Factors intrinsic and extrinsic to blood hamper the development of a routine blood test for human prion diseases.

Development of numerous advanced techniques in recent years have allowed detection of the pathological prion protein (PrP(TSE)), the unique marker of transmissible spongiform encephalopathies (TSEs, or prion diseases), in the blood of animals and humans; however, an ante mortem screening test that can be used for the routine diagnosis of human prion diseases remains unavailable. A critical, analytical review of all the diagnostic assays developed to date will allow an evaluation of progress in this field and may facilitate the identification of the possible reasons for this delay. Thus, in this review, I provide a detailed overview of the techniques currently available for detecting PrP(TSE) and other markers of the disease in blood, as well as an analysis of the significance, feasibility, reliability and application spectrum for these methods. I highlight that factors intrinsic and extrinsic to blood may interfere with the detection of PrP(TSE)/prions, and that this is not yet taken into account in current tests. This may inspire researchers in this field to not only aspire to increase test sensitivity, but also to adopt other strategies in order to identify and overcome the limitations that hamper the development of a successful routine blood test for prion diseases.

Detection of exosomal prions in blood by immunochemistry techniques.

In most forms of prion diseases, blood is infectious, but detection by immunochemistry techniques of the only available marker of infection (the misfolded prion protein, PrPTSE) in blood remains elusive. We developed a novel method for the detection of PrPTSE in blood of prion-infected rodents based on the finding that PrPTSE is associated with plasma exosomes. However, further purification of the exosomes on a sucrose gradient was necessary to remove plasma immunoglobulins, which interfere with PrPTSE, masking its detection by immunochemistry. Finally, we report that about 20% of plasma infectivity is associated with exosomes.

Detection of water-soluble disease-associated PrP species in blood and brain of scrapie-infected hamster.

The high-speed supernatant (S(HS)) of scrapie-infected hamster brain homogenate contains a soluble infectivity similar to that of the plasma that escapes leukodepletion and can transmit prion infection. This recent finding highlights the fact that soluble prion infectivity could be relevant for prion disease propagation and progression. PrP(Sc) is essential in prion disease pathogenesis, but little to nothing is known about the PrP(Sc) species that may be associated with this form of prion infectivity. Scrapie-infected hamster plasma and S(HS) were subjected to biochemical analysis, and the results demonstrate for the first time that soluble infectivity is associated with a water-soluble PrP(Sc) species with substantially different properties from classical PrP(Sc), the concentration of which seems to correlate with the magnitude and efficiency of the soluble infectivity. Such characteristics suggest that this species might represent the soluble prion agent itself or its vehicle, highlighting the need to adequately revise the strategies involved in prion removal, diagnosis, and therapy.

Assessment of prion reduction filters in decreasing infectivity of ultracentrifuged 263K scrapie-infected brain homogenates in "spiked" human blood and red blood cells.

BACKGROUND: The safety of red blood cells (RBCs) is of concern because of the occurrence of four transfusion-transmitted variant Creutzfeldt-Jakob disease (vCJD) cases in the United Kingdom. The absence of validated screening tests requires the use of procedures to remove prions from blood to minimize the risk of transmission. These procedures must be validated using infectious prions in a form that is as close as possible to one in blood. STUDY DESIGN AND METHODS: Units of human whole blood (WB) and RBCs were spiked with high-speed supernatants of 263K scrapie-infected hamster brain homogenates. Spiked samples were leukoreduced and then passed through prion-removing filters (Pall Corporation). In another experiment, RBCs from 263K scrapie-infected hamsters were treated as above, and residual infectivity was measured by bioassay. RESULTS: The overall removal of infectivity by the filters from prion-spiked WB and RBCs was approximately two orders of magnitude. No infectivity was detected in filtered hamster RBCs endogenously infected with scrapie. CONCLUSION: The use of prion-removing filters may help to reduce the risk of transfusion-transmitted vCJD. To avoid overestimation of prion removal efficiency in validation studies, it may be more appropriate to use supernates from ultracentrifugation of scrapie-infected hamster brain homogenate rather than the current standard brain homogenates.

Feasibility of Using a Type I IFN-Based Non-Animal Approach to Predict Vaccine Efficacy and Safety Profiles.

Animal-based tests are used for the control of vaccine quality. However, because highly purified and safe vaccines are now available, alternative approaches that can replace or reduce animal use for the assessment of vaccine outcomes must be established. In vitro tests for vaccine quality control exist and have already been implemented. However, these tests are specifically designed for some next-generation vaccines, and this makes them not readily available for testing other vaccines. Therefore, universal non-animal tests are still needed. Specific signatures of the innate immune response could represent a promising approach to predict the outcome of vaccines by non-animal methods. Type I interferons (IFNs) have multiple immunomodulatory activities, which are exerted through effectors called interferon stimulated genes (ISGs), and are one of the most important immune signatures that might provide potential candidate molecular biomarkers for this purpose. This paper will mainly examine if this idea might be feasible by analyzing all relevant published studies that have provided type I IFN-related biomarkers for evaluating the safety and efficacy profiles of vaccines using an advanced transcriptomic approach as an alternative to the animal methods. Results revealed that such an approach could potentially provide biomarkers predictive of vaccine outcomes after addressing some limitations.

Comparison of nanofiltration efficacy in reducing infectivity of centrifuged versus ultracentrifuged 263K scrapie-infected brain homogenates in "spiked" albumin solutions.

BACKGROUND: The safety of plasma-derived products is of concern for possible transmission of variant Creutzfeldt-Jakob disease. The absence of validated screening tests requires the use of procedures to remove or inactivate prions during the manufacture of plasma-derived products to minimize the risk of transmission. These procedures need proper validation studies based on spiking human plasma or intermediate fractions of plasma fractionation with prions in a form as close as possible to that present in blood. STUDY DESIGN AND METHODS: Human albumin was spiked with low-speed or high-speed supernatants of 263K scrapie-infected hamster brain homogenates. Spiked albumin was then passed through a cascade of filters from 100 nm down to 20 to 15 nm. Residual infectivity was measured by bioassay. RESULTS: The overall removal of infectivity spiked into albumin through serial nanofiltration steps was 4 to 5 logs using low-speed supernatant and 2 to 3 logs with high-speed supernatant. CONCLUSION: These findings confirm the utility of nanofiltration in removing infectivity from plasma (or other products) spiked with scrapie brain homogenate supernatants. However, efficiency is diminished using supernatants that have been ultracentrifuged to reduce aggregated forms of the infectious agent. Thus, filtration removal data based on experiments using "standard" low-speed centrifugation supernatants might overestimate the amount of prion removal in plasma or urine-derived therapeutic products.

The Potential of Liquid Biopsy of the Brain Using Blood Extracellular Vesicles: The First Step Toward Effective Neuroprotection Against Neurodegenerative Diseases.

Early diagnosis and biomarker-based ante-mortem tests are essential in efforts against the development of neurodegenerative diseases and can be considered primary neuroprotective measures. Blood is the ideal biofluid for a routine ante-mortem screening test. However, biomarker discovery in the blood is particularly difficult because of interference from factors both intrinsic and extrinsic to blood with the detection of hallmark neurodegenerative biomarkers, such as the pathological prion protein, amyloid-ß, and others. Blood extracellular vesicles (EVs), such as exosomes, are cell-derived vesicles released into the blood from all parts of the body (including the brain and spinal cord). They are an enriched source of neural-derived EVs containing neurodegenerative biomarkers that mirror (in the blood) the condition present in the brain. The feasibility of using, and the reliability of, neural-derived blood EVs (NDBEVs) as a method of diagnosing Alzheimer disease and other neurodegenerative diseases has been assessed in strong proof-of-concept studies. Results from these studies strongly suggest that NDBEVs might represent the right strategy for specific, reliable, and early diagnosis of neurodegenerative diseases. Based on these results, NDBEVs might enable the creation of an ante-mortem blood test (liquid biopsy of the brain) for neurodegenerative diseases. This would enormously accelerate the therapy of neurodegenerative diseases. This review highlights the powerful potential of liquid biopsy of the brain using NDBEVs for early diagnosis and treatment of neurodegenerative diseases, and the challenges and limitations related to the identification of clinically applicable EV (exosomal) biomarkers using blood are discussed.

PrPTSE in muscle-associated lymphatic tissue during the preclinical stage of mice infected orally with bovine spongiform encephalopathy.

The involvement of muscles in the pathogenesis of transmissible spongiform encephalopathies (TSEs) is irregular and unpredictable. We show that the TSE-specific protein (PrP(TSE)) is present in muscles of mice fed with a mouse-adapted strain of bovine spongiform encephalopathy as early as 100 days post-infection, corresponding to about one-third of the incubation period. The proportion of mice with PrP(TSE)-positive muscles and the number of muscles involved increased as infection progressed, but never attained more than a limited distribution, even at the clinical stage of disease. The appearance of PrP(TSE) in muscles during the preclinical stage of disease was probably due to the haematogenous/lymphatic spread of infectivity from the gastrointestinal tract to lymphatic tissues associated with muscles, whereas in symptomatic animals, the presence of PrP(TSE) in the nervous system, in neuromuscular junctions and in muscle fibres suggests a centrifugal spread from the central nervous system, as already observed in other TSE models.

Efficacy of phthalocyanine tetrasulfonate against mouse-adapted human prion strains.

In vitro and in vivo studies have shown that phthalocyanine tetrasulfonate (PcTS), a cyclic tetrapyrrole compound, is an efficient antiscrapie drug. To investigate the spectrum of PcTS against prion diseases, we tested the effect of PcTS on two mouse-adapted human strains. We also tested PcTS in rodents infected with two scrapie strains (139A and 263K). PcTS treatment significantly prolonged mean survival times of all infected animals. These results show that PcTS is effective on different prion strains, confirming its potential use for prion therapy.

Analysis of the Glycosylation Profile of Disease-Associated Water-Soluble Prion Protein Using Lectins.

The disease-associated water-soluble form of hamster prion protein (ws-PrPSc) has recently been found to be less stable than classical PrPSc. Since the stability of PrP to degradation correlates with its glycosylation level, the aim of this study was to investigate whether there are differences between the glycosylation of ws-PrPSc and classical PrPSc of hamster which might account for the ws-PrPSc minor stability compared with that of the classical PrPSc. Thus, ws-PrP and classical PrP were captured from noninfected or scrapie-infected hamster brain homogenate [high-speed supernatant (SHS) and high-speed pellet (PHS)] and blood plasma by anti-PrP antibodies (3F4 and 6H4) and subjected to screening for glycans by lectins under denaturing or nondenaturing procedures in a sandwich lectin-ELISA. Glycans have been found in minor quantities and differently exposed on ws-PrPSc from SHS and plasma compared with classical PrPSc from PHS. These differences have been shown to be potentially responsible for the instability of ws-PrPSc. Treatment of infected blood with GdnHCl significantly (P<0.01) increased the detection of ws-PrPSc in ELISA, reflecting an increase in its stability, and showed efficacy in removing high-abundance proteins in silver-stained gels. This increase in ws-PrPSc stability is due to an interaction of GdnHCl not only with high-abundance proteins but also with the ws-PrPSc glycosylation with particular regard to the mannose sugar. Analysis of lectins immunoreactivity toward total proteins from plasma collected before and at different time points after infection revealed that mannose might exert a stabilizing effect toward all of hamster blood glycoproteins, regardless of scrapie infection. Since low levels of ws-PrPSc/soluble-infectivity have been estimated both in blood and brain of hamster, this glycosylation-related instability may have negatively influenced the propensity of ws-PrPC to convert to ws-PrPSc both in blood and the brain. Therefore, PrPC glycosylation characteristics may provide a tool for the determination risk of prion transmissibility.

Neurodegenerative Disorders Treatment: The MicroRNA Role.

Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease and prion disease are not timely and effectively treated using conventional therapies. This emphasizes the need for alternative therapeutic approaches. In this respect, gene-based therapies have been adopted as potentially feasible alternative therapies, where the microRNA (miRNA) approach has experienced a great explosion in recent years. Because miRNAs have been shown to be implicated in the pathogenesis of several diseases including neurodegenerative diseases, they are intensely studied as candidates for diagnostic and prognostic biomarkers, as predictors of drug response and as therapeutic agents. In this review, we evaluate the feasibility of both direct and indirect miRNA mimics and inhibitors toward the regulation of neurodegenerative-related genes both in vivo and in vitro models, highlight the advantages and drawbacks associated with miRNA-based therapy, and summarize the relevant techniques and approaches attempted to deliver miRNAs to the central nervous system for therapeutic purposes, with particular regard to the exosomes. Additionally, we describe a new approach that holds great promise for the treatment of a wide range of diseases including neurodegenerative disorders. This approach is based on addressing the incorporation of miRNAs into exosomes to increase the quantity and quality of miRNA packed and delivered to the central nervous system and other sites of action.

Capillary electrophoresis as a tool for the characterization of pentosan nanoparticles.

Because capillary zone electrophoresis (CZE) showed higher resolution for highly charged large carbohydrates and complex structures when compared to other chromatographic separation methods, it was chosen for the characterization of nanoparticles (NPs) of pentosan polysulfate (PPS). Thus, using the CZE technique, we developed a reliable, sensitive and rapid protocol that allowed the detection and characterization of PPS NPs. This protocol was able to determine the profile of both the NPs and the species of PPS entrapped into them, and to quantify free and bound PPS showing high reproducibility, acceptable accuracy and a good degree of precision. Moreover, it allowed the evaluation of the size and charge of the NPs. This protocol might be suitable for the characterization of other kinds of NPs also.

The pathological prion protein forms ionic conductance in lipid bilayer.

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative pathologies characterized by the accumulation of amyloid fibrils mainly composed of the pathological isoform of the prion protein (PrP(TSE)). PrP(TSE) pre-amyloid fibrils are supposed to induce neurodegenerative lesions possibly through the alteration of membrane permeability. The effect of PrP(TSE) on cellular membranes has been modeled in vitro by synthetic peptides that are, however, only partially representative of PrP(TSE) isoforms found in vivo. In the present work we show that a synthetic membrane exposed to PrP27-30 extracted from TSE-infected hamster brains changes its permeability because of the formation of molecular pores that alter the conductance of the synthetic lipid bilayer. Synthetic membrane challenged with the recombinant prion peptide PrP90-231 shows a much lower conductance. Elevation of calcium ion concentration not only increases the current amplitude due to the action of both PrP27-30 and PrP90-231 on the membrane, but also amplifies the interaction of PrP90-231 with the lipid bilayer.