Journal: Scientific Reports

Article Title: Distinct systemic microbiome and microbial translocation are associated with plasma level of anti-CD4 autoantibody in HIV infection

doi: 10.1038/s41598-018-31116-y

Figure Lengend Snippet: Demographic and clinical characteristics of the participants.

Article Snippet: Human soluble CD4 protein (sCD4, Progenics Tarrytown, NY) or human soluble CD8B/P37/LEU2 protein (sCD8, Sino Biological Inc. Beijing, China) were diluted at the concentration of 16 μg/ml and added to microtiter wells, and incubated at 4 °C overnight.

Techniques: Cell Counting

Journal: Scientific Reports

Article Title: Distinct systemic microbiome and microbial translocation are associated with plasma level of anti-CD4 autoantibody in HIV infection

doi: 10.1038/s41598-018-31116-y

Figure Lengend Snippet: Plasma level of anti-CD4 IgG and its association with microbial translocation in HIV+ subjects. sCD4 and sCD8 proteins were used to detect plasma anti-CD4 IgGs ( A ) and anti-CD8 IgGs ( B ) by ELISA. Plasma levels of LPS were detected by limulus amebocyte assay ( C ), bacterial 16S rDNA were detected by qPCR ( D ), sCD14 ( E ) and LBP ( F ) by ELISA in healthy controls and HIV+ subjects with plasma anti-CD4 IgG > 50 ng/mL and ≤50 ng/mL. Non-parametric Mann-Whitney tests.

Article Snippet: Human soluble CD4 protein (sCD4, Progenics Tarrytown, NY) or human soluble CD8B/P37/LEU2 protein (sCD8, Sino Biological Inc. Beijing, China) were diluted at the concentration of 16 μg/ml and added to microtiter wells, and incubated at 4 °C overnight.

Techniques: Translocation Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Distinct systemic microbiome and microbial translocation are associated with plasma level of anti-CD4 autoantibody in HIV infection

doi: 10.1038/s41598-018-31116-y

Figure Lengend Snippet: Circulating microbiome relative abundance analysis in healthy controls and HIV+ subjects. Microbial DNA was extracted from plasma and V4 variable region of bacterial 16S rDNA gene was amplified. The relative abundance of phylum ( A ), class ( B ), order ( C ), family ( D ), and genus ( E ) level bacteria (>1%) were shown in plasma from healthy controls, HIV+ subjects with plasma anti-CD4 IgG level ≤ 50 ng/mL and HIV+ subjects with anti-CD4 IgG > 50 ng/mL. The plasma enrichment of Alphaproteobacteria class was significantly higher in the low anti-CD4 IgG patient group compared to the high anti-CD4 IgG patient group after controlling for FDR.

Article Snippet: Human soluble CD4 protein (sCD4, Progenics Tarrytown, NY) or human soluble CD8B/P37/LEU2 protein (sCD8, Sino Biological Inc. Beijing, China) were diluted at the concentration of 16 μg/ml and added to microtiter wells, and incubated at 4 °C overnight.

Techniques: Amplification

Journal: Scientific Reports

Article Title: Distinct systemic microbiome and microbial translocation are associated with plasma level of anti-CD4 autoantibody in HIV infection

doi: 10.1038/s41598-018-31116-y

Figure Lengend Snippet: Reduced diversity was associated with increased plasma level of anti-CD4 autoantibody in HIV+ subjects. Box and whiskers plots of the Simpson ( A ) and Shannon ( B ) diversity indexes of plasma samples from HIV+ subjects with anti-CD4 IgG levels ≤ 50 ng/mL, >50 ng/mL and healthy controls. The top and bottom boundaries of each box indicate the 3 rd and 1 st quartile values, respectively. The central horizontal line represents the median values. The dot represents Simpson and Shannon diversity index of each sample. Non-parametric Mann-Whitney U tests. Correlations between the Simpson diversity index and plasma anti-CD4 IgG levels in healthy controls ( C ) and HIV+ subjects ( D ). Spearman correlation tests.

Article Snippet: Human soluble CD4 protein (sCD4, Progenics Tarrytown, NY) or human soluble CD8B/P37/LEU2 protein (sCD8, Sino Biological Inc. Beijing, China) were diluted at the concentration of 16 μg/ml and added to microtiter wells, and incubated at 4 °C overnight.

Techniques: MANN-WHITNEY

Journal: Scientific Reports

Article Title: Distinct systemic microbiome and microbial translocation are associated with plasma level of anti-CD4 autoantibody in HIV infection

doi: 10.1038/s41598-018-31116-y

Figure Lengend Snippet: Nonmetric multidimensional scaling ordination (NMDS) plot of the OTUs with fitted vectors of clinical variables ( A ), and based on the abundance of bacterial phyla ( B ). Dots with different colors represent data from each plasma sample in HIV+ subjects with anti-IgG level ≤ 50 ng/mL (red) and HIV+ subjects with anti-CD4 IgG > 50 ng/mL (green). Ellipses denote the standard deviation of the weighted average NDMS score of anti-IgG level ≤ 50 ng/mL group (red) and anti-CD4 IgG > 50 ng/mL group (green). Community differences were verified by PERMONOVA test (Adonis, P < 0.05). Arrows represent the direction and magnitude of correlation of each clinical variable ( A ) and the abundance of bacterial phyla ( B ) with the ordination axes.

Article Snippet: Human soluble CD4 protein (sCD4, Progenics Tarrytown, NY) or human soluble CD8B/P37/LEU2 protein (sCD8, Sino Biological Inc. Beijing, China) were diluted at the concentration of 16 μg/ml and added to microtiter wells, and incubated at 4 °C overnight.

Techniques: Standard Deviation

Journal:

Article Title: Differential Inhibition of Human Immunodeficiency Virus Type 1 Fusion, gp120 Binding, and CC-Chemokine Activity by Monoclonal Antibodies to CCR5

doi:

Figure Lengend Snippet: Inhibition of CCR5 coreceptor function by anti-CCR5 MAbs. Inhibition of cell-cell fusion by anti-CCR5 MAbs was tested in the RET assay (a). A total of 0 to 250 μg of PA8 to PA12 per ml or 0 to 25 μg of PA14 or 2D7 per ml was added to a mix of HeLa-EnvJR-FL+ and PM1 cells. Results are mean RET values from three independent experiments and are expressed as percent inhibition of fusion = [1 − (% RET in the presence of MAb/% RET in the absence of MAb)] × 100%. Inhibition of HIV-1 entry by anti-CCR5 MAbs was tested in a single-round replication luciferase-based entry assay (b). U87-CD4+ CCR5+ cells were infected with NLLuc+ Env− reporter virus carrying the JR-LF envelope in the presence of 0 to 250 μg of PA8 to PA12 per ml or 0 to 25 μg of PA14 or 2D7 per ml. Luciferase activity (RLU) was measured in cell lysates 72 h postinfection. Results are from a representative experiment and are expressed as percent inhibition of entry = [1 − (RLU in the presence of MAb/RLU in the absence of MAb)] × 100%. Shown is binding of biotinylated (b) gp120, sCD4, and b-gp120-CD4 complexes to L1.2-CCR5+ cells (c). Strong binding is observed when gp120 derived from the R5 virus HIV-1JR-LF is complexed with an equimolar amount of sCD4. No binding is observed in the absence of sCD4 or for gp120 derived from the X4 virus HIV-1LAI. Background binding to CCR5-L1.2 cells has been subtracted from all curves. Inhibition of gp120-sCD4 binding to L1.2-CCR5+ cells was tested in the presence of varying concentrations of each antibody (d). Cells were preincubated in 96-well plates with an anti-CCR5 MAb followed by an incubation with a saturating concentration of biotinylated gp120-sCD4. Finally, binding of PE-labeled streptavidin to cells was measured with a fluorescence plate reader. Results are from a representative experiment and are expressed as percent inhibition of gp120-sCD4 binding = [1 − (MFI in the presence of MAb/MFI in the absence of MAb)] × 100%.

Article Snippet: CD4-immunoglobulin G2 (IgG2) ( 2 ), sCD4 ( 3 ), and recombinant HIV-1 JR-FL gp120 and HIV-1 LAI gp120 ( 55 ) were produced by Progenics Pharmaceuticals, Inc. CD4-IgG2 is an antibody-like recombinant fusion protein in which the D1 and D2 domains of human CD4 are linked to the heavy and light chain constant regions of human IgG2. sCD4 contains the extracellular domains D1 to D4 of CD4.

Techniques: Inhibition, Luciferase, Infection, Activity Assay, Binding Assay, Derivative Assay, Incubation, Concentration Assay, Labeling, Fluorescence

Journal:

Article Title: Differential Inhibition of Human Immunodeficiency Virus Type 1 Fusion, gp120 Binding, and CC-Chemokine Activity by Monoclonal Antibodies to CCR5

doi:

Figure Lengend Snippet: Potency of anti-CCR5 MAbs in inhibiting HIV-1 envelope-mediated membrane fusion, viral entry, gp120-CCR5 binding, and chemokine signaling a

Article Snippet: CD4-immunoglobulin G2 (IgG2) ( 2 ), sCD4 ( 3 ), and recombinant HIV-1 JR-FL gp120 and HIV-1 LAI gp120 ( 55 ) were produced by Progenics Pharmaceuticals, Inc. CD4-IgG2 is an antibody-like recombinant fusion protein in which the D1 and D2 domains of human CD4 are linked to the heavy and light chain constant regions of human IgG2. sCD4 contains the extracellular domains D1 to D4 of CD4.

Techniques: Binding Assay, Inhibition

Journal:

Article Title: Properties of the Surface Envelope Glycoprotein Associated with Virulence of Simian-Human Immunodeficiency Virus SHIV SF33A Molecular Clones

doi: 10.1128/JVI.76.4.1588-1599.2002

Figure Lengend Snippet: Neutralization of pseudotyped viruses expressing SHIV envelope glycoproteins. HIV-1-luciferase viruses pseudotyped with Env33, EnvA2, and EnvA5 were incubated with serial dilutions of antibodies before being added to HOS-CD4-CXCR4 target cells. Viral entry was monitored by measuring the luciferase activity at day 3 postinfection. Neutralization was determined by measuring the percentages of entry inhibition at different antibody concentrations. Antibodies used in neutralization assays: SHIVSF33 serum from animal 25814 at week 52 (A); MAb IgG1b12, which recognizes the CD4 binding site (B); the IgG-CD4 chimeric protein (C); MAb 17b, which recognizes a CD4i epitope (D); MAb17b in the presence of 0.02 μg of sCD4/ml (E); and MAb 17b (F). For panel F, target cells expressed rhesus macaque CXCR4 instead of human CXCR4.

Article Snippet: The human IgG-CD4 chimeric protein was obtained from Progenics (Tarrytown, N.Y.), and the soluble form of CD4 (sCD4) was obtained from Chiron (Emeryville, Calif.).

Techniques: Neutralization, Expressing, Luciferase, Incubation, Activity Assay, Inhibition, Binding Assay

Journal:

Article Title: Properties of the Surface Envelope Glycoprotein Associated with Virulence of Simian-Human Immunodeficiency Virus SHIV SF33A Molecular Clones

doi: 10.1128/JVI.76.4.1588-1599.2002

Figure Lengend Snippet: Binding of IgG-CD4 to soluble and virion-associated SHIV envelope glycoproteins. (A) Binding of chimeric protein IgG-CD4 to soluble SHIV gp120. Supernatants of cells transfected with Env expression vectors were normalized for gp120 content and then incubated with increasing IgG-CD4 concentrations. The gp120-IgG-CD4 complexes were captured on a D6205-coated plate and quantitated by ELISA. (B) Binding of IgG-CD4 to the surface of SHIV virions. Sucrose-purified virion preparations were incubated with increasing concentrations of IgG-CD4. The virion-IgG-CD4 complexes were separated from unbound IgG-CD4 by centrifugation. Pelleted viruses were lysed in 1% NP-40, which did not disrupt the gp120-CD4 complexes. The lysates were added to D6205-coated plates, and the amount of gp120 bound to CD4 was quantitated by ELISA. neg, mock virion preparation. The results are representative of at least three independent experiments.

Article Snippet: The human IgG-CD4 chimeric protein was obtained from Progenics (Tarrytown, N.Y.), and the soluble form of CD4 (sCD4) was obtained from Chiron (Emeryville, Calif.).

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