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Blood, Vol. 110, Issue 9, 3426-3435, November 1, 2007
Src-family kinase–dependent disruption of endothelial barrier function by Plasmodium falciparum merozoite proteins
Blood Gillrie et al.
110: 3426
Supplemental materials for: Gillrie et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 71.2 KB)
- Table S1. Clinical P. falciparum parasite isolates used in studies (JPG, 83.9 KB)
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- Figure S1. Lack of effect of intact P. falciparum–infected erythrocytes (IRBCs) on endothelial permeability (JPG, 77.6 KB)
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IRBCs at mid to late trophozoite stages from three clinical isolates that have been shown to adhere to HDMECs were purified on a Percoll gradient and added at 0.2%, 1.0% and 5% hematocrit and 50% parasitemia, i.e. 0.5 or 2.5 × 107 or 1.25 × 108 parasites/cm², to the upper chamber of transwells with 3-day post-confluence HDMECs in RPMI-1640 medium and 5% normal human AB serum. Normal erythrocytes (NRBC) were added at identical hematocrits as negative controls. The corresponding sonicate from each parasite isolate was used at 1 × 107 parasite equivalents/cm². No significant change in endothelial permeability compared to medium alone was seen as indicated by (A) flux of FITC-albumin or (B) TER. Blood smears (stained with Field stain and examined with a 100× objective under oil immersion) at (C) T = 0 h and (D) T = 24 h showed normal development of parasites in the coculture. * = P < 0.05 compared to control values by Student’s paired t-test.
- Figure S2. P. falciparum food vacuoles induce endothelial permeability in HDMECs (JPG, 57.7 KB)
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HDMECs were incubated with 0.01 to 1.0 µL/cm² of purified P. falciparum food vacuoles for 24 h. (A) A significant increase in FITC-albumin flux was seen (n= 4). (B) Compared to controls, immunofluorescence staining of HDMEC monolayers incubated with food vacuoles showed a complete loss of cortical ZO-1 staining and absent or irregular nuclei consistent with apoptosis. Phase contrast micrographs of the same field show the widespread presence of food vacuoles remaining after the disruption of the endothelial monolayer. All micrographs are of 3-day post-confluent HDMEC monolayers incubated with or without 0.5 µL/cm² of food vacuoles for 24 hours prior to fixation. Results are representative of 3 independent experiments. ** = P < 0.01 and *** = P < 0.001compared to control values by Student’s paired t-test.
- Figure S3. Lack of the activity of P. falciparum hemozoin (JPG, 54.5 KB)
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(A) Purified hemozoin (n = 4) and (B) synthetic hemozoin (n = 4) had no effect on FITC-albumin flux after incubation with HDMECs for 24 h. (C) Pre-treatment of HDMECs with 10 µM chloroquine, an inhibitor of endosomal acidification and hence TLR-9 activation, for 30 min did not inhibit the ability of parasite sonicates to induce endothelial permeability at 24 hours (n = 4). * = P < 0.05, ** = P < 0.01 compared to control values by Student’s paired t-test.
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