The current classification of human sporadic prion diseases recognizes six major phenotypic subtypes with distinctive clinicopathological features, which largely correlate at the molecular level with the genotype at the polymorphic codon 129 (methionine, M, or valine, V) in the prion protein gene and with the size of the protease-resistant core of the abnormal prion protein, PrPSc (i.e. type 1 migrating at 21 kDa and type 2 at 19 kDa). We previously demonstrated that PrPSc typing by Western blotting is a reliable means of strain typing and disease classification. Limitations of this approach, however, particularly in the interlaboratory setting, are the association of PrPSc types 1 or 2 with more than one clinicopathological phenotype, which precludes definitive case classification if not supported by further analysis, and the difficulty of fully recognizing cases with mixed phenotypic features. In this study, we tested the inter-rater reliability of disease classification based only on histopathological criteria. Slides from 21 cases covering the whole phenotypic spectrum of human sporadic prion diseases, and also including two cases of variant Creutzfeldt–Jakob disease (CJD), were distributed blindly to 13 assessors for classification according to given instructions. The results showed good-to-excellent agreement between assessors in the classification of cases. In particular, there was full agreement (100 %) for the two most common sporadic CJD subtypes and variant CJD, and very high concordance in general for all pure phenotypes and the most common subtype with mixed phenotypic features. The present data fully support the basis for the current classification of sporadic human prion diseases and indicate that, besides molecular PrPSc typing, histopathological analysis permits reliable disease classification with high interlaboratory accuracy.

The α-synuclein gene (SNCA) plays a major role in the aetiology of Lewy body disease (LBD) including Parkinson's disease (PD). Point mutations and genetic alterations causing elevated gene expression are causally linked to familial PD. To what extent epigenetic changes play a role in the regulation of α-synuclein expression and may contribute to the aetiology of sporadic LBD is a matter of debate. We analysed the methylation state of the promoter region and a CpG-rich region of intron 1 of α-synuclein in several brain regions in sporadic LBD and controls using 454 GS-FLX-based high-resolution bisulphite sequencing. Our results indicate that there are significant differences in the level of methylation between different brain areas. The overall methylation levels in the promoter and intron 1 of α-synuclein are rather low in controls and-in contrast to previously reported findings-are not significantly different from LBD. However, single CpG analysis revealed significant hyper- and hypomethylation at different positions in various brain regions and LBD stages. A slight overall increase in methylation related to LBD patients' age was detected.

Since researchers identified α-synuclein as the principal component of Lewy bodies and Lewy neurites, studies have suggested that it plays a causative role in the pathogenesis of dementia with Lewy bodies and other ‘synucleinopathies’. While α-synuclein dyshomeostasis likely contributes to the neurodegeneration associated with the synucleinopathies, few direct biochemical analyses of α-synuclein from diseased human brain tissue currently exist. In this study, we analysed sequential protein extracts from a substantial number of patients with neuropathological diagnoses of dementia with Lewy bodies and corresponding controls, detecting a shift of cytosolic and membrane-bound physiological α-synuclein to highly aggregated forms. We then fractionated aqueous extracts (cytosol) from cerebral cortex using non-denaturing methods to search for soluble, disease-associated high molecular weight species potentially associated with toxicity. We applied these fractions and corresponding insoluble fractions containing Lewy-type aggregates to several reporter assays to determine their bioactivity and cytotoxicity. Ultimately, high molecular weight cytosolic fractions enhances phospholipid membrane permeability, while insoluble, Lewy-associated fractions induced morphological changes in the neurites of human stem cell-derived neurons. While the concentrations of soluble, high molecular weight α-synuclein were only slightly elevated in brains of dementia with Lewy bodies patients compared to healthy, age-matched controls, these observations suggest that a small subset of soluble α-synuclein aggregates in the brain may drive early pathogenic effects, while Lewy body-associated α-synuclein can drive neurotoxicity.

Viral encephalitis is a life-threatening condition with prognosis mainly depending on viral pathogenicity, host immunologic state, and availability of virostatic therapy [1]. The Toscana virus (TOSV) is an arthropod-borne virus transmitted by sandflies and typically causes a transient febrile illness [2]. Recently, reports have emerged that TOSV causes not only aseptic meningitis but also serious meningoencephalitis or encephalitis without meningitis [2][3][4][5][6][7][8]. We present a patient who travelled around Tuscany and developed progressive fatal encephalitis. Serological positive TOSV antibodies were detected, and the autopsy revealed lymphocytic viral encephalitis.The general condition of a 73-year-old previously healthy man deteriorated rapidly a couple of days after returning from a 3 week trip to Tuscany. First symptoms were dizziness and disturbance of balance. Internal medical assessment was unremarkable at that time. As symptoms progressed he was transferred to our department of neurology. On admission he was alert but disoriented, and initially psychomotorically retarded, later agitated. Meningeal signs were negative. Cranial nerve functions and reflexes were normal except for hearing impairment on both sides. Walking and standing showed a tendency to fall to the left and were only possible with help. No impairment of the motor nerves and no sensory disturbances were found.Repeated neuroimaging examinations (cCT/cMRI) were unremarkable. EEG-studies showed generally slowed activity, but no epileptiform discharges. The body temperature fluctuated between 37-39.5°C.The laboratory studies revealed a slightly elevated CRP without a rise in WBC count. The CSF showed a slightly elevated cell count (8/3; normal \3/3) with elevated protein (757 mg/dl; normal \450), indicating a disturbance of the blood-CSF barrier. PCR for HSV, VZV and FSME virus were negative; antibody specificity tests for HSV, VZV, and FSME were within normal ranges. Serological studies on Legionella, Treponema pallidum, HIV, Hepatitis C, Borreliose were negative as were blood cultures. Serologically IgG antibodies against TOSV were positive 1:640 and IgM antibodies against TOSV were positive 1:40 using IIFT. In the CSF IgG antibodies against TOSV were tested positive 1:10. With PCR TOSV could not be detected in the CSF.As encephalitis was clinically assumed, an anti-infectious treatment course with broad antibiotics and aciclovir was implemented without clinical benefit. The patient gradually deteriorated and progressed to an akinetic-mutistic state, which persisted 2 weeks after, leading to coma and death. Post-mortem examination revealed a grossly normal brain with no signs of brain swelling. Histologic examination showed dominant perivascular cuffing of mainly lymphocytes in H & E stained sections in various brain regions (neocortex, basal ganglia, hypothalamus, thalamus, limbic areas, brain stem) and sparse infiltration of inflammatory cells in the leptomeninges, typical of viral meningoencephalitis (Fig. 1). Immunohistochemistry (...