Expression of MSRVenv and syncytin-1 by PBMC subsets exposed to EBVgp350 or proinflammatory cytokines.

<p><b>A.</b> Levels of MSRV<i>env</i> and syncytin-1 mRNAs of PBMC from MSRV(+) HD treated overnight with 1–100 ng/ml of EBVgp350, either as such or separated in T, B, NK and monocyte subsets. <b>B.</b> Comparison of the MSRVenv and syncytin-1 mRNA levels of monocytes and MDM after overnight EBVgp350 treatment. A and B: Data are the means of three experiments run in duplicate, calculated by the 2^−ΔΔCt method; <b>C.</b> Expression of the HERV-Wenv protein on the plasma membrane, evaluated by flow cytometry as present env-specific positivity of PBMC treated for 24 h with TNFα (1 ng/ml), IFNγ (1000 IU/ml), or PMA (50 NM); the bars indicate standard deviations.</p

Env genes integrated in the human genome that are recognized by MSRV<i>env</i>- and syncytin-1-specific real-time PCR assays.

a<p>BLAST program, using as query the MSRV<i>env</i> amplicon; * amino acids; § nucleotides; ° Accession number; ^b BLAST program, using as query the syncytin-1 amplicon.</p

Clinical and therapy features of MS patients.

*<p>: n.a.: not applicable.</p

Chromosomal representation of multiple sites of integration in human DNA of HERV-W<i>env</i> loci covering ≥80% of the HERV-Wenv gene.

<p>The <i>in silico</i> analysis of the current version of the human genome was performed by NCBI Basic Local Alignment Search Tool (BLAST) program, using as queries the MSRV<i>env</i> and syncytin-1 sequences (PV14 MSRV clone, GenBank accession number AF331500, and ERVW-1<i>env</i> coding sequence NM_014590.3, respectively). From the initial BLAST-identified regions, only the sequences covering ≥80% of the HERV-Wenv gene were selected as target and are reported. Red dashes: target:score ≥200; mauves dashes: target:score 80–200. Hits: number of loci; Hit GIs: Locus number all matches; MT: mitochondrial chromosome.</p

Basal expression of MSRVenv/ syncytin-1/HERV-Wenv in U87-MG astrocytes and PBMC subsets. A.

<p>Detection of MSRVenv and syncytin-1 mRNAs of U87-MG cells, by real time RT-PCR (means of three experiments run in duplicate, calculated by the 2^−ΔCt method; bars indicate standard deviation. <b>B</b>. Flow cytometry evaluation of the HERV-Wenv protein on U87-MG plasma membrane. Shaded histograms: HERV-Wenv-specific staining; open histograms: isotype control. <b>C.</b> MSRV<i>env</i> and Syncytin-1 mRNA expression on PBMC from MSRV(+) donors as such, and after immunobeads separation in CD3⁺T, CD19⁺ B, CD56⁺/CD19⁻/CD3⁻ NK and CD19⁻/CD3–/CD56⁻ monocyte subsets, and subsequent monocyte differentiation into MDM (bars indicate standard deviation). <b>D</b>. Flow cytometry evaluation of the HERV-Wenv protein on the membrane of PBMC from a representative MSRV(+) donor <i>in toto</i> and after sorting of the CD3⁺T, CD19⁺ B, CD56⁺/CD19⁻/CD3⁻ NK and CD19⁻/CD3⁻/CD56⁻ monocyte subsets. Shaded histograms: HERV-Wenv-specific staining; open histograms: isotype control. <b>E.</b> Cell populations distribution of PBMC from a representative MSRV(+) donor before, and after capture by magnetic beads charged with anti-HERV-Wenv or with an unrelated isotype antibody. The unprocessed PBMC and the cells retained by the immunobeads were sorted for T, B, NK and monocyte markers. The unretained cells were also analysed (not shown). <b>F</b>. Presence of surface HERV-Wenv protein in blood cells from five MSRV(+) HD, five untreated MS patients and three MS patients under effective therapy, evaluated by flow cytometry in PBMC <i>in toto</i> and after sorting of the CD3⁺T, CD19⁺ B, CD56⁺/CD19⁻/CD3⁻ NK and CD19⁻/CD3−/CD56⁻ monocyte subsets. Each dot represents an individual; horizontal bars represent the means. HERV-Wenv positivity of CD3⁺T cells was <2% for all samples (not shown).</p

MSRV-type mRNA expression by PBMC from IM patients and healthy donors (HD).

<p>The amounts of MSRV-type HERV-Wenv transcripts were evaluated by discriminatory real time RT-PCR, and normalized by the 2^−ΔCt method (see Methods for details). Data are expressed as medians (line), with maximum and minimum values (whiskers); boxes represent interquartile range of the samples. Statistical significance was evaluated by the Mann-Whitney U-test for comparison of two groups, and by the Kruskal-Wallis test for three or four groups. (<b>A</b>) Comparison of IM patients (N = 17) and all healthy donors (HD, N = 24). (<b>B</b>) Comparison of IM patients (N = 17) and HD donors stratified according to plasmatic anti-EBNA-1 IgG titers, as EBV-negative (EBV−, N = 9), <600 IU/ml (N = 7), and >600 IU/ml (N = 8). Kruskall-Wallis test gave p = 0.0005 for all four groups, and p = 0.014 for the three HD groups.</p

Demographic and clinical characteristics of patients hospitalized with infectious mononucleosis and of healthy controls.

a<p>data are expressed as EBNA-1 DNA copies/ml of plasma. Detection limits: 100 copies/ml of plasma;</p>b<p>bl, borderline;</p>c<p>not done.</p