human soluble cd4 scd4  (R&D Systems)

Journal: Neuroimmune Pharmacology and Therapeutics

Article Title: The link between chronic cocaine use, B cell perturbations, and blunted immune recovery in HIV-infected individuals on suppressive ART

doi: 10.1515/nipt-2022-0019

Figure Lengend Snippet: Increased plasma levels of anti-CD4 IgG in HIV+ cocaine users and its effect on CD4+ T cell death through ADCC. This study included HIV-negative cocaine users (HIV–/D) and non-users (HIV–/ND), and aviremic ART-treated HIV+ cocaine users (HIV+/D) and non-users (HIV+/ND). The median plasma anti-CD4 IgG levels (A) and CD4+ T cell counts (B) and their correlations in HIV+ subjects (C) were evaluated. (D) The ADCC was performed in CD4+ T and NK cells in the presence of 5 µg/mL of purified anti-CD4 IgGs from ART-treated HIV+ cocaine users (HIV+/D), anti-CD4 IgGs from ART-naive HIV+ cocaine users (Neg1), total IgGs (Neg2) from ART-treated HIV+ cocaine users and an anti-CD4 mAb (Pos). The percent of CD4+ T cytolysis was assessed using flow cytometry. Non-parametric Mann–Whitney U and Spearman correlation tests. *p<0.05; **p<0.01.

Article Snippet: Briefly, we coated the ELISA plate with human soluble CD4 protein (sCD4, Progenics, Tarrytown, NY).

Techniques: Purification, Flow Cytometry, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Elicitation of Neutralizing Antibodies Directed against CD4-Induced Epitope(s) Using a CD4 Mimetic Cross-Linked to a HIV-1 Envelope Glycoprotein

doi: 10.1371/journal.pone.0030233

Figure Lengend Snippet: Surface Plasmon Resonance (SPR) binding analysis of cross-linked gp140-miniCD4 (gp140-S-S-M64U1) complex, in comparison to uncomplexed gp140, to ligands immobilized on CM5 sensor chip: (A) soluble CD4 (sCD4), (B) (CD4 binding site), (C) MAb 2G12 (glycans), and (D) MAb 17b (CD4i site).

Article Snippet: Soluble CD4 (sCD4) was purchased from Progenics Pharmaceuticals (Tarrytown, NY).

Techniques: SPR Assay, Binding Assay

Journal: PLoS ONE

Article Title: Elicitation of Neutralizing Antibodies Directed against CD4-Induced Epitope(s) Using a CD4 Mimetic Cross-Linked to a HIV-1 Envelope Glycoprotein

doi: 10.1371/journal.pone.0030233

Figure Lengend Snippet: Individual rabbit sera from the four groups (immunized with either gp140, miniCD4 M64U1, cross-linked gp140-S-S-M64U1 complex or mixed gp140+M64U1 complex) were tested for neutralizing activity against the isolate 7312A/V434A in the absence (open bars) or presence (filled bars) of sCD4. The ID50 neutralization titers are shown for the pre-immune (black), 2wp3 (red), and 2wp4 (blue) serum samples. The antigens used for immunization are indicated above each graph. Dashed lines indicate the lowest dilution tested.

Article Snippet: Soluble CD4 (sCD4) was purchased from Progenics Pharmaceuticals (Tarrytown, NY).

Techniques: Activity Assay, Neutralization

Journal: BMC Infectious Diseases

Article Title: Anti-HIV-1 activity of cellulose acetate phthalate: Synergy with soluble CD4 and induction of "dead-end" gp41 six-helix bundles

doi: 10.1186/1471-2334-2-6

Figure Lengend Snippet: Binding of HIV-1 and HIV-1-CD4 complexes to CAP coated wells. Suspensions (50 μl) of purified HIV-1 IIIB (6.8 × 10 9 virus particles/ml) and of HIV-1 BaL (2.5 × 10 9 virus particles/ml), respectively, in 0.1 M sodium acetate pH 7.0 were mixed with 5 μg of sCD4 and incubated for 30 min at 20°C. sCD4 was not added to control virus preparations. The suspensions were cooled on ice and polyethylene glycol 6000 (PEG) was added to a final concentration of 3% to separate HIV-1 from sCD4 (which does not precipitate in 3% PEG). After 90 min at 4°C, the mixtures were centrifuged at 10,000 rpm, the supernatant fluids removed and the pellets washed once with 3% PEG in PBS containing 10 mg/ml BSA. The pellets were resuspended in sodium acetate buffer pH 7.0 containing 25 μg/ml BSA, and serial dilutions (indicated on the abscissa) were added to CAP coated and control wells, respectively. After incubation for 5 h at 4°C, the supernatant fluids were removed, the wells washed, and the bound virus quantitated by ELISA for p24 antigen. The amount of HIV-1 IIIB and HIV-1 BaL bound to control wells was ≤ 2.1% and ≤ 13%, respectively, of that bound to CAP coated wells. All experiments were done at least in triplicate.

Article Snippet: Recombinant soluble CD4 (sCD4) was from Genentech Inc., South San Francisco, CA.

Techniques: Binding Assay, Purification, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: BMC Infectious Diseases

Article Title: Anti-HIV-1 activity of cellulose acetate phthalate: Synergy with soluble CD4 and induction of "dead-end" gp41 six-helix bundles

doi: 10.1186/1471-2334-2-6

Figure Lengend Snippet: Synergism between CAP and sCD4 for virucidal activity against HIV-1 IIIB. sCD4 was added to 50 μl of HIV-1 IIIB (6.8 × 10 9 virus particles/ml) in PBS. After 30 min at 20°C, CAP was added to final concentrations between 0 and 1250 μg/ml and the mixtures were incubated for 5 min at 37°C. CAP at the same concentrations described above was also added to virus not pretreated with sCD4. Virus suspensions were put on ice and PEG was added to a final concentration of 3% to separate HIV-1 from CAP and sCD4. After 90 min at 4°C, the mixtures were centrifuged at 10,000 rpm, the supernatant fluids removed and the pellets washed twice with 3% PEG in PBS containing 10 mg/ml BSA. The final washed pellets were resuspended in tissue culture medium, and serial 2-fold dilutions in the same medium were added to MT-2 cells. Infection was monitored by ELISA for p24 antigen. The gray horizontal line corresponds to virus inactivation (80.2 ± 1.3% of residual infectivity) caused by treatment with sCD4 in the absence of CAP. All experiments were done at least in triplicate.

Article Snippet: Recombinant soluble CD4 (sCD4) was from Genentech Inc., South San Francisco, CA.

Techniques: Activity Assay, Incubation, Concentration Assay, Infection, Enzyme-linked Immunosorbent Assay

Journal: BMC Infectious Diseases

Article Title: Anti-HIV-1 activity of cellulose acetate phthalate: Synergy with soluble CD4 and induction of "dead-end" gp41 six-helix bundles

doi: 10.1186/1471-2334-2-6

Figure Lengend Snippet: Treatment of HIV-1 with CAP enhances the expression of binding sites for mAb NC-1. Purified HIV-1 IIIB (6.8 × 10 9 virus particles) and HIV-1 BaL (2.5 × 10 9 virus particles), respectively, each in 100 μl PBS were incubated for 30 min at 20°C in the presence (5 μg) or absence of sCD4. Each sample was divided into two equal portions. CAP in 0.1 M acetate pH 6.0 was added to one aliquot (final concentration 5 mg/ml). An equivalent amount of acetate without CAP was added to the second aliquot. After incubation for 5 min at 37°C, the suspensions were cooled on ice and PEG was added to a final concentration of 3% to separate HIV-1 from sCD4 and CAP. After 90 min at 4°C, the mixtures were centrifuged at 10,000 rpm, the supernatant fluids removed and the pellets washed once with 3% PEG in PBS containing 10 mg/ml BSA. The final washed pelleted viruses were suspended in PBS containing 100 μg/ml of each BSA and gelatin, and dilutions (indicated on the abscissa) in the same buffer were added to mAb NC-1 coated and control wells. After 5 h at 4°C, the wells were washed and bound virus was quantitated by ELISA for p24 antigen. The amount of HIV-1 IIIB and HIV-1 BaL, respectively, bound to control wells was ≤ 2.1% and ≤ 6.1% of that bound to mAb NC-1 coated wells. All experiments were done at least in triplicate.

Article Snippet: Recombinant soluble CD4 (sCD4) was from Genentech Inc., South San Francisco, CA.

Techniques: Expressing, Binding Assay, Purification, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: BMC Infectious Diseases

Article Title: Anti-HIV-1 activity of cellulose acetate phthalate: Synergy with soluble CD4 and induction of "dead-end" gp41 six-helix bundles

doi: 10.1186/1471-2334-2-6

Figure Lengend Snippet: Binding of biotinyl-gp120, biotinyl-sCD4 and biotinyl-gp120-sCD4 complexes to CAP coated wells. Graded quantities of biotinyl-gp120 (indicated on the abscissa) and of biotinyl-gp120-sCD4 complexes were added to CAP coated and control wells. The complexes were prepared by mixing 1 μg of biotinyl-gp120 with 500 ng of sCD4 for 30 min at 20°C in PBS. To determine the effect of sCD4/gp120 ratios on gp120 binding to CAP, constant amounts (500 ng) of biotinyl-gp120 were mixed with graded quantities of sCD4 (0 to 1,000 ng) (insert) and the mixtures were further handled as described above. Binding of biotinyl-gp120 did not increase at sCD4/biotinyl-gp120 weight ratios >1 (data not shown). In control experiments, graded quantities of biotinyl-sCD4 in the absence of gp120 were added to the wells. After incubation at 4°C overnight, the wells were washed and bound biotinylated proteins were detected from subsequent binding of HRP-streptavidin. Absorbance corresponding to biotinylated proteins bound to control wells was in the range of 0–0.026 and was substracted from the absorbance corresponding to biotinylated proteins bound to CAP coated wells.

Article Snippet: Recombinant soluble CD4 (sCD4) was from Genentech Inc., South San Francisco, CA.

Techniques: Binding Assay, Incubation

Journal: BMC Infectious Diseases

Article Title: Anti-HIV-1 activity of cellulose acetate phthalate: Synergy with soluble CD4 and induction of "dead-end" gp41 six-helix bundles

doi: 10.1186/1471-2334-2-6

Figure Lengend Snippet: Synergism between CAP and sCD4 in inhibiting infection by HIV-1 IIIB.

Article Snippet: Recombinant soluble CD4 (sCD4) was from Genentech Inc., South San Francisco, CA.

Techniques: Infection

Journal: BMC Infectious Diseases

Article Title: Anti-HIV-1 activity of cellulose acetate phthalate: Synergy with soluble CD4 and induction of "dead-end" gp41 six-helix bundles

doi: 10.1186/1471-2334-2-6

Figure Lengend Snippet: Synergism between CAP and sCD4 in inhibiting infection by HIV-1 BaL.

Article Snippet: Recombinant soluble CD4 (sCD4) was from Genentech Inc., South San Francisco, CA.

Techniques: Infection

Journal: BMC Infectious Diseases

Article Title: Anti-HIV-1 activity of cellulose acetate phthalate: Synergy with soluble CD4 and induction of "dead-end" gp41 six-helix bundles

doi: 10.1186/1471-2334-2-6

Figure Lengend Snippet: Induction of gp41 six-helix bundles by HIV-1 treatment with sCD4, CAP, CCR5 S-peptide and heat. HIV-1 IIIB and HIV-1 BaL, respectively, were treated with CAP and sCD4 as described in the legend for Fig. or exposed to 60°C for 10 min. HIV-1 BaL was also treated with the S-peptide and a control non-sulfated peptide from CCR5 as described in the legend for Fig. . The treated virus preparations and untreated control virus were treated with lysis buffer (see Methods) for 30 min at 20°C. CAP in lysis buffer (0.078 to 10 mg/ml) was also tested; the results for a 5 mg/ml concentration are shown (identical results were obtained for all other concentrations). The lysates were tested by a sandwich ELISA for the gp41 six-helix bundle (see Methods). All experiments were done at least in triplicate.

Article Snippet: Recombinant soluble CD4 (sCD4) was from Genentech Inc., South San Francisco, CA.

Techniques: Lysis, Concentration Assay, Sandwich ELISA

Journal: BMC Infectious Diseases

Article Title: Anti-HIV-1 activity of cellulose acetate phthalate: Synergy with soluble CD4 and induction of "dead-end" gp41 six-helix bundles

doi: 10.1186/1471-2334-2-6

Figure Lengend Snippet: Electrostatic potential maps of gp120 and the gp120-CD4 complex. Electrostatic potentials are shown for the solvent accessible surfaces. (A) The electrostatic potential map on gp120. Blue indicates electropositive areas whereas red represents electronegative areas. (B) After CD4 binds to gp120, the electropositive surface area (blue) increases markedly while the most negatively charged (red) area on gp120 (arrow) becomes blocked by sCD4.

Article Snippet: Recombinant soluble CD4 (sCD4) was from Genentech Inc., South San Francisco, CA.

Techniques:

Journal: Viruses

Article Title: HIV-1 Envelope Glycoprotein Cell Surface Localization Is Associated with Antibody-Induced Internalization

doi: 10.3390/v13101953

Figure Lengend Snippet: Antibody-internalization of HIV-1 Env from the cell surface can be accelerated upon the selective opening of Env. ( A – D ) Cell surface staining of primary CD4+ T cells infected in vitro with ( A ) CH58 T/F virus, ( B ) JRFL WT virus, ( C ) CH58 T/F WT or L193A virus, and ( D ) CH58 T/F virus defective for Nef and Vpu expression was performed 48 h post-infection. ( E , F ) Primary CD4+ T cells from at least three different HIV-1-infected individuals were isolated and reactivated with PHA-L for 48 h, followed by incubation with IL-2 to expand the endogenous virus. Cell surface staining of endogenously infected primary CD4+ T cells was performed upon reactivation. ( A – F ) Antibody binding was detected using Alexa Fluor 647-conjugated anti-human secondary Abs. (Top) Quantification of remaining antibody–Env complexes on the cell surface over different timepoints is expressed as percentage of the MFI relative to the 0 min timepoint control. (Bottom) Areas under the curve (AUC) were calculated based on MFI data sets using GraphPad Prism software. Error bars indicate means ± the SEM. Statistical significance was tested using an unpaired t test or a Mann-Whitney U test based on statistical normality (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Article Snippet: To investigate whether the differences in antibody-mediated Env internalization is due to distinct populations of Env on the cell surface being targeted, we utilize ligands such as small CD4-mimetics (CD4mc) and soluble CD4 (sCD4) to ‘force’ open the otherwise ‘closed’ Env populations and evaluate the rates of nNAb-mediated internalization.

Techniques: Staining, Infection, In Vitro, Virus, Expressing, Isolation, Incubation, Binding Assay, Control, Software, MANN-WHITNEY

Journal: Viruses

Article Title: HIV-1 Envelope Glycoprotein Cell Surface Localization Is Associated with Antibody-Induced Internalization

doi: 10.3390/v13101953

Figure Lengend Snippet: Antibody-induced internalization of Env from the surface of transfected cells. ( A ) Cell surface staining of 293T cells transfected with plasmid encoding JRFL Env alone or together with an expressor of the human CD4 receptor was performed 48-h post-transfection. Ab binding was quantified at 0-, 60-, 120- and 180-min using flow cytometry. Histograms depict representative staining of transfected cells and untransfected (gray) with 17b or 19b Abs. ( B , C ) The 293T cells were transfected with a plasmid encoding JRFL Env alone or together with an expressor of the human CD4 receptor and were stained with ( B ) 19b conjugated with Alexa Fluor 647 or ( C ) 17b conjugated with Alexa Fluor 594 for confocal microscopy analyses to visualize internalization. Alternatively, staining with 17b-sCD4 was performed in combination with the goat anti-human IgG Alexa Fluor 594 secondary Ab. ( B , C , Left panels) Images show the localization of antibody–Env complexes at different time points (0 and 120 min). Images represent a single confocal z-section through the middle of the cell; at least 25 cells were imaged per condition, and representative images are shown. Scale bar, 10 μm. ( B , C , Right panels) The remaining cell surface antibody–Env complexes over different time points are expressed as percentages of the surface fluorescence relative to the 0 min time point control. Error bars indicate means ± the SEM. Statistical significance was tested using an unpaired t test or a Mann–Whitney U test based on statistical normality (**, p < 0.01; ***, p < 0.001). mCD4; membrane-anchored CD4.

Article Snippet: To investigate whether the differences in antibody-mediated Env internalization is due to distinct populations of Env on the cell surface being targeted, we utilize ligands such as small CD4-mimetics (CD4mc) and soluble CD4 (sCD4) to ‘force’ open the otherwise ‘closed’ Env populations and evaluate the rates of nNAb-mediated internalization.

Techniques: Transfection, Staining, Plasmid Preparation, Binding Assay, Flow Cytometry, Confocal Microscopy, Fluorescence, Control, MANN-WHITNEY, Membrane

Journal: Viruses

Article Title: HIV-1 Envelope Glycoprotein Cell Surface Localization Is Associated with Antibody-Induced Internalization

doi: 10.3390/v13101953

Figure Lengend Snippet: Differential localization of antibody–Env complexes visualized by lipid microdomain fractionation. The 293T cells were transfected with a plasmid encoding JRFL Env alone or together with an expressor of the human CD4 receptor and 48-h post-transfection all cell surface proteins were biotinylated. ( A ) Immunoprecipitation of cell surface biotinylated Env from cells transfected to express the (left) JRFL Env alone or (right) with the human CD4 receptor after incubation with 19b. Further, cell lysates were fractionated on a sucrose density gradient as described in Materials and Methods. ( B ) Equal volumes of individual fractions were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with HRP-conjugated CTx-B to detect DRM marker ganglioside GM1 or with OKT-9 antibody to detect DSM marker CD71. ( C – F ) Immunoprecipitation of cell surface biotinylated Env from individual sucrose gradient fractions using ( C ) PGT151, ( D ) 19b, ( E ) 19b with 5μM BNM-III-170 from cells expressing the JRFL Env only and ( F ) 19b from cells expressing both the JRFL Env and human CD4 receptor. Values represent densities of respective band intensities quantified using ImageJ normalized to the bottom fractions. ( B – F ) Representative blots from at least three independent experiments are shown. mCD4; membrane-anchored CD4.

Article Snippet: To investigate whether the differences in antibody-mediated Env internalization is due to distinct populations of Env on the cell surface being targeted, we utilize ligands such as small CD4-mimetics (CD4mc) and soluble CD4 (sCD4) to ‘force’ open the otherwise ‘closed’ Env populations and evaluate the rates of nNAb-mediated internalization.

Techniques: Fractionation, Transfection, Plasmid Preparation, Immunoprecipitation, Incubation, SDS Page, Membrane, Marker, Expressing