Journal: Nature Communications

Article Title: NETosis and thrombosis in vaccine-induced immune thrombotic thrombocytopenia

doi: 10.1038/s41467-022-32946-1

Figure Lengend Snippet: a PF4 and PF4-heparin ELISA experiment of VITT serum and controls. The cut-off, 0.50 OD units (Vax, VTE n = 7; AB, VITT Pt and HIT n = 3). b 14 C-serotonin release assay for VITT samples with buffer alone, PF4 (10 μg/mL), 0.1 or 100 U/mL heparin or IV.3 antibody (50 μg/mL). Each dot represents the mean of assays done in triplicate. The cut-off was set at 20% CPM. c Platelet aggregation responses. Purified IgG from VITT patients induced aggregation in platelet-rich plasma (red, blue and black traces). Blockage of FcγRIIa with IV.3 inhibited aggregation (purple, green and light blue traces). d Nucleosomal CitH3 (H3R8Cit ELISA, * p = 0.03), e myeloperoxidase (ELISA, ** p = 0.01), and f cfDNA (PicoGreen fluorescence assay, **** p < 0.0001) levels in VITT patients’ plasma ( n = 7) relative to controls ( n = 7) were determined. g Representative side and forward scatter flow cytometry plot backgated for neutrophils (yellow) and monocytes (blue) from VITT patient’s and vaccine control blood. LDG are indicated. h LDG quantitated as number of LDG events relative to 200 monocytes ( n = 7, except VITT n = 3; * p = 0.02). i Representative plot of NPA from VITT and vaccine control blood. j Quantification of NPA in VITT ( n = 7, except VITT n = 3; ** p = 0.004). k Representative plot of NETs from VITT and vaccine control blood. Quantification of NETs in VITT in l whole blood ( n = 7, except VITT n = 3; ** p = 0.005) and in m LDG population ( n = 6, except VITT n = 3; * p = 0.02). MPO + , CitH3 + double positive cells within the CD15 + population were defined as neutrophils undergoing NETosis. The percentage of gated events is indicated in each quadrant. Statistics: Kruskal-–Wallis ANOVA with Dunn’s correction. Data are presented as a , j , l , m mean ± SD; h mean ± SEM. OD optical density units, CPM counts per minute, Vax ctrl healthy vaccinated subject IgG; VTE venous thromboembolism patient IgG, ICU intensive care unit patient IgG, NPA neutrophil-platelet aggregates, LDG low density granulocytes, cfDNA cell-free DNA, CitH3 citrullinated histone H3, Pt patient. Source data are provided in the Source Data file.

Article Snippet: Plasma levels of myeloperoxidase and citrullinated histone H3 were determined using the human myeloperoxidase ELISA kit (ab119605, Abcam) and H3R8Cit ELISA Capture and Detection kit (R&D143002, EpiCypher) , respectively, following the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Release Assay, Purification, Clinical Proteomics, Fluorescence, Flow Cytometry, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Control of HIV Infection In Vivo Using Gene Therapy with a Secreted Entry Inhibitor

doi: 10.1016/j.omtn.2017.08.017

Figure Lengend Snippet: Promoter Analysis (A) Overview of the SIN lentiviral vectors with different internal promoters. (B) Flow cytometric analysis of transduced cell lines and primary cells. Cells were transduced with LV-CMV-sCD4, LV-UCMV-sCD4, or LV-EF1α-sCD4 as described in the . HSPCs were analyzed 4 days post-transduction. All other cell types were analyzed 14 days post-transduction. The percentage of gene-modified cells is indicated, and the median fluorescence intensity is shown in parentheses. (C) Western blot analysis for the presence of sCD4 in culture media of 293T or Jurkat cells transduced with the LV-CMV-sCD4 (CMV), LV-UCMV-sCD4 (UCMV), or LV-EF1α-sCD4 (EF1α) at 2–3 weeks post-transduction. (D) Signal peptide analysis. The native CD4 signal peptide was substituted by the alpha-1 antitrypsin (AAT) signal peptide sequence. Jurkat cells were transduced with LV-EF1α-sCD4 (CD4) or LV-EF1α-AAT-sCD4 (AAT), and culture media were analyzed by anti-His-tag ELISA. A two-tailed unpaired t test was used to determine statistical significance; *p < 0.05. Data are means and SEM representative of two independent experiments performed in duplicates. Ψ, packaging signal; cPPT, central polypurine tract; IRES, internal ribosome entry site; RRE, Rev response element; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; ΔU3, deletion in the U3 promoter.

Article Snippet: Western blot analysis was performed as previously described., His-tag ELISA (Genscript) was performed according to the manufacturer’s instructions.

Techniques: Transduction, Modification, Fluorescence, Western Blot, Sequencing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Virus

Journal: Molecular Therapy. Nucleic Acids

Article Title: Control of HIV Infection In Vivo Using Gene Therapy with a Secreted Entry Inhibitor

doi: 10.1016/j.omtn.2017.08.017

Figure Lengend Snippet: Antiviral Effect of sCD4 (A and B) Primary CD4 + T cells or HSPCs were transduced with LV-EF1α-AAT-sCD4. Unmodified cells served as a control. 5 × 10 5 cells/mL were cultured for 4 days, and the culture supernatants (sups) were harvested. (A) T cell and HSPC culture supernatants were analyzed by His-tag ELISA for the presence of sCD4. (B) Single-round infection assays with HIV JRFL were performed in the presence of T cell and HSPC culture supernatants. The number of infected TZM-bl cells was determined as described in the . Data are means and SEM from two independent experiments performed in duplicate. (C and D) Primary CD4 + T cells were transduced with LV-EF1α (control) or LV-EF1α-AAT-sCD4 (∼60% gene modification) and infected with HIV IIIB . (C) Culture supernatants from infected T cell cultures were analyzed by p24 ELISA. (D) Culture supernatants from infected T cell cultures were used to infect TZM-bl cells in single-round infection assays. Data are means and SEM from two independent experiments performed in duplicate. (E and F) 293T cells were transduced with LV-CMV-AAT-CD4. Unmodified 293T cells (control) or gene-modified 293T cells (sCD4) were transfected with plasmids for the production of replication-incompetent HIV JRFL . (E) Culture supernatants from transfected 293T cells were analyzed by p24 ELISA. (F) Culture supernatants from transfected 293T cells were used to infect TZM-bl cells in single-round infection assays. Data are means and SEM from three independent experiments. (G) TZM-bl cells were transduced with LV-EF1α-AAT-sCD4. Unmodified (control) or gene-modified TZM-bl cells (sCD4) were co-cultured with 293T cells transiently expressing HIV JRFL Env. Syncytia formation was analyzed by light microscopy, and the surface area covered by fused cells was determined. A two-tailed unpaired t test was used to determine statistical significance; *p < 0.05. Data are means and SEM from two independent experiments performed in duplicates. (H) Representative microscope images of fused cells from the 15-h time point are shown. The dotted lines indicate the area of fused cells.

Article Snippet: Western blot analysis was performed as previously described., His-tag ELISA (Genscript) was performed according to the manufacturer’s instructions.

Techniques: Transduction, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Infection, Modification, Transfection, Expressing, Light Microscopy, Two Tailed Test, Microscopy

Journal: Molecular Therapy. Nucleic Acids

Article Title: Control of HIV Infection In Vivo Using Gene Therapy with a Secreted Entry Inhibitor

doi: 10.1016/j.omtn.2017.08.017

Figure Lengend Snippet: Generation of Humanized Mice Capable of Expressing sCD4 NSG mice were engrafted with HSPCs transduced with LV-EF1α (control) or LV-EF1α-AAT-sCD4 (sCD4). (A–C) 13–19 weeks post-injection, peripheral blood was analyzed for the presence of human CD45 + , CD19 + , CD3 + , CD4 + , and CD8 + cells and the presence of gene-modified cells within the same cell populations. CD19 + and CD3 + cells were pre-gated for CD45. CD4 + and CD8 + cells were pre-gated for CD45 and CD3. (A) Representative flow cytometry images for a mouse from the sCD4 group. (B) Human cell population in humanized mice. (C) Gene marking across cell populations in humanized mice. (D) The concentration of sCD4 in the peripheral blood of humanized mice was analyzed by ELISA. All data are expressed as means and SEM (n = 9 for the control group; n = 12 for the sCD4 group).

Article Snippet: Western blot analysis was performed as previously described., His-tag ELISA (Genscript) was performed according to the manufacturer’s instructions.

Techniques: Expressing, Transduction, Control, Injection, Modification, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay