Table 1.
Preclinical active and passive immunization against prion disease.
Fig 1.
(A) Magnetic resonance DWI images at 48h after injection of antibody D13 (2, 6 and 12 μg; respectively) into the left hippocampus. For control, D13 was preincubated with a three-fold molar excess of recombinant mouse PrP (residues 90–230; D13+rmPrP) and injected into the right hippocampus. Whereas both D13 and D13+rmPrP induced minor traumatic lesions in the neighborhood of the injection site, only D13 induced extensive hyperintensity throughout the hippocampus. (B) Haematoxylin and eosin (HE) stained sections from the mice shown in panel A. Asterisks: needle track (only visible on select sections). Rectangles denote regions magnified in panel C. The bilateral cortical incisions (crosses) were introduced post-mortem as landmarks in order to properly orient the paraffin blocks for histology. (C) Higher magnification of the Cornu ammonis, sector 1 (CA1). Left panels: D13 injections (2, 6 and 12 μg). Right panels: D13+rmPrP. D13-exposed tissue displayed vacuoles indicative of edema (arrowhead). Numerous neurons showed condensed chromatin, hypereosinophilic cytoplasm (asterisks), and nuclear disintegration (arrow). Injection of 12 μg induced neuropil coarsening indicative of severe lesions. (D) DWI-based volumetric quantification of lesions depicted on a log10 scale after 2, 6 and 12 μg of D13. Statistical analysis revealed significant lesion induction at 6 and 12 μg of D13 in contrast to injection at 2 μg of D13 and D13 (12 μg) preincubated with rmPrP (grey). N = 5, mean±sd of log10 values, one-Way Anova with Dunnett’s post-hoc test, **P<0.01, *P<0.05. (E) TUNEL-stained paraffin sections of the CA1 region of the mouse shown in panels A-C (injection of 6 μg D13). Quantitation showed 41±19% TUNEL+ cells (n = 30 fields at 20x) after exposure to D13 and ca. 0.5% TUNEL+ cells after exposure to D13+recPrP. Blue: nuclear counterstaining with 4’,6-diamidino-2-phenylindole (Dapi). (F) Confocal laser scanning microscopy of paraffin sections immunostained for activated caspase-3 revealed little to no caspase activity after injection of 6 μg D13.
Fig 2.
ICSM18 trigger mouse hippocampal neurotoxicity.
(A) DWI showing a hyperintense lesion (arrowhead) at 24h after injection of ICSM18 (6 μg) into the left CA1 region of a BL10 female mouse. No signal alteration was found on the contralateral side injected with IgG1 isotype control. Arrowheads point to hyperintense lesions. Asterisks: needle tracks. (B) HE-stained histological sections of the brain shown in panel A (48h p.i.). Asterisks: location of needle track. Rectangles: regions magnified in panel C. (C) High-magnification images of the regions identified by the red and yellow rectangles, respectively, on panel B. Neurons with condensed nuclei and hypereosinophilic cytoplasm were found (star) in the area corresponding to the DWI hyperintensity (left panel). In contrast, the mechanical lesions induced by the needle track were characterized by cellular debris (right panel, white arrowhead). (D) Representative DWI images 24h after stereotaxic injection of 6 μg ICSM18 versus BRIC222 into the CA3 region of BL6 females, BL10 males, or BL10 females (as indicated). Arrowheads point to hyperintense lesions. Asterisks: needle tracks (only visible on select planes). (E) HE-stained histological sections of the brains depicted in panel D. Mice were sacrificed at 48h p.i. Rectangles: regions magnified in Panel F. (F) High-magnification images of the regions identified on panel E. Numerous dying neurons are seen in the dentate gyrus after exposure to 6 μg ICSM18 (red rectangle), but not to 6 μg BRIC222 (yellow rectangle). Occasional "dark neurons" were found in BRIC222-treated samples at frequencies similar to those of untreated mice, and were interpreted as fixation artifacts. (G) 24h after CA3 administration of 6 μg POM1, the findings were similar to those after ICSM18 administration. For control, we blocked POM1 by pre-incubation with a three-fold molar excess of the antigen rmPrP. (H) HE micrograph from the mouse depicted in panel G (48h p.i.). Rectangles: regions magnified in Panel I. (I) POM1 related tissue damage was morphologically similar to the ICSM18-induced neurotoxicity shown in panel F. (J) Representative HE images at high and low resolution 48h p.i. of 2 μg ICSM18 versus 2 μg BRIC222. No lesions were found. (K) Significant lesion induction was found after injection of 6 μg ICSM18 into the CA3 region of female BL10 mice, but not after injection into the CA1 region and not after injection of 2 μg. Lesion volumes were slightly larger in BL6 mice of either gender (two-tailed Student’s t-test). Stereotaxic injection of POM1 (same dose as ICSM18) induced damage similarly to the injection of ICSM18 in BL6 mice. Log10 scale; n = 5 for CA1 region injections and n = 4 for CA3 injections; mean ±SD of log10 values; Multi column comparison (sample three to five) with one-way Anova with Tukey’s post-hoc test, comparing of two samples with two-tailed Student’s t-test, **P<0.01, *P<0.05, ns: not significant.
Fig 3.
Penetration of monoclonal antibodies after stereotaxic injection.
(A) Representative frozen sections of mouse brains at 24h p.i. of Cy5-POM2 conjugate (2 μg or 6 μg) into the CA3 region. Conjugated holoantibody diffuses within a well-defined region of the hippocampus, but did not penetrate into other neuroanatomical regions. DG: dentate gyrus; CA: cornu ammonis. (B) Higher-magnification images of the dentate gyrus illustrate the distribution of Cy5-POM2 around the neurons of the granular cell layer (GCL), the molecular layer (ML), and the hilus. (C) Mean distribution volumes of 1.8 mm3 and 5 mm3 were found after administration of 2 μg and 6 μg of Cy5-POM2 conjugate, respectively.
Fig 4.
Tissue damage after chronic exposure to the toxic antiprion antibody POM1.
(A) Representative DWI images of mouse brains 4 days after the implantation of a mini-osmotic Alzet pump delivering a single-chain variable fragment (scFv) of POM1. A large region of restricted diffusion became visible in tga20 mice (right), whereas no lesion was detected in Prnp°/° mice (left). (B) Images of the same mice as in panel A, at 11 days p.i. The hyperintense signal in the tga20 mouse was no longer visible, indicating that the acute phase of tissue damage had subsided. (C) Coronal manganese-enhanced MRI (MEMRI) images of the tga20 mice shown in panels A and B. A reduction of the MEMRI signal was visible in the dentate gyrus (asterisk) and CA1 region. Moreover, there was a conspicuous enlargement of the ventricles (v) indicating parenchymal loss. No lesion was detected in Prnp°/° mice. (D) Representative HE sections from the mice shown in panels A at 21 p.i. Gross damage to the brain architecture, especially to the hippocampus of tga20 mice. Numbered rectangles: regions magnified in supplemental S4 Fig. (E) Low-magnification micrographs of glial fibrillary acidic protein (GFAP) stainings of consecutive sections, illustrating tissue damage in both hemispheres, particularly in the hippocampal region.
Table 2.
PrPC epitopes and toxicity of anti-PrPC antibodies.