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In vivo immune response and preventive effect activated by FI‐mOMVs. (a) Schematic diagram of immunization, sample collection and challenge schedule. BALB/c mice were immunized s.c. with the indicated formulations on Days 0, 14, 28 ( n = 8), and a PBS control was set up ( n = 5). (b) Serum was collected 7, 14, 21, 28, 35 and 42 days after initial vaccination, and the FI antigen–specific IgG antibody titre was detected by ELISA. (c–e) Detection of antigen‐specific cellular immune response. The spleen lymphocytes were isolated 6 weeks after the initial immunization. The proliferation levels were detected by CCK8 (c), and the expression levels of IFN‐γ (d) and IL‐4 (e) in the culture supernatant were determined by ELISA. (f, g) Percentage of CD3 + <t>CD4</t> + T lymphocytes (f), and CD3 + CD8 + T lymphocytes (g) in the spleen on Day 42 after immunization ( n = 8). Mice without immunization were used as controls ( n = 5). (h) Survival curves of the mice infected with PAO1 (1 × 10 8 CFU/mouse, i.n.) after different treatments ( n = 12). (i, j) Quantification of the number of bacteria in the lung (i) and the pathological changes of the lung (j) of the mice infected with a sublethal dose of PAO1 (5 × 10 6 CFU/mouse, i.n.) after different treatments ( n = 6). Scale bar, 200 µm. The data are presented as mean ± SEM. Statistical analysis was performed by a two‐tailed unpaired Student's t ‐test (b–g), log‐rank (Mantel–Cox) test (h), or two‐tailed Mann–Whitney U ‐test (i), and differences were considered significant at p < 0.05. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001.
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In vivo immune response and preventive effect activated by FI‐mOMVs. (a) Schematic diagram of immunization, sample collection and challenge schedule. BALB/c mice were immunized s.c. with the indicated formulations on Days 0, 14, 28 ( n = 8), and a PBS control was set up ( n = 5). (b) Serum was collected 7, 14, 21, 28, 35 and 42 days after initial vaccination, and the FI antigen–specific IgG antibody titre was detected by ELISA. (c–e) Detection of antigen‐specific cellular immune response. The spleen lymphocytes were isolated 6 weeks after the initial immunization. The proliferation levels were detected by CCK8 (c), and the expression levels of IFN‐γ (d) and IL‐4 (e) in the culture supernatant were determined by ELISA. (f, g) Percentage of CD3 + <t>CD4</t> + T lymphocytes (f), and CD3 + CD8 + T lymphocytes (g) in the spleen on Day 42 after immunization ( n = 8). Mice without immunization were used as controls ( n = 5). (h) Survival curves of the mice infected with PAO1 (1 × 10 8 CFU/mouse, i.n.) after different treatments ( n = 12). (i, j) Quantification of the number of bacteria in the lung (i) and the pathological changes of the lung (j) of the mice infected with a sublethal dose of PAO1 (5 × 10 6 CFU/mouse, i.n.) after different treatments ( n = 6). Scale bar, 200 µm. The data are presented as mean ± SEM. Statistical analysis was performed by a two‐tailed unpaired Student's t ‐test (b–g), log‐rank (Mantel–Cox) test (h), or two‐tailed Mann–Whitney U ‐test (i), and differences were considered significant at p < 0.05. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001.
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In vivo immune response and preventive effect activated by FI‐mOMVs. (a) Schematic diagram of immunization, sample collection and challenge schedule. BALB/c mice were immunized s.c. with the indicated formulations on Days 0, 14, 28 ( n = 8), and a PBS control was set up ( n = 5). (b) Serum was collected 7, 14, 21, 28, 35 and 42 days after initial vaccination, and the FI antigen–specific IgG antibody titre was detected by ELISA. (c–e) Detection of antigen‐specific cellular immune response. The spleen lymphocytes were isolated 6 weeks after the initial immunization. The proliferation levels were detected by CCK8 (c), and the expression levels of IFN‐γ (d) and IL‐4 (e) in the culture supernatant were determined by ELISA. (f, g) Percentage of CD3 + <t>CD4</t> + T lymphocytes (f), and CD3 + CD8 + T lymphocytes (g) in the spleen on Day 42 after immunization ( n = 8). Mice without immunization were used as controls ( n = 5). (h) Survival curves of the mice infected with PAO1 (1 × 10 8 CFU/mouse, i.n.) after different treatments ( n = 12). (i, j) Quantification of the number of bacteria in the lung (i) and the pathological changes of the lung (j) of the mice infected with a sublethal dose of PAO1 (5 × 10 6 CFU/mouse, i.n.) after different treatments ( n = 6). Scale bar, 200 µm. The data are presented as mean ± SEM. Statistical analysis was performed by a two‐tailed unpaired Student's t ‐test (b–g), log‐rank (Mantel–Cox) test (h), or two‐tailed Mann–Whitney U ‐test (i), and differences were considered significant at p < 0.05. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001.
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In vivo immune response and preventive effect activated by FI‐mOMVs. (a) Schematic diagram of immunization, sample collection and challenge schedule. BALB/c mice were immunized s.c. with the indicated formulations on Days 0, 14, 28 ( n = 8), and a PBS control was set up ( n = 5). (b) Serum was collected 7, 14, 21, 28, 35 and 42 days after initial vaccination, and the FI antigen–specific IgG antibody titre was detected by ELISA. (c–e) Detection of antigen‐specific cellular immune response. The spleen lymphocytes were isolated 6 weeks after the initial immunization. The proliferation levels were detected by CCK8 (c), and the expression levels of IFN‐γ (d) and IL‐4 (e) in the culture supernatant were determined by ELISA. (f, g) Percentage of CD3 + <t>CD4</t> + T lymphocytes (f), and CD3 + CD8 + T lymphocytes (g) in the spleen on Day 42 after immunization ( n = 8). Mice without immunization were used as controls ( n = 5). (h) Survival curves of the mice infected with PAO1 (1 × 10 8 CFU/mouse, i.n.) after different treatments ( n = 12). (i, j) Quantification of the number of bacteria in the lung (i) and the pathological changes of the lung (j) of the mice infected with a sublethal dose of PAO1 (5 × 10 6 CFU/mouse, i.n.) after different treatments ( n = 6). Scale bar, 200 µm. The data are presented as mean ± SEM. Statistical analysis was performed by a two‐tailed unpaired Student's t ‐test (b–g), log‐rank (Mantel–Cox) test (h), or two‐tailed Mann–Whitney U ‐test (i), and differences were considered significant at p < 0.05. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001.
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In vivo immune response and preventive effect activated by FI‐mOMVs. (a) Schematic diagram of immunization, sample collection and challenge schedule. BALB/c mice were immunized s.c. with the indicated formulations on Days 0, 14, 28 ( n = 8), and a PBS control was set up ( n = 5). (b) Serum was collected 7, 14, 21, 28, 35 and 42 days after initial vaccination, and the FI antigen–specific IgG antibody titre was detected by ELISA. (c–e) Detection of antigen‐specific cellular immune response. The spleen lymphocytes were isolated 6 weeks after the initial immunization. The proliferation levels were detected by CCK8 (c), and the expression levels of IFN‐γ (d) and IL‐4 (e) in the culture supernatant were determined by ELISA. (f, g) Percentage of CD3 + <t>CD4</t> + T lymphocytes (f), and CD3 + CD8 + T lymphocytes (g) in the spleen on Day 42 after immunization ( n = 8). Mice without immunization were used as controls ( n = 5). (h) Survival curves of the mice infected with PAO1 (1 × 10 8 CFU/mouse, i.n.) after different treatments ( n = 12). (i, j) Quantification of the number of bacteria in the lung (i) and the pathological changes of the lung (j) of the mice infected with a sublethal dose of PAO1 (5 × 10 6 CFU/mouse, i.n.) after different treatments ( n = 6). Scale bar, 200 µm. The data are presented as mean ± SEM. Statistical analysis was performed by a two‐tailed unpaired Student's t ‐test (b–g), log‐rank (Mantel–Cox) test (h), or two‐tailed Mann–Whitney U ‐test (i), and differences were considered significant at p < 0.05. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001.
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In vivo immune response and preventive effect activated by FI‐mOMVs. (a) Schematic diagram of immunization, sample collection and challenge schedule. BALB/c mice were immunized s.c. with the indicated formulations on Days 0, 14, 28 ( n = 8), and a PBS control was set up ( n = 5). (b) Serum was collected 7, 14, 21, 28, 35 and 42 days after initial vaccination, and the FI antigen–specific IgG antibody titre was detected by ELISA. (c–e) Detection of antigen‐specific cellular immune response. The spleen lymphocytes were isolated 6 weeks after the initial immunization. The proliferation levels were detected by CCK8 (c), and the expression levels of IFN‐γ (d) and IL‐4 (e) in the culture supernatant were determined by ELISA. (f, g) Percentage of CD3 + CD4 + T lymphocytes (f), and CD3 + CD8 + T lymphocytes (g) in the spleen on Day 42 after immunization ( n = 8). Mice without immunization were used as controls ( n = 5). (h) Survival curves of the mice infected with PAO1 (1 × 10 8 CFU/mouse, i.n.) after different treatments ( n = 12). (i, j) Quantification of the number of bacteria in the lung (i) and the pathological changes of the lung (j) of the mice infected with a sublethal dose of PAO1 (5 × 10 6 CFU/mouse, i.n.) after different treatments ( n = 6). Scale bar, 200 µm. The data are presented as mean ± SEM. Statistical analysis was performed by a two‐tailed unpaired Student's t ‐test (b–g), log‐rank (Mantel–Cox) test (h), or two‐tailed Mann–Whitney U ‐test (i), and differences were considered significant at p < 0.05. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001.

Journal: Journal of Extracellular Vesicles

Article Title: High‐Yield Outer Membrane Vesicles Derived From Probiotics as a Nanoplatform for Precise Treatment and Prophylaxis of Pseudomonas aeruginosa Infection

doi: 10.1002/jev2.70194

Figure Lengend Snippet: In vivo immune response and preventive effect activated by FI‐mOMVs. (a) Schematic diagram of immunization, sample collection and challenge schedule. BALB/c mice were immunized s.c. with the indicated formulations on Days 0, 14, 28 ( n = 8), and a PBS control was set up ( n = 5). (b) Serum was collected 7, 14, 21, 28, 35 and 42 days after initial vaccination, and the FI antigen–specific IgG antibody titre was detected by ELISA. (c–e) Detection of antigen‐specific cellular immune response. The spleen lymphocytes were isolated 6 weeks after the initial immunization. The proliferation levels were detected by CCK8 (c), and the expression levels of IFN‐γ (d) and IL‐4 (e) in the culture supernatant were determined by ELISA. (f, g) Percentage of CD3 + CD4 + T lymphocytes (f), and CD3 + CD8 + T lymphocytes (g) in the spleen on Day 42 after immunization ( n = 8). Mice without immunization were used as controls ( n = 5). (h) Survival curves of the mice infected with PAO1 (1 × 10 8 CFU/mouse, i.n.) after different treatments ( n = 12). (i, j) Quantification of the number of bacteria in the lung (i) and the pathological changes of the lung (j) of the mice infected with a sublethal dose of PAO1 (5 × 10 6 CFU/mouse, i.n.) after different treatments ( n = 6). Scale bar, 200 µm. The data are presented as mean ± SEM. Statistical analysis was performed by a two‐tailed unpaired Student's t ‐test (b–g), log‐rank (Mantel–Cox) test (h), or two‐tailed Mann–Whitney U ‐test (i), and differences were considered significant at p < 0.05. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001.

Article Snippet: The cell suspensions (100 μL) were taken and stained with FITC‐conjugated anti‐mouse CD4 (cat# FITC‐65104, Proteintech, USA), PE‐conjugated anti‐mouse CD8 (cat# PE‐65069, Proteintech, USA) and APC‐conjugated anti‐mouse CD3 (cat# APC‐65077, Proteintech, USA) for 30 min at 37°C in the dark.

Techniques: In Vivo, Control, Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Infection, Bacteria, Two Tailed Test, MANN-WHITNEY