Similar Biochemical Signatures and Prion Protein Genotypes in Atypical Scrapie and Nor98 Cases, France and Norway

Similarities raise questions regarding the origin of these recently described cases

Beyond PrPres Type 1/Type 2 Dichotomy in Creutzfeldt-Jakob Disease

Sporadic Creutzfeldt-Jakob disease (sCJD) cases are currently subclassified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the proteinase K (PK) digested abnormal prion protein (PrPres) identified on Western blotting (type 1 or type 2). These biochemically distinct PrPres types have been considered to represent potential distinct prion strains. However, since cases of CJD show co-occurrence of type 1 and type 2 PrPres in the brain, the basis of this classification system and its relationship to agent strain are under discussion. Different brain areas from 41 sCJD and 12 iatrogenic CJD (iCJD) cases were investigated, using Western blotting for PrPres and two other biochemical assays reflecting the behaviour of the disease-associated form of the prion protein (PrPSc) under variable PK digestion conditions. In 30% of cases, both type 1 and type 2 PrPres were identified. Despite this, the other two biochemical assays found that PrPSc from an individual patient demonstrated uniform biochemical properties. Moreover, in sCJD, four distinct biochemical PrPSc subgroups were identified that correlated with the current sCJD clinico-pathological classification. In iCJD, four similar biochemical clusters were observed, but these did not correlate to any particular PRNP 129 polymorphism or western blot PrPres pattern. The identification of four different PrPSc biochemical subgroups in sCJD and iCJD, irrespective of the PRNP polymorphism at codon 129 and the PrPres isoform provides an alternative biochemical definition of PrPSc diversity and new insight in the perception of Human TSE agents variability

Rapid Typing of Transmissible Spongiform Encephalopathy Strains with Differential ELISA

A strain-typing ELISA distinguishes bovine spongiform encephalopathy from other scrapie strains in small ruminants

PK Resistance ELISA in Frontal Cortex from sCJD and French or UK iCJD (Growth Hormone and Dura Mater Cases).

<p>sCJD (solid line) and i-CJD (dashed line) frontal cortex samples were investigated by PK resistance assay. (A) MM1 sCJD cases (open circles, hexagons and squares), MM1 iCJD UK dura mater cases (open diamonds), MM1 French GH cases (open triangles). (B) MV2 sCJD cases (filled circles, hexagons and squares) and MV2 UK GH case (filled triangles). (C) MV1 sCJD (open circles, diamonds and squares) and MV1 French GH (open triangles) cases. (D) VV2 sCJD (filled circles and hexagons) and VV2 UK GH (filled triangles) cases. (E) VV1 sCJD case (open circles) and VV1 dura mater French case (open triangles). (F) MM2 sCJD cases (filled circles and triangles).</p

PrP^res Profiles in sCJD Patients.

<p>(A) PrP^res from cerebellum (lane 1), caudate nucleus (lane 2), frontal cortex (lane 3) and occipital cortex (lane 4) of a single MV patient was extracted and submitted to WB. Detection with antibody Sha31 reveals different PrP^res profiles. (B–D) PrP^res in cerebellum from seven different patients using Sha31 mAb (B), 8G8 mAb (C), and 12B2 mAb (D). Because of their epitope, Sha31 and 8G8 can detect both type 1 and type 2 PrP^res while 12B2 detects type 1 only. Lane 1, MM patient; lane 2, MV patient; lane 3, VV patient; lane 4, MV patient; lane 5, MV patient; lane 6, VV patient; lane 7, MM patient; line 8, sheep BSE control. (E, F) PrP^res profile in temporal cortex from three MV patients revealed by Sha31 mAb (E) or 12B2 mAb (F).</p

PrP^res Profiles in iCJD Patients.

<p>PrP^res (frontal cortex) was revealed in WB by antibodies Sha31 (A) and 12B2 (B). Lane 1, MM UK dura mater patient; lane 2, VV French dura mater patient; lane 3, MM GH French patient; lane 4, French MV GH patient; lane 5, MV UK GH patient; lane 6, VV UK GH patient.</p

Abnormal PrP Properties as Assessed by Western Blot, PK Digestion ELISA and Strain Typing ELISA in 41 French Sporadic CJD Patients and 12 Iatrogenic CJD Patients Originating either from France or the United Kingdom

<p>Sp, Sporadic; DM, iatrogenic cases linked to dura mater grafts; GH, iatrogenic cases linked to growth hormone treatment.</p>a<p>Patients originating from the United Kingdom.</p>b<p>Express the PK concentration for which, when increasing PK concentration, the ELISA PrP^Sc signal reach an arbitrary cut-off value set at 20% of the signal observed with a PK concentration of 50 µg/ml.</p>c<p>Express the ratio of ELISA signal obtained (A/A′) after PrP^sc PK digestion differential PK digestions in a non-perturbing detergent mixture (A), and a denaturing (SDS) detergent mixture (A′).</p

PrPsc PK Resistance and Molecular Strain Variations in sCJD and iCJD Brain Samples.

<p>Each investigated brain sample was initially characterized by WB using antibody Sha31. Symbol patterns represent type 1 in white, type 1+2 in grey and type 2 in black. (A) Results from PK resistance ELISA carried out on three different brain areas (cerebellum, caudate nucleus and temporal cortex) from a MM1 (open circles), a VV1 (open triangles), a VV2 (inverted filled triangle) and a MM2 (filled squares) sCJD patient. Values obtained are expressed as percentage of signal obtained with the lowest PK concentration (50 µg/mL). (B) Results from CEA strain typing ELISA (one symbol per patient—3 to 5 different areas by patients). PrP^Sc signal intensity was measured after PK digestion into two different detergent solutions. Normalized A/A′ ratio was calculated for each sample (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000029#s2" target="_blank">Methods</a> section). MM1 and MV1 had a low ratio indicating an absence of alteration of PrP^sc PK sensitivity linked to the modification detergent digestion conditions. This ratio was higher in MV2, VV2, MV1+2, and VV1+2, while, in the unique VV1 case, an intermediate ratio was observed. In MM2 patients, the huge ratio indicated a strong increase in PK sensitivity by modification of detergent conditions. (C, D) PK resistance assay in three areas from a (C) VV (triangles) or MM (circles) patient and in (D) a MV (triangles) patient harbouring distinct PrP^Sc WB type in their different brain areas. Artificial mixtures of MM2/VV1 or MM1/VV2 samples were prepared. All homogenates were first equilibrated by dilution into negative brain homogenate to obtain an equal PrP^Sc signal in ELISA. (E, F) Mixtures were then tested by Western Blot (200 µg PK digestion—Sha31 anti PrP antibody). (E) Lane 1: MM1 100%; Lane 2: MM1 75%/VV2 25%; Lane 3: MM1 50%/VV2 50%; Lane 4: MM1 25%/VV2 75%; Lane 5: VV2 100%. (F) Lane 1: VV1 100%; Lane 2: VV1 75%/MM2 25%; Lane 3: VV1 50%/MM2 50%; Lane 4: VV1 25%/MM2 75%; Lane 5: MM2 100%. (G, H) Same mixtures were tested in the PK resistance ELISA assay. (G) VV2 100% (filled circles), VV2 75%/MM1 25% (filled triangles), VV2 50%/MM1 50% (filled inverted triangles), VV2 25%/MM1 75% (open triangles), MM1 100% (open circles). (H) MM2 100% (filled circles), MM2 75%/VV1 25% (filled triangles), MM2 50%/VV1 50% (filled inverted triangles), MM2 25%/VV1 75% (open triangles), VV1 100% (open circles).</p