ABSTRACT
To explore the protective immune effect induced by mucosal delivery heparin-binding hemagglutinin (HBHA)-a candidate vaccine antigen of Mycobacterium tuberculosis. Female C57BL/6 mice were between 6 and 8 weeks of age before experimental use. Thirty mice received different immunization strategies and were randomly divided into the control group, the early secreting antigen target-6 (ESAT-6) intranasal immunization group, the HBHA intranasal immunization group, the BCG priming PBS control group, or BCG priming HBHA boost group, 6 mice in each group. In order to analyzed the immune effect, the concentrations of plasma Interleukin-17A (IL-17A) and other cytokines were measured by ELISA. Quantitative real-time PCR analyses were performed to detect the relative quantity (RQ) mRNA of IL-17A in the lung. The lung tissue sections were stained to detect the formation of the tertiary lymphoid structures. The chemokines contributed to formation of the tertiary lymphoid structures were also measured. Flow cytometry was used to detect the frequency of Th1 and Th17 cells in the system. Sixty mice in the BCG priming PBS control group and the BCG priming HBHA boost group were sacrificed at different time points after infection to count the lung bacterial burden. The concentrations of plasma IL-17A and relative quantity of lung IL-17A mRNA were highest in the BCG priming HBHA boost group [(14.76±4.73) pg/mL,RQ (12.27±6.71)], which was significantly higher than the control group [(5.57±2.95) pg/mL,RQ (1.30±0.97)] (t=4.213, P<0.001; t=5.984, P<0.001), and also significantly higher than the BCG priming PBS control group [(6.81±2.18) pg/mL,RQ (1.44±1.16)] (t=3.646 P=0.001; t=6.185 P<0.001). Compared with the BCG priming PBS control group (0.38±0.38)% the frequency of spleen Th17 cells were also significantly increased (t=-0.280 , P=0.048) in the BCG-primary HBHA boost group (1.02±0.34)%. In addition, HBHA boosting could promote better formation of the tertiary lymphoid structures in the lung, and decrease the bacterial load on the early stage after BCG challenge. Collectively, mucosal delivery of HBHA can effectively enhance the protective effect after BCG vaccination, and it is a potential candidate vaccine component.
Subject(s)
Animals , Female , Humans , Mice , Antigens, Bacterial , Bacterial Proteins , Immunization, Secondary , Interleukin-17 , Lectins , Mice, Inbred C57BL , Mycobacterium tuberculosis , Tuberculosis/prevention & control , Tuberculosis VaccinesABSTRACT
<p><b>OBJECTIVE</b>To study the radiobiological effects of fast neutron/photon mixed irradiation on human cancer cell in vitro and to discuss the mechanism in relation with cell cycle and apoptosis, thus to provide experimental support for the further application of fast neutron radiotherapy of cancer.</p><p><b>METHODS</b>Exponentially growing human nasopharyngeal cancer cell line CNE-1 was irradiated in vitro with 35 MeV p-->Be fast neutron and 6 MV-X ray in grading doses (0 cGy, 40 cGy, 80 cGy, 120 cGy, 160 cGy, 240 cGy, 320 cGy and 400 cGy for neutron, and 0 cGy, 100 cGy, 200 cGy, 300 cGy, 400 cGy, 600 cGy, 800 cGy and 1000 cGy for X ray). Clonogenic assay was performed, and relative biological effectiveness (RBE) of fast neutron was determined with D(10) by means of cell survival curves. Isoeffective doses of 35 MeV p-->Be fast neutron and 6 MV-X ray were obtained according to the RBE. The cells were assigned into two irradiation regimens, (1) the one-week-fractionation regimen, which adopted the radiation pattern of X x 5, N x 2 and X-N-X-X-N. After irradiation the clonogenic assay was performed to compare their survival fractions; (2) the two-dose regimen, with the radiation pattern of X + N, N + X and X + X. Flow cytometry was done at different time points after irradiation to analyze cell cycle distribution and apoptosis. Fast neutron dose was delivered on Tuesday and Friday, and all the other irradiation intervals were 24 h.</p><p><b>RESULTS</b>The RBE of fast neutron to X ray in CNE-1 cells according to the D(10) ratio was 2.40. The neutron isoeffective dose for a single dose of 200 cGy of 6 MV-X ray was approximately 80 cGy. In clonogenic assay, the cell survival fractions were significantly lower in X-N-X-X-N group (0.0079) than those in X x 5 (0.018) and N x 2 (0.017) groups. The flow cytometry suggested a higher percentage of apoptotic cells after mixed irradiation, and different sequence of X ray and neutron irradiations caused varying changes in cell cycle arrest.</p><p><b>CONCLUSION</b>Mixed irradiation of fast neutron and X ray showed a synergic effect in vitro on CNE-1 cell killing. Cell cycle arrest and apoptosis may play some role in the radiation damage repair mechanisms of mixed beam irradiation.</p>