Effects of human PrPSc type and PRNP genotype in an in-vitro conversion assay

Prion protein type and codon 129 genotype are thought to be major determinants of susceptibility and phenotype in human prion diseases. Using an in-vitro system (protein misfolding cyclic amplification) we have attempted to model human prion protein conversion using the abnormal prion protein associated with each of the major sporadic Creutzfeldt–Jakob disease subtypes, in substrates containing the normal cellular form of the prion protein of each of the three possible human PRNP codon 129 polymorphic genotypes. The prion protein type is converted with fidelity in these amplification reactions, but the efficiency of conversion depends both on the methionine/valine polymorphic status of the sporadic Creutzfeldt–Jakob disease seed and substrate homogenate, and on the abnormal prion protein type

In vitro amplification and detection of variant Creutzfeldt-Jakob disease PrPSc

Variant Creutzfeldt–Jakob disease (vCJD) poses a serious risk of secondary transmission and the need to detect infectivity in asymptomatic individuals is therefore of major importance. Following infection, it is assumed that minute amounts of disease‐associated prion protein (PrPSc) replicate by conversion of the host cellular prion protein (PrPC). Therefore, methods of rapidly reproducing this conversion process in vitro would be valuable tools in the development of such tests. We show that one such technique, protein misfolding cyclic amplification (PMCA), can amplify vCJD PrPSc from human brain tissue, and that the degree of amplification is dependent upon the substrate PRNP codon 129 polymorphism. Both human platelets and transgenic mouse brain are shown to be suitable alternative substrate sources, and amplified PrPSc can be detected using a conformation‐dependent immunoassay (CDI), allowing the detection of putative proteinase K sensitive forms of PrPSc

Human platelets as a substrate source for the in vitro amplification of the abnormal prion protein (PrPSc) associated with variant Creutzfeldt-Jakob disease

BACKGROUND: Four recent cases of transfusion‐related transmission of variant Creutzfeldt‐Jakob disease (vCJD) highlight the need to develop a highly sensitive and specific screening test to detect infectivity in the blood of asymptomatic infected individuals. Protein misfolding cyclic amplification (PMCA), a method for the amplification of minute amounts of disease‐associated abnormal prion protein (PrPSc) to readily detectable levels, could be incorporated into such a test provided that a suitable substrate source for routine use in human PMCA reactions can be found. STUDY DESIGN AND METHODS: With the use of seed sources from individuals with variant and sporadic CJD, the use of human platelets (PLTs) as a PMCA substrate source was evaluated. The effects of seed/substrate prion protein gene (PRNP) codon 129 genotype compatibility on amplification efficiency and freeze‐thaw on a substrate's ability to support amplification and the degree of amplification achieved by serial PMCA (sPMCA) were investigated. RESULTS: Seed/substrate PRNP codon 129 compatibility was found to have a major influence on PrPSc amplification efficiency. Individual substrates, of the same PRNP codon 129 genotype, could be pooled and stored frozen for use in subsequent PMCA reactions. A consistent 10‐fold increase in PrPSc detection sensitivity was achieved after each round of sPMCA, resulting in a 10,000‐fold increase in detection sensitivity after four rounds, with no evidence of de novo PrPSc production detected in the unseeded PLT substrate. CONCLUSIONS: Providing issues of seed/substrate PRNP codon 129 compatibility are taken into consideration human PLTs are a suitable, readily available, renewable substrate source for use in human PMCA applications

Diagnostik und Therapie der Endometriose

Die Endometriose ist eine häufige aber wenig bekannte Erkrankung, unter der schätzungsweise 1 von 10 Frauen in ihren fruchtbaren Jahren leidet. Es handelt sich um eine chronische Erkrankung, die durch das Auftreten von Endometriumgewebe ausserhalb des Cavum Uteri gekennzeichnet ist und häufig bereits in der Adoleszenz beginnt. Typischerweise betroffen sind Ovar, Peritoneum, Douglas-Raum, Blase und Rektum. Verschiedene pathogenetische Mechanismen werden diskutiert, die genaue Ursache ist jedoch nach wie vor unbekannt. Grundsätzlich handelt es sich um eine benigne, aber lokal infiltrierende Erkrankung

Targeted therapy in renal cancer

Renal cell cancer (RCC) has an increasing incidence internationally and is a disease for which there have been limited therapeutic options until recently. The last decade has seen a vastly improved understanding of the biological and clinical factors that predict the outcome of this disease. We now understand some of the different molecular underpinnings of renal clear cell carcinoma by mutation or silencing of the von Hippel Lindau (VHL) gene and subsequent deregulated proliferation and angiogenesis. Survival in advanced disease is predicted by factors (performance status, anemia, hypercalcemia, and serum lactate dehydrogenase, time from diagnosis to recurrence) incorporated into the Memorial Sloan Kettering Cancer Center (MSKCC) criteria (also referred to as ‘Motzer’ criteria). These criteria allow classification of patients with RCC into good, intermediate and poor risk categories with median overall survivals of 22 months, 12 months and 5.4 months, respectively. Predicated upon these advances, six new targeted drugs (sorafenib, sunitinib, temsirolimus, everolimus, bevacizumab and pazopanib) have been tested in well-designed phase III trials, selected or stratified for MSKCC risk criteria, with positive results. All of these new drugs act at least in part through vascular endothelial growth factor (VEGF) mediated pathways with other potential therapeutic impact on platelet-derived growth factor (PDGF), raf kinase and mammalian target of rapamycin (mTOR) pathways. Importantly, data from each of these trials show a consistent doubling of progression-free survival (PFS) over prior standard of care treatments. In addition, sorafenib, sunitinib and temsirolimus, have demonstrated significant overall survival (OS) benefits as well; further follow-up is required to determine whether the disease control exhibited by everolimus and pazopanib will translate into a survival advantage. These drugs are generally well tolerated, as demonstrated by quality-of-life improvement in clinical trials, and result in clinical benefit for in excess of 70% of patients treated. They have challenged the traditional outcomes of clinical trial design by achieving their benefits with relatively few radiographic responses, but high rates of disease stability. The unique side-effect profile coupled with the chronicity of therapy requires increased vigilance to maximize exposure to the drugs while maintaining quality of life and minimizing toxicity. This review focuses on the background, clinical development and practical use of these new drugs in RCC