Figures & data
Figure 1 The effects of LPPC on the antigen uptake ability of P338D1 cells. The fluorescent protein (BSA-FITC) was absorbed on LPPC as a reporter antigen and was incubated with P338D1 cells. With (open bar) or without (closed bar) the addition of trypan blue, the fluorescence was analyzed using FACS. The cells with a fluorescence higher than that of the untreated cells were counted as positive cells. The fluorescent rate is presented as follows: (the number of positive cells divided by the number of untreated cells) multiplied by 100%.
Abbreviations: BSA, bovine serum albumin; FITC, fluorescein Isothiocyanate; LPPC, liposome-polyethylene glycol (PEG)-polyethyleneimine (PEI) complex.
Figure 2 The surface maker expression of mouse phagocytes after LPPC treatment. The surface marker expression of P338D1 cells was analyzed by FACS following the incubation of BSA antigen alone or BSA antigen in combination with LPPC. The fold of fluorescence mean was evaluated using the following equation: the mean fluorescence intensity of the sample divided by the mean fluorescence intensity of the untreated group.
Note: A significant difference between BSA antigen in combination with LPPC and the BSA alone group is indicated by *P < 0.05 and **P < 0.01.
Abbreviations: MHC, major histocompatibility complex; PBS, phosphate-buffered saline; BSA, bovine serum albumin, LPPC, liposome-polyethylene glycol (PEG)-polyethyleneimine (PEI) complex.
Figure 3 The effects of LPPC on the presentation efficiency of mouse antigen presenting cells. After incubation of splenocytes from naive mice (solid columns) or BSA-immunized mice (open columns) with LPPC complex, the concentration of the cytokines (A) interleukin (IL)-2, (B) IL-4, (C) interferon-gamma (IFN-γ), and (D) IL-10 in the supernatants was determined by ELISA.
Notes: Data are expressed as the mean ± standard deviation, and experiments were carried out in duplicate (n = 6). A significant difference compared to the BSA group is indicated by *P < 0.05 and **P < 0.01.
Abbreviations: PBS, phosphate-buffered saline; BSA, bovine serum albumin; LPPC, liposome-polyethylene glycol (PEG)-polyethyleneimine (PEI) complex.
Table 1 The effects of LPPC on the expressions of cytokines in PBMCs or mouse splenocytes
Figure 4 The adjuvant effects of LPPC on the production of antibody in vivo. (A) The total titers of anti-HpHsp60 antibody were determined every 3 days starting 21 days after immunization and (B) the antibody isotypes (IgM, IgG1, IgG2a, IgG2b, and IgA) were determined 21 days after immunization.
Notes: Data are expressed as the mean ± SD (n = 4). A significant difference compared to the CFA+IFA/Hsp60 group is indicated by *P < 0.05.
Abbreviations: CFA, complete Freund’s adjuvant; IFA, incomplete Freund’s adjuvant; LPPC, liposome-polyethylene glycol (PEG)-polyethyleneimine (PEI) complex; PBS, phosphate-buffered saline; Ig, immunoglobulin.
Figure 5 The adjuvant effects of LPPC on cytokine release ex vivo. Following different treatments, splenocytes were harvested from mice, were incubated with HpHsp60 and supernatants were analyzed for expression of (A) IL-2, (B) IFN-γ, (C) TNF-α, (D) IL-4, (E) IL-10, and (F) IL-1β cytokines.
Notes: *P < 0.05; **P < 0.01.
Abbreviations: CFA, complete Freund’s adjuvant; IFA, incomplete Freund’s adjuvant; IFN-γ, interferon-gamma; IL, interleukin; LPPC, liposome-polyethylene glycol (PEG)-polyethyleneimine (PEI) complex; PBS, phosphate-buffered saline; TNF-α, tumor necrosis factor-alpha.
Figure 6 The adjuvant effects of LPPC on the differentiation of T cells in vivo. The numbers of (A) interferon-gamma (IFN)-γ or (B) interleukin (IL)-4-expressing T cells responding to HpHsp60 in treated mice were evaluated by ELISPOT kit. The spots were observed, and the data were calculated and expressed as the mean ± SD.
Notes: n = 6; **P < 0.05; #P < 0.0001.
Abbreviations: CFA, complete Freund’s adjuvant; IFA, incomplete Freund’s adjuvant; LPPC, liposome-polyethylene glycol (PEG)-polyethyleneimine (PEI) complex; PBS, phosphate-buffered saline; SD, standard deviation.
Figure 7 The adjuvant effects of LPPC on CTL. Following immunization with different treatments, the cytotoxicity of splenocytes from different mice against (A) pCJ-HpHsp60-or (B) pCJ-HpUrease B-transfected Balb/3T3 cells was determined. The lysis rate was calculated using the following equation: the survival rate of the phosphate-buffered treated group (100%) minus the survival rate of individual group. Statistically significant differences were found for the 150 μg LPPC/Hsp60 group compared with the CFA+IFA/ Hsp60 group.
Notes: n = 6; *P < 0.05; **P < 0.01.
Abbreviations: CFA, complete Freund’s adjuvant; IFA, incomplete Freund’s adjuvant; LPPC, liposome-polyethylene glycol (PEG)-polyethyleneimine (PEI) complex; PBS, phosphate-buffered saline.
Figure 8 The effects of LPPC in combination with different immunomodulators on antibody isotype expression. LPPC adsorbed with HpHsp60 and different immunomodulators (LPS or CpG ODN) was used to immunize and boost mice. (A) The total titer of anti-HpHsp60 antibody was determined every 3 days starting on day 21 after immunization and (B) the antibody isotype titers (IgM, IgG1, IgG2a, IgG2b, and IgA) were determined 21 days after immunization.
Notes: Data are expressed as the mean ± SD (n = 4). A significant difference compared to the CFA+IFA/Hsp60 group is indicated by *P < 0.05.
Abbreviations: CFA, complete Freund’s adjuvant; IFA, incomplete Freund’s adjuvant; LPPC, liposome-polyethylene glycol (PEG)-polyethyleneimine (PEI) complex; PBS, phosphate-buffered saline; LPS, lipopolysaccharides; Ig, immunoglobulin.
Figure 9 The effects of LPPC in combination with different immunomodulators on the cytotoxicity of mouse splenocytes. Following immunization with LPPC adsorbed with different immunomodulators (LPS or CpG ODN), the cytotoxicity of splenocytes from different mice against (A) pCJ-HpHsp60- or (B) pCJ-HpUrease B-transfected Balb/3T3 cells was determined. The lysis rate was calculated using the following equation: the survival rate of the nontreated group (100%) minus the survival rate of the individual group. Significant differences were found for the LPPC+CpG/Hsp60 group compared with the LPPC+LPS/Hsp60 group.
Notes: n = 6; *P < 0.05; **P < 0.01.
Abbreviations: LPPC, liposome-polyethylene glycol (PEG)-polyethyleneimine (PEI) complex; PBS, phosphate-buffered saline; LPS, lipopolysaccharides.
Figure 10 The effects of LPPC in combination with different immunomodulators on cytokine expression. Following treatment, mouse splenocytes were harvested, incubated with HpHsp60 and the supernatants were analyzed for cytokine concentration. The expression of (A) IL-4, (B) IL-5, (C) IFN-γ, and (D) TGF-β was evaluated.
Notes: *P < 0.05; **P < 0.01.
Abbreviations: CFA, complete Freund’s adjuvant; IFA, incomplete Freund’s adjuvant; IL, interleukin; IFN-γ, interferon-gamma; LPPC, liposome-polyethylene glycol (PEG)-polyethyleneimine (PEI) complex; LPS, lipopolysaccharides; TGF-β, transforming growth factor-beta.
Figure 11 The effects of LPPC in combination with different immunomodulators on the changes in antibody isotypes of mice immunized with Freund’s adjuvant. Mice were immunized with HpHsp60 that was mixed with Freund’s adjuvant and were further treated with LPPC with or without immunomodulators (LPS or CpG ODN). IgG1, IgG2a, or IgA expression was determined every 7 days and the kinetics are shown (in A, B, and C, respectively).
Note: A significant difference compared to the Hsp60 alone group is indicated by *P < 0.05 and **P < 0.01.
Abbreviations: Ig, immunoglobulin; LPPC, liposome-polyethylene glycol (PEG)-polyethyleneimine (PEI) complex; LPS, lipopolysaccharides.
