Figure 7

In vivo and in vitro assays for NF-κB activation. (A–B) An in vivo assay as performed to measure NF-κB activation levels during malaria infection. In particular, PBMCs were isolated from monkeys in the two groups at different time points for each phase. We counted the cells with NF-κB nuclear staining and classified them as cells with NF-κB activation. (A) NF-κB nuclear transport was measured by immunofluorescence to determine the activation of NF-κB in PBMCs. DAPI staining was used to identify the nuclear region to assess gross cell morphology. The white arrows show the NF-κB-positive nuclei in PBMCs. These cells were considered to contain activated NF-κB. (B) The ART + Pc group maintained higher levels of NF-κB activation in PBMCs during malaria infection, as indicated by the percentages of NF-κB-positive nuclei in the PBMCs (16.23 ± 3.12% vs. 9.69 ± 1.74%). The data in this figure are presented as the means, and the error bars show the SD. (C-D) In vitro assays were performed to detect specific factors associated with malaria that activate NF-κB. PBMCs isolated from monkeys were stimulated with Pf extract or 25 ng/ml PHA plus 1 μg/ml LPS. NF-κB activation in the cells was then measured by ELISA. (C) Pf soluble extract induced an increase in activated NF-κB in J-Lat cells at 5 μg/ml after a 2- or 4-hour incubation. (D) Pf soluble extract induced an increase in activated NF-κB in RM PBMCs at 50 μg/ml after a 2- or 4-hour incubation. The percentages of NF-κB+ PBMCs at different time points of the same phase in the same group were combined for statistical analyses. The Mann–Whitney U test was utilized to examine the difference in this parameter between the two groups. One-way ANOVA was used to compare the variables in C and D. The data in this figure are presented as the means, and the error bars indicate the SD.