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Neuropathology 35

12b

12b. Immunocytochemistry for prion protein in the cerebellum in kuru shows intense labelling of the kuru plaques in the molecular layer and granular layer, and in addition shows multiple smaller plaque-like structures within the granular layer and a fine background granular/synaptic pattern of accumulation.

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12a

12a. The cerebellum in kuru shows moderate spongiform change in the molecular layer with a large kuru plaque composed of a fibrillary amyloid structure with a dense eosinophilic core and a paler periphery within the molecular layer (lower centre). H&E stain.

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11d

11d. Immunocytochemistry for prion protein in the cerebral cortex in a case of iCJD in a
human dura mater graft recipient shows a widespread granular/synaptic pattern of accumulation, with no plaque-like structures identified.

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11b

11b. Immunocytochemistry for prion protein in the cerebellum in a case of iCJD in a human growth hormone recipient shows intense labelling of kuru plaques in the granular layer and molecular layer, with smaller plaque-like aggregates also present.

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11a

11a. The cerebellum in a case of iCJD in a human growth hormone recipient shows occasional small kuru plaques (centre) within the granular layer and mild spongiform change in the
molecular layer (right) H&E stain.

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10b

10b. Immunocytochemistry for glial fibrillary acidic protein in fatal familial insomnia shows intense labelling of the reactive astrocytes within the affected regions of the thalamus. No significant spongiform change is present and Immunohistochemistry for prion protein
in this region of the brain gives a negative reaction.

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10a

10a. The thalamus in fatal familial insomnia shows intense neuronal loss and gliosis, particularly in the medial and dorsal nuclei. There is no spongiform change present and no amyloid plaques are evident. H&E stain.

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9b

9b. Immunocytochemistry for prion protein in GSS (P102L) shows intense labelling of the large multicentric plaques which can be identified the molecular layer, the granular layer and even in the subcortical white matter.

Neuropathology 20

7h

7h. Immunocytochemistry for prion protein in the putamen in variant CJD shows numerous  small rounded aggregates often arranged in a linear distribution, apparently  representing peri-axonal accumulation. No florid plaques are present in this field.

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7f

H&E stain. 7f. Immunocytochemistry for glial fibrillary acidic protein in the pulvinar in variant CJD shows intense labelling of the large reactive astrocytes. A minor degree of vacuolation is present in the upper of this image, but no florid plaques are present.

Neuropathology 17

7e

7e. The thalamus in variant CJD shows severe neuronal loss and gliosis, particularly in the pulvinar and in the dorsomedial nucleus. There is very little spongiform change and florid plaques are not present.

Neuropathology 17

7d

7d. Immunocytochemistry for prion protein in the cerebellar cortex in variant CJD shows intense staining of the florid plaques and the smaller plaques in the granular layer, with some amorphous accumulation in the molecular layer.

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7c

7c. The cerebellar cortex in variant CJD contains florid plaques in the molecular (centre), surrounded by spongiform change. There is widespread vacuolation in the molecular layer, and numerous amyloid plaques are also present in the granular layer. H&E stain.

Neuropathology 15

7b

7b. Immunocytochemistry for PrP in the cerebral cortex in variant CJD shows intense labelling of the large florid plaques, but also demonstrates multiple smaller plaque-like structures not evident on the H&E stain. In addition there is a widespread amorphous accumulation of PrP around smaller neurons and occasional blood vessels.

Neuropathology 14

7a

7a. The characteristic pathological feature in variant CJD is the florid plaque (centre) composed of large radiating fibrils of amyloid with a dense core and paler periphery, surrounded by a halo of spongiform change. Multiple smaller plaques are present elsewhere in this image and there is severe neuronal loss with accompanying astrocytosis. H&E stain.

Neuropathology 13

6b

6b. Immunocytochemistry for prion protein in the cerebral cortex of the VV2 subtype of sCJD shows a characteristic perineuronal and granular/synaptic pattern of accumulation, with decoration of large pyramidal neurons and their apical ascending dendrites (centre) in layer 5 of the cortex.

Neuropathology 9

4b

4b Immunocytochemistry for prion protein in the MV2 subtype of sCJD shows intense labelling of the kuru plaques in the granular layer of the cerebellum (centre). Numerous smaller plaque-like structures not evident on the H&E stain are also visualised by this technique.

Neuropathology 7

4a

4a The cerebellum in the MV2 subtype of sCJD shows characteristic small kuru plaques (centre and bottom) in the granular layer. Spongiform change is present to a mild degree in the molecular layer (right). H&E stain.

Neuropathology6

3b

3b. Immunocytochemistry for prion protein in the MM2 (cortical) subtype of sCJD shows widespread perivacuolar accumulation. A fine background granular/synaptic pattern of abnormal prion protein accumulation is also evident.

Neuropathology5

3a

3a. The MM2 (cortical) subtype of sCJD is characterised by widespread confluent spongiform change, where large-cyst like structures are present in the neuropil in an irregular distribution.
The size of these vacuoles is much larger that in the MM1 subtype of sCJD (see figure 1A). H&E stain.

Neuropathology4

2b

2b. Immunohistochemistry for glial fibrillary acidic protein shows the widespread reactive
astrocytosis present in the affected thalamic nuclei in the MM2 (thalamic) subtype of sCJD. There is relatively little spongiform change present in this tissue.

Neuropathology3

2a

2a. Severe neuronal loss and gliosis in the MM2 (thalamic) subtype of sCJD is a typical neuropathological finding, most evident in the medial and anterior thalamic nuclei. There is relatively little spongiform change present in the affected areas. H&E stain.

PrPres analysis Western Blot Image

 

prp

Detection and typing of abnormal disease-associated prion protein (PrPSc) isoforms by partial proteolytic degradation and Western blot analysis.

Western blot analysis of protease resistant prion protein (PrPres) in post-mortem brain tissue homogenates of cerebral cortex samples from the two most common subtypes of sporadic Creutzfeldt-Jakob disease (sCJD MM1 and sCJD VV2), compared with that of variant Creutzfeldt-Jakob disease (vCJD), and similar analysis of brain stem from a case of bovine spongiform encephalopathy (BSE).

In each example three bands are present, representing non-glycosylated (o), mono-glycosylated (*) and di-glycosylated (**) PrPres. PrPres type 1 differs from type 2 in terms of electrophoretic mobility, most obviously seen by comparing the mobility of the non-glycosylated band. The type 2 PrPres characteristic of vCJD (type 2B) has a predominant diglycosylated band (**), distinguishing it from the type 2 PrPres found in sCJD (type 2A). The predominance of the di-glycosylated band is a feature shared by vCJD and BSE.

The monoclonal antibody 3F4 (which can be obtained from Dako or Signet) is CE marked and was used for the detection and typing of the human specimens shown here. Bovine PrP is not recognised by 3F4, and the vCJD and BSE comparison shown employed the monoclonal antibody 6H4 (from Prionics).

The PrP typing nomenclature is that of Parchi & Gambetti. (Parchi et al., Typing prion isoforms. Nature 1997;386:232-234). The cases of CJD originate from the UK and the Western blot image was provided by Dr. Mark Head (National CJD Surveillance Unit). The BSE tissue was supplied by the Veterinary Laboratory Agency’s TSE Archive (UK) and the Western blot image was provided by Ms Zuzana Krejciova (National CJD Surveillance Unit).

For more information please contact Mark Head m.w.head@ed.ac.uk

Genetics 2

gen2

Codon 129: Codon 129 genotyping by PCR amplification of PRNP and restriction enzyme digest with Nsp1.

(Mw: 100bp molecular weight ladder (dark band 600bp); MV, MM, VV: codon 129 genotypes)

For more information please contact Richard Knight r.knight@ed.ac.uk

CSF 14-3-3 Western blot

 

csf

Results

Lanes 1, 2, 3 are positive for CSF 14-3-3

Lanes 4, 5, 6 are weakly positive for CSF 14-3-3

Lanes 7, 8 and 9 are negative for CSF 14-3-3

Lanes 1 and 2 are from two patients with sporadic Creutzfeldt-Jakob disease (CJD)

Lanes 3 and 4 are from patients who have had a stroke

Lanes 5, 6, 7, 8 and 9 contain CSF samples from patients who do not have CJD

In the United Kingdom weakly positive CSF 14-3-3 results are not considered to be supportive of a diagnosis of sporadic CJD.

Method
SDS-PAGE gel:
12% SDS-PAGE running gel with 4% stacking gel
CSF volume:
15μl of CSF sample per well
Electrophoretic conditions:
40mA constant current for 1 hour 20 minutes
Transfer conditions:
0.72mA/cm2 gel for 2 hours
Primary antibody:
Mouse monocional anti-14-3-3b (Santa Cruz Code: sc-1657)1: 1000 dilution incubated overnight
Secondary antibody:
Rabbit anti-mouse lg HRP (DAKO Code:P0260)1: 1000dilution for 1 hour
ECl reagent:
ECL Plus (GE Healthcare Code: RPN2132)
Exposure time:
2 minutes

For more information please contact Alison Green alison.green@ed.ac.uk