competitive enzyme linked immunoassay  (R&D Systems)

Journal: bioRxiv

Article Title: Cannabidiol prevents mucosal HIV-1 transmission by targeting Langerhans cells, dendritic cells, macrophages and T-cells

doi: 10.64898/2026.01.07.697949

Figure Lengend Snippet: (A) Graphical representation of the assay evaluating HIV-1 transfer from MDLCs to autologous CD4 + T-cells, cumulating the early and late phases of transfer. (B) MDLCs were left untreated or pre-treated for 24h with the indicated molar concentrations of CBD, AEA, ACEA, JWH133 or CGRP, pulsed with HIV-1 R5 ADA for 4h, and washed. Cells were then co-cultured with autologous CD4 + T-cells, and the supernatants were evaluated 7 days post infection (dpi) for p24 content by ELISA. Graph shows mean±SD (n=3-9) percentages of HIV-1 transfer inhibition, calculated against untreated cells serving as set point. (C) MDLCs were left untreated or pre-treated for 24h with AEA (10 -6 M), in the absence or presence of the TRPV1 antagonist A425619 (10 -6 M) added 15min before AEA. HIV-1 transfer was then evaluated as above, and graph shows mean±SD percentages of HIV-1 transfer inhibition. (D) MDLCs were left untreated or pre-treated for 24h with agonists (10 -5 M). The cells were then washed, cultured in fresh medium for additional 48h, and secretion of CGRP into the culture supernatants was evaluated using a competitive CGRP EIA. Graph shows mean±SD levels of pg/ml secreted CGRP. (E) MDLCs were pre-treated for 24h with CBD, AEA or CGRP (10 -6 M), in the absence or presence of the CGRP receptor antagonist BIBN (10 -6 M) added 15min before agonists. HIV-1 transfer was then evaluated and is presented as above. (F) MDDCs were pre-treated for 24h with CBD, ACEA, JWH133 (10 -5 M) or CGRP (10 -6 M), and HIV-1 transfer was then evaluated and is presented as above. (G) Graphical representation of the assay evaluating temporally the early and late phases of HIV-1 transfer from MDLCs to GFP-reporter T-cells. (H, I) MDLCs were left untreated or pre-treated for 24h with the indicated molar concentrations of CBD, AEA or CGRP, pulsed with HIV-1 R5 ADA for 4h, and washed. Cells were then co-cultured with CD4 high CCR5 high GFP-reporter T-cells, added immediately after pulse (H) or 48h later (I), followed by flow cytometry 48h later. Graph show mean±SD (n=3-8) percentages of HIV-1 transfer inhibition, calculated against untreated cells serving as set point. (J-L) MDLCs were left untreated or pre-treated for 24h with agonists (10 -5 M) or CGRP (10 -6 M), followed by flow cytometry. Graphs show mean±SD percentages of positive cells expressing langerin (J), or proportions (out of langerin + cells) of cells expressing CCR5 (K) or CD4 (L). In all graphs, dots denote different experiments using cells prepared from different human donors; *p<0.05, **p<0.005 and ***p<0.0005, Student’s t-test.

Article Snippet: The culture supernatants were collected immediately following treatment and CGRP levels were measured using a competitive CGRP enzyme immunoassay kit (EIA; Phoenix Pharmaceuticals) according to the manufacturer’s instructions.

Techniques: Cell Culture, Infection, Enzyme-linked Immunosorbent Assay, Inhibition, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Cannabidiol prevents mucosal HIV-1 transmission by targeting Langerhans cells, dendritic cells, macrophages and T-cells

doi: 10.64898/2026.01.07.697949

Figure Lengend Snippet: (A) Representative flow cytometry overlay histograms showing CB1 and CB2 surface expression in primary blood CD4 + T-cells. Numbers and graph show mean±SD percentages of CB1 + or CB2 + T-cells. (B) PHA+IL-2-activated primary blood CD4 + T-cells were pre-treated for 24h with the indicated molar concentrations of CBD, AEA, JWH133, ACEA or CGRP. The cells were then washed, pulsed with HIV-1 R5 JRCSF or X4 LAI, and HIV-1 replication was evaluated 4 days later by p24 intracellular staining and flow cytometry. Graphs show mean±SD (n=4-6) percentages of HIV-1 infection inhibition, normalized against untreated cells serving as set point. (C, D) Primary blood CD4 + T-cells were activated for 48h with PHA+IL-2, alone or in the presence of CBD, AEA, JWH133 or ACEA (10 -5 M). The cells were then evaluated by flow cytometry for surface expression of the activation marker CD69 and CD25. In (C) shown are representative flow cytometry dot plots. In (D), graph shows mean±SD percentages of CD69 + CD25 + double-positive CD4 + T-cells. (E, F) Primary blood CD4 + T-cells were left untreated or treated for 24h with agonists (10 -5 M), followed by flow cytometry evaluating surface expression of CD4 and CCR5. Graphs show mean±SD of CD4 MFI (E) or percentages of CCR5 + CD4 + T-cells (F). In all graphs, dots denote different experiments using cells prepared from different human donors; *p<0.05, **p<0.005 and ***p<0.0005, Student’s t-test.

Article Snippet: The culture supernatants were collected immediately following treatment and CGRP levels were measured using a competitive CGRP enzyme immunoassay kit (EIA; Phoenix Pharmaceuticals) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Expressing, Staining, Infection, Inhibition, Activation Assay, Marker

Journal: bioRxiv

Article Title: Cannabidiol prevents mucosal HIV-1 transmission by targeting Langerhans cells, dendritic cells, macrophages and T-cells

doi: 10.64898/2026.01.07.697949

Figure Lengend Snippet: (A) Representative flow cytometry overlay histograms showing CB1 and CB2 surface expression in MDMs. Numbers and graph show mean±SD percentages of CB1 + or CB2 + MDMs. (B, C) MDMs were left untreated or pre-treated for 24h with CBD, AEA, JWH133, ACEA or CGRP (10 -6 M). The CGRP receptor antagonist BIBN (10 -6 M) was added 15 min before addition of agonists. The cells were then washed, pulsed with HIV-1 R5 ADA for 4h, washed again, and HIV-1 content in the culture supernatants was evaluated at 7 dpi by p24 ELISA. Graphs show mean±SD percentages of HIV-1 infection inhibition, normalized against untreated cells serving as set point. In all graphs, dots denote different experiments using cells prepared from different human donors; *p<0.05, **p<0.005 and ***p<0.0005, Student’s t-test.

Article Snippet: The culture supernatants were collected immediately following treatment and CGRP levels were measured using a competitive CGRP enzyme immunoassay kit (EIA; Phoenix Pharmaceuticals) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Infection, Inhibition

Journal: bioRxiv

Article Title: Cannabidiol prevents mucosal HIV-1 transmission by targeting Langerhans cells, dendritic cells, macrophages and T-cells

doi: 10.64898/2026.01.07.697949

Figure Lengend Snippet: (A, B) Inner foreskin epidermal cell suspensions were labeled with Viobility Fixable Dye to distinguish Live/Dead (L/D) cells, stained for surface expression of CD45, CD1a/CD207 (langerin) or CD3/CD8, CB1 and CB2, and examined by flow cytometry. Shown are representative dot plots of viable CD45 + cells subsequently separated into CD1a hi CD207 hi LCs and CD1a low CD207 low/neg EpiDCs (A) or CD3 + CD8 - T-cells (B). Also shown are representative flow cytometry overlay histograms of CB1 and CB2 surface expression in CD1a hi CD207 hi LCs (A) and CD3 + CD8 - T-cells (B). Numbers and graph show mean±SD percentages of CB1 + or CB2 + cells. (C) Schematic representation of the different experimental steps, showing treatment with CBs followed by polarized HIV-1 infection of inner foreskin tissue explants. (D-F) Inner foreskin tissue explants were submerged in culture media, and either left untreated or treated with CBD or AEA (10 -5 M). In (D), media were collected 24h later and the levels of secreted CGRP were determined using an EIA. Shown are mean±SD pg/ml secreted levels of CGRP. In (E, F), untreated or CBD-treated inner foreskin tissue explants were next washed, transferred to two-chamber Transwell inserts, inoculated in a polarized manner with either non-infected or HIV-1-infected cells, and 4h later the cell-associated HIV-1 inoculum was washed out. In (E), explants were immediately digested with dispase and trypsin to obtain epidermal cell suspensions. After gating out cell debris and FSC highest SSC highest keratinocytes, cells were further gated on viable CD45 + CD3 + CD8 − T-cells, and the percentages of FSC hi conjugates with CD1a hi langerin hi LCs were determined by flow cytometry. Shown are mean±SD folds increase in conjugate formation, calculated as [(% conjugates following inoculation with HIV-1-infected PBMCs) / (% conjugates following inoculation with non-infected PBMCs). In (F), explants were further incubated for 3 additional days submerged in fresh culture media, before digestion with dispase and collagenase/DNase to obtain dermal cell suspensions. Cells were gated on viable FSC low SSC low lymphocytes, and the percentages of CD3 + CD8 - p24 + cells were determined by flow cytometry. Shown are mean±SD percentages of HIV-1-infected T-cells, calculated as [(%CD3 + CD8 - p24 + cells following inoculation with HIV-1-infected PBMCs) - (%CD3 + CD8 - p24 + cells following inoculation with non-infected PBMCs)]. In all graphs, dots denote different experiments using tissues from different human donors; *p<0.05, **p<0.005 and ***p<0.0005, Student’s t-test.

Article Snippet: The culture supernatants were collected immediately following treatment and CGRP levels were measured using a competitive CGRP enzyme immunoassay kit (EIA; Phoenix Pharmaceuticals) according to the manufacturer’s instructions.

Techniques: Labeling, Staining, Expressing, Flow Cytometry, Infection, Incubation