Acta Neuropathol (2009) 118:659–671 663 Fig. 1 a mAb 1E4 shows a relative higher afﬁnity for PrPSc type 2 than for PrPSc type 1. Western blot proﬁles of PrPSc from 3 sCJD MM subjects showing various relative proportions of PrPSc type 1 and type 2. Each sample has been loaded twice at the same concentration and probed either with 3F4 (left) and 1E4 (right). Lane 1 sample with dominant type 1 and only 3–5% of type 2; Lane 2 sample with 60/ 40% type1/type2 ratio; Lane 3 sample with pure type 2. b Western blot proﬁles of PrPSc (3F4 antibody) from sCJD subjects with concurrent PrPSc types 1 and 2 carrying different codon 129 PRNP genotypes (lanes 2–4). PrPSc type 1 from a MM1 case (lane 1), and PrPSc type 2 from a VV2 case (lane 6) are included as controls. The PrPSc proﬁle associated with the sCJD MV2 phenotype with kuru plaques (sCJD MV2K) is shown in lane 5. The upper band of the PrPSc doublet in lane 5 migrates faster than the type 1 band. Frontal cortex homogenates were treated with PK and probed with 3F4. Approximate molecular masses are in kilodaltons Table 1 PRNP genotype and PrPSc type in a unselected series of 200 sCJD cases Typea Type 1 Types 1?2 concurrence Type 2 Total MM 67 (52) 56 (43) 6 (5)b 129 MV 2 (6) 7 (23)b 22 (71)b 31 VV 2 (5) 6 (15) 32 (80) 40 Total 71 (35) 69 (35) 60 (30) 200 MV 1?2 includes 2 cases with kuru plaques and 5 without, MM2 includes 4 MM 2T, and 2 MM 2C cases, whereas MV2 includes 20 cases with kuru plaques and 2 without % are expressed in brackets and refer to each single column a Typing results refer to both 3F4 and 1E4 mAbs b Phenotypically heterogenous patient groups type 2 detectable only focally in the cerebral neocortex or thalamus (Fig. 2a), whereas only a small group of subjects showed a predominant PrPSc type 2 ‘‘load’’ (Fig. 2b). The two groups will be indicated as MM 1?2C and MM 2C?1 throughout the manuscript, MM 2C?1 being deﬁned as a case in which the mean total amount of type 2 (total type 2 ‘‘load’’) expressed in % of total PrPSc signal and calculated by averaging the results obtained from all brain regions examined, is higher than that of type 1 (i.e. [51%). The detailed regional PrPSc typing in the MM 1?2C group provided the following results. When 3F4 was used, 74% of cases showed type 2 in 7 or fewer of the 21 regions analyzed, and 42% of them in only 1 or 2 (Fig. 3). In the latter group, 3F4 detected the PrPSc types mixture only in the cerebral cortex or thalamus in 37 of the 504 samples analyzed (Fig. 3). However, when 1E4 was used, the number of mixed samples in the cerebral cortex or thala- mus raised about 2.6 times to 98 (data not shown). Noteworthy, the number of cases showing type 2 in only one or two areas dropped from 24 to 8. Furthermore, 1E4 detected PrPSc type 2 in 6 out of 7 subjects in which 3F4 failed to demonstrate the type 2 protein despite its presence was strongly suggested by the results of PrP immunohis- tochemistry (see below). Finally, in the remaining samples from subcortical areas, 1E4 conﬁrmed the results obtained with 3F4 and failed to reveal a signiﬁcant type 2 accumulation. In the group of MM 1?2C with type 2 in up to 7 brain regions (i.e. 74% of cases), the amount of PrPSc type 1 was higher than that of type 2 in 84% of the mixed samples, while type 2 had a stronger signal than type 1 in the remaining 16%. The mean total type 2 ‘‘load’’ in this group, calculated on immunoblots probed with 3F4, was 3.4% (range 0.6–14%). The remaining 26% of MM 1?2C cases showed a more widespread PrPSc type 2 accumulation involving from 10 to 16 different brain regions. In this group, PrPSc type 1 was predominant over type 2 in 43% of the mixed samples, while type 2 had a stronger signal than type 1 in the remaining 57%. The mean total type 2 ‘‘load’’, again cal- culated on immunoblots probed with 3F4 raised to 30% (range 18–38%). The study of the regional protein distribution in MM 1?2C subjects showed that PrPSc type 2 was overall more frequently seen in the cerebral cortex than in subcortical areas (Fig. 3). The temporal, parietal, and occipital neo- cortices were the cortical areas which preferentially accumulated the type 2 protein (Fig. 3). Among subcortical areas, the thalamus was by far the most frequent site of type 2 accumulation, whereas the brainstem and, to a greater extent, the cerebellum (Fig. 3) were the regions with the lowest frequency (1 and 0 cases, respectively) of type 2 detection. At variance with MM 1?2C cases, in MM 2C?1 sub- jects type 2 was the only detectable protein species in some areas (Fig. 2b, Table 2) and its amount was higher than that of type 1 also in most areas where the two proteins coexisted (Table 2). 123