Sn expression on CD14⁺ monocytes from HIV seropositive individuals.

<p>(A) Representative frequency histograms of relative Sn expression on CD14⁺ monocytes isolated from subjects with high viral load (HVL, 214,000 RNA copies/ml, thick black line), low viral load (LVL, 6,350 RNA copies/ml, thin black line) and a seronegative control (dotted line). The isotype-matched control mAb is shown in the shaded profile. (B) Correlation analysis of Sn expression and viral load. Sn expression on CD14⁺ monocytes from HIV seropositive subjects (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001967#pone-0001967-t001" target="_blank">Table 1</a>, n = 24) was determined by flow cytometry and quantified as a geometric mean for each subject. Pearson's correlation analysis showed statistical significance between Sn expression and the log of the subject's viral load (p<0.0017). (C) Correlation analysis of Sn expression and CD4 (counts/ml) revealed no significant relationship (p<0.08).</p

Sn binds HIV-1 <i>in vitro</i>.

<p>(A) TSn binds HIV-1 in an Sn-dependant manner. TSn and THP-1 were incubated with lab-adapted HIV-1_NL4-3, an HIV-1 clade B primary isolate or clade C primary isolate for 1 h at 37°C, washed and then assayed for HIV-1 p24 by ELISA. HIV-1 binding to Sn was abrogated by pretreatment of cells with Sn mAb 7D2 or by pretreatment of HIV-1 with broad-spectrum sialidase. Pretreatment with IgG1 isotype control or CD4 mAbs did not reduce binding. Data presented are the average of 3 separate experiments. (B) IFN-α-treated CD14⁺ monocytes bind HIV-1 in an Sn-dependant manner. HIV-1 binding assays were also performed on CD14⁺ monocytes from seronegative controls treated with 500 U/ml IFN-α to induce Sn expression. Pretreatment with Sn mAb 7D2 and sialidase dramatically reduced HIV-1 binding while pretreatment with IgG1 isotype control or CD4 mAbs had little effect. Data represents monocytes from four separate donors. Error bars represent SD.</p

Sn-expressing cells capture HIV-1_NL4-3 and enhance infectivity.

<p>TZM-bl cell cultures were seeded with various concentrations of HIV-1_NL4-3 (800–8000 pg/ml). Monocytic cells, TSn or THP-1 cells, were added and cocultured for 48 h. The capacity of Sn to capture HIV-1_NL4-3 in the cell culture medium and <i>trans</i> infect TZM-bl cells was analyzed for TSn, THP-1 and cell-free virus. Luciferase expression in HIV-1_NL4-3-infected TZM-bl cells was quantified as relative light units (RLU). Results were compiled from 3 separate experiments. Error bars represent SD.</p

Constitutive Sn expression in THP-1 by gene transduction (TSn).

<p>(A) Immunoblot analysis of Sn protein expression. THP-1 cell line was transduced with a plasmid encoding Sn cloned downstream of the high-level constitutive promoter CMV. Cell lysates were standardized, reduced with DTT and 10 µg of protein were loaded into each well: monocytes (lane 1), IFN-α-induced monocytes (lane2), THP-1 (lane 3) and TSn (lane 4) (M, molecular size marker). (B) Histogram of relative distribution of Sn on the cell surface of TSn clone. TSn (thick black line) and THP-1 cells (thin black line) were evaluated for Sn expression by flow cytometry using anti-Sn mAb 7D2 relative to the background isotype-matched control mAb (shaded region).</p

Interferon-α and -γ induce Sn expression on CD14⁺ monocytes and THP-1 cells.

<p>Cells were cultured in 500 U/ml IFN-α, 100 U/ml IFN-γ or 10 ng/ml TNF-α at 37°C for 48 h and analyzed for Sn expression by flow cytometry. Sn expression on IFN-α-, IFN -γ- or TNF-α-treated cells (thick black lines) and untreated cells (thin black line) were relative to an isotype-matched mAb control (shaded region). Results shown are representative histograms from three independent experiments using monocytes from three seronegative donors.</p

Sn-dependent <i>trans</i> infection of reporter cells TZM-bl.

<p>(A) Sn-expressing cells, TSn and IFN-α-induced monocytes, bind HIV-1 and infect TZM-bl cells in <i>trans</i>. Cells were incubated with HIV-1_NL4-3 for 1 h, washed and then cocultured with TZM-bl cells for 48 h. HIV-1 infection of TZM-bl was defined by luciferase expression and quantified as relative light units (RLU). Cells pretreated with Sn mAb 7D2 showed significantly reduced capacity to facilitate <i>trans</i> infection of TZM-bl cells while mAb IgG1 isotype control had no effect. (B) HIV-1 receptor inhibitors block <i>trans</i> infection. Receptor and coreceptor requirements for <i>trans</i> infection of TZM-bl cells were tested by incubating TZM-bl cells with receptor inhibitors including mAbs to CD4, CXCR4 or CCR5, and small molecules AMD3100 or TAK779 prior to adding TSn with bound HIV-1_NL4-3. The CD4, CXCR4, CCR5, AMD3100 and TAK779 receptor inhibitors were tested individually with TZM-bl and did not induce luciferase expression (data not shown). As a control, productive infection of TSn cells was prevented by addition of indinavir (100 µM). Data presented are the average of three separate experiments. Error bars represent SD.</p

Clinical data and Sn expression levels from controls and HIV-1 seropositive subjects.

<p>Data for controls (C, n = 10), HIV-1 seropositive subjects (V, n = 24)</p><p>Data sorted on increasing viral load</p>a<p>Sialoadhesin (Sn) expression on CD14+ monocytes by flow cytometry quantified as the geometric mean</p>b<p>HIV RNA copies/ml</p>c<p>CD4 positive cells/ml</p>d<p>On (+) or off (−) highly active antiretroviral therapy; Structured treatment interruption (STI)</p

Subject clinical and demographic information.

<p>Mean (± SD) except for ethnicity which is count.</p>a<p>HIV_HVL ≥10,000 copies/ml.</p>b<p>HIV_UD = undetectable viral load (<50 copies/ml).</p>c<p>HIV is younger than HCV (anova p = 0.026, Tukey posthoc p = 0.011).</p>d<p><i>X</i>₂ = 11.9, p = 0.919.</p>e<p>Student t test p = 0.103.</p>f<p>Anova p<0.001, HIV/HCV, HIV and HIV_UD were lower than HIV-Controls (Tukey posthoc p<0.001). HIV was lower than HIV/HCV and HIV_UD (Tukey posthoc p<0.05).</p><p>NA: not applicable.</p

Subject T scores for neuropsychological domains and combined global deficit scores.

<p>Mean (SD).</p>a<p>Healthy controls.</p>b<p>HIV-suppressed (<50 copies/ml) coinfected.</p>c<p>HIV undetected (<50 copies/ml).</p

Upregulation of HIV-related monocyte genes correlated with GDS in HIV-suppressed coinfected subjects.

<p>Global deficit scores were calculated from neuropsychological tests assessing seven domains where GDS >0.5 designates cognitive impairment. (<b>A</b>) Expression of six genes identified by cDNA microarrays were validated by qPCR and reanalyzed for correlated expression with GDS. Gene expression (x axis) was determined relative to GAPDH and transformed to log₂. All genes tested correlated with GDS (Pearson coefficient analysis). (<b>B</b>) Plasma endotoxin (EU/ml) in HIV-suppressed coinfected subjects showed no correlation with GDS (Spearman rank correlation).</p