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1.
Figure 2

Figure 2. Leishmania parasites are internalized more efficiently in uninfected bystander MDMs compared to virus-infected cells.. From: HIV-1 Promotes Intake of Leishmania Parasites by Enhancing Phosphatidylserine-Mediated, CD91/LRP-1-Dependent Phagocytosis in Human Macrophages.

The total numbers of complement-opsonized Alexa488-labeled zymosan particles or GFP-expressing Leishmania parasites internalized were determined in mock-infected control, uninfected bystander, or productively HIV-1-infected MDMs, by fluorescence microscopy, as previously illustrated in . shown are of a single representative donor out of a grand total of nine donors (mean number of targets ± SEM).

Robert Lodge, et al. PLoS One. 2012;7(3):e32761.
2.
Figure 8

Figure 8. HIV-1-mediated increase in Leishmania phagocytosis in uninfected bystander MDMs is inhibited by LRPAP/RAP.. From: HIV-1 Promotes Intake of Leishmania Parasites by Enhancing Phosphatidylserine-Mediated, CD91/LRP-1-Dependent Phagocytosis in Human Macrophages.

Mock-infected or 6 day HIV-1-infected MDMs were treated for 4 hours with the CD91/LRP-1-ligand antagonist, LRPAP/RAP, prior and during phagocytosis of Leishmania parasites (panel A) or zymosan particles (panel B), for 4 hours. MDMs were then fixed, stained and mounted for confocal microscopy analysis as described in . The numbers of internalized targets were then determined. shown are the means of four distinct donors ± SEM.

Robert Lodge, et al. PLoS One. 2012;7(3):e32761.
3.
Figure 7

Figure 7. HIV-1 infection enhances CD91/LRP-1 expression in macrophages.. From: HIV-1 Promotes Intake of Leishmania Parasites by Enhancing Phosphatidylserine-Mediated, CD91/LRP-1-Dependent Phagocytosis in Human Macrophages.

MDMs were infected for 6 days with HIV-1, and cell surface expression of CD91/LRP-1 was determined by flow cytometry as described in . The graphs shown is from results obtained from a donor (depicted with an asterisk) chosen from the three reported in the small table below the graph. The percentage of CD91 positive cells and mean fluorescence intensity (MFI) (shown in parentheses) are illustrated for the three different donors tested.

Robert Lodge, et al. PLoS One. 2012;7(3):e32761.
4.
Figure 5

Figure 5. Enhanced intake of Leishmania parasites in uninfected bystander MDMs is dependent on a surface macrophage receptor for phosphatidylserine.. From: HIV-1 Promotes Intake of Leishmania Parasites by Enhancing Phosphatidylserine-Mediated, CD91/LRP-1-Dependent Phagocytosis in Human Macrophages.

Alexa488-labeled zymosan particles or GFP-expressing Leishmania parasites were first pre-incubated for 30 min in annexin V-binding buffer with the listed concentrations of annexin V, and added to MDMs previously treated or not with Tat. The amount of internalized zymosan particles or Leishmania parasites was determined as described in . are the means of a donor representative of three (mean number of targets ± SEM).

Robert Lodge, et al. PLoS One. 2012;7(3):e32761.
5.
Figure 6

Figure 6. Tat and TGF-β enhance CD91/LRP-1 surface expression in macrophages.. From: HIV-1 Promotes Intake of Leishmania Parasites by Enhancing Phosphatidylserine-Mediated, CD91/LRP-1-Dependent Phagocytosis in Human Macrophages.

MDMs were treated for 24 hours with either Tat (panel A) or TGF-β (panel B), and cell surface CD91/LRP-1 determined by flow cytometry as described in . Graphs shown are from results obtained from a donor (depicted with an asterisk) chosen from the three reported in the small table below each graph. Different donors were used in panels A and B. The percentage of CD91 positive cells and mean fluorescence intensity (MFI) (shown in parentheses) are illustrated for the six different donors tested.

Robert Lodge, et al. PLoS One. 2012;7(3):e32761.
6.
Figure 3

Figure 3. MDMs productively infected with HIV-1 secrete a factor that is necessary for inducing enhanced Leishmania parasite intake.. From: HIV-1 Promotes Intake of Leishmania Parasites by Enhancing Phosphatidylserine-Mediated, CD91/LRP-1-Dependent Phagocytosis in Human Macrophages.

MDMs were treated for 24 hours with cell-free supernatants from 6-day old virus-infected macrophages or uninfected MDM supernatants supplemented with EFZ. Mock-treated cells or MDMs treated for 24 hours with IL-10 were used as controls. Next, MDMs were pulsed for 1 hour either with complement-opsonized Alexa488-labeled zymosan particles or GFP-expressing Leishmania parasites. Thereafter, excess zymosan particles/Leishmania parasites were washed out and MDMs cultured for another 3 hours. After fixing and mounting the cells, the numbers of internalized zymosan particles or Leishmania parasites were then determined by fluorescence microscopy. are from a donor representative of three (mean number of targets ± SEM).

Robert Lodge, et al. PLoS One. 2012;7(3):e32761.
7.
Figure 9

Figure 9. HIV-1 infection of MDMs activates serum TGF-β, leading to enhanced cell surface CD91/LRP-1 expression.. From: HIV-1 Promotes Intake of Leishmania Parasites by Enhancing Phosphatidylserine-Mediated, CD91/LRP-1-Dependent Phagocytosis in Human Macrophages.

HIV-1-infected MDMs were treated either with MVC or EFZ (panel A) or either neutralizing TGF-β antibodies or Furin inhibitor I (panel B) during HIV-1 infection for 8 hours, and cell surface CD91/LRP-1 determined by flow cytometry as described in . Both uninfected and untreated HIV-1-infected MDMs were used as controls. Graphs shown are from results obtained from a donor (shown with an asterisk) chosen from the three reported in the small table below each graph. Different donors were used in panels A and B. The percentage of CD91 positive cells and mean fluorescence intensity (MFI) (shown in parentheses) are illustrated for the six different donors tested.

Robert Lodge, et al. PLoS One. 2012;7(3):e32761.
8.
Figure 4

Figure 4. Tat and TGF-β mediate enhanced Leishmania parasite intake in MDMs.. From: HIV-1 Promotes Intake of Leishmania Parasites by Enhancing Phosphatidylserine-Mediated, CD91/LRP-1-Dependent Phagocytosis in Human Macrophages.

Cells were either left untreated, or treated for 24 hours with uninfected MDM supernatants, Tat or TGF-β, prior to a 1 hour exposure to Alexa488-labeled zymosan particles or GFP-expressing Leishmania parasites. In some cases, anti-Tat, or anti-TGF-β neutralizing antibodies, were added at the time of incubation with either Tat or TGF-β, respectively. Alternatively, anti-Tat was added to supernatants from HIV-1-infected macrophages, and this mix added to MDMs at 24 hours before performing the phagocytosis assay. Thereafter, excess zymosan particles/Leishmania parasites were washed out and MDMs cultured for another 3 hours. Numbers of internalized zymosan particles or Leishmania parasites were determined as described in . are the means obtained from a donor representative of three (mean number of targets ± SEM).

Robert Lodge, et al. PLoS One. 2012;7(3):e32761.
9.
Figure 1

Figure 1. HIV-1 infection exerts a different effect on MDM phagocytosis of zymosan particles or Leishmania parasites.. From: HIV-1 Promotes Intake of Leishmania Parasites by Enhancing Phosphatidylserine-Mediated, CD91/LRP-1-Dependent Phagocytosis in Human Macrophages.

Mock-infected control MDMs (panels A and C) or cells infected for 6 days with NL4-3-Bal-IRES-HSA reporter virus (panels B and D) were put in contact either with complement-opsonized Alexa488-tagged zymosan particles (panels A and B) or GFP-expressing Leishmania parasites (panels C and D) (both shown in green) for 1 hour. Next, excess zymosan particles/Leishmania amastigotes were washed out and MDMs cultured for an additional 3 hours. Cells were then fixed, mounted and immunostained for HSA (shown in red) and DNA (using DRAQ5, shown in blue) to detect HIV-1-infected cells and Leishmania DNA/host cell DNA, respectively. Shown are representative images obtained by confocal microscopy. Arrows indicate uninfected bystander MDMs displaying numerous internalized Leishmania parasites.

Robert Lodge, et al. PLoS One. 2012;7(3):e32761.

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