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1.
FIG. 6.

FIG. 6. From: Perforin and Gamma Interferon Expression Are Required for CD4+ and CD8+ T-Cell-Dependent Protective Immunity against a Human Parasite, Trypanosoma cruzi, Elicited by Heterologous Plasmid DNA Prime-Recombinant Adenovirus 5 Boost Vaccination .

Phenotypic characterization of splenic specific CD8 T cells induced by immunization of C57BL/6 WT or perforin KO mice with the heterologous prime-boost vaccination regimen. C57BL/6 WT or perforin KO mice were immunized with pIgSPCl.9/AdASP-2. Fourteen days after the final immunizing dose, these mice had their CD8+ splenic cells purified and stained with APC-labeled anti-CD8, biotin-labeled H-2Kb-VNHRFTLV, and the indicated marker-specific antibody labeled with FITC prior to analysis by FACS. The histograms show the expression of the markers on CD8+ H-2Kb-VNHRFTLV+ T cells (blue lines) or control naive CD8+ spleen cells (red lines). Representative analyses from three or more mice studied are shown.

Bruna C. G. de Alencar, et al. Infect Immun. 2009 October;77(10):4383-4395.
2.
FIG. 7.

FIG. 7. From: Perforin and Gamma Interferon Expression Are Required for CD4+ and CD8+ T-Cell-Dependent Protective Immunity against a Human Parasite, Trypanosoma cruzi, Elicited by Heterologous Plasmid DNA Prime-Recombinant Adenovirus 5 Boost Vaccination .

In vivo cytotoxic activity against target cells coated with peptides representing the CD8 epitope and trypomastigote-induced parasitemia and mortality of IFN-γ KO mice immunized with heterologous prime-boost regimen. IFN-γ KO mice were immunized as described in the legend of Fig. 3. (A) Two weeks after the final immunizing dose, mice were injected with splenic syngeneic cells labeled with CFSE and coated with the peptide VNHRFTLV. The specific in vivo cytotoxic activity was estimated after 20 h as described in Materials and Methods. The results are expressed as medians (bars) and each individual mouse (dots, n = 3). An asterisk denotes significantly higher (P < 0.01) specific in vivo cytotoxic activity of ASP-2-immunized IFN-γ KO mice than control pcDNA-3/Adβ-gal-injected mice. (B) Two weeks after the final immunizing dose, mice were challenged i.p. with 104 bloodstream trypomastigotes. The parasitemia for each mouse group is represented as mean ± the SD (n = 6). (C) The graph shows the Kaplan-Meier curves for survival of the mice groups immunized and challenged as described above (n = 6). These experiments were performed twice with similar results.

Bruna C. G. de Alencar, et al. Infect Immun. 2009 October;77(10):4383-4395.
3.
FIG. 2.

FIG. 2. From: Perforin and Gamma Interferon Expression Are Required for CD4+ and CD8+ T-Cell-Dependent Protective Immunity against a Human Parasite, Trypanosoma cruzi, Elicited by Heterologous Plasmid DNA Prime-Recombinant Adenovirus 5 Boost Vaccination .

In vivo cytotoxic activity against target cells coated with a peptide representing the CD8 epitope in mice immunized with different protocols before or after challenge with T. cruzi. A/Sn mice were injected with the different immunization protocols depicted at the side of the charter. The immunizing doses were injected i.m. at 0 and 3 weeks. Two weeks after the final immunizing dose, half of the mice were injected with splenic syngeneic cells labeled with CFSE and coated with peptide TEWETGQI. The remaining half of the animals was challenged i.p. with 150 blood forms of T. cruzi. Thirteen days later, these mice were also injected with splenic syngeneic cells labeled with CFSE and coated with the peptide TEWETGQI. The specific in vivo cytotoxic activity was estimated after 20 h as described in Materials and Methods. Results are expressed as medians (bars), and each dot represents an individual mouse. Symbols denote significantly higher (P < 0.01) specific in vivo cytotoxic activity compared to control naive or pcDNA-3/Adβ-gal injected mice prior to infection (asterisks) or after a challenge with T. cruzi (crosses). The results are representative of experiments performed twice with similar results.

Bruna C. G. de Alencar, et al. Infect Immun. 2009 October;77(10):4383-4395.
4.
FIG. 5.

FIG. 5. From: Perforin and Gamma Interferon Expression Are Required for CD4+ and CD8+ T-Cell-Dependent Protective Immunity against a Human Parasite, Trypanosoma cruzi, Elicited by Heterologous Plasmid DNA Prime-Recombinant Adenovirus 5 Boost Vaccination .

ICS of CD8 T cells from C57BL/6 WT or perforin KO mice immunized with the heterologous prime-boost vaccination regimen. C57BL/6 WT or perforin KO mice were immunized with pcDNA3/Adβ-gal or pIgSPCl.9/AdASP-2. Fourteen days after the final immunizing dose, these mice had their splenic cells cultured in the presence of anti-CD28 and brefeldin A, with or without the peptide VNHRFTLV. After 12 h, cells were stained with APC-labeled anti-CD8, fixed, permeabilized, and stained with APC-Cy7-labeled anti-CD3, PE-Cy7-labeled anti-IFN-γ, and Alexa 488-labeled anti-TNF-α. (A and B) Examples of splenic CD3+ CD8+ cells from immunized C57BL/6 WT or perforin KO mice stained for IFN-γ and TNF-α. (C) Frequency of each cell population. Asterisks indicate that C57BL/6 WT mice immunized with IgSPCl.9/AdASP-2 had a higher frequency of CD3+ CD8+ T cells expressing either IFN-γ or TNF-α or both cytokines than perforin KO mice immunized with pIgSPCl.9/AdASP-2 (P ≤ 0.01 in all cases). (D) We calculate the frequency of each cell population in relation to the total amount of cells expressing any cytokine. An asterisk indicates that C57BL/6 WT mice immunized with of pIgSPCl.9/AdASP-2 had higher frequencies of CD3+ CD8+ T cells expressing both IFN-γ and TNF-α than perforin KO mice immunized with pIgSPCl.9/AdASP-2 (P < 0.01 in all cases). A cross indicates that perforin KO mice immunized with IgSPCl.9/AdASP-2 had a higher frequency of CD3+ CD8+ T cells expressing only IFN-γ than C57BL/6 WT mice immunized with pIgSPCl.9/AdASP-2. The results are presented as medians (bars) and each individual mouse (dots) and are representative of experiments performed twice with similar results.

Bruna C. G. de Alencar, et al. Infect Immun. 2009 October;77(10):4383-4395.
5.
FIG. 3.

FIG. 3. From: Perforin and Gamma Interferon Expression Are Required for CD4+ and CD8+ T-Cell-Dependent Protective Immunity against a Human Parasite, Trypanosoma cruzi, Elicited by Heterologous Plasmid DNA Prime-Recombinant Adenovirus 5 Boost Vaccination .

Trypomastigote-induced parasitemia and mortality in C57BL/6 WT, perforin KO, CD4-depleted, or CD8 KO mice immunized with the heterologous prime-boost regimen. C57BL/6 WT, perforin KO, CD4-depleted, or CD8 KO mice were immunized with pIgSPCl.9, followed by AdASP-2 both injected i.m., at 0 and 3 weeks, respectively. Control mice were injected with pcDNA3, followed by Adβ-gal both injected i.m., at 0 and 3. Two weeks after the last immunizing dose, mice were challenged i.p. with 104 bloodstream trypomastigotes. (A to D) The parasitemia for each mouse group is represented as the mean ± the SD (n = 5 to 8). Asterisks denote that parasitemia was significantly lower (P < 0.01) for ASP-2-vaccinated mice (▪) than for controls nonimmune mice (•). The results are representative of experiments performed twice with similar results. (E and F) The graphs show the Kaplan-Meier curves for survival of the mouse groups immunized and challenged as described above (A to D). For C57BL/6 WT and perforin KO mice, the total number of animals was 13. In the case of CD4-depleted or CD8 KO mice, the number of animals was 10 or 8, respectively. Statistical analysis showed that (i) ASP-2-vaccinated C57BL/6 WT mice survived longer than all of the other mouse groups (P < 0.0001 in all cases), (ii) ASP-2-vaccinated perforin KO mice survived longer than control nonimmune perforin KO mice or CD8 KO mice (P < 0.001 in both cases), (iii) ASP-2-vaccinated CD4-depleted mice survived longer than control nonimmune CD4-depleted mice (P < 0.001, and (iv) ASP-2-vaccinated CD8 KO mice survived longer than control nonimmune CD8 KO mice (P < 0.01).

Bruna C. G. de Alencar, et al. Infect Immun. 2009 October;77(10):4383-4395.
6.
FIG. 4.

FIG. 4. From: Perforin and Gamma Interferon Expression Are Required for CD4+ and CD8+ T-Cell-Dependent Protective Immunity against a Human Parasite, Trypanosoma cruzi, Elicited by Heterologous Plasmid DNA Prime-Recombinant Adenovirus 5 Boost Vaccination .

Cell mediated response of C57BL/6 WT or perforin KO mice after immunization with the heterologous prime-boost vaccination regimen. C57BL/6 WT or perforin KO mice were immunized as described in the legend of Fig. 3. Two weeks after the final immunizing dose, splenic cells were restimulated in vitro in the presence of medium (Med), recombinant glutathione S-transferase (GST; 10 μg/ml) or recombinant ASP-2 (His-65 kDa; 10 μg/ml [A]). When we compared all mouse groups, ASP-2-immunized WT spleens cells secreted more IFN-γ upon recombinant protein stimulation (asterisk, P < 0.05 in all cases). We estimated the frequency of specific splenic cells by staining with anti-CD8 and the multimer H-2Kb/VNHRFTLV (B) or the number of splenic IFN-γ spot-forming cells (SFC) by the ex vivo ELISPOT assay (C). When we compared groups of mice immunized with pIgSPCl.9/AdASP-2, immunized C57BL/6 WT mice had a higher frequency of SFC than the immunized perforin KO mice (asterisk, P < 0.001). Alternatively, these mice had their splenic cells cultured in the presence of anti-CD28 and FITC-labeled anti-CD107a, with or without the peptide VNHRFTLV. (D) After 12 h, cells were stained with APC-labeled anti-CD8, fixed, permeabilized, and stained with APC-Cy7-labeled anti-CD3, PE-Cy7-labeled anti-IFN-γ. When we compared groups of mice immunized with pIgSPCl.9/AdASP-2, immunized C57BL/6 WT mice had a higher frequency of CD3+CD8+ T cells expressing either IFN-γ or double-stained for CD107a/IFN-γ than the immunized perforin KO mice (asterisks, P < 0.01 in all cases). Finally, we estimated the in vivo cytotoxic activity by injecting immunized mice with CFSE-labeled splenic cells labeled with CFSE and coated with the peptide VNHRFTLV. (E) After 4 or 20 h, the in vivo cytotoxic activity was determined. When we compared groups of mice immunized with pIgSPCl.9/AdASP-2, immunized C57BL/6 WT mice displayed significantly higher in vivo cytotoxicity than perforin KO mice (asterisk, P < 0.01 in both cases). The results are expressed as medians (bars) and each individual mouse (dots) and are representative of experiments performed at least twice with similar results.

Bruna C. G. de Alencar, et al. Infect Immun. 2009 October;77(10):4383-4395.
7.
FIG. 1.

FIG. 1. From: Perforin and Gamma Interferon Expression Are Required for CD4+ and CD8+ T-Cell-Dependent Protective Immunity against a Human Parasite, Trypanosoma cruzi, Elicited by Heterologous Plasmid DNA Prime-Recombinant Adenovirus 5 Boost Vaccination .

Trypomastigote-induced parasitemia and mortality in A/Sn mice immunized with different combinations of plasmid DNA and/or replication defective human adenovirus type 5 expressing the ASP-2 of T. cruzi. (A) A/Sn mice were immunized as described. Priming and boosting immunizations were performed with the indicated plasmid or adenovirus i.m. at 0 and 3 weeks, respectively. Two weeks after the final immunizing dose, mice were challenged i.p. with 150 bloodstream trypomastigotes. (B) The peak parasitemia for each mouse group is represented as the mean ± the standard deviation (SD) (n = 6). Asterisks denote that the parasitemia of mice from groups 3, 4, and 5 was significantly lower than that of mice from groups 1 and 2 (P < 0.01 in all cases). Mice from groups 4 and 5 had a significantly lower parasitemia than mice from group 3 (P < 0.05). (C) The graph shows the Kaplan-Meier curves for survival of the mice groups immunized and challenged as described above (n = 9). Pooled results from two experiments are shown. Mice from group 3 survived significantly longer than animals from group 1 and 2 (P < 0.0001 in both cases). Mice from groups 4 and 5 survived significantly longer than the other groups (P < 0.0001 in all cases). (D) A/Sn mice were immunized as described. Two weeks or 98 days after the final immunizing dose, mice were challenged i.p. with 150 bloodstream trypomastigotes. (E) The parasitemia for each mouse group is represented as the mean ± the SD (n = 6). Asterisks indicate that ASP-2-vaccinated mice had a significantly lower parasitemia (P < 0.01) than nonimmune animals. (F) The graph shows the Kaplan-Meier curves for survival of the mouse groups immunized and challenged as described above (n = 6). Mice from groups vaccinated with ASP-2 survived significantly longer than nonimmune control animals (P < 0.001). (G) A/Sn mice were immunized as described. Before and after challenge i.p. with 150 bloodstream trypomastigotes, mice were treated as described in Materials and Methods with rat IgG or anti-CD4 MAb. (H) The parasitemia for each mouse group is represented as the mean ± the SD (n = 12). Asterisks indicate that ASP-2-vaccinated mice treated with rat IgG had a significantly lower parasitemia (P < 0.01) than nonimmune animals or ASP-2-vaccinated mice treated with anti-CD4 MAb. (I) The graph shows the Kaplan-Meier curves for survival of mouse groups immunized and challenged as described above (n = 12). ASP-2-immunized mice treated with rat IgG survived significantly longer than nonimmune animals or ASP-2-immunized mice treated with anti-CD4 MAb (P < 0.0001 in both cases). ASP-2-immunized mice treated with anti-CD4 MAb survived longer than nonimmune animals. The results are pooled from two independent experiments. (J) A/Sn mice were immunized as described. Before and after challenge i.p. with 150 bloodstream trypomastigotes, mice were treated as described in Materials and Methods with rat IgG or anti-CD8 MAb. (K) The parasitemia for each mouse group is represented as the mean ± the SD (n = 6). Asterisks indicate that ASP-2-immunized mice treated with rat IgG had a significantly lower parasitemia (P < 0.01) than nonimmune animals or ASP-2-vaccinated mice treated with anti-CD8 MAb. (L) The graph shows the Kaplan-Meier curves for survival of mouse groups immunized and challenged as described above (n = 8 to 9). ASP-2-immunized mice treated with rat IgG survived significantly longer (P < 0.0001) than nonimmune animals or ASP-2-immunized mice treated with anti-CD8 MAb. The results are representative of two independent experiments.

Bruna C. G. de Alencar, et al. Infect Immun. 2009 October;77(10):4383-4395.

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