Journal: EMBO Molecular Medicine

Article Title: Antioxidant nanozyme counteracts HIV‐1 by modulating intracellular redox potential

doi: 10.15252/emmm.202013314

Figure Lengend Snippet: A CEM‐GFP cells were pre‐treated with 50 ng/μl of Vs for 15 min and infected with 0.1 moi of CXCR4‐using HIV‐1 (NL‐4.3), and GFP fluorescence was measured at 488 nm as an indicator of HIV LTR activity. Vs treatment was repeated every 24 h for the experiment. B–D A similar assay was performed using Jurkat (CD4 + T‐cell line), and viral replication was assessed by (B) gag RT–PCR, (C) p24 ELISA in the culture supernatant, and (D) immunoblotting for p24 (viral capsid protein) in the whole cell lysate. E U937 (promonocytes) were pre‐treated with 50 ng/μl of Vs for 15 min followed by infection with 1 moi of CCR5 using HIV‐1 (NL‐AD8), and viral replication was measured by gag RT–qPCR at 24 h post‐infection (hpi). F Primary CD4 + T cells purified from human PBMCs (3 healthy donors) were activated, pre‐treated with 25 ng/μl Vs for 15 min, and infected with 0.05 moi of HIV‐1 NL‐4.3. Virus released in supernatant was quantified by p24 ELISA. Vs treatment was repeated every 48 h. Data information: All figures except (B) and (E) were analyzed by 2‐way ANOVA. (B), and (E) were analyzed by Mann–Whitney test. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05. Data are representative of results from three independent experiments performed in triplicate (mean ± SD). Source data are available online for this figure.

Article Snippet: Briefly, 50 × 10 6 PBMCs were thawed and CD4 + T cells were isolated using EasySep human CD4 + T‐cell isolation kit (Stem Cell Technologies, Canada).

Techniques: Infection, Fluorescence, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR, Purification, Virus, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Antioxidant nanozyme counteracts HIV‐1 by modulating intracellular redox potential

doi: 10.15252/emmm.202013314

Figure Lengend Snippet: Human monocyte‐derived macrophages (HMDMs) were pre‐treated with 12.5 ng/µl of Vs for 15 min, followed by infection with HIV‐1 NL‐AD8. Viral release in supernatant was quantified by p24 ELISA at 7 and 14 dpi. Vs treatment was repeated every 72 h. Data are obtained from one healthy donor in duplicate (mean ± SD). Survival of HIV‐infected primary CD4 + T cells was monitored by Annexin V/PI staining at 3 dpi in presence or absence of Vs treatment. Percentage of necrotic (PI + ), early apoptotic (Annexin V + ), and late apoptotic (Annexin V + /PI + ) cells were plotted . Data are aggregated from three healthy donors (mean ± SEM). Data information: *** P < 0.001, ** P < 0.01, ns—non‐significant analyzed by 2‐way ANOVA.

Article Snippet: Briefly, 50 × 10 6 PBMCs were thawed and CD4 + T cells were isolated using EasySep human CD4 + T‐cell isolation kit (Stem Cell Technologies, Canada).

Techniques: Derivative Assay, Infection, Enzyme-linked Immunosorbent Assay, Staining

Journal: EMBO Molecular Medicine

Article Title: Antioxidant nanozyme counteracts HIV‐1 by modulating intracellular redox potential

doi: 10.15252/emmm.202013314

Figure Lengend Snippet: Schematic representation of generation of expanded CD4 + T cells and reactivation. CD4 + T cells were sorted from PBMCs of ARV‐suppressed HIV‐infected individuals and expanded in presence of PHA, IL‐2, and autologous feeder PBMCs from healthy donor. Expanded CD4 + T cells from three patients were cultured in presence of IL‐2 and ARVs, with and without 25 ng/µl Vs for 21 days. Vs treatment was given for 15 min every 3 rd day. HIV transcripts were quantified by RT–qPCR at day 14, day 21, and at 24 h post‐stimulation of cells cultured for 21 days by prostratin. Limit of detection for RT–qPCR was 3 viral transcripts per million cells. At day 21, cells were stimulated with 1 µM prostratin for 24 h and HIV transcripts were quantified by RT–qPCR. Reduction in viral stimulation in Vs‐treated samples are represented as percentage values. ND—non‐determined. Aggregate plot for 3 patients from data (C). Total HIV‐1 DNA was determined up to 21 days in cells treated with ARVs or Vs + ARVs. Data Information: (B), (D), and (E) were analyzed by one‐way ANOVA with Tukey’s multiple correction. * P < 0.05, ns—non‐significant. Data are aggregated from three ARV‐suppressed HIV‐infected human subjects (mean ± SD).

Article Snippet: Briefly, 50 × 10 6 PBMCs were thawed and CD4 + T cells were isolated using EasySep human CD4 + T‐cell isolation kit (Stem Cell Technologies, Canada).

Techniques: Infection, Cell Culture, Quantitative RT-PCR

Journal: Nature biotechnology

Article Title: Development and function of human innate immune cells in a humanized mouse model

doi: 10.1038/nbt.2858

Figure Lengend Snippet: a , Percentages of human myeloid cells (hCD33 + ) among human hematopoietic cells (hCD45 + ) in the blood of the indicated recipient mice, engrafted as newborns by intra-hepatic injection of fetal liver CD34 + cells after X-ray preconditioning. Each symbol represents an individual mouse and the red bars indicate mean values (n=20-113; statistical analysis is shown in ). b , Composition of human white blood cells in the same mice as in a (n=20-113 mice/group; n=8 human donors; error bars indicate SEM). c , Immunohistological staining of human myeloid cells (hCD68 + ) in non-lymphoid tissues of the indicated recipient mice. The black bar represents 20 μm, and the images shown are representative of at least three mice analyzed per group. d-e , Representative flow cytometry analysis ( d ) and frequencies ( e ) of human monocyte subsets, identified by expression of CD14 and CD16 among hCD45 + CD33 + cells in the blood of recipient mice (n=8-12 mice/group; error bars indicate SEM). Dot plots in d are gated on CD33 hi SSC lo cells to show the subset distribution among monocytic cells. f , Human monocytes in the blood of MISTRG recipients and human monocytes from a human donor were stained with the indicated antibodies. Staining with isotype control antibodies is shown in red and specific antibodies in blue.

Article Snippet: Human CD33 + cells were enriched by magnetic isolation (EasySep CD33 selection kit, StemCell Technologies).

Techniques: Injection, Staining, Flow Cytometry, Expressing, Control