Journal: PLoS ONE

Article Title: Interaction of Treponema pallidum , the syphilis spirochete, with human platelets

doi: 10.1371/journal.pone.0210902

Figure Lengend Snippet: (A) Flow cytometry isolates the co-localized platelet and treponeme populations by SSC and FSC gating. Dot plots of (B) CFSE-labeled heat-treated treponemes demonstrate reduced binding to human platelets labeled with PE/Cy5 anti-CD41a compared with (C) CSFE-labeled viable treponemes. (D) CFSE-labeled viable treponemes bound significantly more human platelets (mean = 55.13% ± 2.91 [SD] *P<0.0001) compared with heat-treated treponemes (mean = 19.05% ± 2.29 [SD]) following co-incubation for 16 hours at 37˚C and ~ 5% oxygen. Results represent the mean of three independent experiments with statistical significance computed by two-way ANOVA, with a minimum of 3 replicates per sample type per experiment. (E) Darkfield microscopy FOV counts (20 random locations/slide) demonstrate viable treponemes bind significantly more human platelets (mean = 31.73% ± 1.19 [SD] *P<0.0001) than heat-treated treponemes (mean = 6.47% ± 1.19 [SD]) following co-incubation at 5% oxygen at 34˚C for 48 hours. Results represent the mean of three independent experiments with statistical significance computed by unpaired two-tailed Student’s t test, n = 3. (F) Heat-treated treponemes (top) are non-motile yet morphologically similar to viable treponemes (bottom). (G) Darkfield microscopy at 1000x magnification demonstrates platelet interactions are reversible. Image capture from video micrographs show edgewise attachment of a treponeme to an activated platelet (0.01 s to 8.10 s) which then detaches and moves away (9.05 s).

Article Snippet: Software was set to log scale for both forward scatter (FSC E-1) and side scatter (SSC), and gating was aided by using the SPHERO Flow Cytometry Size Standard Kit (containing polystyrene beads between 2.0 and 9.96 μm from Spherotech, Inc., Lake Forest, IL).

Techniques: Flow Cytometry, Labeling, Binding Assay, Incubation, Microscopy, Two Tailed Test

Journal: PLoS ONE

Article Title: Interaction of Treponema pallidum , the syphilis spirochete, with human platelets

doi: 10.1371/journal.pone.0210902

Figure Lengend Snippet: The potential for treponemes to activate platelets was assessed by flow cytometry (A) and using a plate-based assay (B). (A) Following 18 hours co-incubation at 37°C the results of two independent flow cytometry assays quantified platelet activation by the median fluorescence intensity (MFI) associated with CD41a receptor up-regulation. Platelets co-incubated with treponemes (hatched bar) demonstrated a significantly higher MFI (mean = 806.0 ± 37.76 [SEM], *P = 0.0118, n = 25) compared to resting platelets (white bar) (mean = 545.5 ± 104.4 [SEM], n = 18). There was no significant difference between treponeme co-incubated platelets and agonist pre-activated platelets (black bar) (mean = 746.5 ± 124.3 [SEM], n = 18). (B) Following co-incubation for 18 hours at 34°C the results of two independent platelet activation experiments were assessed by measuring the absorbance at 600 nm as a surrogate measure of fibrin clot formation. Platelets co-incubated with T . pallidum (hatched bar) had a higher absorbance (mean = 0.4137 ± 0.007 [SEM], *P = 0.0221, n = 20) compared to resting platelets (white bar) (mean = 0.3730 ± 0.018 [SEM], n = 12). There was no significant difference between treponeme-co-incubated platelets and activated platelets (black bar) (mean = 0.3791 ± 0.027 [SEM], n = 7).

Article Snippet: Software was set to log scale for both forward scatter (FSC E-1) and side scatter (SSC), and gating was aided by using the SPHERO Flow Cytometry Size Standard Kit (containing polystyrene beads between 2.0 and 9.96 μm from Spherotech, Inc., Lake Forest, IL).

Techniques: Flow Cytometry, Incubation, Activation Assay, Fluorescence