Figure 6. Evaluation of in vivo therapeutic activity of silvestrol in hu-PBL SCID model.

SCID mice depleted of murine NK cells were injected intraperitoneally with 5×10⁷ PBMC from a healthy EBV-seropositive human donor. Treatment with vehicle or 1.5 mg/kg silvestrol (N=14 per group) every other day by the IP route began two weeks post-engraftment. (A) Human Ig ELISA on peripheral blood from all mice (N=28) 4 weeks after engraftment; averages for both groups are shown. Bars show mean ± SD. Differences were not significant (ns). (B) Spleens are shown from two mice from each group 8 weeks post-engraftment, with weights in mg. (C) Spleen weights were recorded from all mice upon euthanasia or end of study (day 140 post-engraftment) (N=9 for vehicle control and N=7 for silvestrol group). Bars show mean ± SD; differences were significant (p=0.008). (D) Kaplan-Meier analysis of overall survival (vehicle control N=9; silvestrol-treated N=7; p<0.01). (E) Specific lymphocyte subsets were depleted from PBMC directly prior to engraftment using immunomagnetic bead depletion for CD8 (cytotoxic T cells), CD14 (monocytes), or CD56 (NK cells). Biotin-only conjugated beads were used for the control (mock-depleted) condition. Efficiency of depletion was verified by flow cytometric analyses and show to be greater than 90%. Equivalent numbers of depleted PBMC preparations (5×10⁷ cells in the mock-depleted condition) were engrafted by intraperitoneal injection into murine NK cell-depleted SCID mice (N=5 per group). After 4 weeks, human Ig levels were assessed by ELISA, and only mice showing engraftment by this parameter were included in the study. Kaplan-Meier analysis of overall survival was then performed. The differences between the non-depleted vehicle control (N=3) vs. non-depleted silvestrol-treated (N=4) groups, and between the CD8-depleted silvestrol-treated vs. non-depleted silvestrol-treated groups (N=4 each), were significant (p=0.029 each). The difference between CD56-depleted silvestrol-treated (N=5) vs. non-depleted silvestrol-treated (N=4) was not significant. Also, there was no difference between CD8-depleted vs. non-depleted vehicle controls (not shown).