Figure 6.
P falciparum histones are toxic factors in CM patient sera which are necessary to induce brain endothelial disruption ex vivo. (A) Time course of barrier disruption of primary HBMEC by P falciparum histones in a Transwell dual chamber system treated with 100 μg/ml of purified plasmodial histones compared with cells with media alone or cells treated with histones plus 200 μg/ml of antihistone single chain variable Fragment (ahscFv). Barrier disruption was assessed by measuring the optical density (OD) of the liquid in the lower part of the Transwell system (measures horseradish peroxidase pass through, detected by reaction with tetramethylbenzidine substrate, 3 biological replicates per condition). By 2-way ANOVA there was significant barrier disruption in the histone group when compared with the media group (P = .0045). This was abrogated by treatment with ahscFv, which was not significantly different from the media-only group (P = .093). (B) Serum-induced barrier disruption by patient samples. Transwell permeability changes of HBMEC monolayer are expressed as fold changes in horseradish peroxidase pass-through over 1 hour. Data are expressed as violin plots and analyzed with the Kruskal-Wallis test with Dunn’s test to adjust for multiple comparisons. Compared with healthy control serum there was a significant increase in permeability induced by the patient serum from Ret+CM cases (P = .0123), but not with any of the other patient groups (n = 3 biological replicates for all conditions): mild febrile illness (MF; P = .423), uncomplicated malaria (UM; P = .580), non-CM comatose illness (non-CM; P > .99), and Ret−CM (P > .99). (C) Histone-induced disruption is abrogated by ahscFv (200 μg/ml, P = .44 when compared with media-only condition). (D) HBEC were grown on gold-electrode–coated plates in an impedance system (xCELLigence) that enables real-time noninvasive measurement of TEER. Confluent cells were treated with purified P falciparum histones with or without heparin (200 μg/ml) or histones preincubated with magnetic beads coated with ahscFv (200 μg/ml), with beads removed by magnet prior to incubation. (E) The effect of histone concentration on barrier disruption was measured as downward slope, reflecting changes in cell impedance. Compared with the media-only control there was no significant difference at 25 µg/ml histone (P = .35); however, significant disruption was observed at histone concentrations of 50 µg/ml (P = .011) and 100 µg/ml (P = .0009) (Kruskal-Wallis test with Dunn’s test). (F) These differences were abrogated by prior treatment with heparin (P = .23). (G) HBMECs were treated for 1 hour with serum from patients with or without ahscFv or nonanticoagulant heparin (200 μg/ml). Cell toxicity was determined by propidium iodide staining using flow cytometry. Data are expressed as medians and IQR relative to cells treated with media alone (set to 0%) and compared using the Kruskal-Wallis with Dunn’s test to adjust for multiple comparisons. Compared with serum from the healthy control (HC; n = 8), serum from Ret+CM cases (n = 13) induced significant toxicity (P = .0012), whereas serum from other patient groups did not: UM (n = 5; P = .50), MF (n = 5; P = .23), non-CM (n = 5; P = .99), Ret−CM; (n = 6; P = .69). (H) Among Ret+CM cases, serum from cases with histones >100 μg/ml (n = 8) caused higher toxicity than those with histone <25 μg/ml (n = 5, P = .0016,); Mann-Whitney U test. (D) This was abrogated by treatment with ahscFv (n = 3, P = .205) or heparin (n = 5, P = .115).

P falciparum histones are toxic factors in CM patient sera which are necessary to induce brain endothelial disruption ex vivo. (A) Time course of barrier disruption of primary HBMEC by P falciparum histones in a Transwell dual chamber system treated with 100 μg/ml of purified plasmodial histones compared with cells with media alone or cells treated with histones plus 200 μg/ml of antihistone single chain variable Fragment (ahscFv). Barrier disruption was assessed by measuring the optical density (OD) of the liquid in the lower part of the Transwell system (measures horseradish peroxidase pass through, detected by reaction with tetramethylbenzidine substrate, 3 biological replicates per condition). By 2-way ANOVA there was significant barrier disruption in the histone group when compared with the media group (P = .0045). This was abrogated by treatment with ahscFv, which was not significantly different from the media-only group (P = .093). (B) Serum-induced barrier disruption by patient samples. Transwell permeability changes of HBMEC monolayer are expressed as fold changes in horseradish peroxidase pass-through over 1 hour. Data are expressed as violin plots and analyzed with the Kruskal-Wallis test with Dunn’s test to adjust for multiple comparisons. Compared with healthy control serum there was a significant increase in permeability induced by the patient serum from Ret+CM cases (P = .0123), but not with any of the other patient groups (n = 3 biological replicates for all conditions): mild febrile illness (MF; P = .423), uncomplicated malaria (UM; P = .580), non-CM comatose illness (non-CM; P > .99), and Ret−CM (P > .99). (C) Histone-induced disruption is abrogated by ahscFv (200 μg/ml, P = .44 when compared with media-only condition). (D) HBEC were grown on gold-electrode–coated plates in an impedance system (xCELLigence) that enables real-time noninvasive measurement of TEER. Confluent cells were treated with purified P falciparum histones with or without heparin (200 μg/ml) or histones preincubated with magnetic beads coated with ahscFv (200 μg/ml), with beads removed by magnet prior to incubation. (E) The effect of histone concentration on barrier disruption was measured as downward slope, reflecting changes in cell impedance. Compared with the media-only control there was no significant difference at 25 µg/ml histone (P = .35); however, significant disruption was observed at histone concentrations of 50 µg/ml (P = .011) and 100 µg/ml (P = .0009) (Kruskal-Wallis test with Dunn’s test). (F) These differences were abrogated by prior treatment with heparin (P = .23). (G) HBMECs were treated for 1 hour with serum from patients with or without ahscFv or nonanticoagulant heparin (200 μg/ml). Cell toxicity was determined by propidium iodide staining using flow cytometry. Data are expressed as medians and IQR relative to cells treated with media alone (set to 0%) and compared using the Kruskal-Wallis with Dunn’s test to adjust for multiple comparisons. Compared with serum from the healthy control (HC; n = 8), serum from Ret+CM cases (n = 13) induced significant toxicity (P = .0012), whereas serum from other patient groups did not: UM (n = 5; P = .50), MF (n = 5; P = .23), non-CM (n = 5; P = .99), Ret−CM; (n = 6; P = .69). (H) Among Ret+CM cases, serum from cases with histones >100 μg/ml (n = 8) caused higher toxicity than those with histone <25 μg/ml (n = 5, P = .0016,); Mann-Whitney U test. (D) This was abrogated by treatment with ahscFv (n = 3, P = .205) or heparin (n = 5, P = .115).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal