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: Virology Journal

Article Title: Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen

doi: 10.1186/1743-422x-9-180

Figure Lengend Snippet: Figure 1 Schematic illustrations of two biobarcode amplification (BCA) assays and ELISA assay to detect HIV-1 p24 antigen. (A) ELISA, (B) Microplate [22] and (C) MMPs methods. The detailed description of the methods is described in Materials and Methods. The enzyme (E) used in the ELISA assay was horse radish peroxidase.

Article Snippet: HIV-1 p24 recombinant antigen was purchased from GenWay Biotech (San Diego, CA).

Techniques: Amplification, Enzyme-linked Immunosorbent Assay

Journal: Virology Journal

Article Title: Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen

doi: 10.1186/1743-422x-9-180

Figure Lengend Snippet: Figure 2 Relative HIV-1 p24 antigen detection sensitivity using 2 BCA-based assays and an in-house ELISA assay. The X axis represents serial dilutions (log10) of HIV-1 p24 antigen, the left Y axis is OD450 value for ELISA, and the right Y axis is the DNA amounts (pmoles) calculated from gel electrophoresis band of samples for the two BCA assays. Each data point represents the average of 3 independent determinations. Squares : ELISA, circles: microplate method, and triangles: MMPs method.

Article Snippet: HIV-1 p24 recombinant antigen was purchased from GenWay Biotech (San Diego, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Nucleic Acid Electrophoresis

Journal: Virology Journal

Article Title: Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen

doi: 10.1186/1743-422x-9-180

Figure Lengend Snippet: Figure 3 Linear dynamic p24 detection ranges of three assays: (A) in-house ELISA, (B) BCA-based microplates and (C) BCA-based MMPs. Each data point represents the average of three independent determinations. Statistical analysis of linear regression model between the signal value of each methods and the concentration of HIV-1 p24 are carried out with SPSS 16.0.

Article Snippet: HIV-1 p24 recombinant antigen was purchased from GenWay Biotech (San Diego, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

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

Article Title: Sensitivity of a rapid point of care assay for early HIV antibody detection is enhanced by its ability to detect HIV gp41 IgM antibodies.

doi: 10.1016/j.jcv.2015.08.005

Figure Lengend Snippet: Fig. 2. The ability of INSTI HIV-1 gp41 antigens to bind anti-HIV-1 IgM antibody. Anti-HIV-1

Article Snippet: Anti-HIV-1 IgM enzyme-linked immunosorbent assay (ELISA) Fig. 1) In this ELISA method, 0.5 l of gp41 recombinant protein at a oncentration of 1 mg/ml (GenWay Biotech Inc., San Diego, CA) as spotted onto unbacked Hi-Flow (HF-180) nitrocellulose memranes (EMD Millipore Billerica, MD, USA).

Techniques:

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

Article Title: Sensitivity of a rapid point of care assay for early HIV antibody detection is enhanced by its ability to detect HIV gp41 IgM antibodies.

doi: 10.1016/j.jcv.2015.08.005

Figure Lengend Snippet: Fig. 3. Relative sensitivity of INSTI gp41 antigen to HIV-IgM antibody levels. The

Article Snippet: Anti-HIV-1 IgM enzyme-linked immunosorbent assay (ELISA) Fig. 1) In this ELISA method, 0.5 l of gp41 recombinant protein at a oncentration of 1 mg/ml (GenWay Biotech Inc., San Diego, CA) as spotted onto unbacked Hi-Flow (HF-180) nitrocellulose memranes (EMD Millipore Billerica, MD, USA).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Universal amplification-free molecular diagnostics by billion-fold hierarchical nanofluidic concentration.

doi: 10.1073/pnas.1904513116

Figure Lengend Snippet: Fig. 6. Protein detection by HOLMES. (A) Schematic of selective protein enrichment in HOLMES. (B) Schematic and (C) micrographs of protein detection by HOLMES. (D) Detection of HIV p24 protein by HOLMES. (E) Correlation of HOLMES and ELISA in the detection of human cardiac troponin I in patient plasma samples. Each data point represents the mean and SD of triplicate runs. LOD, limit of detection; nd, not detected.

Article Snippet: The HIV p24 Ab pair and recombinant HIV p24 protein were from the HIV-1 Gag p24 DuoSet ELISA kit (R&D Systems).

Techniques: Protein Enrichment, Enzyme-linked Immunosorbent Assay, Clinical Proteomics