Band-shift assay for HMGB1 detection in cervicovaginal secretions from HSV-2-infected women.

<p>Ten microliters of cervicovaginal specimens (#1–18) collected from 18 women seropositive for HSV-2 were mixed with radiolabeled hemicatenated DNA (hcDNA). HMGB1-hcDNA shifted complexes were analyzed by electrophoresis on nondenaturing polyacrylamide gel, using a band-shift assay, as described in (30). The amount of HMGB1 was calculated from the percentage of shifted hcDNA, quantified with ImageJ software (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016145#pone-0016145-t001" target="_blank">table 1</a>).</p

HMGB1 mobility is altered by HSV-2 infection.

<p>HEC-1 cells were treated with TNF-α (2 ng/ml) and CHX (10 µg/ml). At various times post-induction, western blot was performed on whole cell extracts to detect PARP and pro-caspase 3. A nuclear fraction obtained after NP-40-induced membrane permeabilization was also subjected to western blot to detect chromatin-bound HMGB1 (bottom). (A) Time-course analysis of HMGB1 in the cytoplasm and nucleus after HSV-2 infection of HEC-1 cells at 0.1 and 1.0 pfu/cell. M  =  mock-infected cells. (B) Time-course analysis of mobile (M) and chromatin-bound (CB) HMGB1 during HSV-2 infection, following membrane permeabilization by NP40. (C) Measurement of extracellular HMGB1 by ELISA after infection of HEC-1 cells by HSV-2. Results are representative of 3 independent experiments.</p

HMGB1 transcription and expression during HSV-2 infection.

<p>HEC-1 cells were infected at 0.1 or 1.0 pfu/cell. A time-course analysis of HMGB1 mRNA (A) and protein (B) was performed by quantitative RT-PCR and western blot, respectively, with 18S rRNA and GAPDH protein as controls. RNA quantification was performed according to the 2^{- ΔΔCT} method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016145#pone.0016145-Livak1" target="_blank">[50]</a>. (C) HMGB1 was detected more than 4 days in HEC-1 cells treated with actinomycin D. GADPH and P53 were used as controls. M  =  mock-infected cells. Results are representative of 3 independent experiments.</p

Correlations between HMGB1 concentrations and HSV-2 shedding in cervico-vaginal samples collected from HSV-2-infected women.

<p>Eighteen genital samples were collected from women seropositive for HSV-2. Viral culture was performed on Vero cells, and HSV-2 DNA was quantified with real-time PCR. HMGB1 was quantified with a commercial ELISA and a band-shift assay, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016145#pone.0016145-Chen1" target="_blank">[32]</a>.</p

HEC-1 cell death during HSV-2 infection.

<p>HEC-1 cells, infected at 0.1 or 1.0 pfu/cell, were analyzed at various times post-infection for (A) HSV-2 productive infection, assessed by plaque assay, (B) cell viability, assessed by trypan blue exclusion, (C) apoptosis, assessed by western blot against PARP and pro-caspase 3, and (D) necrosis, assessed by extracellular LDH release over total LDH. (E) Necrosis was measured in mock and HSV-2-infected cells in the presence and absence of Z-VAD, an inhibitor of apoptosis. Results are either representative of at least 3 independent experiments (A, B, C) or are means and SD from 3 independent experiments (D, E).</p

HMGB1 released by HSV-2-infected cells activates fibroblast migration and stimulates HIV-1 expression.

<p>MRC5 fibroblasts were subjected to migration assays in the presence of (A) human recombinant HMGB1 or (B) supernatants from mock (M) or HSV-2-infected HEC-1 cells. HMGB1 concentrations reached 270 ng/ml in the supernatants collected from infected cells, versus 50 ng/ml in mock-infected cell media. Control experiments used glycyrrhizin or neutralizing antibodies against HMGB1 or its receptors RAGE, TLR-4 and TLR-2. Data are expressed as -fold increases in cell migration compared to non treated control cells. ACH-2 cells, that contain a latent HIV-1 provirus, were grown in the presence of (C) human recombinant HMGB1 or (D) supernatants from mock (M) or HSV-2-infected HEC-1 cells. P24 antigen was measured by ELISA after 36 h. Data are expressed as the -fold increase in p24 antigen compared with non treated control cells. Virion-free supernatants were obtained by ultracentrifugation. Results are either representative of several experiments (A, C, D) or expressed as the mean and SD of 3 independent experiments (B).</p