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1.
Figure 4

Figure 4. Detection of dengue viral RNA in infected engrafted NOD-scid IL2rγnull mice.. From: Dengue Virus Infection and Virus-Specific HLA-A2 Restricted Immune Responses in Humanized NOD-scid IL2rγnull Mice.

(A) RNA was extracted from the livers of two infected and two mock infected mice by the s.c. route. RNA was also extracted from DENV-2 NGC and used as a positive control. Viral RNA was amplified using Lanciotti primers as described in . (B) Normalized viral RNA levels on a subset of liver samples that were positive by RT-PCR.

Smita Jaiswal, et al. PLoS One. 2009;4(10):e7251.
2.
Figure 7

Figure 7. A2 restricted human T cell responses in NOD-scid HHD Tg(HLA–A2)mice.. From: Dengue Virus Infection and Virus-Specific HLA-A2 Restricted Immune Responses in Humanized NOD-scid IL2rγnull Mice.

Splenocytes from infected NOD-scid HHD Tg(HLA–A2)mice were stimulated for 6 hrs with 10 µg/ml of NS4b 2353(111–119), NS4b 2423(181–189), and NS4a 2148(56–64). A) Representative dot plots indicating the frequencies (upper right quadrant) of IFN-γ+, TNF-α+ and IL-2+ T cells by intracellular cytokine staining. B) Frequencies of hCD45+CD3+CD8+ IFN-γ+ secreting T cells in splenocytes of infected NOD-scid IL2rγnull Tg(HLA–A2/Huβ2M) and C) Representative plots demonstrating double cytokine secretion of antigen-specific T cells in response to stimulation with 10 µg/ml of NS4a 2148(56–64).

Smita Jaiswal, et al. PLoS One. 2009;4(10):e7251.
3.
Figure 1

Figure 1. Human hematopoiesis in organs of engrafted NOD-scid IL2rγnull mice.. From: Dengue Virus Infection and Virus-Specific HLA-A2 Restricted Immune Responses in Humanized NOD-scid IL2rγnull Mice.

(A) Approximately 3 months after engraftment of NOD-scid IL2rγnull mice with human T cell-depleted umbilical cord blood cells, reconstitution was assessed in target organs using flow cytometry. Values on the Y-axis represent frequencies of hCD45+ and mCD45+ cells within the mononuclear cell gates in the PBMC (n = 26), spleen (n = 31) and all cells in the bone marrow (n = 31). Error bars represent standard deviation from the mean value. (B,C) Representative flow cytometry plots demonstrating human hematopoiesis in the bone marrow (B) and spleen (C) of an engrafted mouse.

Smita Jaiswal, et al. PLoS One. 2009;4(10):e7251.
4.
Figure 3

Figure 3. Expression of viral antigen in target tissues of mice 3–7 days after in vivo infection.. From: Dengue Virus Infection and Virus-Specific HLA-A2 Restricted Immune Responses in Humanized NOD-scid IL2rγnull Mice.

Spleen and bone marrow cells were isolated from mice infected by the s.c. or i.p. route (n = 17) with DENV or mock infected (n = 5) mice 3–7 days post infection. Splenocytes and bone marrow cells were assessed for the expression of the envelope protein of DENV-2 using the zenon conjugated 3H5 mAb (A and B) or a non structural protein 1 of DENV-2 using the zenon conjugated 7E11 mAb (C and D). Data shown are frequencies of 3H5+, 7E11+ or ctrl Ab+ cells within the human CD45+ lymphocyte gate.

Smita Jaiswal, et al. PLoS One. 2009;4(10):e7251.
5.
Figure 6

Figure 6. Human T cells secrete IFN-γ in response to dengue antigen stimulation.. From: Dengue Virus Infection and Virus-Specific HLA-A2 Restricted Immune Responses in Humanized NOD-scid IL2rγnull Mice.

(A) Splenocytes from a mouse immunized two weeks prior with DENV-2 NGC were stimulated with an inactivated DENV-2 Ag, control vero antigen, PMA (0.1 µg/ml) + ionomycin (1 µg/ml) or media. Data shown are frequencies of human CD4+ T cells that produced IFN-γ in response to stimulation. (B and C) Frequencies of human CD45+, hCD4+ and hCD8+ IFN-γ secreting cells from DENV-2 infected engrafted NOD- scid IL2rγnull mice at 1 or 2 weeks post infection. The Wilcoxin signed rank test was used to assess significance.

Smita Jaiswal, et al. PLoS One. 2009;4(10):e7251.
6.
Figure 5

Figure 5. Detection of IgM-specific antibodies in the sera of infected engrafted NOD-scid IL2rγnull mice.. From: Dengue Virus Infection and Virus-Specific HLA-A2 Restricted Immune Responses in Humanized NOD-scid IL2rγnull Mice.

Sera were obtained from DENV-infected engrafted (n = 8 at prebleed; n = 19 at 1 week; n = 15 at 2 weeks), non-engrafted (n = 8) and mock infected (n = 6) NOD-scid IL2rγnull mice. IgM antibodies against DENV-2 E protein were detected by ELISA. Open circles represent OD values obtained from sera of mice obtained at a single time point. Closed connected circles represent values obtained from sera of the same mice that were bled at 1 and 2 weeks after infection with DENV-2 NGC. Open triangles represent values obtained from mock infected mice. The Wilcoxin signed rank test was used to assess significance.

Smita Jaiswal, et al. PLoS One. 2009;4(10):e7251.
7.
Figure 2

Figure 2. Bone marrow cells are susceptible to DENV-2 infection.. From: Dengue Virus Infection and Virus-Specific HLA-A2 Restricted Immune Responses in Humanized NOD-scid IL2rγnull Mice.

Bone marrow cells (2×106) from engrafted NOD-scid IL2rγnull mice were infected (m.o.i. = 2) in vitro with DENV-2 for 48 hrs. (A) Gating strategy used to identify cells with low and high side scatter. (B) Cells within the hCD45+ cells in gate 1 were further analyzed for viral antigen using the 3H5 mAb in uninfected and infected bone marrow cells. (C) Summary of DENV antigen expression in hCD45+ cells after in vitro infection with DENV-NGC. Values represent frequencies of hCD45+3H5+ cells within the respective gates. Median values are denoted by horizontal lines. Immunoperoxidase staining was performed to detect viral antigen in (D) DENV-2 infected bone marrow cells and (E) uninfected bone marrow cells using the 3H5 antibody. (F) Persistently infected Raji cells (positive control) and (G) uninfected Raji cells (negative control) were used to assess the specificity of staining.

Smita Jaiswal, et al. PLoS One. 2009;4(10):e7251.

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