Histopathological features and biomarker expression in the skin of Buruli ulcer patients.

<p>The distribution of the data were assessed using the D'Agostino & Pearson normality test with a cut-off value of P<0.05. Semi-quantitative scoring was carried out as described in Materials and Methods. TM is normally expressed in the keratinocytes of the epidermis and the endothelial cells of the dermis and subcutis. Necrosis was scored according to the appearance of the tissue in H&E staining.</p><p>¹ TM staining was only scored in areas of lesions that had stained positively for SMA (endothelial cell marker), and a combined score derived from both the coverage and intensity was used.</p><p>²A major effect was defined as a score <1 for SMA and keratinocyte TM, <4 for endothelial TM combined score and >2 for fibrin staining and appearance of necrosis.</p><p>³A moderate effect was defined as a score of 1–2.5 inclusive for SMA and keratinocyte TM, combined endothelial TM score of 4–8 inclusive and 0.5–2 inclusive for fibrin staining and appearance of necrosis.</p><p>⁴No effect was defined as patients with maximum (SMA and TM) or zero (fibrin staining and appearance of necrosis) scores.</p><p>⁵In 11 (25%) of patients, TM was considered to be absent (combined TM score <1).</p><p>⁶Four of these patients did not show evidence of SMA staining in the subcutis and so TM was not quantified in these biopsies due to lack of intact endothelium.</p><p>⁷In 18 (50%) of patients, TM was considered to be absent (combined TM score <1)</p><p>Histopathological features and biomarker expression in the skin of Buruli ulcer patients.</p

Clinical features of the BU patients from whom punch biopsy samples were analysed in this work.

<p>The WHO category for each lesion was as follows: Category 1; a single small lesion or ulcer <5 cm in diameter, category 2; a single lesion 5–15 cm in diameter, category 3 a single lesion > 15 cm, multiple small lesions or lesions on the face. Data are n (%) or median (IQR).</p><p>Clinical features of the BU patients from whom punch biopsy samples were analysed in this work.</p

Mycolactone causes detachment of endothelial cells, apoptosis in 3–4 days, and depletes CD31 (PECAM-1) and VE-cadherin the surface.

<p>Human dermal microvascular endothelial cells were exposed to various concentrations of mycolactone (MYC), 10ng/ml TNF, 1μM staurosporin or 0.025% DMSO (solvent control) as appropriate. A. Both attached and detached cells were subjected to Calcein/EtBr staining for live/dead cells. The proportion of cells that were either attached or detached and alive (Calcein +/EtBr-), and attached or detached and dead (Calcein-/EtBr +) are expressed as a % of the total population of cells. mean±SEM, n = 3. B. HDMVECs were exposed to 7.8ng/ml mycolactone over a timecourse. Cells were then analysed by confocal microscopy following staining of cells with no-wash reagents. CellEvent caspase-3/7 green detection reagent identified cells undergoing apoptosis, alongside PI and DRAQ5. The number of cells in late apoptosis (positive for both active caspase 3/7 and PI) were counted in 3 fields and expressed as a proportion of total cells (DRAQ5 stained) Mean±SEM (n = 3). C. Cells were harvested and subjected to flow cytometry for VE-Cadherin and CD31 (PECAM-1). MFIs are expressed as % of untreated cells (mean±SEM, n = 4). Unstained and isotype bars are for untreated cells. **, P<0.01; ***, P<0.001.</p

Spearman’s correlation of histopathology/immunohistochemistry scoring.

<p>Spearman’s correlations and associated <i>P</i> value for dermis (bottom left of the table, below the cells marked ‘*‘) and subcutis (top right of the table, above the cells marked ‘*‘)</p><p>Spearman’s correlation of histopathology/immunohistochemistry scoring.</p

Mycolactone does not affect thrombin generation <i>per se</i> but profoundly inhibits the ability of endothelial cells to activate protein C.

<p>A. Thrombin generation was measured by calibrated automated thrombography. Thrombin generation was quantified in human pooled plasma containing various concentrations of mycolactone or 0.013% DMSO as a control. The experiment was initiated with 4pM tissue factor, 4μM phospholipid vesicles, and 16.6mM CaCl₂. Thrombin generation was monitored using 0.42mM of the fluorogenic substrate Z-GlyArg-AMC-HCl as described in the text. B. Platelet aggregation was assessed using an optical platelet aggregometer by the addition of 1μg/ml collagen to washed human platelets that had been treated with various concentrations of mycolactone or 0.1% DMSO as a control, and are expressed relative to an untreated control. Mean±SEM n = 3 (except for resting platelets where n = 1). C. Platelet activation was determined by quantifying fibrinogen binding to, and P-selectin exposure on, human platelets by flow cytometry. Washed human platelets were treated with various concentrations of mycolactone or 0.1% DMSO as a control then stimulation with 1μg/ml CRP-XL, and are expressed relative to an untreated control. Mean±SEM n = 3. D and E. Protein C activation over human dermal microvascular endothelial cells. Protein C was added to cells in the presence of Ca²⁺ and Mg²⁺. Activation was initiated by the addition of 13.5nM thrombin and proceeded for 30 mins at which point the reaction was stopped 5μg antithrombin and 3U heparin. Activated protein C was quantified by assessing the rate of chromogenic substrate S2366 cleavage compared to an APC standard curve. D. Michaelis-Menton curve of protein C activation. E. Cells were exposed to various concentrations of mycolactone (MYC), 10ng/ml IL-1β or 0.025% DMSO (solvent control) for 24 hours. In one case, untreated cells were exposed to a function-blocking antibody (CTM) for 1hr prior to the assay. Protein C (5nM) activation was assessed as before and is expressed as % of untreated cells. Mean±SEM n = 3.</p

Thrombomodulin (TM) expression is dysregulated in Buruli ulcer patient skin and tissue.

<p>Histological sections were stained with α-TM, α-CD31 (PECAM-1) or α-SMA (smooth muscle actin) antibodies and counterstained with Haematoxylin or with Haematoxylin-Eosin (B1). Slides were analyzed with a DM2500 Microscope (Leica). Pictures were taken either with an Aperio scanner (B1) or a Leica DFC 420 camera and the Leica application Suite V4 software. Comparative staining on healthy skin sample from an unaffected donor (A) or 4mm punch biopsies from laboratory confirmed BU patients (B and C). Typical results are shown. A. TM staining of healthy skin. A1, Low magnification image; A2 and A3, higher magnification showing strong TM staining of endothelial cells and keratinocytes, respectively. B1. Scan of a HE stained BU punch biopsy (E; Epidermis, D; Dermis, S; Subcutis). B2 –B7, higher magnification of subcutaneous tissue showing reduced TM staining in the endothelium (B2 and B3). This patient had a combined score of 2 in the dermis (coverage 2, intensity 1) and 2 in the subcutis (coverage 2, intensity 1). Endothelial cells in the region still showed reasonable staining for CD31 (B4 and B5) and strong staining for αSMA (B6 and B7), scoring 2.5 in the dermis and 3 in the subcutis. C. Higher magnification of the epidermis showing variable reduction in TM staining in the keratinocytes of BU patient skin, ranging from reduced (C1) to no staining (C3). Hyperplasia of the epidermis, as seen in these three patients, is typical of BU. D and E. TM staining in the dermis, subcutis and epidermis was scored as described in Materials and Methods. The score for each individual biopsy analysed is shown, consisting of 31 patients with ulcers (red circles) and 9 patients with plaque lesions (green circles). In all cases, error bars show the median and 25–75% percentile scores for the all BU patients. D. Scores of dermis and subcutis are for endothelial TM and were obtained by multiplying two scores (each 0–3) of intensity of staining and the coverage, (healthy tissue scored the maximum 9). Numbers for subcutical staining are slightly lower since those patients that had no intact endothelium in the subcutis according to SMA staining were excluded. E. Score for epidermal TM (keratinocytes) are relative to a maximum of 3 (intensity only).</p

Abundant fibrin deposition in the skin of patients with Buruli ulcer.

<p>A and B. Histological sections were stained with an α-fibrin antibody and counterstained with Haematoxylin. Slides were analyzed with a DM2500 Microscope (Leica). Pictures were taken with an Aperio scanner. Comparative staining of a healthy skin sample from an unaffected donor (A) or 4mm punch biopsies from 8 different laboratory confirmed BU patients (B) showing the variability in fibrin staining observed ranging from small isolated fibrin depositions (B1-B2), to large deposition seen only in the dermis or subcutis (B3-B6) and finally to extensive depositions covering the whole tissue sample (B7-B8). C and D. Scoring was carried out as described in Materials and Methods and are relative to a maximum of 3. The score for each individual biopsy analysed is shown, consisting of 31 patients with ulcers (red circles) and 9 patients with plaque lesions (green circles). The expected score for healthy tissue was 0 for both fibrin and necrosis (see A). In all cases, error bars show the median and 25–75% percentile scores. C. Fibrin staining in the dermis and subcutis. D. The degree of necrosis in each biopsy was scored according to appearance of the entire biopsy.</p

Mycolactone causes a profound depletion of thrombomodulin (TM) endothelial cell surfaces.

<p>Human dermal microvascular endothelial cells were exposed to various concentrations of mycolactone (MYC), 10ng/ml IL-1β or 0.025% DMSO (solvent control, equivalent to that in 125ng/ml MYC) for 24 hours (or other times as indicated). Cells were harvested and subjected to flow cytometry for TM and EPCR. A. Forward and side scatter plots. B. Histogram plots for TM and EPCR. Untreated unstained cells; filled grey, untreated cells with isotype control; dotted black line, untreated cells with TM or EPCR-PE; black line; MYC treated cells (7.8ng/ml) with TM or EPCR-PE, red line; IL-1β treated cells with TM or EPCR-PE, green line. C. Quantitation of TM and EPCR surface expression. MFIs are expressed as % of untreated cells with TM or EPCR-PE (mean±SEM, n = 3). Unstained and isotype bars are for untreated cells. D Quantitation of TM and EPCR surface expression over time in the presence of 7.8ng/ml mycolactone. MFIs are expressed as % of untreated cells with TM or EPCR-PE. DMSO control (0.08%) is after 48hrs exposure (mean±SEM, n = 3). *, P<0.05; **, P<0.01; ***, P<0.001.</p