Figure 3
Figure 3. Endothelial activation and decreased EPCR and TM ex vivo in biopsies from children with CM. (A) Immunofluorescence-labeled needle biopsies samples of subcutaneous tissue from healthy children and children with CM. Nuclei appear blue (DAPI) and endothelial receptors green (Alexafluor 488). Micrographs (40× lens) show microvessels from healthy children as indicated by morphology, elliptical endothelial nuclei, and bright staining for CD31, ICAM-1, TM, and EPCR. Vessels from a CM case show IE sequestration (arrows) associated with low TM and EPCR staining. Needle biopsy samples were digested, labeled, and then analyzed by flow cytometer to examine the receptor expression of endothelial cells from microvessels in the sample. (B) Flow cytometry gating strategy for samples to distinguish endothelial cells as single, live, CD31+CD45− cells. (C) Histograms for 3 different endothelial receptors: ICAM-1, TM, and EPCR; representative plots from a single case for CM (blue), healthy children (HC; red), and antibody isotype control (IC, gray). ICAM-1 and TM staining show low overlap with the isotype control and receptor expression was determined by mean fluorescence intensity (MFI), whereas EPCR staining overlapped considerably with the isotype and expression was determined by percentage positive events. (D) Scatterplots for the endothelial expression levels of ICAM-1, TM, and EPCR as determined by flow cytometry in 17 CM cases and 20 HCs. (E) Scatterplots for levels of soluble ICAM-1, soluble TM, and soluble EPCR as determined by enzyme-linked immunosorbent assay in plasma samples in HC, CM, and aparasitemic febrile controls (FC) who were noncomatose patients that had a lumbar puncture taken because of clinical suspicion of meningitis. (F) Scatterplots for levels of soluble ICAM-1, soluble TM, and soluble EPCR in paired CSF samples in FC and CM; CSF samples were not taken from healthy children. Horizontal lines indicate geometric means and bars 95% CIs. Significance determined by one-way analysis of variance with the Tukey honestly significant difference test to adjust for multiple comparisons in E. * P < .05, ** P < .01, *** P < .001. Fluorescence micrographs were taken using a Leica DM1L microscope and a Leica DFC300FX camera using Leica Application Suite version 2.6.0 software.

Endothelial activation and decreased EPCR and TM ex vivo in biopsies from children with CM. (A) Immunofluorescence-labeled needle biopsies samples of subcutaneous tissue from healthy children and children with CM. Nuclei appear blue (DAPI) and endothelial receptors green (Alexafluor 488). Micrographs (40× lens) show microvessels from healthy children as indicated by morphology, elliptical endothelial nuclei, and bright staining for CD31, ICAM-1, TM, and EPCR. Vessels from a CM case show IE sequestration (arrows) associated with low TM and EPCR staining. Needle biopsy samples were digested, labeled, and then analyzed by flow cytometer to examine the receptor expression of endothelial cells from microvessels in the sample. (B) Flow cytometry gating strategy for samples to distinguish endothelial cells as single, live, CD31+CD45− cells. (C) Histograms for 3 different endothelial receptors: ICAM-1, TM, and EPCR; representative plots from a single case for CM (blue), healthy children (HC; red), and antibody isotype control (IC, gray). ICAM-1 and TM staining show low overlap with the isotype control and receptor expression was determined by mean fluorescence intensity (MFI), whereas EPCR staining overlapped considerably with the isotype and expression was determined by percentage positive events. (D) Scatterplots for the endothelial expression levels of ICAM-1, TM, and EPCR as determined by flow cytometry in 17 CM cases and 20 HCs. (E) Scatterplots for levels of soluble ICAM-1, soluble TM, and soluble EPCR as determined by enzyme-linked immunosorbent assay in plasma samples in HC, CM, and aparasitemic febrile controls (FC) who were noncomatose patients that had a lumbar puncture taken because of clinical suspicion of meningitis. (F) Scatterplots for levels of soluble ICAM-1, soluble TM, and soluble EPCR in paired CSF samples in FC and CM; CSF samples were not taken from healthy children. Horizontal lines indicate geometric means and bars 95% CIs. Significance determined by one-way analysis of variance with the Tukey honestly significant difference test to adjust for multiple comparisons in E. * P < .05, ** P < .01, *** P < .001. Fluorescence micrographs were taken using a Leica DM1L microscope and a Leica DFC300FX camera using Leica Application Suite version 2.6.0 software.

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