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: Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology

Article Title: Performance evaluation of the CHEMBIO DPP ® (dual path platform) HIV-1/2 assay in early and established infections

doi: 10.1016/j.jcv.2015.07.005

Figure Lengend Snippet: Sensitivity of assay reactivity during early HIV-1 infections as number of days before first positive WB when 50% of specimens tested with each test became positive. The names, abbreviations, and sources, of the HIV assays previously evaluated 11,12 are as follows: APTIMA HIV-1 Quantitative assay (Aptima, Gen-Probe, Inc., San Diego, CA); ARCHITECT® HIV Ag/Ab Combo assay (Architect; Abbott Diagnostics, Wiesbaden Germany; CE marked version was used as the US version was not available when testing was conducted); GS HIV Combo Ag/Ab (BioRad Combo; Bio-Rad Laboratories, Redmond, WA); DetermineTM HIV-1/2 Ag/Ab Combo (Determine Combo (rapid test); Alere Medical Co., Ltd. Scarborough, ME); GS HIV-1/HIV-2 PLUS O EIA (GS + O; Bio-Rad Laboratories, Redmond, WA); VITROS anti-HIV 1 + 2 assay (Vitros; Ortho-Clinical Diagnostics, Buckinghamshire, UK); ADVIA Centaur HIV 1/O/2 enhanced assay (Advia; Bayer, Tarrytown, NY); Abbott HIVAB HIV-1/2 (rDNA) EIA (Abbott; Abbott Laboratories, Abbott Park, IL); Avioq HIV-1 Microelisa system (Avioq; Avioq, Inc, Rockville, MD); Multispot HIV-1/HIV-2 rapid test (Multispot (rapid test); Bio-Rad Laboratories); Clearview HIV-1/2 STAT-PAK (Statpak (rapid test); Inverness Medical, Princeton, NJ); Clearview Complete HIV-1/2 (Complete (rapid test); Inverness Medical); Reveal G2 and G3 Rapid HIV-1 antibody tests (Reveal G2 or G3 (rapid test); MedMira Laboratories, Inc.; Halifax, Nova Scotia, Canada); OraQuick ADVANCE Rapid HIV-1/2 antibody test (Oraquick (rapid test); OraSure Technologies, Inc.; Bethlehem, PA); Uni-Gold Recombigen HIV (Unigold (rapid test); Trinity Biotech USA, St. Louis, MO). These assays have manufacturer reported point estimates for sensitivity ranging from 99.60% to 100.00% and point estimates for specificity ranging from 98.60% to 99.90%. The Genetic Systems HIV-1 Western blot (WB; Bio-Rad Laboratories) and Cambridge Biotech HIV-1 Western blot (WB; Maxim Biomedical Inc., Rockville, MD) have been shown to give concordant interpretations in studies conducted to qualify use in our clinical laboratory and were used interchangeably.

Article Snippet: The names, abbreviations, and sources, of the HIV assays previously evaluated 11,12 are as follows: APTIMA HIV-1 Quantitative assay (Aptima, Gen-Probe, Inc., San Diego, CA); ARCHITECT ® HIV Ag/Ab Combo assay (Architect; Abbott Diagnostics, Wiesbaden Germany; CE marked version was used as the US version was not available when testing was conducted); GS HIV Combo Ag/Ab (BioRad Combo; Bio-Rad Laboratories, Redmond, WA); DetermineTM HIV-1/2 Ag/Ab Combo (Determine Combo (rapid test); Alere Medical Co., Ltd. Scarborough, ME); GS HIV-1/HIV-2 PLUS O EIA (GS + O; Bio-Rad Laboratories, Redmond, WA); VITROS anti-HIV 1 + 2 assay (Vitros; Ortho-Clinical Diagnostics, Buckinghamshire, UK); ADVIA Centaur HIV 1/O/2 enhanced assay (Advia; Bayer, Tarrytown, NY); Abbott HIVAB HIV-1/2 (rDNA) EIA (Abbott; Abbott Laboratories, Abbott Park, IL); Avioq HIV-1 Microelisa system (Avioq; Avioq, Inc, Rockville, MD); Multispot HIV-1/HIV-2 rapid test (Multispot (rapid test); Bio-Rad Laboratories); Clearview HIV-1/2 STAT-PAK (Statpak (rapid test); Inverness Medical, Princeton, NJ); Clearview Complete HIV-1/2 (Complete (rapid test); Inverness Medical); Reveal G2 and G3 Rapid HIV-1 antibody tests (Reveal G2 or G3 (rapid test); MedMira Laboratories, Inc.; Halifax, Nova Scotia, Canada); OraQuick ADVANCE Rapid HIV-1/2 antibody test (Oraquick (rapid test); OraSure Technologies, Inc.; Bethlehem, PA); Uni-Gold Recombigen HIV (Unigold (rapid test); Trinity Biotech USA, St. Louis, MO).

Techniques: Sensitive Assay, Western Blot

Journal: Virology

Article Title: Epitope mapping and characterization of a novel CD4-induced human monoclonal antibody capable of neutralizing primary HIV-1 strains

doi: 10.1016/S0042-6822(03)00521-X

Figure Lengend Snippet: Fig. 1. CD4 induction of the binding of the E51 antibody to HIV-1 gp120. The YU2 HIV-1 gp120 glycoprotein was captured on an ELISA plate using the D7324 antibody, which is directed against the gp120 carboxyl-termi- nus. The binding of the 17b and E51 antibodies to the captured gp120 glycoprotein was studied in the absence (open circles) or presence (filled circles) of sCD4 (8 g/ml).

Article Snippet: The ELISA plate was coated with sheep antibody D7324 (Aalto BioReagents, Dublin, Ireland), which recognizes the gp120 carboxylterminus.

Techniques: Binding Assay, 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: Retrovirology

Article Title: Immune regulator ABIN1 suppresses HIV-1 transcription by negatively regulating the ubiquitination of Tat

doi: 10.1186/s12977-017-0338-5

Figure Lengend Snippet: ABIN1 knockdown increases HIV-1 replication. a Jurkat T cells were transfected with siRNAs targeting ABIN1 (siABIN1-1 or siABIN1-2) or scramble siRNA (siNC) as described in “ ”. 36 h post transfection (hpt), cells were counted and challenged with HIV-1 strain NL4-3. At 1, 3, 5 days post infection, the cells and supernatants were harvested to measure viral production by p24 ELISA of the supernatants, against a standard curve. Cells were lysed and subjected to Western Blots to determine the knockdown efficiency of ABIN1. The lower panel showed the ABIN1 knockdown efficiency at day 5 as the representative. b The experiments were conducted similarly as in ( a ), except that human primary CD4+ T lymphocytes were used. c , d Determination of ABIN1 mRNA level after viral infection. Jurkat cells were challenged with HIV(NL4-3) as in a , b , at 0, 24, 72 hpi, cells were harvested for RNA extraction. The mRNA levels of ABIN1 and HIV-1 gag was measured by real time PCR, normalized to cellular GAPDH. Data are shown as mean ± SD of triplicate samples and are representative of at least three independent experiments. p values were calculated based on unpaired t test and significant changes relative to siNC transfected cells or samples collected at 0 hpi. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The antibodies used were as follows: mouse antibodies to Flag (Sigma), Myc (Roche) and β-actin (Proteintech), rabbit antibodies to ABIN1 (Proteintech), Tat (Abcam), and HRP-conjugated anti-ubiquitin antibody (ENZO Life Sciences).

Techniques: Knockdown, Transfection, Infection, Enzyme-linked Immunosorbent Assay, Western Blot, RNA Extraction, Real-time Polymerase Chain Reaction

Journal: Retrovirology

Article Title: Immune regulator ABIN1 suppresses HIV-1 transcription by negatively regulating the ubiquitination of Tat

doi: 10.1186/s12977-017-0338-5

Figure Lengend Snippet: ABIN1 depletion stimulates the activity of HIV-1 promoter (Ghost-CXCR4 (X4) cells). a – e Ghost-X4 cells stably transfected a HIV-1 LTR driving GFP expression were treated with siNC, siABIN1-1, or siABIN1-2 before challenged with HIV-1(NL4-3). 48 hpi, cells were harvested and subjected to flowcytometry analysis. The effect of ABIN1 depletion on HIV-1 replication was determined by calculating the percentage of GFP positive cells, the relative mean fluorescence intensity (MFI) and relative median of GFP intensity of these GFP positive cells, over 10,000 cells were measured for each sample. a Indicates the basal level of GFP in non-infected Ghost-X4 cells. b – d Comparison of the cell counts at different GFP intensity after viral infection following ABIN1 knockdown. e The emphasis of the comparison. f , g Changing fold of MFI ( f ) and GFP intensity median ( g ) in infected cells following transfection of siRNAs targeting ABIN1 relative to siNC. The basal MFI and median of GFP intensity in siNC transfected cells were both set to 1000. h Representative of ABIN1 knockdown efficiency in Ghost-X4 cells. Data are shown as mean ± SD of triplicate samples and are representative of at least three independent experiments. p values were calculated based on unpaired t test and significant changes relative to siNC indicated. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The antibodies used were as follows: mouse antibodies to Flag (Sigma), Myc (Roche) and β-actin (Proteintech), rabbit antibodies to ABIN1 (Proteintech), Tat (Abcam), and HRP-conjugated anti-ubiquitin antibody (ENZO Life Sciences).

Techniques: Activity Assay, Stable Transfection, Transfection, Expressing, Fluorescence, Infection, Comparison, Knockdown

Journal: Retrovirology

Article Title: Immune regulator ABIN1 suppresses HIV-1 transcription by negatively regulating the ubiquitination of Tat

doi: 10.1186/s12977-017-0338-5

Figure Lengend Snippet: ABIN1 depletion stimulates the activity of HIV-1 promoter (HeLa cells). a – e HeLa cells were transfected with siNC, siABIN1-1, or siABIN1-2 for 36 h, then cells were challenged with VSV-G pseudotyped luciferase reporter HIV-1(Luc). Cells were harvested at the indicated time points post infection for luciferase activity analysis, DNA or RNA extraction. a Luciferase activities of the infected cells were monitored at 48 hpi ( upper panel ) and ABIN1 knockdown efficiency was determined by Western Blots as the representative ( lower panel ). b – d Total DNA of the infected cells were extracted and quantified for subsequent real-time PCR at b 12 hpi for late RT levels, c 24 hpi 2-LTR-circle levels, d 48 hpi proviral DNA levels. e Total RNA was extracted at 48 hpi, and used for quantitation of HIV-1 mRNA. f The effect of overexpressed ABIN1 on LTR promoter activity were determined by luciferase assays after co-transfection of Flag-ABIN1 expressing plasmid or vector control together with pNL4-3.Luc.R-.E-, and pRL-TK in HEK-293T cells for 24 h. g Expression of Flag-ABIN1 was determined by Western Blots. The real-time PCR analysis of DNA and RNA extract were normalized to cellular β-globin and GAPDH, respectively. The primer pairs used were as described in “ ”. Data are represented as mean ± SD of triplicate samples and are representative of at least three independent experiments. p values were calculated based on unpaired t test and significant changes relative to siNC or NC indicated. * p < 0.05; ** p < 0.01; *** p < 0.001, ns not significant

Article Snippet: The antibodies used were as follows: mouse antibodies to Flag (Sigma), Myc (Roche) and β-actin (Proteintech), rabbit antibodies to ABIN1 (Proteintech), Tat (Abcam), and HRP-conjugated anti-ubiquitin antibody (ENZO Life Sciences).

Techniques: Activity Assay, Transfection, Luciferase, Infection, RNA Extraction, Knockdown, Western Blot, Real-time Polymerase Chain Reaction, Quantitation Assay, Cotransfection, Expressing, Plasmid Preparation, Control

Journal: Retrovirology

Article Title: Immune regulator ABIN1 suppresses HIV-1 transcription by negatively regulating the ubiquitination of Tat

doi: 10.1186/s12977-017-0338-5

Figure Lengend Snippet: ABIN1 suppresses HIV-1 Tat ubiquitination via its ubiquitin binding property. a HEK-293T cells were transfected with siRNAs targeting ABIN1 (siABIN1-1, siABIN1-2) or siNC for 24 h, then a second round transfection of Flag-Tat expressing constructs was performed for another 24 h, and cells were then harvested and assayed as described in “ ”. b After transfection with Myc-ABIN1 and Flag-Tat expressing plasmids for 24 h as described in “ ”, the ubiquitination of Tat in HEK-293T cells was analyzed by Flag IP under denaturing conditions as in ( a ). c HEK-293T cells were co-transfected with plasmids encoding Myc tagged wild-type ABIN1 or ABIN1-QE2 mutant and Flag-Tat for 24 h, cells were then harvested and analyzed as in ( a ). d The effect of overexpressed ABIN1 and QE2 mutant were determined by luciferase assays after HIV-1(Luc) infection following ABIN1, QE2 or vector control (NC) transfection in HeLa cells. The panel below indicates the expression of ABIN1 or its mutant. β-actin was detected as sample loading control. Data are represented as mean ± SD of triplicate samples, all data and Western Blots are representative of at least three independent experiments. p values were calculated based on unpaired t test and significant changes relative to NC indicated. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The antibodies used were as follows: mouse antibodies to Flag (Sigma), Myc (Roche) and β-actin (Proteintech), rabbit antibodies to ABIN1 (Proteintech), Tat (Abcam), and HRP-conjugated anti-ubiquitin antibody (ENZO Life Sciences).

Techniques: Ubiquitin Proteomics, Binding Assay, Transfection, Expressing, Construct, Mutagenesis, Luciferase, Infection, Plasmid Preparation, Control, Western Blot

Journal: Retrovirology

Article Title: Immune regulator ABIN1 suppresses HIV-1 transcription by negatively regulating the ubiquitination of Tat

doi: 10.1186/s12977-017-0338-5

Figure Lengend Snippet: ABIN1 negatively regulates the K63-linked polyubiquitination of HIV-1 Tat. a To determine the effect of ABIN1 on the stability of Tat, HEK-293T cells were transfected with Myc-ABIN1 and Flag-Tat encoding plasmids as indicated for 24 h, followed by treatment of CHX at the final concentration of 10 μg/ml for 6 h to arrest cell translation. The levels of Tat protein were detected by Western Blot. b To elucidate whether ABIN1 targeting Tat for proteasome-dependent degradation, HEK-293T cells were transfected with siABIN1-1 or siNC for 24 h, followed by a second round transfection of Flag-Tat encoding plasmids, cells were then treated with MG-132 at the final concentration of 10 ng/ml for 2 h to block proteasomal degradation. The accumulation of Flag-Tat were detected by IB. c The type of ubiquitination regulated by ABIN1 was explored in HEK-293T or HEK-293T ABIN1-KO cells by immunoprecipitation as in Fig. a after transfected with Flag-Tat, together with wild-type HA–Ub, HA–Ub K63R or HA–Ub K48R mutant expression vectors for 24 h. β-actin was detected as sample loading control. The blots were representatives of at least three independent experiments achieving similar results

Article Snippet: The antibodies used were as follows: mouse antibodies to Flag (Sigma), Myc (Roche) and β-actin (Proteintech), rabbit antibodies to ABIN1 (Proteintech), Tat (Abcam), and HRP-conjugated anti-ubiquitin antibody (ENZO Life Sciences).

Techniques: Transfection, Concentration Assay, Western Blot, Blocking Assay, Ubiquitin Proteomics, Immunoprecipitation, Mutagenesis, Expressing, Control

Journal: Retrovirology

Article Title: Immune regulator ABIN1 suppresses HIV-1 transcription by negatively regulating the ubiquitination of Tat

doi: 10.1186/s12977-017-0338-5

Figure Lengend Snippet: ABIN1 specifically decreases the ubiquitination of Tat at Lys71. a HEK-293T cells were treated with siRNAs and plasmids similar as in Fig. a as indicated, except that Flag-Tat-K71R mutant was used to assess the ubiquitination of wild-type Tat and Tat K71R mutant in the presence or absence of ABIN1. b HeLa cells were infected with HIV-1(Luc) or HIV-1(Luc-K71R) at the same MOI of 0.2, replication activity (Repl. activity) was assessed by luciferase assays 48 hpi to evaluate the importance of Lys71 in Tat. c , d HEK-293T cells were treated with siRNAs targeting ABIN1 (siABIN1-1, siABIN1-2) or siNC ( c ) or transfected with wild-type ABIN1 or QE2 mutant encoding vectors and challenged with HIV-1(Luc) or HIV-1(Luc-K71R) ( d ), then subjected to luciferase assays 24 hpi to monitor the replication efficiency, and the cell lysates were collected and cleared for detection of knockdown efficiency and overexpression level of ABIN1/mutant. The blots are representatives of at least three independent experiments achieving similar results

Article Snippet: The antibodies used were as follows: mouse antibodies to Flag (Sigma), Myc (Roche) and β-actin (Proteintech), rabbit antibodies to ABIN1 (Proteintech), Tat (Abcam), and HRP-conjugated anti-ubiquitin antibody (ENZO Life Sciences).

Techniques: Ubiquitin Proteomics, Mutagenesis, Infection, Activity Assay, Luciferase, Transfection, Knockdown, Over Expression

Journal: Retrovirology

Article Title: Immune regulator ABIN1 suppresses HIV-1 transcription by negatively regulating the ubiquitination of Tat

doi: 10.1186/s12977-017-0338-5

Figure Lengend Snippet: ABIN1 regulates the ubiquitination of Tat by modulating the distribution of HDM2. a HEK-293T ABIN1-KO cells were transfected with plasmids encoding Myc-ABIN1 or Myc-QE2, together with Myc-HDM2, Flag-Tat or plasmid vectors as indicated, and subjected to IP as in Fig. a. b HeLa cells were transfected with expression vectors encoding or Myc-ABIN1, Myc-QE2, Myc-HDM2, Flag-HDM2 or Flag-Tat individually ( upper panel ) or in combination ( middle and lower panel ) as indicated for 24 h. Then cells were washed with PBS and harvested for Immunofluorescence analysis. Mouse anti-Myc and rabbit anti-Flag antibodies were used as primary antibodies, FITC-conjugated goat anti-rabbit IgG and Rhodamine-conjugated goat anti-mouse IgG were used to detect the two proteins. The consensus scale bar was 20 μm

Article Snippet: The antibodies used were as follows: mouse antibodies to Flag (Sigma), Myc (Roche) and β-actin (Proteintech), rabbit antibodies to ABIN1 (Proteintech), Tat (Abcam), and HRP-conjugated anti-ubiquitin antibody (ENZO Life Sciences).

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Expressing, Immunofluorescence