Effect of heparin activated ATIII (hep-ATIII) on gene expression in acutely HIV infected PBMC.

<p>(<b>A</b>) Gene up-regulation of acutely infected PBMC after treatment with different doses of hep-ATIII, each dose compared to infected hep-ATIII untreated vehicle control. (<b>B</b>) Gene down-regulation of acutely infected PBMC after treatment with different doses of hep-ATIII, each dose compared to infected hep-ATIII untreated vehicle control. For signal transduction gene analysis, 10⁵ PBMC were infected with a 0.01 MOI of primary isolate HIV-1 (HIV 89.6) for 2 hrs at 37°C. Cells were washed and treated with 0.09, 0.17 and 0.4 µM hep-ATIII for 48 h. Total RNA was purified and a RT-PCR expression array was performed. The expression levels of genes of 20 different signal transduction pathways (84 genes) were analyzed. Genes with significant changes in gene expression (<i>p</i><0.05, n = 3) compared to controls are show. Significance was calculated using the ΔΔC_t method for three independent experiments.</p

Effect of ATIII on gene expression in acutely HIV infected PBMC.

<p>(<b>A</b>) Gene up-regulation of acutely infected PBMC after treatment with different doses of ATIII, each dose compared to infected untreated control. (<b>B</b>) Gene down-regulation of acutely infected PBMC after treatment with different doses of ATIII, each dose compared to an infected untreated control. For signal transduction gene analysis, 10⁵ PBMC were infected with a 0.01 MOI of primary isolate HIV-1 (HIV 89.6) for 2 h at 37°C. Cells were washed and treated with 6.8, 34 and 68 µM ATIII for 48 h. Total RNA was purified and a RT-PCR expression array was performed. The gene expression levels from 20 different signal transduction pathways were analyzed. Genes with significant changes in gene expression (<i>p</i><0.05, n = 3) compared to controls are shown. Significance was calculated using the ΔΔC_t method for three independent experiments.</p

Effect of ATIII on gene expression in uninfected PBMC.

<p>Gene-regulation of uninfected PBMC treated with different doses of ATIII, each dose compared to an uninfected, ATIII untreated vehicle control. For signal transduction gene analysis, 10⁵ PBMC were infected with a 0.01 MOI of primary isolate HIV-1 (HIV 89.6) for 2 h at 37°C. Cells were washed and treated with 6.8, 34 and 68 µM ATIII for 48 h. Total RNA was purified and a RT-PCR expression array was performed. The expression of 84 genes from 20 different signal transduction pathways was analyzed. Genes with significant changes in gene expression (<i>p</i><0.05, n = 3) compared to controls are shown. Significance was calculated using the ΔΔC_t method for three independent experiments.</p

Inhibition of HIV replication by PTGS2.

<p>(<b>A</b>) Viral replication measured in RNA copy number per ml of mock-transfected (empty vector) and pCMV-PTGS2-transfected (PTGS2) PBMC. (<b>B</b>) Protein expression of Western-blot of 20 µg lysate protein of mock-transfected (lane 1) or PTGS2-transfected PBMC (lane 2). PBMC were transiently transfected with plasmid DNA. After 2 days cells were infected with a 0.01 MOI of the HIV-1 primary isolate (HIV 89.6) for 2 hrs at 37°C. Cells were washed and supernatant was collected after 48 hr. HIV-1 RNA levels were measured using the COBAS Ampliprep/COBAS Taqman 48 system for three independent experiments. For the Western analysis, transfected Jurkat cellular homogenates (20 µg/lane) were separated by SDS-PAGE. Target proteins were identified by Western analysis using polyclonal antibody generated against the PTGS2 protein. Representative data of three independent experiments are shown. (<b>C</b>) PBMC were infected with HIV-1 primary isolate (HIV 89.6) at a MOI of 0.01. Hep-ATIII was added at concentrations of 0.09, 0.17 and 0.4 µM. After 48 hours, PTGS2 was quantified using the PTGS2-specific ELISA.</p

Effect of acute HIV infection on PBMC gene expression.

<p>Gene expression profiling of PBMC acutely infected with HIV-1. For signal transduction gene analysis, 10⁵ PBMC were infected with a 0.01 MOI of the primary isolate HIV-1 (HIV 89.6), for 2 h at 37°C. Total RNA was purified two days after infection. A RT-PCR expression array was performed and gene expression of 84 genes from 20 different signal transduction pathways was analyzed. Genes with significant changes in gene expression (<i>p</i><0.05, n = 3) compared to uninfected vehicle treated controls are shown. Significance was calculated using the ΔΔC_t method for three independent experiments.</p

Highest scoring network after interactive network analysis of gene expression changes induced after hep-ATIII treatment of SIV-infected rhesus macaques.

<p>The highest scoring primary transcriptional network activated by hep-ATIII treatment of chronically infected rhesus macaques, at a time point when viral replication is inhibited by hep-ATIII (day 7), is shown. Rx (orange): potential medication treatment options, BM (green): possible biomarkers. Network analysis was performed using Ingenuity 8.0 software. Explanation for symbols is given in <b>Figure S2</b>.</p

Characterization of heparin-activated ATIII.

<p>(<b>A</b>) Sepharcyl S100 ÄKTA FPLC of purified hep-ATIII. A 9–14 ml fraction was separated, termed as hep-ATIII and used for our experiments. Detection at 260 nm for protein detection and 280 nm for heparin detection is shown. (<b>B</b>) Hep-ATIII purity and molecular weight were also determined by SDS-PAGE and silver staining (Bio-Rad kit) of a 15% slab gel. For molecular weight determination a low-range protein molecular weight marker (Bio-Rad) was used. Lane 1∶2 µg hep-ATIII; lane 2: phosphorylase B (97 kDa), serum albumin (69 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), aprotinin (6.5 kDa).</p

<i>In vivo</i> anti-viral activity of non-activated and heparin-activated ATIII in rhesus macaques.

<p>(<b>A</b>) Viral loads as RNA copies/ml of chronically SIV_mac239 infected rhesus macaques treated with 0.8 µmol/kg non-activated ATIII (n = 3) and (<b>B</b>) corresponding log₁₀ reduction of viral load of same treatment group. (<b>C</b>) Viral loads as RNA copies/ml of chronically SIV_mac239 infected rhesus macaques treated with 0.6 µmol/kg heparin-activated ATIII (n = 3) and (<b>D</b>) corresponding log₁₀ reduction of viral load of same treatment group. Administration via i. v. inoculation is shown at indicated time points depicted by arrows. Viral load was measured and compared to animals before treatment (day 0). *, <i>P</i><0.05, paired T-test, compared to pre-treatment (day 0). Data are shown as mean ± S.E.</p

Effect of heparin-activated ATIII in pseudovirus inhibition assay.

<p>Pseudoviruses with clade B envelopes (in <b>A</b> and <b>C</b>) and clade C envelopes (in <b>B</b> and <b>D</b>) were treated with three fold dilutions of heparin-activated ATIII (hep-ATIII), unmodified ATIII and heparin. Percentage of inhibition was calculated by comparing of residual luciferase activity and untreated control. Experiments were done in triplicates. Data are shown as mean ± S.E.</p

<i>In vivo</i> anti-viral activity of ET-ATIII in rhesus macaques.

<p>Chronically SIV_mac239 infected rhesus macaques (n = 2) were treated with 1.5 ml ET-ATIII (0.3 nmol/kg encapsulated hep-ATIII) at indicated time points depicted by arrows. (<b>A</b>) Viral load of ET-ATIII treated animals as RNA copies/ml. (<b>B</b>) Log₁₀ reduction of viral load. Vehicle liposomes were used as a control. Viral load was measured and compared to animals before treatment (day 0). Data are shown as mean ± S.E.</p