18 research outputs found
Prion type 2 selection in sporadic Creutzfeldt-Jakob disease affecting peripheral ganglia
In sporadic Creutzfeldt–Jakob disease (sCJD), the pathological changes appear to be restricted to the central nervous system. Only involvement of the trigeminal ganglion is widely accepted. The present study systematically examined the involvement of peripheral ganglia in sCJD utilizing the currently most sensitive technique for detecting prions in tissue morphologically. The trigeminal, nodose, stellate, and celiac ganglia, as well as ganglia of the cervical, thoracic and lumbar sympathetic trunk of 40 patients were analyzed with the paraffin-embedded tissue (PET)-blot method. Apart from the trigeminal ganglion, which contained protein aggregates in five of 19 prion type 1 patients, evidence of prion protein aggregation was only found in patients associated with type 2 prions. With the PET-blot, aggregates of prion protein type 2 were found in all trigeminal (17/17), in some nodose (5 of 7) and thoracic (3 of 6) ganglia, as well as in a few celiac (4 of 19) and lumbar (1 of 5) ganglia of sCJD patients. Whereas aggregates of both prion types may spread to dorsal root ganglia, more CNS-distant ganglia seem to be only involved in patients accumulating prion type 2. Whether the prion type association is due to selection by prion type-dependent replication, or due to a prion type-dependent property of axonal spread remains to be resolved in further studies
PrPSc spreading patterns in the brain of sheep linked to different prion types
Scrapie in sheep and goats has been known for more than 250 years and belongs nowadays to the so-called prion diseases that also include e.g. bovine spongiform encephalopathy in cattle (BSE) and Creutzfeldt-Jakob disease in humans. According to the prion hypothesis, the pathological isoform (PrPSc) of the cellular prion protein (PrPc) comprises the essential, if not exclusive, component of the transmissible agent. Currently, two types of scrapie disease are known - classical and atypical/Nor98 scrapie. In the present study we examine 24 cases of classical and 25 cases of atypical/Nor98 scrapie with the sensitive PET blot method and validate the results with conventional immunohistochemistry. The sequential detection of PrPSc aggregates in the CNS of classical scrapie sheep implies that after neuroinvasion a spread from spinal cord and obex to the cerebellum, diencephalon and frontal cortex via the rostral brainstem takes place. We categorize the spread of PrPSc into four stages: the CNS entry stage, the brainstem stage, the cruciate sulcus stage and finally the basal ganglia stage. Such a sequential development of PrPSc was not detectable upon analysis of the present atypical/Nor98 scrapie cases. PrPSc distribution in one case of atypical/Nor98 scrapie in a presumably early disease phase suggests that the spread of PrPSc aggregates starts in the di- or telencephalon. In addition to the spontaneous generation of PrPSc, an uptake of the infectious agent into the brain, that bypasses the brainstem and starts its accumulation in the thalamus, needs to be taken into consideration for atypical/Nor98 scrapie
Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Quantification of ARID1A in Tissue Lysates
ARID1A is a subunit of the mammalian SWI/SNF complex, which is thought to regulate
gene expression through restructuring chromatin structures. Its gene ARID1A is frequently mutated
and ARID1A levels are lowered in several human cancers, especially gynecologic ones. A functional
ARID1A loss may have prognostic or predictive value in terms of therapeutic strategies but has not
been proposed based on a quantitative method. Hardly any literature is available on ARID1A levels in
tumor samples. We developed an indirect enzyme-linked immunosorbent assay (ELISA) for ARID1A
based on the current EMA and FDA criteria. We demonstrated that our ELISA provides the objective,
accurate, and precise quantification of ARID1A concentrations in recombinant protein solutions, cell
culture standards, and tissue lysates of tumors. A standard curve analysis yielded a ‘goodness of
fit’ of R2 = 0.99. Standards measured on several plates and days achieved an inter-assay accuracy of
90.26% and an inter-assay precision with a coefficient of variation of 4.53%. When tumor lysates were
prepared and measured multiple times, our method had an inter-assay precision with a coefficient of
variation of 11.78%. We believe that our suggested method ensures a high reproducibility and can
be used for a high sample throughput to determine the ARID1A concentration in different tumor
entities. The application of our ELISA on various tumor and control tissues will allow us to explore
whether quantitative ARID1A measurements in tumor samples are of predictive value
Bisulfite profiling of the MGMT promoter and comparison with routine testing in glioblastoma diagnostics
Background: Promoter methylation of the DNA repair gene O6
-methylguanine-DNA methyltransferase (MGMT) is an
acknowledged predictive epigenetic marker in glioblastoma multiforme and anaplastic astrocytoma. Patients with
methylated CpGs in the MGMT promoter beneft from treatment with alkylating agents, such as temozolomide, and
show an improved overall survival and progression-free interval. A precise determination of MGMT promoter methyla‑
tion is of importance for diagnostic decisions. We experienced that diferent methods show partially divergent results
in a daily routine. For an integrated neuropathological diagnosis of malignant gliomas, we therefore currently apply a
combination of methylation-specifc PCR assays and pyrosequencing.
Results: To better rationalize the variation across assays, we compared these standard techniques and assays to deep
bisulfte sequencing results in a cohort of 80 malignant astrocytomas. Our deep analysis covers 49 CpG sites of the
expanded MGMT promoter, including exon 1, parts of intron 1 and a region upstream of the transcription start site
(TSS). We observed that deep sequencing data are in general in agreement with CpG-specifc pyrosequencing, while
the most widely used MSP assays published by Esteller et al. (N Engl J Med 343(19):1350–1354, 2000. https://doi.org/
10.1056/NEJM200011093431901) and Felsberg et al. (Clin Cancer Res 15(21):6683–6693, 2009. https://doi.org/10.1158/
1078-0432.CCR-08-2801) resulted in partially discordant results in 22 tumors (27.5%). Local deep bisulfte sequencing
(LDBS) revealed that CpGs located in exon 1 are suited best to discriminate methylated from unmethylated samples.
Based on LDBS data, we propose an optimized MSP primer pair with 83% and 85% concordance to pyrosequencing
and LDBS data. A hitherto neglected region upstream of the TSS, with an overall higher methylation compared to
exon 1 and intron 1 of MGMT, is also able to discriminate the methylation status.
Conclusion: Our integrated analysis allows to evaluate and redefne co-methylation domains within the MGMT pro‑
moter and to rationalize the practical impact on assays used in daily routine diagnostics
Presence and Seeding Activity of Pathological Prion Protein (PrPTSE) in Skeletal Muscles of White-Tailed Deer Infected with Chronic Wasting Disease
Chronic wasting disease (CWD) is a contagious, rapidly spreading transmissible spongiform encephalopathy (TSE), or prion disease, occurring in cervids such as white tailed-deer (WTD), mule deer or elk in North America. Despite efficient horizontal transmission of CWD among cervids natural transmission of the disease to other species has not yet been observed. Here, we report for the first time a direct biochemical demonstration of pathological prion protein PrPTSE and of PrPTSE-associated seeding activity, the static and dynamic biochemical markers for biological prion infectivity, respectively, in skeletal muscles of CWD-infected cervids, i. e. WTD for which no clinical signs of CWD had been recognized. The presence of PrPTSE was detected by Western- and postfixed frozen tissue blotting, while the seeding activity of PrPTSE was revealed by protein misfolding cyclic amplification (PMCA). Semi-quantitative Western blotting indicated that the concentration of PrPTSE in skeletal muscles of CWD-infected WTD was approximately 2000-10000 -fold lower than in brain tissue. Tissue-blot-analyses revealed that PrPTSE was located in muscle-associated nerve fascicles but not, in detectable amounts, in myocytes. The presence and seeding activity of PrPTSE in skeletal muscle from CWD-infected cervids suggests prevention of such tissue in the human diet as a precautionary measure for food safety, pending on further clarification of whether CWD may be transmissible to humans
Types and Strains: Their Essential Role in Understanding Protein Aggregation in Neurodegenerative Diseases
Morgagnian cataract resulting from a naturally occurring nonsense mutation elucidates a role of CPAMD8 in mammalian lens development
<div><p>To investigate the genetic basis of hereditary lens opacities we analyzed 31 cases of bilateral congenital cataract in Red Holstein Friesian cattle. A genome-wide association study revealed a significant association on bovine chromosome 7 at positions 6,166,179 and 12,429,691. Whole genome re-sequencing of one case and four relatives showed a nonsense mutation (g.5995966C>T) in the PZP-like, alpha-2-macroglobulin domain containing 8 (<i>CPAMD8</i>) gene leading to a premature stop codon (CPAMD8 p.Gln74*) associated with cataract development in cattle. With immunohistochemistry we confirmed a physiological expression of CPAMD8 in the ciliary body epithelium of the eye in unaffected cattle, while the protein was not detectable in the ciliary body of cattle with cataracts. RNA expression of <i>CPAMD8</i> was detected in healthy adult, fetal and cataractous lenses.</p></div
Tissue samples for RNA analyses of <i>CPAMD8</i>, including ddPCR results and RNA integrity number (RIN) from RNA quality measurement using RNA 6000 Pico Kit (Agilent Technologies, Waldbronn, Germany).
<p>Tissue samples for RNA analyses of <i>CPAMD8</i>, including ddPCR results and RNA integrity number (RIN) from RNA quality measurement using RNA 6000 Pico Kit (Agilent Technologies, Waldbronn, Germany).</p