Plasma GrM and VWF/FVIII ratios positively correlate in patients with meningococcal disease.

<p>(A) Representative example of GrM in plasma of patients with meningococcal disease without shock (1) and with shock (2–5), using immunoblotting with a monoclonal antibody against GrM. (B) VWF and FVIII levels in plasma were determined by ELISA and chromogenic activity, respectively. VWF/FVIII ratios were determined in meningococcal shock patients with or without detectable plasma GrM, patients with meningococcal disease without shock, and healthy controls. P values are indicated. (C) VWF/FVIII ratios are plotted against plasma GrM levels, as quantified by western blot. R_spearman = 0.92, p<0.001.</p

All forty <i>Cryptococcus</i> strains induce low amounts of IL-17, but high amounts of IL-22.

<p>IL-17 and IL-22 production after 7 d by PBMCs stimulated with RPMI+, either one of 40 different heat-killed <i>Cryptococcus</i> strains [10⁷ microorganisms/mL] or heat-killed <i>Candida albicans</i> [10⁵ microorganisms/mL] is shown respectively. Mean values ± SE (n = 5) of three independent experiments are presented.</p

Details of 11 additional <i>C. neoformans</i> var <i>grubii</i> isolates, arranged by Microsatellite Complex (MC) [29].

<p>Details of 11 additional <i>C. neoformans</i> var <i>grubii</i> isolates, arranged by Microsatellite Complex (MC) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055579#pone.0055579-IllnaitZaragozi1" target="_blank">[29]</a>.</p

GrM slightly affects VWF binding to collagen.

<p>Purified plasma VWF (1 µg/mL) was incubated with indicated concentrations of GrB, GrB-SA, GrM, or GrM-SA in Tris (pH 7.4) for 1 hour at 37°C. Samples were assessed for collagen binding, using an immunosorbent assay. Data represent the mean +/− SD of three independent experiments. GrM (25 nM) versus GrM-SA (25 nM), p = 0.02; GrB (25 nM) versus GrB-SA (25 nM), p<0.001.</p

GrM cleaves VWF.

<p>(A) Purified plasma VWF (5 µg/mL) was incubated with various purified recombinant granzymes (300 nM) in TBS for 1 hour at 37°C and analyzed by immuno blot, using an antibody against VWF. Mature VWF and VWF cleavage fragments (*) are indicated by the arrows. (B) Purified plasma VWF (2 µg/well) was immobilized onto plastic and incubated with various purified recombinant granzymes (300 nM) in TBS for 1 hour at 37°C and analyzed by immuno blot, using an antibody against VWF. (C) Purified plasma VWF (5 µg/mL) was incubated with different concentrations of GrM (0–800 nM) or GrM-SA (800 nM) in TBS for 4 hours at 37°C and analyzed by immuno blot, using an antibody against VWF. (D) Purified plasma VWF (2 µg/well) was immobilized onto plastic and incubated with GrM (0–800 nM) or GrM-SA (800 nM) in TBS for 1 hour at 37°C and analyzed by immuno blot, using an antibody against VWF. (E) Purified plasma VWF (5 µg/mL) was incubated with GrM (100 nM) for 1 hour at 37°C in Tris (pH 7.4) in the presence or absence of increasing concentrations of NaCl (0–600 mM) and analyzed by immuno blot, using an antibody against VWF. (F) GrM, GrB, and ADAMTS13 cleavage sites in VWF are schematically indicated. Data are representative of at least three independent experiments.</p

Comparison of cytokine production by PBMCs induced by clinical or environmental cryptococcal isolates.

<p>Heat killed clinical isolates of <i>C. gattii</i> are compared to environmental <i>C. gattii</i> isolates and to clinical isolates of <i>C. neoformans</i>. The clinical isolates of <i>C. gattii</i> genotype AFLP6/VGII are depicted separately. Mean values (n = 5 to 7) ± SE values of three independent experiments are presented. *, p 0.01 to 0.05; **, p 0.001 to 0.01; ***, p<0.001. The horizontal line represents the lower detection limit.</p

Successful experimental infection of goats with <i>Coxiella burnetii</i> 3262.

<p>Eleven out of seventeen goats were intranasally infected with <i>C. burnetii</i> 3262. The remaining six were kept naive. During the infection, antibodies against <i>C. burnetii</i> were measured weekly in the serum of all goats using the LSIVET RUMINANT milk/serum Q-fever ELISA kit. Grey squares represent naive goats, black squares represent infected goats. Median values with SEM are shown. Parturition is indicated for each individual goat using an arrow. Abortion, defined as birth of dead lambs, is indicated with an asterisk. The final two remaining pregnant infected goats were culled for other purposes, and therefore not included in this graph.</p

GrM disrupts FVIII binding to VWF.

<p>(A) Purified plasma VWF (1 µg/mL) VWF was incubated with indicated concentration of GrM, GrB, GrM-SA or GrB-SA in Tris (pH 7.4) for 1 hour at 37°C and subsequent FVIII binding was assessed in an ELISA setup. Data represent the mean +/− SD of three independent experiments. GrM (25 nM) versus GrM-SA (25 nM), p = 0.009; GrB (25 nM) versus GrB-SA (25 nM), p = 0.009. (B) VWF (30 ng) was pre-incubated in the presence or absence of GrM (100 nM) or GrM-SA (100 nM) in 5 mM Tris (pH 7.4) for 1 hour at 37°C, followed by incubation with purified FVIII heterodimer in TBS, 10 mM CaCl₂ for 2 hours at 37°C. Protein mixtures were immunoprecipitated by ProtA/G sepharose pre-treated with anti-VWF antibody. Samples were analyzed by Western blotting, using antibodies against VWF and FVIII heterodimer. Full-length VWF, its GrM-cleaved form, and the FVIII heavy chain (HCh) and light chain (LCh) are indicated by the arrows. IP, immunoprecipitation; WB, Western blot. Data are representative of at least three independent experiments.</p

The role of TLR2, TLR4 and TLR9 in IL-1β and IL-17 induction by <i>C. gattii</i>.

<p>Cytokine production by human PBMCs preincubated for one hour with culture medium (white bar) or PRR blocking reagents (dark gray bars) or their control (light gray bar) prior to stimulation with heat-killed <i>C. gattii</i> (strain B5742) [10⁷/ml]. IL-1β is determined after 24 h incubation, IL-17 is determined after 7 d incubation. Mean values ± SE of eight individuals in 4 independent experiments (IL-17) or six individuals in 5 independent experiments (IL-1β) (with exclusion of additional four individuals with undetectable cytokine induction by <i>C. gattii</i>) are presented. *, p 0.01 to 0.05. The horizontal line represents the lower detection limit.</p

Cytokine profile of <i>C. burnetii</i> stimulated PBMCs from naive and <i>C. burnetii</i> infected goats.

<p><b>3A/B:</b> EDTA blood was sampled at day 7, 35 and 56 of the study from six naive goats (grey bars) and eleven infected goats (dark grey bars). The white bars show the mRNA expression at day zero. PBMCs were isolated and incubated for 4 h or 24 h with either medium (<b>3A</b>), heat killed <i>C. burnetii</i> NM 1×10⁷/ml (<b>3B</b>) or <i>E. coli</i> LPS 10 ng/ml (<b>3E</b>). mRNA expression of TNF-α, IL-1β, IL-10 and IFN-γ was determined using qPCR. <b>3C/D</b>: IFN-γ protein was measured in the supernatant of PBMCs stimulated for 24 h with nil (<b>3C</b>) or <i>C. burnetii</i> NM 1×10⁷/ml (<b>3D</b>). IFN-γ was measured using the Bovigam Elisa kit. Grey bars represent six naive goats, dark grey bars represent eleven infected goats. Box and Whisker plots are shown. Outliers are indicated as open circles. NS; not significant *p<0.05; **p<0.01; ***p<0.001, Mann-Whitney U-test.</p