Plasma total prion protein as a potential biomarker for neurodegenerative dementia: diagnostic accuracy in the spectrum of prion diseases.

AIMS: In the search for blood-based biomarkers of neurodegenerative diseases, we characterized the concentration of total prion protein (t-PrP) in the plasma of neurodegenerative dementias. We aimed to assess its accuracy in this differential diagnostic context. METHODS: Plasma t-PrP was measured in 520 individuals including healthy controls (HC) and patients diagnosed with neurological disease control (ND), Alzheimer's disease (AD), sporadic Creutzfeldt-Jakob disease (sCJD), frontotemporal dementia (FTD), Lewy body dementia (LBD) and vascular dementia (VaD). Additionally, t-PrP was quantified in genetic prion diseases and iatrogenic CJD. The accuracy of t-PrP discriminating the diagnostic groups was evaluated and correlated with demographic, genetic and clinical data in prion diseases. Markers of blood-brain barrier impairment were investigated in sCJD brains. RESULTS: Compared to HC and ND, elevated plasma t-PrP concentrations were detected in sCJD, followed by FTD, AD, VaD and LBD. In sCJD, t-PrP was associated neither with age nor sex, but with codon 129 PRNP genotype. Plasma t-PrP concentrations correlated with cerebrospinal fluid (CSF) markers of neuro-axonal damage, but not with CSF t-PrP. In genetic prion diseases, plasma t-PrP was elevated in all type of mutations investigated. In sCJD brain tissue, extravasation of immunoglobulin G and the presence of swollen astrocytic end-feet around the vessels suggested leakage of blood-brain barrier as a potential source of increased plasma t-PrP. CONCLUSIONS: Plasma t-PrP is elevated in prion diseases regardless of aetiology. This pilot study opens the possibility to consider plasma t-PrP as a promising blood-based biomarker in the diagnostic of prion disease.

Magnetic resonance spectroscopy in intracranial tumours of glial origin.

BACKGROUND AND PURPOSE: To determine in vivo magnetic resonance spectroscopy (MRS) characteristics of intracranial glial tumours and to assess MRS reliability in glioma grading and discrimination between different histopathological types of tumours. MATERIAL AND METHODS: Analysis of spectra of 26 patients with glioblastomas, 6 with fibrillary astrocytomas, 4 with anaplastic astrocytomas, 2 with pilocytic astrocytoma, 3 with oligodendrogliomas, 3 with anaplastic oligodendrogliomas and 17 control spectra taken from healthy hemispheres. RESULTS: All tumours' metabolite ratios, except for Cho/Cr in fibrillary astrocytomas (p = 0.06), were statistically significantly different from the control. The tumours showed decreased Naa and Cr contents and a high Cho signal. The Lac-Lip signal was high in grade III astrocytomas and glioblastomas. Reports that Cho/Cr ratio increases with glioma's grade whereas Naa/Cr decreases were not confirmed. Anaplastic astrocytomas compared to grade II astrocytomas had a statistically significantly greater mI/Cr ratio (p = 0.02). In pilocytic astrocytomas the Naa/Cr value (2.58 ± 0.39) was greater, whilst the Cho/Naa ratio was lower (2.14 ± 0.64) than in the other astrocytomas. The specific feature of oligodendrogliomas was the presence of glutamate/glutamine peak Glx. However, this peak was absent in two out of three anaplastic oligodendrogliomas. Characteristically, the latter tumours had a high Lac-Lip signal. CONCLUSIONS: MRS in vivo cannot be used as a reliable method for glioma grading. The method is useful in discrimination between WHO grade I and WHO grade II astrocytomas as well as oligodendrogliomas from other gliomas.

Differential diagnosis of intracranial meningiomas based on magnetic resonance spectroscopy.

BACKGROUND AND PURPOSE: To determine in vivo magnetic resonance spectroscopy (MRS) characteristics of intracranial meningiomas and to assess MRS reliability in meningioma grading and discrimination from tumours of similar radiological appearance, such as lymphomas, schwannomas and haemangiopericytomas. MATERIAL AND METHODS: Analysis of spectra of 14 patients with meningiomas, 6 with schwannomas, 2 with lymphomas, 2 with haemangiopericytomas and 17 control spectra taken from healthy hemispheres. RESULTS: All the patients with meningiomas had a high Cho signal (long TE). There were very low signals of Naa and Cr in the spectra of 10 patients. A reversed Ala doublet was seen only in 2 cases. Four patients had a negative Lac signal, whereas 3 had high Lac-Lip spectra. Twelve spectra showed high Cho signals (short TE). In one case the Cho signal was extremely low. All spectra displayed a very low Cr signal, but high Glx and Lac-Lip signals. Ala presence was found only in 3 patients. The mean Cho/Cr ratio (PRESS) was 5.97 (1.12 in normal brain, p < 0.05). Lac-Lip was present in all the meningiomas (STEAM). The Ala signal was seen only in 2 spectra with long TE and in 3 sequences of the short TE sequences. There were both ß/γ-Glx and α-Glx/glutathione signals in all 14 meningiomas. CONCLUSIONS: MRS is unable to discriminate low and high grade meningiomas. The method seems to be helpful in discriminating lymphomas (absent Glx signal), schwannomas (mI signal in the short TE sequences) and haemangiopericytomas (presence of mI band) from meningiomas.

Melatonin-induced glycosaminoglycans augmentation in myocardium remote to infarction.

Elevated levels of collagen as well as transient increases of glycosaminoglycans (GAG) have been shown in the myocardium remote to the infarction. The aim of the study is to observe the effect of melatonin on the accumulation of collagen and GAG in the left ventricle wall, remote to the infarction. A second aim is to determine whether the effect of the pineal indole is mediated by the membrane melatonin receptors of heart fibroblasts. Rats with myocardial infarction induced by ligation of the left coronary artery were treated with melatonin at a dose of 60 µg/100 g b.w. or vehicle (2% ethanol in 0.9% NaCl). The results were compared with an untreated control. In the second part of the study, the fibroblasts from the non-infarcted part of myocardium were isolated and cultured. Melatonin at a range of concentrations from 10(-8) M to 10(-6) M was applied to the fibroblast cultures. In the final part of the study, the influence of luzindole (10(-6) M), the melatonin membrane receptor inhibitor, on melatonin-induced GAG augmentation was investigated. Both collagen and GAG content were measured in the experiment. Melatonin elevated GAG content in the myocardium remote to the infarcted heart. Collagen level was not changed by pineal indoleamine. Fibroblasts isolated from the myocardium varied in shape from fusiform to spindle-shaped. Moreover, the pineal hormone (10(-7)M and 10(-6)M) increased GAG accumulation in the fibroblast culture. Luzindole inhibited melatonin-induced elevation of GAG content at 10(-6)M. Melatonin increased GAG content in the myocardium remote to infarction. This effect was dependent on the direct influence of the pineal indole on the heart fibroblasts. The melatonin-induced GAG elevation is blocked by luzindole, the melatonin membrane receptors inhibitor, indicating a direct effect of this indole.

Glioma cells showing IDH1 mutation cannot be propagated in standard cell culture conditions.

BACKGROUND: It has recently been reported by several sources that original (i.e., present in vivo) glioma cell phenotypes or genotypes cannot be maintained in vitro. For example, glioblastoma cell lines presenting EGFR amplification cannot be established. METHODS AND RESULTS: IDH1 sequencing and loss of heterozygosity analysis was performed for 15 surgery samples of astrocytoma and early and late passages of cells derived from those and for 11 archival samples. We were not able to culture tumour cells presenting IDH1 mutations originating from currently proceeded 10 tumours; the same results were observed in 7 samples of archival material. CONCLUSION: The IDH1 mutation is expected to be almost mutually exclusive with EGFR amplification, so glioma cells with IDH1 mutations seem to represent a new group of tumour cells, which cannot be readily analysed in vitro because of their elimination. The reasons for this intriguing phenomenon should be investigated since its understanding can help to define a new therapeutic approach based on simulating in vivo conditions, responsible for tumour cells elimination in vitro. Moreover, a new model for culturing glioma cells in vitro should be designed since the current one does not provide conditions corresponding to in vivo growth.

Ultrastructural study of florid plaques in variant Creutzfeldt-Jakob disease: a comparison with amyloid plaques in kuru, sporadic Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker disease.

BACKGROUND: Although the histological features of the amyloid plaques in variant Creutzfeldt-Jakob disease (vCJD) are distinct from those in other forms of prion disease [kuru, sporadic Creutzfeldt-Jakob disease (sCJD) and Gerstmann-Sträussler-Scheinker disease (GSS)], their ultrastructural features have only been described in a single case report. AIMS: To study vCJD plaques systematically and compare them with plaques in kuru, sCJD, GSS and Alzheimer disease (AD). METHODS: Amyloid plaques were studied by transmission electron microscopy and image analysis in five cases of vCJD, three cases of GSS, two cases of sCJD, one case of kuru and five cases of AD. Immunohistochemistry was performed on paraffin sections from one case of vCJD, two cases of GSS, one case of kuru and two cases of sCJD. RESULTS: The florid plaques in vCJD were either compact or more diffuse; in both forms, the radiating fibrils were organized into thick 'tongues', in contrast to kuru plaques. Dystrophic neurites (DNs) containing lysosomal electron-dense bodies or vesicles surrounded florid plaques. Microglial cells were found within florid plaques; occasional amyloid fibrils were identified in membrane-bound pockets of microglial cells. In vCJD, there was significant tau immunoreactivity in DNs around florid plaques while, in sCJD, GSS and kuru, minimal tau immunoreactivity was observed around plaques. CONCLUSIONS: The ultrastructure of the florid plaques and DNs in vCJD is more reminiscent of neuritic plaques in AD than kuru or multicentric plaques. These findings may reflect differences both in the strains of the transmissible agents responsible for these disorders and in host factors.

Kuru: its ramifications after fifty years.

Kuru was the first human neurodegenerative disease in the group of transmissible spongiform encephalopathies, prion diseases or, in the past, slow unconventional virus diseases. It was reported to Western medicine in 1957 by Gajdusek and Zigas. Kuru was spread by endocannibalism and because of this the ratio of affected women and children to men was excessive. The hallmark of kuru neuropathology is the amyloid plaque. We may speculate what would happen if kuru had not been discovered or did not exist. The infectious nature of Creutzfeldt-Jakob disease (CJD) would probably not have been suspected until the beginning of the variant CJD (vCJD) outbreak in the UK. Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker disease would have remained for decades as obscure neurodegenerations of merely academic interest. The familial forms of CJD would not have benefited from PRNP gene (a gene encoding for prion protein) analysis, but only later would have been studied by linkage analysis and reverse genetics probably. The study of kuru would have probably been of minimal interest to veterinarians and anthropologists until the bovine spongiform encephalopathy (BSE) epidemic began to exert its devastating effect. The discovery of vCJD would have been delayed, as no surveillance would have been initiated for CJD. And perhaps most importantly, the realization of 'protein-misfolding diseases', including not only the neurodegenerative but also an increasing number of non-neurological disorders, would have been delayed by decades.

BCR expression is decreased in meningiomas showing loss of heterozygosity of 22q within a new minimal deletion region.

Neurofibromin 2 (NF2), located on chromosome arm 22q, has been established as a tumor suppressor gene involved in meningioma pathogenesis. In our study, we investigated 149 meningiomas to determine whether there are additional tumor suppressor genes localized on chromosome 22q, apart from NF2, that might be involved in meningioma pathogenesis. The LOH analysis on chromosome 22q identified two regions of deletion: the first one, which is limited to the NF2 gene locus, and the second one, which is outside this location. The new minimal deletion region (MDR) included the following genes: BCR (breakpoint cluster region), RAB36 (a member of RAS oncogene family), GNAZ [guanine nucleotide binding protein (G protein), alpha-z polypeptide], and RTDR1 (rhabdoid tumor deletion region gene 1). The expression levels of all these genes, including NF2, were subsequently analyzed by quantitative real-time polymerase chain reaction. We observed a significantly lowered expression level of NF2 in meningiomas with 22q loss of heterozygosity (LOH) within NF2 region compared to the one in meningiomas with 22q retention of heterozygosity (ROH, P<0.05). Similarly, BCR showed a significantly lowered expression in meningiomas with 22q LOH within the new MDR compared to cases with 22q ROH (P<0.05). Our data, together with the already published information considering BCR function suggest that BCR can be considered as a candidate tumor suppressor gene localized on chromosome 22q which may be involved in meningioma pathogenesis.

Elimination of wild-type P53 mRNA in glioblastomas showing heterozygous mutations of P53.

We screened 50 glioblastomas for P53 mutations. Five glioblastomas showed heterozygous mutations, while three were putatively heterozygous. Six of these eight glioblastomas showed elimination of wild-type P53 mRNA. These results strongly suggest that some sort of mechanism(s) favouring mutated over wild-type P53 mRNA exists in glioblastoma cells with heterozygous mutations of this gene.

The infrequent simultaneous genetic alterations in glioblastoma multiforme (LOH 10, 17, 19q, TP53 mutation and EGFR amplification) with short clinical course.

We described the case of an unusual, complex genetic alteration in 57 year-old male patient with glioblastoma multiforme (GBM) with short survival (6 and half months). Alterations consisted of p53 mutation, LOH 10, LOH 17, LOH 19q and EGFR amplification. LOH1p, LOH 9 and LOH 13 were negative. Immunohistochemical study did not correlate with molecular results. The overexpression of TP53 protein and RB protein was detected only in small percentage of cells and interestingly the overexpression of EGFR was present only focally. Immnunostainings for PTEN, P16, PI3-K were negative. Additionally, we observed an overexpression of IGFB2 protein. This case indicates the accumulation of molecular changes in glioblastoma multiforme in patient with short survival.

Disease-specific particles without prion protein in prion diseases - phenomenon or epiphenomenon?

The search for the cause of transmissible spongiform encephalopathies (TSEs) has a long and tortuous history. In a recent paper, 25-nm virus-like particles were identified that were consistently observed in cell cultures infected with Creutzfeldt-Jakob disease (CJD) and scrapie; they are similar to, or even identical with, the virus-like tubulovesicular structures (TVS) found in experimental scrapie as early as in 1968, and subsequently in all naturally occurring and experimentally induced TSEs. These particles have been viewed with caution by the scientific community because of the unverified or uninterpretable record of virus-like structures reported over the years in TSEs. TVS are spherical or tubular particles of approximate diameter 25-37 nm. They are smaller than synaptic vesicles, but larger than many particulate structures of the central nervous system, such as glycogen granules. Their electron density is higher compared with synaptic vesicles, and in experimental murine scrapie, they form paracrystalline arrays. None of these observations distinguish between TVS as an entity critical to the infectious process, or as a highly specific ultrastructural epiphenomenon, but their consistent presence in all TSEs demands further research.

Molecular analysis of chromosome 1, 10 and 19 abnormalities in human oligodendroglial tumors: relationship between frequency of LOH grade, age and gender.

BACKGROUND: Loss of heterozygosity (LOH) on 1p and 19q is observed in most oligodendroglial tumors. LOH on 10q appears to be less common in these tumors as compared to other gliomas. PATIENTS AND METHODS: We reviewed 14 patients with oligodendroglial tumors (10 low-grade and 4 anaplastic oligodendroglioma) to evaluate the frequency of LOH on 1p, 10q and 19q and correlate it with tumor grade and patients' age and gender; 5 loci on 1p and 5 on 19q as well as 4 on 10q were analyzed for LOH using PCR techniques. RESULTS: LOH on 1p together with 19q was detected in 6 tumors, 1 tumor showed deletion of 19q accompanied with deletion on 10q. Deletion on 1p was associated with deletion of 19q (p < 0.005) and mutual associations among deletions at loci on 19q (p < 0.05) were found. Patients with LOH on 1p were younger on average than patients with retained heterozygosity (p = 0.05). Grade II oligodendrogliomas predominated among younger patients (p < 0.01) while grade III oligodendrogliomas predominated among women (p < 0.005). No association between LOH on 1p nor 19q and tumor grade or patients' gender was found. CONCLUSION: Our study provides several clinically interesting findings and further supports the hypothesis of chromosome 1p and 19q involvement in the oligodendroglial cancerogenesis.

Ultrastructural pathology of prion diseases revisited: brain biopsy studies.

We report here a detailed ultrastructural comparison of brain biopsies from 13 cases of Creutzfeldt-Jakob disease (CJD) and from one case of fatal familial insomnia (FFI). The latter disease has not heretofore benefited from ultrastructural study. In particular, we searched for tubulovesicular structures (TVS), 35-nm particles regarded as the only disease-specific structures at the level of thin-section electron microscopy. Our material consisted of brain biopsies obtained by open surgery from one FFI case from a new French family, one case of variant CJD (vCJD), nine cases of sporadic CJD (sCJD), two cases of iatrogenic (human growth hormone) CJD and one case of hereditary CJD (Val203Iso). The ultrastructural picture of the cerebral cortex of the FFI patient was virtually indistinguishable from that of CJD. TVS were found, albeit only after prolonged search. Typical spongiform change was observed, consisting of intracellular membrane-bound vacuoles containing secondary chambers (vacuoles within vacuoles) and amorphous material. Neuronal degeneration was widespread: some processes contained degenerating mitochondria and lysosomal electron-dense bodies and these met the criteria for neuroaxonal dystrophy. Other processes contained branching cisterns; still others were filled with electron-dense masses and amorphous vesicles. The overall ultrastructural appearance of variant CJD was similar to that of FFI cerebral cortex, except for a much higher number of cellular processes containing TVS. We detected TVS in the majority of sCJD cases that, in addition to typical spongiform change and robust astrocytic reaction, showed widespread neuritic and synaptic degeneration and autophagic vacuoles. We conclude that TVS are readily found in FFI, vCJD and sCJD and that widespread neuritic degeneration is a part of ultrastructural pathology in prion diseases.

Molecular abnormalities in pediatric embryonal brain tumors--analysis of loss of heterozygosity on chromosomes 1, 5, 9, 10, 11, 16, 17 and 22.

Embryonal tumors, the most common group of malignant brain tumors in childhood, are heterogeneous and have been associated with a large number of genetic abnormalities. The aim of this study was to comprehensively analyze loss of heterozygosity (LOH) on regions harboring suppressor genes (PTCH2, PTCH1, APC, PTEN, DMBT1, SUFU, AXIN1, hSNF5/INI1) and to study chromosomal regions in which deletions have been described most frequently (1p, 1q, 11p, 16p, 17p). Twenty-nine children (17 male and 12 female), aged from 1 year 13 years were included in this study. There were 24 medulloblastomas (MB) and 5 supratentorial primitive neuroectodermal tumors (sPNET). Tissue samples from 29 primary and 11 recurrent tumors were analyzed according to the LOH standard procedures, which were extended to include fluorescence in situ hybridization for detection of isochromosome 17q (i(17q)) and direct sequencing ofTP53 exon 4. LOH on 17p was found in 15 out of 29 tumors. FISH analysis identified the presence of i(17q) in 16 tumors. Comparison of LOH analysis and the FISH data indicated that alterations of 17p were related to be the introduction of an i(17q) formation. LOH on 10q and 9q was observed in 4 and 2 cases, respectively, and was associated with alterations of chromosome 17. These results indicated a connection between alterations of PTCH/SHH genes and abnormalities of chromosome 17. A deleted region on 22q, covering the hSNF5/INI1 locus, was observed in 3 tumors. Progression of the molecular changes occurred in 1 case of recurrent medulloblastoma. LOH on 10q and 17p was found in both primary and recurrent tumor, while losses on 11p, 16p, and 16q occurred only in the recurrent tumor. No evidence of alteration in TP53 exon 4 was identified.

Polymorphisms within the prion (PrP) and prion-like protein (Doppel) genes in AD.

The authors present a study on the association of PRNP and PRND gene polymorphisms with the occurrence and age at onset of Alzheimer's disease (AD). DNA from 79 Polish patients with probable AD and 107 healthy control subjects was studied. The PRNP codon 129 homozygosity seemed to be associated with the occurrence of AD: In AD patients, the percentage of Val/Val and Met/Met genotypes was higher than in the control subjects. A significant difference appeared also between early-onset (<70 years) and late-onset (> or = 70 years) AD patients in the PRND genotypes.

Molecular heterogeneity of meningioma with INI1 mutation.

BACKGROUND: INI1 (hSNF5) mutations are linked to rhabdoid tumours, but mutations in meningiomas with hot spot mutations in position 377 have also been reported. AIMS: To analyse the INI1 gene in meningioma. METHODS: Exons 1, 4, 5, and 9 of the INI1 gene were analysed by the polymerase chain reaction and direct sequencing in 80 meningiomas. For all cases, western blotting of the INI1 protein was performed. RESULTS: Only one of the 80 samples showed a cytosine insertion in codon 376. This mutation changed the open reading frame in almost the whole exon 9 and resulted in a longer hSNF5 protein. Complex analysis of the above described tumour sample by western blotting, DNA sequencing, and loss of heterozygosity (LOH) analysis showed that this particular meningioma consisted of heterogeneic cellular components. One of these components had a mutated INI1 gene, whereas in the other component INI1 was intact. CONCLUSIONS: INI1 mutation is a rare event in the molecular pathology of meningiomas. It is possible for the INI1 gene to be mutated in only a proportion of meningioma cells.

Ultrastructural changes in the optic nerves of rodents with experimental Creutzfeldt-Jakob Disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS) or scrapie.

This report describes the ultrastructural changes in the optic nerves of (1) hamsters infected with the Echigo-1 strain of Creutzfeldt-Jakob disease (CJD), (2) hamsters infected with the 263K or 22C-H strain of scrapie, and (3) mice infected with the Fujisaki strain of Gerstmann-Sträussler-Scheinker disease (GSS). Vacuolation of myelinated fibres was present in the myelin sheaths, with splitting of myelin lamellae. These vacuoles contained typical secondary vacuoles and curled membrane fragments. Myelinated fibre vacuolation was also accompanied by an exuberant cellular reaction consisting of macrophages containing numerous mitochondria, abundant rough endoplasmic reticulum, and secondary lysosomes filled with digested myelin debris and other electron-dense material. Within macrophages, myelin fragments undergoing active digestion, lyre-like bodies and paracrystalline inclusions were frequently noted. Astrocytes and their processes were prominent; glial filaments and many mitochondria were readily detected. Proliferation of inner mesaxons was observed. Cross-sectional profiles of innumerable myelinated fibres contained membranous organelles continuous with the inner lamellae of the oligodendroglial cells. The proliferations of inner mesaxons formed whorls and loops, and intrusion of the membranous tongue of the inner mesaxon into the axoplasm was occasionally observed; dystrophic neurites were relatively numerous. In mice infected with the Fujisaki strain of GSS, fibres had undergone demyelination with stripping of the myelin lamellae, while others showed vesicular myelin degeneration.

Deposition patterns of disease-associated prion protein in captive mule deer brains with chronic wasting disease.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) in captive and free-ranging cervids in the USA; its origin is obscure. Archival formalin-fixed and paraffin-embedded specimens of 16 captive mule deer brains with CWD were analyzed using immunocytochemistry for the disease-associated prion protein (PrP). The most prominent pattern of PrP deposition were plaque-like structures, a substantial proportion of which were florid plaques surrounded by a rim of spongiform vacuoles. The percentage of florid plaques was highly variable according to region, ranging from 0% to 52.7%. The highest percentage was observed in the medulla and basal ganglia, the lowest in the cerebral cortex. Only three brains contained no florid plaques. There were also punctate synaptic-type and perivascular deposits, particularly in areas of severe spongiform change, and subpial and subependymal plaque-like deposits, whereas cerebellar involvement was mild. Thus, CWD brain pathology prominently features florid PrP plaques, as does variant Creutzfeldt-Jakob disease (vCJD), but differs in other characteristics from vCJD.

Codon 129 polymorphism of the PRNP gene in normal Polish population and in Creutzfeldt-Jakob disease, and the search for new mutations in PRNP gene.

Polymorphism at codon 129 of the prion protein gene (PRNP) is implicated both in susceptibility and phenotype of human prion diseases. We characterized the valine and methionine allele frequency at codon 129 in 109 individuals representing the normal Polish population and in 15 Polish CJD cases. The distribution of the genotype was 45% Met/Met, 39% Met/Val, and 16% Val/Val in the control group whereas, of the CJD cases, 73.3% were homozygous for methionine, 13.3% homozygous for valine and 13.3% were heterozygous. The novel missense mutation (ATG-->ACG) at codon 232 was identified in one of the samples with a GSS phenotype.