Journal: ACS Omega

Article Title: Engineering Delivery of Nonbiologics Using Poly(lactic- co -glycolic acid) Nanoparticles for Repair of Disrupted Brain Endothelium

doi: 10.1021/acsomega.0c01517

Figure Lengend Snippet: Internalization and targeting efficiency studies of conjugated and unconjugated PLGA NPs. (A–D) Fluorescence images (green) showing the specificity of PSGL-1 conjugated PLGA NPs to either inactivated or TNF-α activated BECs. (E) Quantitative analysis of the specificity of PSGL-1-conjugated PLGA NPs. Data represent mean ± SD of three to six independent experiments. * p < 0.05 compared to the solid black bar. (F–H) Targeting efficiency study of conjugated PLGA NPs to endothelial cells exposed to mechanical trauma. (F) Control cells incubated with PSGL-1-conjugated PLGA NPs for 1 h. (G) Endothelial cells exposed to mechanical trauma and then incubated with PSGL-1-conjugated PLGA NPs for 1 h. The area of detached cell is apparent and indicated by a dotted line for better illustration. (H) Quantitative analysis of the specificity of PSGL-1-conjugated PLGA NPs. Data represent mean ± SD of three to six independent experiments. * p < 0.05.

Article Snippet: Eighty microliters of 500 μg/mL mouse PSGL-1 antibody (Sino Biological, 50770-MCCH) was added to the NP solution and incubated for 24 h at 4 °C.

Techniques: Fluorescence, Incubation

Journal: Retrovirology

Article Title: P-selectin glycoprotein ligand-1 (PSGL-1/CD162) is incorporated into clinical HIV-1 isolates and can mediate virus capture and subsequent transfer to permissive cells

doi: 10.1186/s12977-022-00593-5

Figure Lengend Snippet: Comparison of PSGL-1 cell surface expression and virus infectivity across model systems. A Schematic depicting the three model systems (HEK293T transfection, T cell line infection, PBMC infection) used to produce virus with various amounts of PSGL-1 in the manuscript. B Cell surface expression of PSGL-1 on HEK293T cells 48 h after co-transfection with HIV-1 pseudovirus constructs alone (PV; grey histogram), or with pseudovirus constructs and 250 ng of a vector encoding PSGL-1 (PV + PSGL-1, blue histogram). Endogenous levels of PSGL-1 detected by cell surface staining and flow cytometry analysis on the T cell lines, H9 (green) and Jurkat (purple), or activated peripheral blood mononuclear cells (PBMC) from two different donors (red and pink histograms) used for HIV-1 propgation. Isotype staining is shown with unshaded histograms. C Viruses produced in the transfected HEK293T cells or HIV IIIB-infected T cell lines and PBMC from B were normalized for equal viral p24 input in TZM-bl cell cultures. After 48 h the level of infectivity was measured using luminescence readout (relative light units; RLU). Results displayed are the merged mean ± SEM of three independent experiments with each condition tested in duplicate wells. P values were determined using an unpaired t test (****P < 0.0001)

Article Snippet: PE-conjugated mouse anti-PSGL-1 mAb (BD Biosciences, Cat#556055) was used for flow virometry labelling.

Techniques: Expressing, Infection, Transfection, Cotransfection, Construct, Plasmid Preparation, Staining, Flow Cytometry, Produced

Journal: Retrovirology

Article Title: P-selectin glycoprotein ligand-1 (PSGL-1/CD162) is incorporated into clinical HIV-1 isolates and can mediate virus capture and subsequent transfer to permissive cells

doi: 10.1186/s12977-022-00593-5

Figure Lengend Snippet: Titration of PSGL-1 expression on virus producing cells and the effect on virus infectivity. A Cell surface expression of PSGL-1 on HEK293T cells as detected by flow cytometry 48 h after co-transfection with HIV-1 pseudovirus constructs and increasing amounts of PSGL-1 pDNA (as outlined in Additional file : Table S1). Isotype staining is shown with empty histograms, and PSGL-1 staining is shown with filled, coloured histograms (blue or gray). B Semi-quantitative comparisons of virion-incorporated PSGL-1 on pseudovirus stocks via immunoblot analysis. The viral capsid protein p24 was used as loading control to ensure equal loading of total virus lysates across all lanes. This immunoblot is representative of three blots performed showing similar results. C Densitometric quantitation of immunoblot data from B . D Virus infection was tested via normalized viral inputs (displayed as 1:1 in graph), followed by three-fold serial dilutions of viruses. All diluted virus stocks were incubated with TZM-bl reporter cells for 48 h before infectivity was measured using luminescence readout (relative light units; RLU). E Infectivity from the RLU reading with the most concentrated amount of virus (1:1) is shown. For C and E the results of unpaired t tests with Bonferroni correction are shown (*P < 0.05; **P < 0.01). Results show the mean ± SEM of three independent experiments

Article Snippet: PE-conjugated mouse anti-PSGL-1 mAb (BD Biosciences, Cat#556055) was used for flow virometry labelling.

Techniques: Titration, Expressing, Infection, Flow Cytometry, Cotransfection, Construct, Staining, Western Blot, Quantitation Assay, Incubation

Journal: Retrovirology

Article Title: P-selectin glycoprotein ligand-1 (PSGL-1/CD162) is incorporated into clinical HIV-1 isolates and can mediate virus capture and subsequent transfer to permissive cells

doi: 10.1186/s12977-022-00593-5

Figure Lengend Snippet: Semi-quantitative comparisons of virion-incorporated PSGL-1 and gp120 on virus stocks via virion capture assay. A Virion capture assays were performed with immunomagnetic beads armed with anti-PSGL-1 or anti-gp120 with normalized inputs (5.25 ng of p24 per capture condition) of pseudovirus (HEK293T), B T cell line viruses, and C PBMC viruses (NL4-3, IIIB, BaL generated in donors 1, 2 and 3 respectively). Bead-associated virus was lysed and HIV-1 p24 Gag was quantified using p24 AlphaLISA as an indicator of the amount of virus capture. Results show the mean ± SEM of three independent experiments in which each condition was tested in duplicate. The results of unpaired t tests with Bonferroni correction are shown (**P < 0.01) for A . Levels of background capture as detected using an isotype control antibody were subtracted from the displayed values

Article Snippet: PE-conjugated mouse anti-PSGL-1 mAb (BD Biosciences, Cat#556055) was used for flow virometry labelling.

Techniques: Generated

Journal: Retrovirology

Article Title: P-selectin glycoprotein ligand-1 (PSGL-1/CD162) is incorporated into clinical HIV-1 isolates and can mediate virus capture and subsequent transfer to permissive cells

doi: 10.1186/s12977-022-00593-5

Figure Lengend Snippet: Detecting PSGL-1-incorporation on the surface of viruses using flow virometry. A Staining of pseudoviruses produced through transfection of HEK293T cells with different amounts of PSGL-1 DNA (0 ng, 2.5 ng, 25 ng, 250 ng for negative, low, medium, and high phenotypes, respectively) with a PE-conjugated anti-PSGL-1 antibody. The horizontal dotted line on the virus dot plots denotes background fluorescence and the limit of instrument detection (~ 10 MESF). B PSGL-1 staining of IIIB viruses produced in the H9, Jurkat, A3R5.7 and PM1 T cells. C PSGL-1 staining of HIV IIIB and NL4-3 viruses propropagated in four independent primary cell (PBMC) donors (D1–D4). Data shown are representative of three replicates for each virus stock

Article Snippet: PE-conjugated mouse anti-PSGL-1 mAb (BD Biosciences, Cat#556055) was used for flow virometry labelling.

Techniques: Staining, Produced, Transfection, Fluorescence

Journal: Retrovirology

Article Title: P-selectin glycoprotein ligand-1 (PSGL-1/CD162) is incorporated into clinical HIV-1 isolates and can mediate virus capture and subsequent transfer to permissive cells

doi: 10.1186/s12977-022-00593-5

Figure Lengend Snippet: Virions circulating in vivo in HIV-infected patients contain PSGL-1 and CD44. A Plasma samples from viremic HIV-infected patients ranging from acute/early to chronic stages of HIV-1 infection were tested in virion capture assays using an isotype control antibody (IgG control), anti-PSGL-1 or anti-CD44. Captured virus was lysed, followed by RNA extraction and quantitative real-time PCR for the detection of HIV-1 genome equivalents (in RNA copies/mL). The sample median for each antibody capture condition is displayed along with results of a Mann–Whitney test (***P < 0.001). Bonferroni correction was used for adjustment of statistical significance. Each unique symbol represents a different patient, with open circles denoting patients in the acute/early stage infection and filled circles as patients in the chronic stage of infection

Article Snippet: PE-conjugated mouse anti-PSGL-1 mAb (BD Biosciences, Cat#556055) was used for flow virometry labelling.

Techniques: In Vivo, Infection, RNA Extraction, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: Retrovirology

Article Title: P-selectin glycoprotein ligand-1 (PSGL-1/CD162) is incorporated into clinical HIV-1 isolates and can mediate virus capture and subsequent transfer to permissive cells

doi: 10.1186/s12977-022-00593-5

Figure Lengend Snippet: Virion-incorporated PSGL-1 retains the ability to bind selectin family receptors. A Virion capture assays were performed with immunomagnetic beads armed with 0.5 μg of recombinant selectins (P-, E-, L-selectin) or MAdCAM-1 with normalized inputs of NL4-3 virus produced through transfection of HEK293T cells with various amounts of PSGL-1 DNA (0 ng, 2.5 ng, 250 ng for negative, low, and high phenotypes respectively). B Viruses produced in T cell lines, and C PBMC (IIIB generated in two separate PBMC donors, D1 and D2) were added to armed beads at their undiluted titre. Bead-associated virus was lysed and HIV-p24 Gag was quantified using p24 AlphaLISA as an indicator of the amount of virus capture. Results show the mean ± SD of two independent experiments in which each condition was tested in duplicate. The results of unpaired t tests with Bonferroni correction are shown (**P < 0.01, ***P < 0.001)

Article Snippet: PE-conjugated mouse anti-PSGL-1 mAb (BD Biosciences, Cat#556055) was used for flow virometry labelling.

Techniques: Recombinant, Produced, Transfection, Generated

Journal: Retrovirology

Article Title: P-selectin glycoprotein ligand-1 (PSGL-1/CD162) is incorporated into clinical HIV-1 isolates and can mediate virus capture and subsequent transfer to permissive cells

doi: 10.1186/s12977-022-00593-5

Figure Lengend Snippet: PSGL-1-positive virions can be selectively captured by cells expressing P-selectin. A Schematic of cell capture assay: virus preparations were added to wells containing HeLa cells transiently transfected to express MAdCAM-1 or P-selectin to allow for virion capture. Post-incubation, wells were washed extensively to remove unbound virus and were assayed for the amount of captured virus using p24 detection. B Experimental results for virus preparations produced through transfection of HEK293T cells with different PSGL-1 phenotypes (0 ng, 2.5 ng, 250 ng of PSGL-1 pDNA for negative, low, and high phenotypes respectively) or through infection ( C ) were used at their undiluted titre. Results displayed are the mean ± SD of two independent experiments with samples tested in duplicate and are representative of three independent experiments. P values were determined using an unpaired t test with Bonferroni correction (***P < 0.001)

Article Snippet: PE-conjugated mouse anti-PSGL-1 mAb (BD Biosciences, Cat#556055) was used for flow virometry labelling.

Techniques: Expressing, Transfection, Incubation, Produced, Infection

Journal: Retrovirology

Article Title: P-selectin glycoprotein ligand-1 (PSGL-1/CD162) is incorporated into clinical HIV-1 isolates and can mediate virus capture and subsequent transfer to permissive cells

doi: 10.1186/s12977-022-00593-5

Figure Lengend Snippet: PSGL-1 positive virions can be captured by P-selectin and transferred to HIV-permissive target cells for infection. A Schematic depicting the experimental workfolw: virus preparations were added to MadCAM-1 and/or P-selectin coated wells to allow for virion capture. Post-incubation, wells were washed extensively to remove unbound virus and then were either assayed for the amount of captured virus using p24 detection (top workflow), or TZM-bl cells were overlayed onto each well for virus transfer, ad infectivity measurements via luminescence readout (bottom workflow). B Experimental results from viruses produced through transfection of HEK293T, C infection of T cell lines, and D PBMC IIIB viruses in plate-based virus capture (left column) and transfer assays (right column). Results displayed are the mean ± SD of two experiments with samples tested in duplicate wells for B and C . P values were determined using an unpaired t test (*P < 0.05; ***P < 0.0001). Results shown in D are representative of two independent experiments tested with duplicate wells

Article Snippet: PE-conjugated mouse anti-PSGL-1 mAb (BD Biosciences, Cat#556055) was used for flow virometry labelling.

Techniques: Infection, Incubation, Produced, Transfection