Journal: AIDS Research and Human Retroviruses

Article Title: HIV Type 1 Nef Is Released from Infected Cells in CD45 + Microvesicles and Is Present in the Plasma of HIV-Infected Individuals

doi: 10.1089/aid.2009.0170

Figure Lengend Snippet: Nef expression and HIV-1 infection induce vesicle formation. TEM image of conditioned media from HEK293 cells transfected with (A) Nef expression plasmid or (B) mock-transfected. Conditioned media collected 3 days posttransfection and subjected to ultracentrifugation (400,000 × g). Pellets were fixed, embedded, and stained with 2% uranyl acetate. Samples were visualized using a JEOL 1200 EX transmission microscope. (C) Culture supernatant from HEK293 transfected with a Nef expression plasmid was subjected high-speed differential centrifugation. Supernatants were centrifuged for 10 min at 300 × g, followed by 30 min at 10,000 × g, and then centrifuged for 1 h at 300,000 × g. Nef was detected in vesicles pelleted at 300,000 by immunoblot using anti-Nef monoclonal. S, supernatants; P, pellet.

Article Snippet: Plasma from HIV-1 + /HCV − /HBV − individuals ( n = 10) and HIV − / HCV − /HBV − controls ( n = 10) was obtained from Bioreclamation (Long Island, NY).

Techniques: Expressing, Infection, Transfection, Plasmid Preparation, Staining, Transmission Assay, Microscopy, Centrifugation, Western Blot

Journal: AIDS Research and Human Retroviruses

Article Title: HIV Type 1 Nef Is Released from Infected Cells in CD45 + Microvesicles and Is Present in the Plasma of HIV-Infected Individuals

doi: 10.1089/aid.2009.0170

Figure Lengend Snippet: Nef vesicles captured using anti-CD45 magnetic bead separation. Supernatants from HIV-1-infected cells were harvested 15 days postinfection and vesicles/virions were concentrated by centrifugation at 200,000 × g for 1 h on a 20% sucrose cushion. The pellet was resuspended in 1 × TNE buffer and subjected to CD45 magnetic bead separation. Each fraction was collected, ultracentrifuged at 400,000 × g for 1 h, and then separated by SDS-PAGE. Fractions (SM = starting material, FT = flow through, W = wash, and E = eluate) were analyzed by Western blot for HIV proteins using anti-HIV sera (upper panel). The singular band present in the eluate was confirmed to be Nef via an immunoblot using an anti-Nef monoclonal antibody (lower panel). Nef+, recombinant Nef protein.

Article Snippet: Plasma from HIV-1 + /HCV − /HBV − individuals ( n = 10) and HIV − / HCV − /HBV − controls ( n = 10) was obtained from Bioreclamation (Long Island, NY).

Techniques: Infection, Centrifugation, SDS Page, Western Blot, Recombinant

Journal: AIDS Research and Human Retroviruses

Article Title: HIV Type 1 Nef Is Released from Infected Cells in CD45 + Microvesicles and Is Present in the Plasma of HIV-Infected Individuals

doi: 10.1089/aid.2009.0170

Figure Lengend Snippet: Production of CD45+ Nef microvesicles is cell-type dependent. Concentrations of p24 (A) microvesicles and Nef (B) were measured by ELISA in culture supernatants and CD45 captured microvesicles of HIV-1-infected Jurkat T-lymphocytes, THP-1 monocytes, and PMA-treated THP-1 (macrophage-like). T-lymphocytes release the most HIV-1 virus as measured by p24 ELISA whereas macrophage-like THP-1 releases the most Nef microvesicles.

Article Snippet: Plasma from HIV-1 + /HCV − /HBV − individuals ( n = 10) and HIV − / HCV − /HBV − controls ( n = 10) was obtained from Bioreclamation (Long Island, NY).

Techniques: Enzyme-linked Immunosorbent Assay, Infection