Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-16 promotes leukotriene C(4) and IL-4 release from human eosinophils via CD4- and autocrine CCR3-chemokine-mediated signaling.

doi: 10.4049/jimmunol.168.9.4756

Figure Lengend Snippet: FIGURE 3. Receptors involved in IL-16-induced activation of eosino- phils. A, CD4 mediates the activation of eosinophils by IL-16. As indi- cated, eosinophils were pretreated for 30 min with inhibitors of IL-16/CD4 interaction (including sCD4 and Fab of OKT4 anti ()-CD4 Ab (or irrel- evant control W6/32 anti-HLA Ab)) or with the Gi protein inhibitor PTX and then were incubated with IL-16 (100 nM) for 1 h for induction of lipid bodies, priming for enhanced LTC4 production (fluid-phase assays), and IL-4 release (solid-phase matrix assays). B, Involvement of Gi protein- coupled CCR3 receptors. As indicated, eosinophils were pretreated for 30 min with antagonists of PAF (CV6209 and BN52021) and CCR3 (Met- RANTES (MetR) and anti-CCR3 neutralizing mAb (or isotype control rat IgG2a)) receptors and then were activated with IL-16. Results are means SD from three independent assays. and , p 0.05 compared with nonstimulated and IL-16-stimulated eosinophils, respectively. B, Values represent the calculated percentage of inhibition with antagonists in com- parison with stimulated increases in lipid body numbers, LTC4 production, and IL-4 release above baselines.

Article Snippet: Eosinophils were stimulated with IL-16 (0.01–100 nM; R&D Systems), RANTES, or eotaxin (6 nM; R&D Systems) for time periods ranging from 5 min to 3 h. For inhibitor studies, cells were pretreated for 30 min with pertussis toxin (PTX) (20 ng/ml; Calbiochem, La Jolla, CA), recombinant soluble CD4 (sCD4) (50 ng/ml; R&D Systems), Fab of anti-CD4 (clone OKT4; American Type Culture Collection, Manassas, VA) and anti-HLA class I (clone W6/32; Sigma-Aldrich) mAbs (1 g/ml), anti-CCR3 mAb (clone 61828.111; R&D Systems), or isotype control rat IgG2a at 10 g/ml (BD PharMingen, San Diego, CA), met-RANTES (60 nM; R&D Systems), the platelet-activating factor (PAF) receptor antagonists CV6209 and BN52021 (10 M; Biomol, Plymouth Meeting, PA), brefeldin A (BFA) (0.1 and 1 g/ml; Biomol), or their vehicles, as indicated.

Techniques: Activation Assay, Control, Incubation, Inhibition

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: BMC Infectious Diseases

Article Title: Cellulose acetate phthalate, a common pharmaceutical excipient, inactivates HIV-1 and blocks the coreceptor binding site on the virus envelope glycoprotein gp120

doi: 10.1186/1471-2334-1-17

Figure Lengend Snippet: Binding of CAP treated and untreated HIV-1 to distinct ligands. The binding of untreated and CAP treated HIV-1 IIIB to wells coated with sCD4 or with distinct mAbs and of HIV-1 BaL to anti-V3 BaL was measured as described for Fig. . The % of binding corresponding to CAP treated virus was calculated based on the formula (% residual binding = [absorbance corresponding to bound CAP treated virus ÷ absorbance corresponding to bound untreated HIV-1] × 100. Absorbance corresponding to p24 antigen (5-fold dilution of the sample) from untreated HIV-1 bound to the respective ligands was in the range of 0.56 to 1.54. Absorbance corresponding to virus captured onto wells coated with control IgG was 0.046. All experiments were done in triplicate. To measure virus binding to the coreceptor CXCR4, treated and control HIV-1 IIIB recovered after PEG precipitation was mixed with 10 μg of sCD4. After 5 min at 20°C, the respective samples were divided into 2 aliquots, each of which was added to 5 × 10 5 GHOST CXCR4 cells suspended in 100 μl PBS containing 100 μg/ml of BSA. Similar experiments were carried out with purified HIV-1 BaL, except that GHOST CCR5 cells were used. After 1 h at 4°C, the cells were pelletted and washed with ice cold PBS containing 100 μg/ml BSA. The pelletted cells were lysed for 30 min at 37°C in PBS with 1% NP40. Serial 5-fold dilutions in PBS (1:5 to 1:4.9 × 10 7 ) were tested by ELISA for the p24 antigen. Changes in HIV-1 binding were determined using calibration curves relating absorbance to virus dilutions.

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

Techniques: Binding Assay, Virus, Control, Purification, Enzyme-linked Immunosorbent Assay

Journal: BMC Infectious Diseases

Article Title: Cellulose acetate phthalate, a common pharmaceutical excipient, inactivates HIV-1 and blocks the coreceptor binding site on the virus envelope glycoprotein gp120

doi: 10.1186/1471-2334-1-17

Figure Lengend Snippet: Binding of sCD4 to CAP treated and untreated gp120. Recombinant gp120 IIIB (5 μg in 400 μl of 0.1 M sodium acetate buffer pH 7.0) was treated with CAP (5 mg/ml) for 5 min at 37°C and then cooled to 0°C. CAP was omitted in control experiments. BSA was added to a final concentration of 25 μg/ml and the samples were filtered using a 2 ml Centricon centrifugal ultrafiltration device with a M w cutoff of 100,000 and centrifuged at 3,500 × g for 30 min. gp120 retained on the filters was washed with PBS, resuspended in PBS, and serially diluted 5-fold. The diluted samples (corresponding to gp120 quantities indicated on the abscissa) were used to coat wells of 96-well polystyrene plates. Binding to the wells of biotin labeled sCD4 was detected from subsequent binding of HRP-streptavidin.

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

Techniques: Binding Assay, Recombinant, Control, Concentration Assay, Labeling

Journal: BMC Infectious Diseases

Article Title: Cellulose acetate phthalate, a common pharmaceutical excipient, inactivates HIV-1 and blocks the coreceptor binding site on the virus envelope glycoprotein gp120

doi: 10.1186/1471-2334-1-17

Figure Lengend Snippet: Binding of sCD4 complexes with CAP treated and control gp120, respectively, to HIV-1 coreceptor expressing cells. CAP treated or control gp120 IIIB or MN (prepared as described for Fig. ) were mixed with biotinylated sCD4 and added to 10 6 MT-2 cells (A) or PBL (B and C). The cells were washed with PBS, treated with phycoerythrin (PE)-labeled streptavidin, fixed in 1% formaldehyde and submitted to flow cytometry analysis.

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

Techniques: Binding Assay, Control, Expressing, Labeling, Flow Cytometry