIL-12 in TLR4 blocking groups (0.646 ± 0.005, 0.592 ± 0.015, 0.638 ± 0.008, 0.595 ± 0.019) were significantly inhibited than those in non-blocking groups (1.120 ± 0.012, 0.946 ± 0.015, 1.254 ± 0.011, 1.112 ± 0.024) (all P < 0.05). CONCLUSION: rBCG regulates the secretion of Th1 cytokines through the TLR4 signaling pathway.

[TLR4 expression of human PBMC treated by BCG and its role of immune activation].

AIM: To investigate TLR4 expression of human peripheral blood mononuclear cells (hPBMC) treated with BCG and its role of immune activation. METHODS: hPBMC were treated with BCG in vitro. TLR4 expression were detected by flow cytometry, IFN-γ and TNF-α expression of hPBMC in both BCG stimulated group and the control group were detected by ELISA. RESULTS: The expression of TLR4 in hPBMC treated with BCG was stronger than the control group significantly (P<0.01) and increased with the time. In 72 h the TLR4 expression of BCG group was (44.73 ± 0.0066)%, while the control group was (1.02 ± 0.0024)%. BCG can promote hPBMC proliferation, and this enhancement was time-dependent. In 24 h, 48 h and 72 h IFN-γ and TNF-α expression of BCG group were significantly higher than the control group(P<0.05), and this enhancement was time-dependent. CONCLUSION: BCG can on enhance TLR4 expression and promote immune activation of hPBMC.

[Effect of recombinant hIFN-alpha-2b-BCG on mouse bladder tumor MB49 cells in vitro].

OBJECTIVE: To investigate the antitumor effect of recombinant IFN-alpha-2b-BCG on mouse bladder cancer MB49 cells in vitro, and to explore its antitumor mechanisms. METHODS: MB49 cells were co-cultured with recombinant BCG or wild BCG, and than were examined by light and transmission electron microscopy. The cell growth was assessed by MTT assay, and apoptosis rate and MHC-I of the MB49 cells was detected by flow cytometry using AO and Hoechst33258 fluorescence immunostaining. RESULTS: The hIFN-alpha-2b-BCG-treated tumor cells showed slow growth, detachment of some cells, and various degree of degeneration. Light microscopy revealed organelle disorganization, chromatin aggregation, nuclear pyknosis, and cytolysis in some cells. Cellular membrane bulged and some bubbles were seen under fluorescence microscope using AO staining. Hoechst33258 assay also depicted frequent apoptosis in the tumor cells. The MTT assay showed that rBCG more actively than the wild BCG inhibited the proliferation of MB49 cells. The apoptosis rate of the recombinant BCG group was 19.7% and 46.6% at the time point of 24 h and 48 h, respectively, significantly higher than 10.8% and 20.9%, respectively, in the wild BCG group. The results of flow cytometry indicated that both types of BCG enhanced the expression of MHC-I in the MB49 cells, but more effective in the recombinant BCG group. CONCLUSION: The recombinant hIFN-alpha-2b-BCG has more strong immuno-modulatory properties, anti-tumor effect on MB49 cells and induces apparent cytotoxicity in the bladder cancer cells in vitro.