Journal: PloS one

Article Title: Inhibition of HIV-1 infection by human α-defensin-5, a natural antimicrobial peptide expressed in the genital and intestinal mucosae.

doi: 10.1371/journal.pone.0045208

Figure Lengend Snippet: Figure 1. Effect of HD5 on HIV-1 replication in purified CD4+ T lymphocytes. (A) Effect of recombinant HD5 on infection by primary isolate HIV-1J176 in cell-free infection assays performed using the spinoculation method. HIV-1 virions were preincubated for 1 hour at 37uC with HD5 at the indicated concentrations in either RPMI-10 (10%FBS), RPMI-0.3-ITS (no serum) or 10 mM phosphate buffer 0.3-ITS (low salt). Gag p24 concentrations in the extracellular supernatant were measured on day 6 post-infection. Neutralizing mAbs directed to gp120 (2G12) or CD4 (Sim4) were used as positive controls to exclude non-specific infection. The data represent mean values (6SD) from 3 experiments each performed in triplicate. (B) Broad- spectrum inhibition of different HIV-1 strains by HD5. Purified CD4+ T lymphocytes were infected with two laboratory-adapted strains (HIV-1IIIB and HIV-1BaL) or 4 primary clinical isolates (HIV-1J1005, HIV-1p36, HIV-1J6195 and HIV-1J176) in the presence of HD5 at 1.4 mM in RPMI-0.3-ITS. Gray bars indicate R5 HIV-1 isolates; solid bars X4 isolates; the striped bar a dualtropic isolate. The data represent mean values (6SD) of three separate experiments, each performed in triplicate. doi:10.1371/journal.pone.0045208.g001

Article Snippet: Bound p24 Ag was then detected using an alkaline phosphatase–conjugated anti-p24 monoclonal antibody (BC 1071-AP; Aalto Bio Reagents) and the AMPAK ELISA amplification system (DAKO A/S, Glostrup, Denmark).

Techniques: Purification, Recombinant, Infection, Inhibition

Journal: Retrovirology

Article Title: Vpx complementation of ‘non-macrophage tropic’ R5 viruses reveals robust entry of infectious HIV-1 cores into macrophages

doi: 10.1186/1742-4690-11-25

Figure Lengend Snippet: Full-length transmitted/founder viruses use low levels of the cells surface CD4 inefficiently. (A) A representative flow cytometry experiment showing CD4 expression levels on minimally ( low ) and maximally ( high ) induced 293-Affinofile cells in comparison with CD4+ primary T cells. (B) Equal amounts of p24 of YU-2 and clinically derived transmitted viruses were used to infect 293-Affinofile cells, which were maximally induced to express high levels of CCR5 and high ( black bars ) or low ( white bars ) levels of CD4. Cells were washed, fixed, permeabilized with saponin and stained with anti-HIV-1 p24 FITC-conjugated monoclonal antibody 48 h post-infection. Percentage infection was determined by flow cytometry. Data are representative of at least two independent experiments and error bars represent the standard deviation. (C) 293-Affinofile cells were induced to express different cell surface levels of CD4 and CCR5. Cells were infected with 50 ng of p24 of YU-2 and ZM247F and percentage of infection determined by flow cytometry. Data shown are representative example of three independent experiments.

Article Snippet: The infection medium was replaced with DMEM complete supplemented with 50 μg/ml of Blasticidin, and the cells were incubated for 48 h. Cells were washed, fixed in 3% paraformaldehyde, permeabilized with saponin and stained with anti HIV-1 p24 FITC-conjugated monoclonal antibody (Insight Biotechnology LTD, UK).

Techniques: Flow Cytometry, Expressing, Comparison, Derivative Assay, Staining, Infection, Standard Deviation

Journal: Science Advances

Article Title: HIV-1 manipulates CD96 on CD4 + T cells to subvert antiviral immunity

doi: 10.1126/sciadv.adx7485

Figure Lengend Snippet: CD96 was knocked out in stimulated primary CD4 + T cells by CRISPR-Cas9. ( A ) Three days later, the KO was confirmed by CD96 cell surface staining, and ( B to D ) cells were infected with equal p24 amounts of HIV-1 NL4-3–IRES–eGFP with or without deletions in nef and vpu . (B) Forty-eight hours postinfection, cells were analyzed for the rate of productive infection by their GFP expression. (C) In addition, supernatant was collected to quantify for HIV-1 production and release via HIV-1 p24 ELISA and (D) determine the infectivity of released HIV-1 particles via reinfection of Jurkat cells with supernatants normalized for HIV-1 p24. Values from four independent experiments were plotted (means ± SD). Significance was tested using a two-way ANOVA with Sidak’s multiple comparison test. ( E ) CD96 KO CD4 + T cells were used as target cells and cocultured with PKH67-stained PBMCs, which served as effector cells for the cellular cytotoxicity assay. The coculture was incubated for 16 hours at an effector:target cell ratio of 40:1. Cell count of target cells was analyzed by flow cytometry. Killing efficiency was calculated by dividing the cell counts of target cells in the absence of effector cells though the cell counts in the presence of effectors relative to mock. Values from four independent experiments were plotted.

Article Snippet: Anti–HIV-1 p24 (rabbit) , ELISA (first antibody) , 1:650 in 10% FBS PBS-T , Eurogentec , -.

Techniques: CRISPR, Staining, Infection, Expressing, Enzyme-linked Immunosorbent Assay, Comparison, Cytotoxicity Assay, Incubation, Cell Counting, Flow Cytometry