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: PLoS Neglected Tropical Diseases

Article Title: Leishmania infantum Amastigotes Trigger a Subpopulation of Human B Cells with an Immunoregulatory Phenotype

doi: 10.1371/journal.pntd.0003543

Figure Lengend Snippet: Purified human CD25 - CD4 + T cells were either left untreated (resting) or activated with plate-bound anti-CD3 and anti-CD28 antibodies. For activated cells, samples were either left untreated (no supernatant), incubated with cell-free supernatants from untreated B cells (control), or incubated with cell-free supernatants from B cells exposed to L . infantum amastigotes at a final parasite:host cell ratio of 3:1 (AMA). Seventy-two hours later, cells were analysed for cell surface expression of CD25 and CD69 by flow cytometry. Dead cells were excluded by 7-AAD staining. A) A representative dot plot is shown for each tested condition. B) Percentages of CD25 + CD69 + Tcells for studies performed with CD25 - CD4 + T cells from 3 different donors incubated with cell-free supernatants from 5 to 7 different B-cell donors. Statistical significance was evaluated by two-tailed Student’s t-test.

Article Snippet: Primary human CD4 + T cells were isolated from PBMCs using a magnetic Easysep CD4 + T-cell enrichment kit (Stemcell Technologies) and CD25 + T cells (both activated CD4 + T cells and Tregs) were depleted with a CD25 positive selection kit (Stemcell Technologies).

Techniques: Purification, Incubation, Control, Expressing, Flow Cytometry, Staining, Two Tailed Test

Journal: PLoS Neglected Tropical Diseases

Article Title: Leishmania infantum Amastigotes Trigger a Subpopulation of Human B Cells with an Immunoregulatory Phenotype

doi: 10.1371/journal.pntd.0003543

Figure Lengend Snippet: Purified human CD25 - CD4 + T cells were first labeled with CFSE and treated as described in . After 5 days, cell proliferation was assessed by CFSE dilution and dead cells were excluded by 7-AAD staining. A) A representative histogram is shown for each tested condition. B) Proliferation index for experiments performed with CD25 - CD4 + T cells from 2 different donors incubated with cell-free supernatants from 6 or 7 different B-cell donors. Statistical significance was evaluated by two-tailed Student’s t-test.

Article Snippet: Primary human CD4 + T cells were isolated from PBMCs using a magnetic Easysep CD4 + T-cell enrichment kit (Stemcell Technologies) and CD25 + T cells (both activated CD4 + T cells and Tregs) were depleted with a CD25 positive selection kit (Stemcell Technologies).

Techniques: Purification, Labeling, Staining, Incubation, Two Tailed Test

Journal: PLoS Neglected Tropical Diseases

Article Title: Leishmania infantum Amastigotes Trigger a Subpopulation of Human B Cells with an Immunoregulatory Phenotype

doi: 10.1371/journal.pntd.0003543

Figure Lengend Snippet: Purified human CD25 - CD4 + T cells were activated for 72 h with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of cell-free supernatants from B cells either left untreated (control) or incubated overnight with L . infantum amastigotes at a final parasite:host cell ratio of 3:1 (AMA). In some cases, cell-free supernatants from B cells incubated with parasites were treated with a soluble IL-10 receptor (1 μg/ml) before being used with activated CD25 - CD4 + T cells. During the last 5 h of incubation, cells were further stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) and Golgiplug™ was added (1 μl per 1 x 10 6 cells). Finally, cells were fixed, permeabilized and stained for intracellular TNF before being analysed by flow cytometry. Dead cells were excluded by 7-AAD staining. Results are expressed as the percentages of TNF + cells multiply by the mean fluorescence intensities (MFI). Data shown are the results from 4 different CD25 - CD4 + T-cell donors incubated with cell-free supernatants from 4 different B-cell donors. Statistical significance was evaluated by two-tailed Student’s t-test.

Article Snippet: Primary human CD4 + T cells were isolated from PBMCs using a magnetic Easysep CD4 + T-cell enrichment kit (Stemcell Technologies) and CD25 + T cells (both activated CD4 + T cells and Tregs) were depleted with a CD25 positive selection kit (Stemcell Technologies).

Techniques: Purification, Control, Incubation, Staining, Flow Cytometry, Fluorescence, Two Tailed Test

Journal: PLoS Neglected Tropical Diseases

Article Title: Leishmania infantum Amastigotes Trigger a Subpopulation of Human B Cells with an Immunoregulatory Phenotype

doi: 10.1371/journal.pntd.0003543

Figure Lengend Snippet: Purified human CD25 - CD4 + T cells were activated for 72 h with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of cell-free supernatants from B cells either left untreated (control) or incubated overnight with L . infantum amastigotes at a final parasite:host cell ratio of 3:1 (AMA). In some cases, cell-free supernatants from B cells incubated with parasites were treated with a soluble IL-10 receptor (1 μg/ml) before being used with activated CD25 - CD4 + T cells. During the last 5 h of incubation, cells were further stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) and Golgiplug™ was added (1 μl per 1 x 10 6 cells). Finally, cells were fixed, permeabilized and stained for intracellular IFNγ before being analysed by flow cytometry. Dead cells were excluded by 7-AAD staining. Results are expressed as the percentages of TNF-positive cells multiply by the mean fluorescence intensities (MFI). Data shown are the results from 3 different CD25 - CD4 + T-cell donors incubated with cell-free supernatants from 3 different B-cell donors. Statistical significance was evaluated by two-tailed Student’s t-test.

Article Snippet: Primary human CD4 + T cells were isolated from PBMCs using a magnetic Easysep CD4 + T-cell enrichment kit (Stemcell Technologies) and CD25 + T cells (both activated CD4 + T cells and Tregs) were depleted with a CD25 positive selection kit (Stemcell Technologies).

Techniques: Purification, Control, Incubation, Staining, Flow Cytometry, Fluorescence, Two Tailed Test