Objective To study the immunoadjuvant effects of chitosan oligosaccharide(COS),including the immune activation and the triggering of lysosomal escape,and to explore whether COS can be used as an adjuvant for attenuated live bacteria vector vaccines.Methods 1)Mouse macrophages RAW264.7 cells were cultured with COS at 0 mg/mL(the control group)and 0.1-4 mg/mL for 24 h and the effect on cell viability was measured by CCK8 assay.Mouse macrophages RAW264.7 were treated with COS at 0(the control group),1,2,and 4 mg/mL for 24 h.Then,the mRNA expression levels of the cytokines,including IFN-γ,IL-10,TGF-β,and TLR4,were determined by RT-qPCR assay.2)RAW264.7 cells were treated with 1 mL of PBS containing different components,including calcein at 50 μg/mL,COS at 2 mg/mL,and bafilomycin A1,an inhibitor,at 1 μmol/mL,for culturing.The cells were divided into the Calcein group,Calcein+COS group,and Calcein+COS+Bafilomycin A1 group accordingly.Laser scanning confocal microscopy was used to observe the phagocytosis and the intracellular fluorescence distribution of calcein,a fluorescent dye,in RAW264.7 cells in the presence or absence of COS intervention to determine whether COS was able to trigger lysosomal escape.3)LM?E6E7 and LI?E6E7,the attenuated Listeria vector candidate therapeutic vaccines for cervical cancer,were encapsulated with COS at the mass concentrations of 0.5 mg/mL,1 mg/mL,2 mg/mL,4 mg/mL,and 8 mg/mL.Then,the changes in zeta potential were measured to select the concentration of COS that successfully encapsulated the bacteria.Phagocytosis of the vaccine strains by RAW264.7 cells was measured before and after LM?E6E7 and LI?E6E7 were coated with COS at 2 mg/mL.Results 1)CCK8 assays showed that,compared with the findings for the control group,the intervention of RAW264.7 cells with COS at different concentrations for 24 h was not toxic to the cells and promoted cell proliferation,with the difference being statistically significant(P＜0.05).According to the RT-qPCR results,compared with those of the control group,the COS intervention up-regulated the mRNA levels of TLR4 and IFN-γ in RAW264.7 cells,while it inhibited the mRNA expression levels of TGF-β and IL-10,with the most prominent effect being observed in the 4 mg/mL COS group(P＜0.05).2)Laser scanning confocal microscopy revealed that the amount of fluorescent dye released from lysosomes into the cells was greater in the Calcein+COS group than that in the Calcein group.In other words,a greater amount of fluorescent dye was released from lysosomes into the cells under COS intervention.Furthermore,this process could be blocked by bafilomycin A1.3)The zeta potential results showed that COS could successfully encapsulate the surface of bacteria when its mass concentration reached 2 mg/mL.Before and after the vaccine strain was encapsulated by COS,the phagocytosis of LM?E6E7 by RAW264.7 cells was 5.70%and 22.00%,respectively,showing statistically significant differences(P＜0.05);the phagocytosis of LI?E6E7 by RAW264.7 cells was 1.55%and 6.12%,respectively,showing statistically significant differences(P＜0.05).Conclusion COS has the effect of activating the immune response of macrophages and triggering lysosomal escape.The candidates strains of coated live attenuated bacterial vector vaccines can promote the phagocytosis of bacteria by macrophages.Further research is warranted to develop COS into an adjuvant for bacterial vector vaccine.

OBJECTIVETo explore the immune response of silicotic rats to sheep red blood cells(SRBC).

METHODSSilicotic rats were immunized with SRBC by tracheal instillation(Group 1) or intraperitoneal injection (Group 2), and non-silicotic rats were immunized by tracheal instillation as normal control(Group 3). The levels of serum hemolytic index(HC50) were measured on 7, 12, 20, 25, and 32 days after primary immunization and 5, 12, 15 days after the second immunization. Special anti-SRBC IgG was measured with ELISA(A490 nm) on 12, 20, 25, 32 days and 5, 12, 15, 27 days respectively. Delayed-type hypersensitivity(DTH) to SRBC was measured 20 days after second immunization and DTH reaction was determined at 24, 48, 72, and 96 h after administration. Total cell count and cell populations in the bronchoalveolar lavage fluid(BALF), lung associated lymph node(LALN) and spleen weight, special IgG secreted from spleen cells were measured at the end of the experiment.

RESULTSThe HC50 of Group 1(47.4 +/- 1.0, 52.2 +/- 4.6, 31.1 +/- 11.9, 43.8 +/- 3.5, 33.6 +/- 16.8, 49.0 +/- 2.3, 92.9 +/- 20.2, 87.7 +/- 5.2) were statistically higher than those of Group 3(40.4 +/- 10.6, 2.8 +/- 2.5, 0.8 +/- 0.6, 6.6 +/- 5.8, 1.4 +/- 0.1, 36.5 +/- 16.5, 53.0 +/- 33.2, 2.6 +/- 2.2). The special anti-SRBC IgG response in Group 1(1.67 +/- 0.19, 1.98 +/- 0.36, 1.12 +/- 0.50, 1.38 +/- 0.30, 2.75 +/- 0.15, 2.60 +/- 0.28, 2.86 +/- 0.10, 2.50 +/- 0.20) were much stronger than those in Group 3 (0.59 +/- 0.30, 0.56 +/- 0.21, 0.21 +/- 0.16, 0.22 +/- 0.01, 0.81 +/- 0.25, 0.74 +/- 0.25, 0.69 +/- 0.26, 1.38 +/- 0.41). Furthermore, the results of DTH showed positive response and the ratios for diameter of skin rash > 5 mm at 24, 48, 72, 96 h were 16/16, 16/16, 16/16, 15/16 respectively in Group 1, while those in Group 3 were 8/15, 1/15, 1/15, 1/15 respectively. Total cell count in the BALF, LALN and spleen weight, and special IgG secreted from spleen cells in Group 1 were higher too. Group 2 expressed almost of the same but with mild immunologic responses as Group 1.

CONCLUSIONSilicosis-induced extremely strong DTH and over-response of humoral immunity to some antigens may contribute to the likelihood of silicosis complicated with tuberculosis.

Animals ; Erythrocytes ; immunology ; Hypersensitivity, Delayed ; etiology ; Immunization ; Immunoglobulin G ; blood ; Rats ; Sheep ; Silicosis ; immunology