Table 8.
Intracerebral inoculation of tg338 mice with PBMC and cell-sorted mononuclear cell subpopulations prepared from VRQ/VRQ sheep orally inoculated with PG127 scrapiea
| Sample type | Results for samples from sheep: |
|||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| D7 |
D9 |
D10 |
D11 |
D12 |
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| Purity (%) | No. of cells inoculated/mouse | tg338 transmission |
Purity (%) | No. of cells inoculated/mouse | tg338 transmission |
Purity (%) | No. of cells inoculated/mouse | tg338 transmission |
Purity (%) | No. of cells inoculated/mouse | tg338 transmission |
Purity (%) | No. of cells inoculated/mouse | tg338 transmission |
||||||
| No. of mice positive for abnormal PrP | Incubation time (days) | No. of mice positive for abnormal PrP | Incubation time (days) | No. of mice positive for abnormal PrP | Incubation time (days) | No. of mice positive for abnormal PrP | Incubation time (days) | No. of mice positive for abnormal PrP | Incubation time (days) | |||||||||||
| PBMC | 4 × 106 | 6/6 | 96 ± 5 | 4 × 106 | 6/6 | 100 ± 3 | 4 × 106 | 6/6 | 86 ± 2 | 4 × 106 | 6/6 | 105 ± 14 | 4 × 106 | 6/6 | 104 ± 14 | |||||
| CD14+ | 98.2 | 1.5 × 105 | 3/6 | 119 ± 17 | 96.4 | 2 × 105 | 3/7 | 107, 133, 138 | 98.2 | 1.8 × 105 | 6/6 | 111 ± 17 | 95.2 | 1.7 × 105 | 4/6 | 123 ± 9 | 96.3 | 1.7 × 105 | 3/6 | 124 ± 17 |
| CD14− | 99 | 1.7 × 106 | 5/6 | 112 ± 14 | 99.3 | 2.8 × 106 | 6/6 | 113 ± 7 | 99.2 | 2.2 × 106 | 6/6 | 107 ± 6 | 99.6 | 3.1 × 106 | 6/6 | 117 ± 9 | 99.7 | 1.5 × 106 | 6/6 | 107 ± 13 |
| CD4/CD8+ | 88.7 | NA | 96.1 | 6 × 105 | 5/6 | 130 ± 15 | 95.3 | 5.8 × 105 | 1/6 | 119 | 95.5 | 7.8 × 105 | 2/6 | 128, 150 | 96.4 | 5.3 × 105 | 2/6 | 117, 119 | ||
| CD4/CD8− | 98.7 | NA | 97.8 | 3.1 × 105 | 4/6 | 124 ± 11 | 98.2 | 5 × 105 | 5/6 | 110 ± 6 | 99.4 | 6.1 × 105 | 4/6 | 123 ± 20 | 98.6 | 3.8 × 105 | 5/6 | 104 ± 8 | ||
| CD45R+ | 91.1 | NA | 95.7 | 3 × 105 | 0/6 | > 200 | 98.4 | 3.5 × 105 | 3/6 | 127 ± 15 | 97.8 | 4 × 105 | 0/6 | >200 | 97.2 | 4.6 × 105 | 1/6 | 112 | ||
| CD45R− | 87.3 | NA | 98.2 | 4 × 105 | 6/6 | 125 ± × 10 | 99.2 | 7.2 × 105 | 4/6 | 112 ± 21 | 97.3 | 8 × 105 | 3/6 | 116 ± 11 | 98.4 | 3.2 × 105 | 7/7 | 101 ± 7 | ||
Five susceptible VRQ/VRQ sheep (identified as D7, D9, D10, D11, and D12) aged between 6 and 10 months were orally challenged with 2 g of brain homogenate (106.6 ID50/g IC in tg338 mice). These animals were sampled at 210 dpi and died at 228, 256, 242, 244, and 248 dpi, respectively. Classical scrapie occurrence was confirmed by histopathology (vacuolar changes in the central nervous system) and the detection of abnormal PrP deposits in the central nervous system and lymphoid tissues. Whole-blood samples (150 ml) were collected for PBMC preparations. CD14+, CD14− (FITC-labeled VPM65 MAb), CD4/CD8+, CD4/CD8− (FITC-labeled SBU/T4 and SBU/T8 MAb), CD45R+, and CD45R− (FITC-labeled 20.29 MAb) subpopulations were sorted using anti-FITC-coated magnetic beads and two passages on magnetically activated cell sorting minicolumns. The purity of sorted cells was assessed by flow cytometry (percentage of FL1-labeled cells for 2 × 104 events). After preparation, cells were counted and pelleted. Each fraction was resuspended in 200 μl of a 5% glucose solution before grinding. Each homogenate was inoculated intracerebrally into tg338 mice (n = 6; 20 μl per mouse). Equivalent numbers of cells inoculated into each mouse are indicated. Mice then were observed until the occurrence of clinical signs or were killed after 200 days postinoculation. Mice were considered positive when abnormal PrP deposition was detected in the brain. Incubation periods are presented as means ± SD except for dilutions with which less than 50% of mice were found positive. In those cases, incubation times of the positive mice are individually presented.