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1.
FIG. 2.

FIG. 2. From: Functionally Distinct Transmission of Human Immunodeficiency Virus Type 1 Mediated by Immature and Mature Dendritic Cells .

DC-target cell contact is required for efficient HIV transmission mediated by iDCs and mDCs. Transmission of HIV-Luc/JRFL with iDCs and mDCs as donors and (A) Hut/CCR5 cells or (B) GHOST/R5 cells as targets was performed as described in the legend to Fig. 1B. Transwell culture plates with membrane pore sizes of 3 μm were used to separate DCs and target cells (Transwell). HIV infection was determined after 2 dpi by measuring the luciferase activity. The data show the means ± standard deviations of triplicate samples. One representative experiment out of three is shown. cps, counts per second.

Jian-Hua Wang, et al. J Virol. 2007 September;81(17):8933-8943.
2.
FIG. 3.

FIG. 3. From: Functionally Distinct Transmission of Human Immunodeficiency Virus Type 1 Mediated by Immature and Mature Dendritic Cells .

Enhanced HIV endocytosis and distinct viral trafficking in mDCs relative to iDCs. DCs were exposed to AT-2-inactivated R5 HIV for 1.5 h, washed thoroughly, fixed, and prepared for electron microscopy. (A, B, and C) Cell surface-bound HIV and internalized viral particles in iDCs. (D, E, and F) HIV internalization is significantly enhanced in mDCs. Arrows indicate DC surface-associated HIV particles or intracellular compartments that trapped intact HIV particles. (G, H, and I) RR labeling of mDC plasma membranes. (I) Higher-magnification image of panel H (a partial area). The open arrows indicate RR-labeled mDC plasma membranes, and the black arrows point to HIV-containing compartments and HIV particles that were not labeled with RR. Scale bars, 0.1 μm (A to F), 0.2 μm (G, I), and 0.5 μm (H).

Jian-Hua Wang, et al. J Virol. 2007 September;81(17):8933-8943.
3.
FIG. 6.

FIG. 6. From: Functionally Distinct Transmission of Human Immunodeficiency Virus Type 1 Mediated by Immature and Mature Dendritic Cells .

mDCs efficiently concentrate HIV at virological synapses. After a 1.5-h exposure to AT-2-inactivated R5 HIV, iDCs and mDCs were washed and cocultured separately with Hut/CCR5 cells for 1 h, fixed, and prepared for electron microscopy. Hut/CCR5 cells exhibit more condensed chromatins; DCs show typical surface dendrites, less-condensed chromatin, and electron-dense lysosome-like granules. (A) Large amount of intact HIV particles concentrated at the mDC-T-cell junction. (B) Higher-magnification images of the boxed areas from panel A. Black arrows indicate HIV particles that were concentrated at the synapses. (C) HIV particles concentrated at the mDC-T-cell junction. Membrane continuity was observed between an HIV-containing compartment and the plasma membrane of an mDC. (D) Higher-magnification images of the boxed areas from panel C. White arrows indicate HIV particles that were concentrated at the mDC-T-cell synapses. (E) Fewer HIV-like particles were observed at the iDC-T-cell junction. (F) A number of intact HIV-like particles were observed at the iDC-T-cell junction. Black arrows indicate HIV-like particles at the synapses. TC, Hut/CCR5 cells; scale bars, 1 μm (A to E) and 0.5 μm (F).

Jian-Hua Wang, et al. J Virol. 2007 September;81(17):8933-8943.
4.
FIG. 5.

FIG. 5. From: Functionally Distinct Transmission of Human Immunodeficiency Virus Type 1 Mediated by Immature and Mature Dendritic Cells .

Effects of trafficking inhibitors on DC-mediated viral transmission. iDCs (A) and mDCs (B) were incubated separately with various inhibitors for 0.5 h and pulsed with HIV-Luc/JRFL in the presence of the inhibitors for 2 h at 37°C. DCs were washed and cocultured with Hut/CCR5 cells for 2.5 days. Medium that contained dissolvent was used as a control. The average relative transmission of four independent experiments using DCs from different donors is shown (medium controls were set at 100%). Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) compared with medium controls. (C) Viability of inhibitor-treated DCs after 3 days in culture. DCs were incubated separately with various inhibitors at 37°C for 2.5 h, washed, and cultured 3 days before staining with 7-amino-actinomycin D. Stained DCs were analyzed by flow cytometry. The average DC viability of three independent experiments is shown (medium controls were set at 100%).

Jian-Hua Wang, et al. J Virol. 2007 September;81(17):8933-8943.
5.
FIG. 7.

FIG. 7. From: Functionally Distinct Transmission of Human Immunodeficiency Virus Type 1 Mediated by Immature and Mature Dendritic Cells .

Intracellular HIV degradation and time course viral transmission mediated by DCs. (A) HIV degradation in DCs. DCs (7.5 × 105) were incubated with AT-2-inactivated R5-tropic HIV (50 ng of p24), washed, and treated with trypsin. Aliquots of DCs were cultured, and DC-associated HIV p24 was measured daily. The p24 result (3,897 pg/ml) for mDCs at day 0 was set at 100%, and relative results are shown. (B) Time course HIV transmission by DCs. Transmission of HIV-Luc/JRFL (multiplicity of infection, 0.2) with iDCs and mDCs as donors and Hut/CCR5 cells as targets was performed as described in the legend to Fig. 1B. At 0, 1, 2, 4, and 6 dpi, aliquots of HIV-pulsed iDCs and mDCs were cocultured separately with Hut/CCR5 cells for an additional 3 days. In parallel, HIV infection of DC-alone controls was determined by measuring the luciferase activity at 3, 4, 5, 7, and 9 dpi. Mock controls of iDCs and mDCs without HIV infection were identical. All data are the means ± standard deviations of triplicate samples. One representative experiment out of three is shown. cps, counts per second.

Jian-Hua Wang, et al. J Virol. 2007 September;81(17):8933-8943.
6.
FIG. 4.

FIG. 4. From: Functionally Distinct Transmission of Human Immunodeficiency Virus Type 1 Mediated by Immature and Mature Dendritic Cells .

mDCs are more potent than iDCs in protecting HIV from proteolysis. (A) mDCs enhance HIV binding and internalization. DCs were incubated with HIV-Luc/JRFL (30 ng of p24) at 4°C or 37°C for 2 h, washed and treated with trypsin or medium, and then lysed for HIV p24 quantification. Asterisks indicate significant differences (P < 0.01) compared with iDCs at the same temperature. (B) DCs protect captured HIV from trypsin cleavage. HIV-pulsed iDCs, mDCs, and Raji/DC-SIGN cells were separately treated with trypsin before coculture with Hut/CCR5 target cells. Transmission of HIV-Luc/JRFL to Hut/CCR5 target cells was performed as described in the legend to Fig. 1B. The average results of four independent experiments are shown. Values for medium controls were set at 100%. Asterisks indicate significant differences (P < 0.05) between trypsin-treated samples and medium controls. (C) DCs protect captured HIV from pronase cleavage. HIV-pulsed iDCs and mDCs were treated with increasing concentrations of pronase before coculture with Hut/CCR5 target cells. The data show the means ± standard deviations of triplicate samples. One representative experiment out of two is shown. cps, counts per second. (D) Decreased surface DC-SIGN levels on DCs after protease treatment. DCs were stained for surface DC-SIGN after separate treatments with trypsin or pronase and analyzed by flow cytometry. Medium treatment was used as a control.

Jian-Hua Wang, et al. J Virol. 2007 September;81(17):8933-8943.
7.
FIG. 1.

FIG. 1. From: Functionally Distinct Transmission of Human Immunodeficiency Virus Type 1 Mediated by Immature and Mature Dendritic Cells .

Mature DCs enhance HIV transmission to different types of target cells independently of C-type lectins. (A) Surface markers of iDCs and mDCs. Monocyte-derived iDCs were cultured with LPS to generate mDCs. Cell surface markers were stained with specific MAbs or isotype-matched IgG controls and analyzed by flow cytometry. The histogram peaks of CD11c staining on iDCs and mDCs were overlapped. (B) Enhanced HIV transmission by mDCs is independent of C-type lectins. DCs and Raji/DC-SIGN cells were preincubated separately with medium, anti-DC-SIGN cocktails, and mannan prior to HIV incubation, as described previously (57). Raji/DC-SIGN cells, iDCs, and mDCs were pulsed separately with single-cycle, luciferase reporter R5-tropic HIV-Luc/JRFL (multiplicity of infection [MOI], 0.2), washed, and cocultured separately with autologous PBLs, the CD4+ T-cell line Hut/CCR5, and the HIV indicator cells GHOST/R5. HIV infection was determined after 2 days by measuring the luciferase activity. (C) No detectable HIV cis-infection in DCs and Raji/DC-SIGN cells. Cells were infected with HIV-Luc/JRFL (MOI, 0.2), and viral infection was determined at 2 dpi. (D) mDCs enhance transmission of HIV pseudotyped with X4-tropic Env (HXB2). Transmission of HIV-Luc/HXB2 with DCs as donors and Hut/CCR5 cells as targets was performed as described for panel B. The asterisk indicates a significant difference (P < 0.05) compared with iDCs. The data show the means ± standard deviations of triplicate samples. One representative experiment out of four is shown. cps, counts per second.

Jian-Hua Wang, et al. J Virol. 2007 September;81(17):8933-8943.

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