Quantitative Detection and Biological Propagation of Scrapie Seeding Activity In Vitro Facilitate Use of Prions as Model Pathogens for Disinfection

Prions are pathogens with an unusually high tolerance to inactivation and constitute a complex challenge to the re-processing of surgical instruments. On the other hand, however, they provide an informative paradigm which has been exploited successfully for the development of novel broad-range disinfectants simultaneously active also against bacteria, viruses and fungi. Here we report on the development of a methodological platform that further facilitates the use of scrapie prions as model pathogens for disinfection. We used specifically adapted serial protein misfolding cyclic amplification (PMCA) for the quantitative detection, on steel wires providing model carriers for decontamination, of 263K scrapie seeding activity converting normal protease-sensitive into abnormal protease-resistant prion protein. Reference steel wires carrying defined amounts of scrapie infectivity were used for assay calibration, while scrapie-contaminated test steel wires were subjected to fifteen different procedures for disinfection that yielded scrapie titre reductions of ≤101- to ≥105.5-fold. As confirmed by titration in hamsters the residual scrapie infectivity on test wires could be reliably deduced for all examined disinfection procedures, from our quantitative seeding activity assay. Furthermore, we found that scrapie seeding activity present in 263K hamster brain homogenate or multiplied by PMCA of scrapie-contaminated steel wires both triggered accumulation of protease-resistant prion protein and was further propagated in a novel cell assay for 263K scrapie prions, i.e., cerebral glial cell cultures from hamsters. The findings from our PMCA- and glial cell culture assays revealed scrapie seeding activity as a biochemically and biologically replicative principle in vitro, with the former being quantitatively linked to prion infectivity detected on steel wires in vivo. When combined, our in vitro assays provide an alternative to titrations of biological scrapie infectivity in animals that substantially facilitates the use of prions as potentially highly indicative test agents in the search for novel broad-range disinfectants

An NF-κB and Slug Regulatory Loop Active in Early Vertebrate Mesoderm

BACKGROUND: In both Drosophila and the mouse, the zinc finger transcription factor Snail is required for mesoderm formation; its vertebrate paralog Slug (Snai2) appears to be required for neural crest formation in the chick and the clawed frog Xenopus laevis. Both Slug and Snail act to induce epithelial to mesenchymal transition (EMT) and to suppress apoptosis. METHODOLOGY & PRINCIPLE FINDINGS: Morpholino-based loss of function studies indicate that Slug is required for the normal expression of both mesodermal and neural crest markers in X. laevis. Both phenotypes are rescued by injection of RNA encoding the anti-apoptotic protein Bcl-xL; Bcl-xL's effects are dependent upon IκB kinase-mediated activation of the bipartite transcription factor NF-κB. NF-κB, in turn, directly up-regulates levels of Slug and Snail RNAs. Slug indirectly up-regulates levels of RNAs encoding the NF-κB subunit proteins RelA, Rel2, and Rel3, and directly down-regulates levels of the pro-apopotic Caspase-9 RNA. CONCLUSIONS/SIGNIFICANCE: These studies reveal a Slug/Snail–NF-κB regulatory circuit, analogous to that present in the early Drosophila embryo, active during mesodermal formation in Xenopus. This is a regulatory interaction of significance both in development and in the course of inflammatory and metastatic disease

Identification of the prion protein allotypes which accumulate in the brain of sporadic and familial Creutzfeldt-Jakob disease patients

A characteristic feature of Creutzfeldt-jakob disease (CJD) is the accumulation in the brain of the amyloid protease-resistant protein PrPres. PrPres derives from a host-encoded, protease-sensitive isoform, PrPsen. Mutations of this protein are linked to familial variants of the disease, and the presence of a methionine or valine residue at the polymorphic position 129 may be critical in sporadic CJD cases. We found that in the brain of patients heterozygous for the mutation in which isoleucine is substituted for valine at codon 210 (Val210Ile), the PrPres is formed by both the wild-type and mutant PrPsen. We also found that in a sporadic CJD patient, who was heterozygous (Met/Val) at position 129, PrPres is also formed by both allotypes. These data associate transmissible spongiform encephalopathies with other amyloidosis, although the nature of the transmissible agent remains unsettled

Clinical and Echocardiographic Correlates of Health Status in Patients with Acute Chest Pain

OBJECTIVE: To assess the ability of echocardiographic data to predict important functional status outcomes in patients with chest pain. DESIGN: Prospective cohort study. SETTING: A large, urban teaching hospital. PATIENTS: Three hundred thirty-three patients admitted from the Emergency Department for evaluation of chest pain. MEASUREMENTS AND MAIN RESULTS: Patients underwent two-dimensional and Doppler echocardiography as well as a face-to-face interview during their initial hospitalization and a telephone interview 1 year thereafter. The interview included the Medical Outcomes Study 36-Item Short Form (SF-36) health inventory, a generic health status instrument with a physical function subscale. The relation between clinical and echocardiographic factors and functional status was explored by univariable and multivariable linear regression and logistic regression analyses. Multiple clinical and echocardiographic factors correlated significantly with functional status measures at 1 year. For the SF-36 score at 1 year, age, male gender, white race, the presence of rales, and a comorbidity score were independently predictors in multivariate analysis; echocardiographic findings of severe left ventricular dysfunction (parameter estimate [PE] −27.6; 95% confidence interval [CI] −43.1, −12.2) and aortic insufficiency (PE −16.7; 95% CI −26.4, −7.0) added independent predictive information. Explanatory power (r(2)) for models using clinical and demographic variables was .27 and increased after inclusion of echocardiographic data to an r (2)of .35. Results in the subset of patients (n =148) with acute coronary syndromes such as unstable angina or myocardial infarction were qualitatively similar. Selected factors (rales on examination, electrocardiographic changes suggestive of ischemia, and moderate to severe mitral regurgitation) also predicted which patients would die or have a decline in their functional status. In multivariate analysis, only rales remained an independent predictor of poor outcome (odds ratio 2.4; 95% CI 1.2, 4.5). CONCLUSIONS: Echocardiographic data are correlated with measures of functional status in patients with chest pain, but the ability to predict future functional status from clinical or echocardiographic information is limited. Because functional status cannot be predicted adequately from either patients' characteristics or echocardiographic testing, it must be assessed directly

The spectrum of mitral regurgitation in idiopathic mitral valve prolapse: a color Doppler study.

To characterize the spectrum of mitral regurgitation in mitral valve prolapse, one hundred patients were studied by color Doppler flow mapping. The findings were correlated with the clinical presentation and with the possible complications. Mitral regurgitation was absent in 46 patients, mild in 26 patients, moderate in 18 patients and severe in 10 patients. The jet orientation was central in 15 patients, antero-medial in 13 patients and postero-lateral in 26 patients. The regurgitation was early systolic in 7 patients, late systolic in 20 patients and holosystolic in 27 patients. A good agreement was observed between the color flow patterns and the presence, timing and radiation of a murmur. Systolic clicks were not predictors of the presence or the severity of regurgitation. The grade of mitral regurgitation was positively correlated with, age, left heart enlargement and valvular redundancy. No sex difference was observed. The prevalence of serious arrhythmias or cerebral ischemic events was not significantly increased when a regurgitation was present.Journal ArticleSCOPUS: ar.jinfo:eu-repo/semantics/publishe