Journal: iScience

Article Title: Sustained type I interferon signaling after human immunodeficiency virus type 1 infection of human iPSC derived microglia and cerebral organoids

doi: 10.1016/j.isci.2024.109628

Figure Lengend Snippet: HIV-1-infected microglia exhibit productive infection, activation, and dysregulated cytokine/chemokine production (A) RT-qPCR analysis of viral gag/pol mRNA after infection with HIV-1 JRFL in microglia monocultures; n = 3 independent infections of 03SF line iPSC-derived microglia; data are presented as the mean ± SEM. (B) ELISA analysis of secreted p24 in the culture supernatant of microglia infected with HIV-1 JR-FL reveals productive infection; n = 3 independent infections of 03SF iPSC-derived microglia; data are presented as the mean ± SEM. (C) RT-qPCR analysis of IL1 β , TNF α and IL-6 mRNA at days 16 and 23 post infection with HIV-1 JRFL; n = 3 independent infections of 03SF iPSC-derived microglia; two-way ANOVA; Šídák multiple comparisons test; ∗ p < 0.05; data are presented as the mean ± SEM. (D) RT-qPCR analysis of CCL2 , CCL3 , and CCL5 mRNA at days 16 and 23 post infection with HIV-1 JRFL; n = 3 independent infections of 03SF iPSC-derived microglia; two-way ANOVA; Šídák multiple comparisons test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; data are presented as the mean ± SEM. (E) RT-qPCR analysis of IBA1 and P2RY12 mRNA at days 16 and 23 post infection with HIV-1 JRFL; n = 3 independent infections of 03SF iPSC-derived microglia; two-way ANOVA; Šídák multiple comparisons test; ∗ p < 0.05; data are presented as the mean ± SEM. (F) Luminex cytokine/chemokine analysis of secreted M-CSF and IP-10/CXCL10 culture supernatant from HIV-1 JRFL-infected microglia; n = 3 independent infections of 03SF microglia, two-way ANOVA or Mixed-effects model, respectively; Šídák or Holm-Šídák multiple comparisons test, respectively; ∗ p < 0.05; data are presented as the mean ± SEM. (G) Representative confocal images of nucleocapsid protein p24 staining in HIV-1 JRFL-infected 03SF-derived microglia (IBA1 + ). Scale bars, 10 μm.

Article Snippet: The amount of viral p24 was determined using the HIV-1 Gag p24 DuoSet ELISA (R &D System, DY7360-05).

Techniques: Infection, Activation Assay, Quantitative RT-PCR, Derivative Assay, Enzyme-linked Immunosorbent Assay, Luminex, Staining

Journal: iScience

Article Title: Sustained type I interferon signaling after human immunodeficiency virus type 1 infection of human iPSC derived microglia and cerebral organoids

doi: 10.1016/j.isci.2024.109628

Figure Lengend Snippet: HIV-1-infected microglia exhibit productive infection, activation, and dysregulated cytokine/chemokine production (A) RT-qPCR analysis of viral gag/pol mRNA at days 9, 16, 23, and 30 post infection with HIV-1 JRFL and HIV-1 YU2; two-way ANOVA; ∗ p < 0.05, ∗∗ p < 0.01; n = 3 independent infections of CD06 iPSC-derived microglia; data are presented as the mean ± SEM. (B) ELISA analysis of secreted p24 in the culture supernatant of microglia infected with HIV-1 JRFL and HIV-1 YU2 at days 9, 15, 21, and 27 reveals productive infection; n = 3 independent infections of CD06 iPSC-derived microglia; two-way ANOVA; ∗∗∗∗ p < 0.0001; data are presented as the mean ± SEM. (C) RT-qPCR analysis of IL1 β and TNFα mRNA at days 9, 16, 23, and 30 post infection with HIV-1 JRFL and HIV-1 YU2; n = 3 independent infections of CD06 iPSC-derived microglia; two-way ANOVA; Holm-Šídák multiple comparisons test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; data are presented as the mean ± SEM. (D) RT-qPCR analysis of CCL2 and CCL3 mRNA at days 9, 16, 23, and 30 post infection with HIV-1 JRFL and HIV-1 YU2; n = 3 independent infections of CD06 iPSC-derived microglia; two-way ANOVA; Holm-Šídák multiple comparisons test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; data are presented as the mean ± SEM. (E) RT-qPCR analysis of IBA1 and P2RY12 mRNA at days 9, 16, 23, and 30 post infection with HIV-1 JRFL and HIV-1 YU2; n = 3 independent infections of CD06 iPSC-derived microglia; two-way ANOVA; Holm-Šídák multiple comparisons test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; data are presented as the mean ± SEM.

Article Snippet: The amount of viral p24 was determined using the HIV-1 Gag p24 DuoSet ELISA (R &D System, DY7360-05).

Techniques: Infection, Activation Assay, Quantitative RT-PCR, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Sustained type I interferon signaling after human immunodeficiency virus type 1 infection of human iPSC derived microglia and cerebral organoids

doi: 10.1016/j.isci.2024.109628

Figure Lengend Snippet: HIV-1-infected microglia incorporate into sliced cerebral organoids (A) Representative confocal images of a day 144 organoid with neurons (MAP2 + ), astrocytes (GFAP + ), and microglia (IBA1 + ). Scale bars, 250 μm. (B) Representative confocal images of day 144 organoids 14 days after the addition of HIV-1 JRFL-infected or mock-infected microglia; (B1) shows a high-magnification image of HIV-1-infected microglia from (B). Scale bars, 250 μm or 20 μm as indicated. (C) High-magnification image of a single HIV-1-infected microglial cell within a sliced organoid. Scale bars, 20 μm. (D) RT-qPCR of gag/pol mRNA from HIV-1 JRFL-infected or mock-infected microglia organoids on day 14; n = 3 independent infections of 03SF microglia; one-tailed t test; ∗∗∗∗ p < 0.0001; data are presented as the mean ± SEM. (E) ELISA analysis of secreted p24 in culture supernatant from HIV-1 JRFL-infected or mock-infected microglia organoid day 14; n = 3 independent infections of 03SF microglia; one-tailed t test; ∗∗ p < 0.01; data are presented as the mean ± SEM. (F) RT-qPCR analysis of ISG15 , RSAD2 , IFITM3 , MX1 , IFI44 , and IFI27 mRNA from HIV-1 JRFL-infected or mock-infected microglia organoids on day 14; n = 3 independent infections of 03SF microglia; one-tailed t test; ∗ p < 0.05; data are presented as the mean ± SEM.

Article Snippet: The amount of viral p24 was determined using the HIV-1 Gag p24 DuoSet ELISA (R &D System, DY7360-05).

Techniques: Infection, Quantitative RT-PCR, One-tailed Test, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Sustained type I interferon signaling after human immunodeficiency virus type 1 infection of human iPSC derived microglia and cerebral organoids

doi: 10.1016/j.isci.2024.109628

Figure Lengend Snippet:

Article Snippet: The amount of viral p24 was determined using the HIV-1 Gag p24 DuoSet ELISA (R &D System, DY7360-05).

Techniques: Virus, Recombinant, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Software

Journal: Nature communications

Article Title: Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.

doi: 10.1038/ncomms4477

Figure Lengend Snippet: Figure 5 | ARF6 depletion does not affect viral budding. (a) Western blot illustrating sustained ARF6 depletion during the time course of the experiment, viral release being measured 6 days after RNAi treatment. (b,c) MCF-7 cells infected with VSV-G-pseudotyped HIV-1 were analysed by confocal fluorescence microscopy to test for their ability to allow viral budding. (b) Viral envelope proteins were stained with anti-Env SUgp120 antibodies (green) and viruses that completed their budding were detected with an antibody recognizing mature matrix protein MAp17 (red). Note the high level of colocalization illustrating that most virions completed their budding at the plasma membrane. Scale bar, 5 mm. Nuclei were stained with DAPI (blue). (c) The total MAp17 fluorescence was divided by the total number of nuclei in each field, calculated for 150 to 200 cells in three independent experiments. Histograms represent the mean fluorescence intensity per nucleus±s.d. as an estimation of the number of mature viruses released per cell. NS, non significant (Student’s t-test). (d) After RNAi treatment, MCF-7 cells were infected with VSV-G-pseudotyped HIV-1. Three days after infection, cells were analysed by confocal fluorescence microscopy using a p24 antibody to stain intracellular Gag and the mature virus. The total p24 fluorescence was divided by the total number of nuclei in each field, calculated for 150 to 200 cells in three independent experiments. Histograms represent the mean fluore- scence intensity per nucleus±s.d. NS, non significant (Student’s t-test). (e) Virus release was determined by CAp24 quantification in the supernatant, using a HIV-1 p24 ELISA kit. p24 release was divided by the total number of cell. Results were normalized to 100% in siCNT cells. NS, non significant (Student’s t-test).

Article Snippet: Virus concentration was determined by CAp24 quantification in the supernatant, using a HIV-1 p24 ELISA kit (PerkinElmer Life Science, Inc.).

Techniques: Western Blot, Infection, Microscopy, Staining, Clinical Proteomics, Membrane, Virus, Enzyme-linked Immunosorbent Assay

Journal: EMBO molecular medicine

Article Title: R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

doi: 10.1002/emmm.201202232

Figure Lengend Snippet: Figure 1. R5 but not X4 HIV-1 induces DCs to migrate through a monolayer of epithelial cells. Caco-2 cells were grown on transwell filter to form a confluent monolayer then DCs were let to adhere to the bottom of the filter. Cell-free HIV-1, LPS (1 mg/ml) or medium (DMEM 10% FCS) were incubated on the apical side of the Caco-2 monolayer for 1.5 h. Filters were processed for CM. A–F. Three-dimensional rendering of representative fields obtained with Volocity 5.0 software. Mouse anti-human E-Cadherin þ Alexafluor488 goat anti-mouse IgG2a (green) depicts interepithelial adherent junctions. Mouse anti-human DC-SIGN-PE (red) labels the DCs, which creep through epithelial cells in response to incubation with R5 HIV-1J6363 (A, at 1 ng of p24 Ag) and HIV-1AD8 (B, at 24 ng of p24 Ag) but do not in response to X4 HIV-1IIIB (D, at 10 ng of p24 Ag) and HIV-1pNL4.3 (E, at 40 ng of p24 Ag). LPS (C) and medium (F) were positive and negative controls. The experiment was repeated three times. G,H. Quantitative analysis of DCs migration across the Caco-2 cell monolayer at the apical (G) and medial (H) level of the cell layer is shown. Results are expressed as percentage of area occupied by DCs compared to that of the whole field. Bars represent mean SD of three or four fields of three different experiments. Statistic analysis was performed as described in Materials and Methods Section. p < 0.05.

Article Snippet: Culture supernatants were collected at consecutive time points up to 21 days and viral growth determined by an HIV-1 p24 Ag ELISA (lower detection limit 4 pg/ml, Aalto Bioreagents, www.aaltobioreagents.ie).

Techniques: Incubation, Software, Migration

Journal: EMBO molecular medicine

Article Title: R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

doi: 10.1002/emmm.201202232

Figure Lengend Snippet: Figure 2. DCs migration does not alter junctional protein expression by Caco-2 cells and preserves the integrity of the HIV-1 treated monolayer. The Caco-2/DCs co-culture system was incubated with medium or R5 HIV-1AD8 at 24 ng of p24 Ag for 1.5 h. A–D. CM cross-sectional images of specimens stained for DCSIGN-PE (red) and for the epithelial junctions (green) JAM (A), E-Cadherin (B) Occludin (C) ZO-1 (D) showed that HIV-1 as well as migrated DCs did not affect the intraepithelial junctions expression in the Caco-2/DC system. DAPI stained the nuclei. Results are from one representative experiment out of three. E. Ultrastructure of Caco-2/DCs culture treated with R5 HIV-1J6363 (at 1 ng of p24 Ag) for 1.5 h. TEM images show DCs adhering to the filter (), DCs protrusions inside the filter membrane pore () and a DC interposed between adjacent Caco-2 cells () Scale bar: 2 mm. Numbers identify the corresponding magnified images displayed in panel (E), and arrows point to interepithelial TJs (1, 2), TJs-like structures between DCs and Caco-2 cells (3–5), contiguity among cells inside the pore (6), and junction like-structures between DCs (7) Scale bar: 500 nm. Results from a representative experiment out of three are shown. F. Semi-thin sections for TEM labelled with Toluidine blue and the corresponding explicative colour mask below (Caco-2 cells red, DCs blue, filter grey) show the morphology and the spatial organization of cells. Caco-2 cells are columnar and polarized, displaying microvilli, dense cytoplasmic granules and vacuoles characteristic of epithelial cells. DCs are disposed along the lower face of the filter, inside the membrane pores and, in HIV-1 treated sample, intercalated in between Caco-2 cells, without destroying epithelial monolayer continuity. G. The permeability of the Caco-2/DCs culture to FD4 (4 kDa, 250 mg/ml) in the presence of medium, R5 HIV-1AD8 (at 24 ng of p24 Ag) and CN54 gp140 protein (100 ng/ml) is shown as percentage of the positive control (i.e. FD4 added in the upper chamber of the transwell without Caco-2 cells). Triton X100 was included as control of barrier disruption. Results are mean values SD of triplicates from a representative experiment out of three.

Article Snippet: Culture supernatants were collected at consecutive time points up to 21 days and viral growth determined by an HIV-1 p24 Ag ELISA (lower detection limit 4 pg/ml, Aalto Bioreagents, www.aaltobioreagents.ie).

Techniques: Migration, Expressing, Co-Culture Assay, Incubation, Staining, Membrane, Permeability, Positive Control, Control, Disruption

Journal: EMBO molecular medicine

Article Title: R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

doi: 10.1002/emmm.201202232

Figure Lengend Snippet: Figure 4. R5 HIV-1 induces migration of DCs through the colonic epithelium. Colonic tissue was either left untreated (A and B) or incubated with X4 HIV-1pNL4.3 (C), R5 HIV-1AD8 (D) or R5 HIV-1J6363 (E–J) (at 50 ng of p24 Ag) for 30 min. CD11cþ cells were detected in the colonic lamina propria of untreated (B) and HIV-1pNL4.3 treated (C) tissues but not in between epithelial cells. Following R5 HIV-1 incubation protruded dendrites (D) or whole DCs (E–H) were observed inside the epithelium. Moreover DCs (DCSIGNþ/CD68

Article Snippet: Culture supernatants were collected at consecutive time points up to 21 days and viral growth determined by an HIV-1 p24 Ag ELISA (lower detection limit 4 pg/ml, Aalto Bioreagents, www.aaltobioreagents.ie).

Techniques: Migration, Incubation

Journal: EMBO molecular medicine

Article Title: R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

doi: 10.1002/emmm.201202232

Figure Lengend Snippet: Figure 5. DCs capture HIV-1 in colonic explant and in vitro Caco-2/DC model. Human colonic tissue was either left untreated (A) or incubated with R5 HIV-1J6363 (B) or R5 HIV-1AD8 (C and D) (at 50 ng of p24 Ag) for 30 min. HIV-1 virions co-localized with CD11cþ DCs that migrate inside the epithelium as well as with DCs closely underlying the epithelium (see white arrows in B and C). Moreover virions were detected at both the apical and basal side as well as penetrating the epithelium. Yellow arrows point to virions entrapped in the mucus. Absence of p24 Ag in the basal medium, determined with ELISA, confirmed the seal integrity of the tissue culture system without any viral leakage (as described in Materials and Methods Section). Panel (D) is a magnification (zoom 3) of the boxed area in C. Cryosections were fixed with 4% PFA and stained with mouse anti-human CD11c-PE (DCs; red), mouse anti-p24 þ Alexafluor488 goat anti-mouse IgG (HIV-1; green), and DAPI (nuclei; blue). Scale bar indicate the magnifications. Each Figure is representative of results from three donor tissues. Caco-2/DCs co-culture was incubated with R5 HIV-1AD8-GFP (at 20 ng of p24 Ag) for 30 min (E), 1.5 h (F) or 4.5 h (G). (A and B) Three-dimensional renderings from CM z-series stacks of representative images (acquired with objective 40, zoom 6.5) showed GFP-expressing virions (green, indicated by arrows) either on migrated (E) as well as non- migrated DCs at the basal side (F, the bottom side of the filter is shown). Cells were stained for DAPI (all cells; blue) and mouse anti-human CD11c þ AlexaFluor594 goat anti-mouse IgG (DCs; red). (G) Three-dimensional rendering from a CM z-series stack of images showed a cluster of DCs (visualized with mouse anti-human DC-SIGN-PE; red) migrated to the Caco-2 side of the culture (Caco-2 cells not shown in the Figure) after incubation with HIV-1 AD8–GFP. GFP-expressing virions (yellow) were associated to migrated DCs.

Article Snippet: Culture supernatants were collected at consecutive time points up to 21 days and viral growth determined by an HIV-1 p24 Ag ELISA (lower detection limit 4 pg/ml, Aalto Bioreagents, www.aaltobioreagents.ie).

Techniques: In Vitro, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Co-Culture Assay, Expressing

Journal: EMBO molecular medicine

Article Title: R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

doi: 10.1002/emmm.201202232

Figure Lengend Snippet: Figure 7. DCs migration is dependent from the viral envelope. In the absence of the HIV-1 env as well as of the V1V3 env region, DCs did not migrate across the Caco-2 monolayer (A and B). The V1V3 env region of an R5 virus completely restored the migratory ability of DCs (C). A–C. The Caco-2/DCs co-culture was incubated apically for 1.5 h with HIV-1 deleted of the env (HIV-1Denv, 6 ng of p24 Ag) (A), the HIV-143DV deleted of the V1V3 region (40 ng of p24 Ag) as negative control (B) and a recombinant HIV-1 with the V1V3 env region of the R5 HIV-1J6363 primary virus recombined with the HIV-143DV backbone (HIV-1J6363-43DV, 20 ng of p24 Ag) (C). Three-dimensional reconstructions from CM z-series image stacks of the Caco-2/DCs culture stained for DAPI (epithelial cells and DCs; blue) and mouse anti-human DC-SIGN-PE (DCs; red) are shown. D,E. The monolayer of the Caco-2/DCs system was incubated for 1.5 h with medium (negative control), HIV-1YU2 (positive control) or YU2 gp120 protein (100 ng/ml). Bar charts represent quantitative analysis of DCs migration across the Caco-2 cell at the apical (D) and medial (E) level of the cell monolayer (as in Fig 1). Results are expressed as percentage of area occupied by DCs compared to that of the whole field. Bars represent mean SD of three different fields of three different experiments. Statistic analysis was performed as described in Materials and Methods Section. p < 0.05.

Article Snippet: Culture supernatants were collected at consecutive time points up to 21 days and viral growth determined by an HIV-1 p24 Ag ELISA (lower detection limit 4 pg/ml, Aalto Bioreagents, www.aaltobioreagents.ie).

Techniques: Migration, Virus, Co-Culture Assay, Incubation, Negative Control, Recombinant, Staining, Positive Control

Journal: EMBO molecular medicine

Article Title: R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

doi: 10.1002/emmm.201202232

Figure Lengend Snippet: Figure 9. HIV-1 penetrates within and in between epithelial cells. A. Virions (stained with mouse anti-p24 þ Alexafluor488 goat anti-mouse IgG, green) were mainly localized at the apical surface of Caco-2 cells at 30 min but also inside the cytoplasm at 90 min of incubation with R5 HIV-1AD8 (20 ng of p24 Ag). Shown are CM single plane cross sectional images of a Caco-2 monolayer (rabbit anti-human Occludin þ Alexafluor594 goat anti-rabbit IgG; red) taken at apical and medial level of the cell layer. B. R5 HIV-1AD8 (red; visualized with human anti-gp120 monoclonal antibody 2G12 þ Alexafluor594 goat anti-human IgG) localized inside the cyto- plasm of epithelial cells and in intrajunctional spaces (indicated by arrows). Shown are four single plane cross sectional images, taken from the apical to the medial plane along the z-axis of the Caco-2 monolayer (mouse anti-human E-Cadherin þ Alexafluor488 rabbit anti-mouse IgG2a; green) incubated with R5 HIV-1J6363 (at 5 ng of p24 Ag) for 90 min. Results are of one representative experiment out of three.

Article Snippet: Culture supernatants were collected at consecutive time points up to 21 days and viral growth determined by an HIV-1 p24 Ag ELISA (lower detection limit 4 pg/ml, Aalto Bioreagents, www.aaltobioreagents.ie).

Techniques: Staining, Incubation

Journal: EMBO molecular medicine

Article Title: R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

doi: 10.1002/emmm.201202232

Figure Lengend Snippet: Figure 10. Transcytosis of cell-free HIV-1 through a tight monolayer of Caco-2 cells. A–E. R5 HIV-1J6363 (A, at 1 ng of p24 Ag), X4 HIV-1IIIB (B, at 10 ng of p24 Ag), R5 93BR029 gp140 protein (C, at 100 ng/ml), R5 HIV-1SF162 (D, at 2 ng of p24 Ag), or medium (E), were added to the Caco-2/DCs cultures for 90 min and immunostained for CM analysis. HIV-1 (red; visualized with human anti-gp120 monoclonal antibody 2G12 þ Alexafluor594 goat-anti-human IgG) was dispersed throughout the cytoplasm but mainly concentrated in the upper part of the Caco-2 cells (visualized with mouse anti-human JAM þ Alexafluor 488 goat-anti-mouse IgG; green). DCs are not shown in the Figure. Transversal xy- and xz-plane visualization from representative fields of the monolayer were obtained with Volocity 5.0 software. 1 unit ¼ 10.3 mm. Results show one representative experiment out of three. F. Transcytosis of HIV-1 R5 and X4 is comparable. The amount of transcytosed virus was evaluated measuring with ELISA the p24 Ag released in the basal chamber after 2.5 h of incubation with cell-free HIV-1 (at 20 ng of p24 Ag), either R5 HIV-1AD8 or X4 HIV-1pNL4.3, on the apical side of the Caco-2 monolayer cultured with or without DCs adherent to the filter. Results are expressed as mean SD of triplicates of three different experiments.

Article Snippet: Culture supernatants were collected at consecutive time points up to 21 days and viral growth determined by an HIV-1 p24 Ag ELISA (lower detection limit 4 pg/ml, Aalto Bioreagents, www.aaltobioreagents.ie).

Techniques: Software, Virus, Enzyme-linked Immunosorbent Assay, Incubation, Cell Culture

Journal: EMBO molecular medicine

Article Title: R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

doi: 10.1002/emmm.201202232

Figure Lengend Snippet: Figure 11. DCs capture virus and transfer infection to target cells. A,B. Caco-2/DCs system was apically incubated for 1.5 h with R5 HIV-1AD8 at 10 or 40 ng (A) or 20 ng of p24 Ag (B). PBMCs (5 105 cells) directly incubated with the same amount of input virus served as positive control (data not shown). (A) R5 virus does efficiently replicated when DCs were cultured with PBMCs (1 106). Low levels of p24 Ag were observed when DCs were cultured alone (inset). (B) DCs transmit infectious virus in the long-term. PBMCs were added to detached DCs immediately or after 3 or 4 days. Results from a representative experiment out of three are expressed as mean of p24 Ag values of triplicate cultures SD. C. DCs transferred both R5 and X4 viruses to PBMCs. Caco-2 cells were treated with R5 HIV-1AD8 (24 ng/ml) and X4 HIV-1pNL4.3 (40 ng/ml) as described above. DCs collected from transwell were co-cultured with PHA-activated PBMCs. Mean SD of three different experiments per- formed in triplicate is shown. p < 0.05.

Article Snippet: Culture supernatants were collected at consecutive time points up to 21 days and viral growth determined by an HIV-1 p24 Ag ELISA (lower detection limit 4 pg/ml, Aalto Bioreagents, www.aaltobioreagents.ie).

Techniques: Virus, Infection, Incubation, Positive Control, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Transcriptomic Profiling Identifies TALAM1 and LINC00702 as HIV-1-Responsive lncRNAs in Microglia

doi: 10.3390/ijms27073271

Figure Lengend Snippet: Polyadenylated transcriptome changes in C20 microglial cells upon HIV-1 infection or TNF-α stimulation compared to mock. ( A ) Experimental workflow for RNA-seq analysis of C20 cells under three conditions: untreated (Mock), HIV-1-infected (HIV, 24 h post-infection), and TNF-α-stimulated (TNF, 24 h). Polyadenylated RNA was then sequenced, aligned, quantified, and classified into protein-coding, lncRNA, and other RNA types. ( B ) Volcano plot showing differential expression of protein-coding and non-coding transcripts in HIV compared to Mock. ( C ) Volcano plot showing differential expression of protein-coding and non-coding transcripts in TNF compared to Mock. ( D ) Gene set enrichment analysis (GSEA) of pathways enriched among differentially expressed transcripts in HIV and TNF conditions. Shared pathways include immune response, inflammation, vesicular trafficking, cell cycle regulation, intracellular signaling, and response to viral infection, highlighting common transcriptional programs activated by viral infection and proinflammatory stimulation. Created in BioRender. Toro-Ascuy, D. (2026) https://BioRender.com/qeljpe0 (accessed on 2 December 2025).

Article Snippet: At 72 h post-transfection, the pseudotyped virus was recovered from the supernatants, filtered through a 0.22 μm filter, and the p24 viral protein was quantified using the HIV-1 Gag p24 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions, as described previously [ ].

Techniques: Infection, RNA Sequencing, Quantitative Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Transcriptomic Profiling Identifies TALAM1 and LINC00702 as HIV-1-Responsive lncRNAs in Microglia

doi: 10.3390/ijms27073271

Figure Lengend Snippet: Effect of TALAM1 and LINC0702 silencing on HIV-1 infection in C20 microglia. ( A , B ) Relative expression of TALAM1 ( A ) and LINC00702 ( B ) in HIV-infected C20 cells after silencing with two independent antisense oligonucleotides (ASOs), compared with transfection control (TC). ( C , D ) Relative HIV-1 genomic RNA (gRNA) levels following TALAM1 ( C ) or LINC00702 ( D ) silencing. ( E , F ) HIV-1 protein expression after TALAM1 ( E ) or LINC00702 ( F ) silencing, showing Gag p55 (55 kDa) and p24 (24 kDa), with GAPDH (37 kDa) as loading control. Data are shown as mean ± s.d.; Unpaired t -test, n = 3, ns ≥ 0.05, p < 0.05.

Article Snippet: At 72 h post-transfection, the pseudotyped virus was recovered from the supernatants, filtered through a 0.22 μm filter, and the p24 viral protein was quantified using the HIV-1 Gag p24 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions, as described previously [ ].

Techniques: Infection, Expressing, Transfection, Control

Journal: International Journal of Molecular Sciences

Article Title: Transcriptomic Profiling Identifies TALAM1 and LINC00702 as HIV-1-Responsive lncRNAs in Microglia

doi: 10.3390/ijms27073271

Figure Lengend Snippet: Functional characterization of TALAM1 and LINC00702 interactomes. ( A , B ) Subcellular distribution of TALAM1 ( A ) and LINC00702 ( B ) in nuclear and cytoplasmic fractions from Mock and HIV-1 infected C20 microglial cells at 24 h post-infection. Values represent the relative percentage of RNA detected in each compartment. Data are shown as mean ± s.d., Two-way ANOVA, n = 3, ns ≥ 0.05, p < 0.05. ( C ) Genomic neighborhood of TALAM1 and LINC00702 visualized using the UCSC Genome Browser. ( D ) Reactome pathways enriched for TALAM1 target genes predicted by NPInter v5.0. ( E ) Reactome pathways enriched for LINC00702 target genes predicted by NPInter v5.0. ( F , G ) Overlap between HIV-1-associated proteins (HIV-1 Human Interaction Database) and predicted target genes of TALAM1 and LINC00702.

Article Snippet: At 72 h post-transfection, the pseudotyped virus was recovered from the supernatants, filtered through a 0.22 μm filter, and the p24 viral protein was quantified using the HIV-1 Gag p24 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions, as described previously [ ].

Techniques: Functional Assay, Infection

Journal: Biology of reproduction

Article Title: CD9 is a surface marker on mouse and rat male germline stem cells.

doi: 10.1095/biolreprod.103.020867

Figure Lengend Snippet: FIG. 1. Expression of CD9 in mouse testis. A) Characterization of anti-CD9 antibody-purified mouse testis cells using flow cytometry. Unselected (upper row) and anti-CD9 antibody-selected (lower row) testis cells were stained with FITC-conjugated streptavidin, PE-conjugated anti-a6-integrin antibody, or APC-conjugated anti-c-kit antibody, and the fluorescence was compared with that of controls. The dotted lines show the range of fluores- cence in the controls. Note the increased percentages of all three antigens in the anti-CD9 antibody-selected cell population. B) Immunohistological staining of CD9 antigen in mouse testis. Positive cells were found on the basement membrane of seminiferous tubules (arrows). Staining was also observed in the interstitial cells. Bar 5 50 mm (section). Stain: DAB followed by hematoxylin.

Article Snippet: Mouse anti-rat CD9 antibody was used with goat anti-mouse IgG microbeads (25 ml; Miltenyi Biotec) to select cells expressing rat CD9 molecules.

Techniques: Expressing, Cytometry, Staining, Membrane

Journal: Biology of reproduction

Article Title: CD9 is a surface marker on mouse and rat male germline stem cells.

doi: 10.1095/biolreprod.103.020867

Figure Lengend Snippet: FIG. 2. Expression of CD9 on mouse spermatogonial stem cells. A) Gross and histological appearance of recipient testes after transplantation of LacZ-marked donor testis cells. Left: Testis from a W recipient mouse 2 mo after transplantation of anti-CD9 antibody-selected cells. Middle: Testis from a W recipient mouse 2 mo after transplantation of unselected testis cells. Right: Histological section from a W recipient testis 2 mo after transplantation of anti-CD9-antibody-selected testis cells. Note the normal appearance and organization of spermatogenesis. Stain: X-gal followed by hematoxylin and eosin. B) Enhanced colonization of recipient testis by anti-CD9 antibody-selected mouse testis cells. The degree of coloni- zation in three experiments is indicated by the number of individual blue colonies. The values (mean 6 SEM) for anti-CD9 antibody-selected cells and unselected cells were 5.5 6 0.8 (n 5 16) and 0.8 6 0.2 (n 5 18) cells per 3 3 104 injected cells, respectively. Bars 5 1 mm (testes) and 50 mm (section).

Article Snippet: Mouse anti-rat CD9 antibody was used with goat anti-mouse IgG microbeads (25 ml; Miltenyi Biotec) to select cells expressing rat CD9 molecules.

Techniques: Expressing, Transplantation Assay, Staining, Injection

Journal: Biology of reproduction

Article Title: CD9 is a surface marker on mouse and rat male germline stem cells.

doi: 10.1095/biolreprod.103.020867

Figure Lengend Snippet: FIG. 3. Expression of CD9 on rat spermatogonial stem cells. A) Characterization of anti-CD9 antibody-purified rat testis cells using flow cytometry. Unselected (left) and anti-CD9 antibody-selected (right) testis cells were stained with Cy5-conjugated anti-mouse IgG antibody, and the fluorescence was compared with similar cell controls. The dotted lines show the control range of fluorescence. Note the increase in percentage of CD9-positive cells after magnetic selection. B) The appearance of recipient testes after transplantation of green fluorescent protein-marked donor testis cells. Seminiferous tubules were dissected using fine forceps. Left: Testis from a busulfan-treated nude recipient mouse 3 mo after transplantation of unselected testis cells (8.2 3 104 cells injected). Right: Testis from a busulfan-treated nude recipient mouse 3 mo after transplantation of anti-CD9 antibody-selected cells (2.7 3 104 cells injected). Note the increase in number of colonies of CD9-positive cells despite the lower concentration of injected cells. Bar 5 100 mm. C) Enhanced colonization of recipient testis by anti-CD9 antibody-selected rat testis cells. The number of individual fluorescent colonies represents the degree of colonization in three experiments. The values (mean 6 SEM) for anti-CD9 antibody-selected and unselected cells were 15.8 6 4.2 (n 5 16) and 3.1 6 0.9 (n 5 13) cells per 105 injected, respectively.

Article Snippet: Mouse anti-rat CD9 antibody was used with goat anti-mouse IgG microbeads (25 ml; Miltenyi Biotec) to select cells expressing rat CD9 molecules.

Techniques: Expressing, Cytometry, Staining, Control, Selection, Transplantation Assay, Injection, Concentration Assay