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Background: A highly purified 10% liquid intravenous immunoglobulin, IQYMUNE ® , has been developed using an innovative manufacturing process including an affinity chromatography step for the removal of anti-A and anti-B hemagglutinins., Objectives: The pathogen (viruses and prions) clearance efficacy of the manufacturing process and its robustness for critical steps were investigated., Methods: The manufacturing process of IQYMUNE ® includes two dedicated complementary virus reduction steps: solvent/detergent (S/D) treatment and 20 nm nanofiltration as well as two contributing steps, namely caprylic acid fractionation and anion-exchange chromatography. The clearance capacity and robustness of these steps were evaluated with a wide range of viruses (enveloped and non-enveloped) and with a model of human transmissible spongiform encephalopathies (TSEs)., Results: The IQYMUNE ® manufacturing process demonstrated a high and robust virus removal capacity with global reduction factors (RFs) of relevant and model viruses: ≥14.8 log 10 for human immunodeficiency virus type 1 (HIV-1), ≥16.9 log 10 for bovine viral diarrhoea virus (BVDV)/Sindbis virus, ≥15.7 log 10 for pseudorabies virus (PRV), ≥12.8 log 10 for encephalomyocarditis virus (EMCV) and 11.0 log 10 for porcine parvovirus (PPV). The process also exhibited a high removal capacity for the TSE agent with an overall RF of ≥12.9 log 10 due to the complementary actions of the caprylic acid fractionation, anion-exchange chromatography and nanofiltration steps., Conclusion: Data from virus and prion clearance studies fully support the high safety profile of IQYMUNE ® , with a minimal reduction of 11 log 10 for the smallest and most resistant non-enveloped virus, PPV, and more than 12 log 10 for the TSE agent.

One intriguing feature of prion diseases is their strain variation. Prion strains are differentiated by the clinical consequences they generate in the host, their biochemical properties, and their potential to infect other animal species. The selective targeting of these agents to specific brain structures have been extensively used to characterize prion strains. However, the molecular basis dictating strain-specific neurotropism are still elusive. In this study, isolated brain structures from animals infected with four hamster prion strains (HY, DY, 139H, and SSLOW) were analyzed for their content of protease-resistant PrP(Sc) Our data show that these strains have different profiles of PrP deposition along the brain. These patterns of accumulation, which were independent of regional PrP(C) production, were not reproduced by in vitro replication when different brain regions were used as substrate for the misfolding-amplification reaction. On the contrary, our results show that in vitro replication efficiency depended exclusively on the amount of PrP(C) present in each part of the brain. Our results suggest that the variable regional distribution of PrP(Sc) in distinct strains is not determined by differences on prion formation, but on other factors or cellular pathways. Our findings may contribute to understand the molecular mechanisms of prion pathogenesis and strain diversity., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Misfolding and aggregation of proteins are common pathogenic mechanisms of a group of diseases called proteinopathies. The formation and spread of proteinaceous lesions within and between individuals were first described in prion diseases and proposed as the basis of their infectious nature. Recently, a similar "prion-like" mechanism of transmission has been proposed in other neurodegenerative diseases such as Alzheimer's disease. We investigated if misfolding and aggregation of corrupted prion protein (PrP(TSE)) are always associated with horizontal transmission of disease. Knock-in transgenic mice (101LL) expressing mutant PrP (PrP-101L) that are susceptible to disease but do not develop any spontaneous neurological phenotype were inoculated with (i) brain extracts containing PrP(TSE) from healthy 101LL mice with PrP plaques in the corpus callosum or (ii) brain extracts from mice overexpressing PrP-101L with neurological disease, severe spongiform encephalopathy, and formation of proteinase K-resistant PrP(TSE). In all instances, 101LL mice developed PrP plaques in the area of inoculation and vicinity in the absence of clinical disease or spongiform degeneration of the brain. Importantly, 101LL mice did not transmit disease on serial passage, ruling out the presence of subclinical infection. Thus, in both experimental models the formation of PrP(TSE) is not infectious. These results have implications for the interpretation of tests based on the detection of protein aggregates and suggest that de novo formation of PrP(TSE) in the host does not always result in a transmissible prion disease. In addition, these results question the validity of assuming that all diseases due to protein misfolding can be transmitted between individuals.

The agents responsible for transmissible spongiform encephalopathies (TSEs), or prion diseases, contain as a major component PrP(Sc), an abnormal conformer of the host glycoprotein PrP(C). TSE agents are distinguished by differences in phenotypic properties in the host, which nevertheless can contain PrP(Sc) with the same amino-acid sequence. If PrP alone carries information defining strain properties, these must be encoded by post-translational events. Here we investigated whether the glycosylation status of host PrP affects TSE strain characteristics. We inoculated wild-type mice with three TSE strains passaged through transgenic mice with PrP devoid of glycans at the first, second or both N-glycosylation sites. We compared the infectious properties of the emerging isolates with TSE strains passaged in wild-type mice by in vivo strain typing and by the standard scrapie cell assay in vitro. Strain-specific characteristics of the 79A TSE strain changed when PrP(Sc) was devoid of one or both glycans. Thus infectious properties of a TSE strain can be altered by post-translational changes to PrP which we propose result in the selection of mutant TSE strains.

The dynamics of the circulation and distribution of transmissible spongiform encephalopathy (TSE) agents in the blood of infected individuals remain largely unknown. This clearly limits the understanding of the role of blood in TSE pathogenesis and the development of a reliable TSE blood detection assay. Using two distinct sheep scrapie models and blood transfusion, this work demonstrates the occurrence of a very early and persistent prionemia. This ability to transmit disease by blood transfusion was correlated with the presence of infectivity in white blood cells (WBC) and peripheral blood mononucleated cells (PBMC) as detected by bioassay in mice overexpressing the ovine prion protein PrP (tg338 mice) and with the identification of abnormal PrP in WBC after using protein misfolding cyclic amplification (PMCA). Platelets and a large variety of leukocyte subpopulations also were shown to be infectious. The use of endpoint titration in tg338 mice indicated that the infectivity in WBC (per ml of blood) was 10(6.5)-fold lower than that in 1 g of posterior brainstem sample. In both WBC and brainstem, infectivity displayed similar resistance to PK digestion. The data strongly support the concept that WBC are an accurate target for reliable TSE detection by PMCA. The presence of infectivity in short-life-span blood cellular elements raises the question of the origin of prionemia.

Prion diseases are fatal, transmissible neurodegenerative diseases of the central nervous system. An abnormally protease-resistant and insoluble form (PrP(Sc)) of the normally soluble protease-sensitive host prion protein (PrP(C)) is the major component of the infectious prion. During the course of prion disease, PrP(Sc) accumulates primarily in the lymphoreticular and central nervous systems. Recent studies have shown that co-infection of prion-infected fibroblast cells with the Moloney murine leukemia virus (Mo-MuLV) strongly enhanced the release and spread of scrapie infectivity in cell culture, suggesting that retroviral coinfection might significantly influence prion spread and disease incubation times in vivo. We now show that another retrovirus, the murine leukemia virus Friend (F-MuLV), also enhanced the release and spread of scrapie infectivity in cell culture. However, peripheral co-infection of mice with both Friend virus and the mouse scrapie strain 22L did not alter scrapie disease incubation times, the levels of PrP(Sc) in the brain or spleen, or the distribution of pathological lesions in the brain. Thus, retroviral co-infection does not necessarily alter prion disease pathogenesis in vivo, most likely because of different cell-specific sites of replication for scrapie and F-MuLV.

Prion diseases or transmissible spongiform encephalopathies are a group of fatal and transmissible disorders affecting the central nervous system of humans and animals. The principal agent of prion disease transmission and pathogenesis is proposed to be an abnormal protease-resistant isoform of the normal cellular prion protein. The microtubule-associated protein tau is elevated in patients with Creutzfeldt-Jakob disease. To determine whether tau expression contributes to prion disease pathogenesis, tau knockout and control wild-type mice were infected with the M1000 strain of mouse-adapted human prions. Immunohistochemical analysis for total tau expression in prion-infected wild-type mice indicated tau aggregation in the cytoplasm of a subpopulation of neurons in regions associated with spongiform change. Western immunoblot analysis of brain homogenates revealed a decrease in total tau immunoreactivity and epitope-specific changes in tau phosphorylation. No significant difference in incubation period or other disease features were observed between tau knockout and wild-type mice with clinical prion disease. These results demonstrate that, in this model of prion disease, tau does not contribute to the pathogenesis of prion disease and that changes in the tau protein profile observed in mice with clinical prion disease occurs as a consequence of the prion-induced pathogenesis.

Soluble receptor-resistant mutant 7 (ssr7) is isolated from a highly neurovirulent mouse hepatitis virus (MHV) JHMV cl-2 strain (cl-2). srr7 exhibits lower virulence than its maternal strain in infected mice, which is typically manifested in a longer lifespan. In this study, during the course of infection with srr7, small spongiotic lesions became apparent at 2 days post-inoculation (pi), they spread out to form spongiform encephalopathy by 8 to 10 days pi. We recently reported that the initial expressions of viral antigens in the brain are detected in the infiltrating monocyte lineage and in ependymal cells. Here, we demonstrate that the next viral spread was observed in glial fibrillary acidic protein-positive cells or nestin-positive progenitor cells which take up positions in the subventricular zone (SVZ). From this restricted site of infection in the SVZ, a large area of gliosis extended deep into the brain parenchyma where no viral antigens were detected but vacuolar degeneration started at 48 h pi of the virus. The extremely short incubation period compared with other experimental models of infectious spongiform degeneration in the brain would provide a superior experimental model to investigate the mechanism of spongiotic lesions formation., (© 2011 The Authors. Pathology International © 2011 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.)

The role of inflammation in the progression of neurodegenerative disease remains unclear. We have shown that systemic bacterial insults accelerate disease progression in animals and in patients with Alzheimer's disease. Disease exacerbation is associated with exaggerated CNS inflammatory responses to systemic inflammation mediated by microglia that become 'primed' by the underlying neurodegeneration. The impact of systemic viral insults on existing neurodegenerative disease has not been investigated. Polyinosinic:polycytidylic acid (poly I:C) is a toll-like receptor-3 (TLR3) agonist and induces type I interferons, thus mimicking inflammatory responses to systemic viral infection. In the current study we hypothesized that systemic challenge with poly I:C, during chronic neurodegenerative disease, would amplify CNS inflammation and exacerbate disease. Using the ME7 model of prion disease and systemic challenge with poly I:C (12 mg/kg i.p.) we have shown an amplified expression of IFN-alpha and beta and of the pro-inflammatory genes IL-1beta and IL-6. Similarly amplified expression of specific IFN-dependent genes confirmed that type I IFNs were secreted and active in the brain and this appeared to have anti-inflammatory consequences. However, prion-diseased animals were susceptible to heightened acute sickness behaviour and acute neurological impairments in response to poly I:C and this treatment also accelerated disease progression in diseased animals without effect in normal animals. Increased apoptosis coupled with double-stranded RNA-dependent protein kinase (PKR) and Fas transcription suggested activation of interferon-dependent, pro-apoptotic pathways in the brain of ME7+poly I:C animals. That systemic poly I:C accelerates neurodegeneration has implications for the control of systemic viral infection during chronic neurodegeneration and indicates that type I interferon responses in the brain merit further study., (Copyright 2010 Elsevier Inc. All rights reserved.)

Scrapie, a transmissible spongiform encephalopathy of sheep and goats, exists in most small ruminant-producing countries of the world. A novel form of this disease was recently recognized and is known by various names, including Nor98, Nor98-like, and atypical scrapie. Differing from classic scrapie in epidemiology, histopathology, and biochemical characteristics, atypical scrapie cases have been identified throughout Europe and in the United States. Enhanced scrapie surveillance efforts recently identified 3 cases of atypical scrapie in Canada.

The variant Creutzfeldt-Jakob disease (vCJD) is a transmissible spongiform encephalopathy (TSE), mainly present in the UK and is associated with the ingestion of bovine products affected with bovine spongiform encephalopathy. Manufacturers of biological products must investigate the ability of their production processes to remove TSE agents. We studied the purification steps in the manufacturing process of two FVIII/VWF concentrates (Alphanate) and Fanhdi in their ability to eliminate an experimental TSE-model agent. Hamster scrapie strain 263K brain-derived materials were spiked into samples of the solutions taken before various stages during its production: 3.5% polyethylene glycol (PEG) precipitation, heparin affinity chromatography and saline precipitation/final filtrations. PEG precipitation and affinity chromatography were studied both as isolated and combined steps. TSE agent removal was determined using a laboratory scale model representative of the industrial manufacturing process. The prion protein (PrP(Sc)) was measured with Western blot and TSE infectivity was measured with bioassay. Western blot results were in agreement with those obtained by bioassay, showing a significant removal capacity in the production process: 3.21-3.43 log(10) for the PEG precipitation; about 3.45 log(10) for the affinity chromatography; and around 2.0 log(10) for the saline precipitation plus final filtrations. PEG precipitation and heparin affinity chromatography were demonstrated to be two complementary TSE-model agent removal mechanisms with total removal being the sum of the two. An overall reduction factor of around 8 log(10) can be deduced. The tests from the production process of FVIII/VWF complex concentrates have demonstrated their potential for eliminating TSE agents.

Transmissible spongiform encephalopathies (TSEs) are a group of diseases that result in progressive and invariably fatal neurologic disease in both animals and humans. TSEs are characterized by the accumulation of an abnormal protease-resistant form of the prion protein in the central nervous system. Transmission of infectious TSEs is believed to occur via ingestion of prion protein-contaminated material. This material is also involved in the transmission of bovine spongiform encephalopathy ("mad cow disease") to humans, which resulted in the variant form of Creutzfeldt-Jakob disease. Abnormal prion protein has been reported in the retina of TSE-affected cattle, but despite these observations, the specific effect of abnormal prion protein on retinal morphology and function has not been assessed. The objective of this study was to identify and characterize potential functional and morphologic abnormalities in the retinas of cattle infected with a bovine-adapted isolate of transmissible mink encephalopathy. We used electroretinography and immunohistochemistry to examine retinas from 10 noninoculated and 5 transmissible mink encephalopathy-inoculated adult Holstein steers. Here we show altered retinal function, as evidenced by prolonged implicit time of the electroretinogram b-wave, in transmissible mink encephalopathy-infected cattle before the onset of clinical illness. We also demonstrate disruption of rod bipolar cell synaptic terminals, indicated by decreased immunoreactivity for the alpha isoform of protein kinase C and vesicular glutamate transporter 1, and activation of Müller glia, as evidenced by increased glial fibrillary acidic protein and glutamine synthetase expression, in the retinas of these cattle at the time of euthanasia due to clinical deterioration. This is the first study to identify both functional and morphologic alterations in the retinas of TSE-infected cattle. Our results support future efforts to focus on the retina for the development of new strategies for the diagnosis of TSEs.

Human CJD, endemic sheep scrapie, epidemic bovine spongiform encephalopathy (BSE), and other transmissible spongiform encephalopathies (TSEs), are caused by a group of related but molecularly uncharacterized infectious agents. The UK-BSE agent infected many species, including humans where it causes variant CJD (vCJD). As in most viral infections, different TSE disease phenotypes are determined by both the agent strain and the host species. TSE strains are most reliably classified by incubation time and regional neuropathology in mice expressing wild-type (wt) prion protein (PrP). We compared vCJD to other human and animal derived TSE strains in both mice and neuronal cultures expressing wt murine PrP. Primary and serial passages of the human vCJD agent, as well as the highly selected mutant 263K sheep scrapie agent, revealed profound strain-specific characteristics were encoded by the agent, not by host PrP. Prion theory posits that PrP converts itself into the infectious agent, and thus short incubations require identical PrP sequences in the donor and recipient host. However, wt PrP mice injected with human vCJD brain homogenates showed dramatically shorter primary incubation times than mice expressing only human PrP, a finding not in accord with a PrP species barrier. All mouse passage brains showed the vCJD agent derived from a stable BSE strain. Additionally, both vCJD brain and monotypic neuronal cultures produced a diagnostic 19 kDa PrP fragment previously observed only in BSE and vCJD primate brains. Monotypic cultures can be used to identify the intrinsic, strain-determining molecules of TSE infectious particles.

Background Information: TSEs (transmissible spongiform encephalopathies) are neurodegenerative disorders affecting humans and animals. PrP(Sc), a conformationally altered isoform of the normal prion protein (PrP(C)), is thought to be the pathogenic agent. However, the biochemical composition of the prion agent is still matter of debate. The potential transmission risk of the prion agent through biological fluids has been shown, but the development of competitive diagnostic tests and treatment for TSEs requires a more comprehensive knowledge of the agent and the cellular mechanisms by which it is disseminated. With this aim, we initiated characterization of the prion agent and the pathways by which it can be propagated using the cellular model system neuroblastoma (N2a)., Results: The present study shows that N2a cells infected with scrapie release the prion agent into the cell culture medium in association with exosome-like structures and viral particles of endogenous origin. We found that both prion proteins and scrapie infectivity are mainly associated with exosome-like structures that contain viral envelope glycoprotein and nucleic acids, such as RNAs., Conclusions: The dissemination of prions in N2a cell culture is mediated through the exosomal pathway.

Prion diseases are fatal neurodegenerative disorders characterized by long incubation periods. To investigate whether concurrent diseases can modify the clinical outcome of prion-affected subjects, we tested the effect of viral infection on the binding and internalization of PrP(Sc), essential steps of prion propagation. To this effect, we added scrapie brain homogenate or purified PrP(Sc) to fibroblasts previously infected with minute virus of mice (MVM), a mouse parvovirus. We show here that the rate of incorporation of PrP(Sc) into MVM-infected cells was significantly higher than that observed for naïve cells. Immunostaining of cells and immunoblotting of subcellular fractions using antibodies recognizing PrP and LysoTracker, a lysosomal marker, revealed that in both control and MVM-infected cells the incorporated PrP(Sc) was associated mostly with lysosomes. Interestingly, flotation gradient analysis revealed that the majority of the PrP(Sc) internalized into MVM-infected cells shifted toward raft-containing low-density fractions. Concomitantly, the MVM-infected cells demonstrated increased levels of the glycosphingolipid GM1 (an essential raft lipid component) throughout the gradient and a shift in caveolin 1 (a raft protein marker) toward lighter membrane fractions compared with noninfected cells. Our results suggest that the effect of viral infection on membrane lipid composition may promote the incorporation of exogenous PrP(Sc) into rafts. Importantly, membrane rafts are believed to be the conversion site of PrP(C) to PrP(Sc); therefore, the association of exogenous PrP(Sc) with such membrane microdomains may facilitate prion infection., ((c) 2008 Wiley-Liss, Inc.)

Prion diseases present unique challenges to healthcare facilities, both in the care and treatment of patients. A significant cause for concern is in the routine reprocessing of medical devices used on patients and how disease transmission can be prevented on the reuse of devices. Investigations have shown that prion disease can be transmitted on medical devices, which can be a concern given the long incubation times associated with these diseases and that guidelines to control transmission only really apply in a small number of known or at risk cases. It is only recently that medical device-associated cleaning, disinfection and sterilization technologies have been investigated and the results of these studies are summarized in this report. The evidence would suggest that many simple decontamination steps can be applied to dramatically reduce the risks to patients, but the research has also given some surprises. Overall, it is reasonable to expect that standard precautions will be able to be applied both today as well as in the future to reduce the risk of prion disease transmission as well as the many other human pathogen concerns, although this may mean changes in some of our practices.

The symptoms associated with slow viral or prion diseases of the central nervous system tend to have multiple neurologic symptoms, and different patients may present with different symptoms. This review discusses the most common slow virus infections and their imaging findings.

Prion infections induce severe disruption of the central nervous system with neuronal vacuolation and extensive glial reactions, and invariably lead to death of affected individuals. The molecular underpinnings of these events are not well understood. To better define the molecular consequences of prion infections, we analyzed the transcriptional response to persistent prion infection in a panel of three murine neural cell lines in vitro. Colony spot immunochemistry assays indicated that 65-100% of cells were infected in each line. Only the Nav1 gene was marginally modulated in one cell line, whereas transcripts previously reported to be derailed in prion-infected cells were not confirmed in the present study. We attribute these discrepancies to the experimental stringency of the current study, which was performed under conditions designed to minimize potential genetic drifts. These findings are at striking variance with gene expression studies performed on whole brains upon prion infections in vivo, suggesting that many of the latter changes represent secondary reactions to infection. We conclude that, surprisingly, there are no universal transcriptional changes induced by prion infection of neural cells in vitro.

Tubulovesicular structures (particles; TVS) are virion-like particles 25-30 nm in diameter found by thin-section electron microscopy in brains of all prion diseases including scrapie, Creutzfeldt-Jakob disease (CJD) fatal familial insomnia (FFI) and Gerstmann-Sträussler-Scheineker disease (GSS) as well as in cell cultures infected with TSE agents. TVS are regarded as a disease-specific ultrastructural marker for TSEs and, by those not completely satisfied with the prion hypothesis, they are even considered to be a possible candidate for the infectious TSE agent itself. A caveat regarding that interpretation stemmed from previous failures to find TVS by electron microscopic studies of tissues from animals infected with the Echigo-1 strain of CJD agent. We now report detecting TVS in brains of hamsters infected with that strain of CJD agent, albeit with a very low frequency.

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.

The transmissible spongiform encephalopathies (TSEs) such as endemic sheep scrapie, sporadic human Creutzfeldt-Jakob disease (CJD), and epidemic bovine spongiform encephalopathy (BSE) may all be caused by a unique class of "slow" viruses. This concept remains the most parsimonious explanation of the evidence to date, and correctly predicted the spread of the BSE agent to vastly divergent species. With the popularization of the prion (infectious protein) hypothesis, substantial data pointing to a TSE virus have been largely ignored. Yet no form of prion protein (PrP) fulfills Koch's postulates for infection. Pathologic PrP is not proportional to, or necessary for infection, and recombinant and "amplified" prions have failed to produce significant infectivity. Moreover, the "wealth of data" claimed to support the existence of infectious PrP are increasingly contradicted by experimental observations, and cumbersome speculative notions, such as spontaneous PrP mutations and invisible strain-specific forms of "infectious PrP" are proposed to explain the incompatible data. The ability of many "slow" viruses to survive harsh environmental conditions and enzymatic assaults, their stealth invasion through protective host-immune defenses, and their ability to hide in the host and persist for many years, all fit nicely with the characteristics of TSE agents. Highly infectious preparations with negligible PrP contain nucleic acids of 1-5 kb, even after exhaustive nuclease digestion. Sedimentation as well as electron microscopic data also reveal spherical infectious particles of 25-35 nm in diameter. This particle size can accommodate a viral genome of 1-4 kb, sufficient to encode a protective nucleocapsid and/or an enzyme required for its replication. Host PrP acts as a cellular facilitator for infectious particles, and ultimately accrues pathological amyloid features. A most significant advance has been the development of tissue culture models that support the replication of many different strains of agent and can produce high levels of infectivity. These models provide new ways to rapidly identify intrinsic viral and strain-specific molecules so important for diagnosis, prevention, and fundamental understanding.

The prion theory postulates that prions are novel infectious agents that are composed largely, if not entirely, of abnormally folded host-encoded prion proteins. However, the existence of different prion strains is enigma, if these novel infectious agents lack a genetic element, such as a nucleic acid. The best proof for this 'protein-only' concept would be the in vitro generation of prions from synthetic sources. Indeed, a substantial body of evidence has meanwhile been accumulated in favour of this postulate. This mini review recapitulates all relevant studies and experimental data on the generation of synthetic prions.

The transmissible spongiform encephalopathies (TSE) that manifest as Creutzfeldt-Jakob disease in humans, as scrapie in sheep and goats, mad cow disease in cattle, or chronic wasting disease in cervids (deer) represent a serious human health crisis and a significant economical problem. Despite much research, the nature of the elusive pathogen directly involved with TSE is currently unresolved. This article reviews current pathogen-cell plasma membrane properties, showing that the primary biochemical marker of the prion disease is used as a receptor by the intracellular bacterium Brucella abortus. Such observation makes plausible the role for the prion in the pathogenesis of TSE, and supports the concept that Spiroplasma, a wall-less bacterium, may be a transmissible agent of TSE. Over the past three decades, we have published convincing evidence that Spiroplasma infection is associated with TSE. The bacterial-prion-receptor concept by other laboratories support a model for TSE wherein a Spiroplasma bacterium can bind to prion receptors (alone or with anchors) on the cell surface lipid raft, allowing entry of the microbe into the cell to initiate infection. The relevance of this new concept is that it offers a new window for future research involving a bacterium in the pathogenesis of TSE. Data from the bacterial-prion-receptor model will aid in the development diagnostic tests and/or treatment protocols for TSE., (2005 Wiley-Liss, Inc.)

In recent years major outbreaks of prion disease linked to oral exposure of the prion agent have occurred in animal and human populations. These disorders are associated with a conformational change of a normal protein, PrP(C) (prion protein cellular), to a toxic and infectious form, PrP(Sc) (prion protein scrapie). None of the prionoses currently have an effective treatment. A limited number of active immunization approaches have been shown to slightly prolong the incubation period of prion infection. Active immunization in wild-type animals is hampered by auto-tolerance to PrP and potential toxicity. Here we report that mucosal vaccination with an attenuated Salmonella vaccine strain expressing the mouse PrP, is effective at overcoming tolerance to PrP and leads to a significant delay or prevention of prion disease in mice later exposed orally to the 139A scrapie strain. This mucosal vaccine induced gut anti-PrP immunoglobulin (Ig)A and systemic anti-PrP IgG. No toxicity was evident with this vaccination approach. This promising finding suggests that mucosal vaccination may be a useful method for overcoming tolerance to PrP and preventing prion infection among animal and potentially human populations at risk.

Sections of medulla oblongata, taken at the level of the obex, palatine tonsil and medial retropharyngeal lymph node from 10,269 captive Rocky Mountain elk (Cervus elaphus nelsoni), were examined by immunohistochemical staining with monoclonal antibody for the prion protein associated with the transmissible spongiform encephalopathy of cervids, chronic wasting disease (PrP(CWD)). The protein was detected in 226 of them. On the basis of the anatomical location of the deposits in the brainstem of 183 elk, four distinct patterns of distribution of PrP(CWD) within the parasympathetic region of the dorsal motor nucleus of the vagus nerve and the adjacent nuclei were observed. Mild gross lesions of chronic wasting disease (serous atrophy of fat) were observed in only three elk, all with spongiform degeneration; the other elk were considered to be in the preclinical stage of the disease. In contrast with the relatively predictable distribution of prion protein (PrP) in the brain and cranial nodes of sheep and mule deer, the distribution of PrP(CWD) in the brain and nodes of the elk was more variable and unrelated to their PrP genotype. One hundred and fifty-five of the 226 positive elk had deposits of PrP(CWD) in the brainstem and lymphoid tissues, 43 had deposits only in the lymphoid tissue and 28 had deposits only in the brainstem.

Some murine retroviruses cause a spongiform neurodegenerative disease exhibiting pathology resembling that observed in transmissible spongiform encephalopathies. The neurovirulence of these "spongiogenic retroviruses" is determined by the sequence of their respective envelope proteins, although the mechanisms of neurotoxicity are not understood. We have studied a highly neurovirulent virus called FrCasE that causes a rapidly progressive form of this disease. Recently, transcriptional markers of endoplasmic reticulum (ER) stress were detected during the early preclinical period in the brains of FrCasE-infected mice. In contrast, ER stress was not observed in mice infected with an avirulent virus, F43, which carries a different envelope gene, suggesting a role for ER stress in disease pathogenesis. Here we have examined in NIH 3T3 cells the cause of this cellular stress response. The envelope protein of F43 bound BiP, a major ER chaperone, transiently and was processed normally through the secretory pathway. In contrast, the envelope protein of FrCasE bound to BiP for a prolonged period, was retained in the ER, and was degraded by the proteasome. Furthermore, engagement of the FrCasE envelope protein by ER quality control pathways resulted in decreased steady-state levels of this protein, relative to that of F43, both in NIH 3T3 cells and in the brains of infected mice. Thus, the ER stress induced by FrCasE appears to be initiated by inefficient folding of its viral envelope protein, suggesting that the neurodegenerative disease caused by this virus represents a protein misfolding disorder.

The prion notion, that a host-encoded protein reacts with itself to become an infectious entity, has become highly favoured as the causal agent in transmissible encephalopathies (TSEs) such as endemic scrapie of sheep, epidemic bovine encephalopathy (BSE), and rare forms of human Creutzfeldt-Jakob Disease (CJD). Much of the claimed "wealth of data" supporting the notion of prions, however, has been irreproducible as well as contradicted by many experimental observations. By 1989, for example, it was already shown that the "infectious prion conformation" could be eliminated without altering either infectivity or agent strain characteristics. Efforts to make the prion protein infectious also continue to fail. All the biological features of TSEs fit better with a conventional viral particle. Furthermore, structural data point to a spherical infectious particle of approximately 25 nm that contains a genome of 1-4 kb in length and probably encodes its own protective nucleocapsid. These viral features can account for the observed host recognition of TSE agents as foreign. Although recognised, TSE agents, like many conventional viruses, can hide in a latent state for many years in lymphoreticular tissues. Previous predictions made by this parsimonious viral interpretation have been accurate, whereas the prion hypothesis has been confounded by several inexplicable realities that have emerged. Recently developed TSE tissue culture models offer a way of identifying rapidly the intrinsic and strain-specific agent molecules so needed for diagnosis and prevention.

Sheep scrapie has been known for at least 200 years and was described as a transmissible disease over 100 years ago. Since then, three groups of transmissible spongiform encephalopathies or TSE diseases have been identified in humans including familial, infectious and sporadic types. The discovery of the prion protein (PrP) in the 1980s greatly accelerated knowledge of the biology and pathogenesis of TSE diseases as this protein was found to play a critical role in disease susceptibility and the TSE species-barrier and may also be a component of the infectious agent itself. Nevertheless, the nature of the TSE agents remains an enigma. Proof of the protein-only hypothesis may require generation of biologically active transmissible agent in a cell-free environment where a virus cannot replicate. Conversely, proof of a viral aetiology will require identification and isolation of a candidate virus. Further understanding of the structure of the disease-associated protease-resistant PrP should help elucidate the mechanism of PrP conversion from the normal to the abnormal form. Such information should open up new approaches to both diagnosis and therapy.

The AIDS or HIV associated dementia is a cognitive-motor disease, characterized by a strong deficit of several cognitive processes such as attention, memory, sensory perception, motor control among others. The HIV associated dementia affects 30% of adult to 50% of infant HIV positive subjects. Since neurons are not infected by HIV, its principal target in the brain is microglia. The pathophysiology of this syndrome, therefore, remains to be disclosed. Several hypothesis have been proposed, one of them suggests that opportunistic infections can affect the brain. Another hypothesis suggests that microglia secretes toxic products as a result of HIV infection and those are the ones causing the damage and finally, the hypothesis, suggesting that the brain is damaged as a result of the insult caused by HIV-derived proteins. In vitro studies suggest that the HIVgp120, a viral surface protein, is highly neurotoxic. For example HIVgp120 increases cytoplasmic Ca+2 by two ways: facilitating glutamate neurotransmission increasing Ca+2 conductance, and activating the IP3 pathway, facilitating Ca+2 release from the smooth endoplasmic reticulum. This Ca+2 in turn, activates several internal signaling pathways such as the MAPK pathway. We use an animal model to test the HIVgp120 effect on neurophysiological signals and behavior as well as several pharmacological approaches to prevent the HIVgp120 neurotoxic effects. This review updates with the most recent literature discussing the potential mechanisms implicated in the pathophysiology of the AIDS dementia complex. We, in addition, hope the reader will be able to correlate the clinical symptoms observed in the HIV infected subjects and the HIVgp120-induced behavioral changes observed in animal models. Likewise, we discuss the new drugs we are testing, in order to offer a new pharmacological treatment to the patient.

Background: A size-exclusion filter (Viresolve 180, Millipore Corp.) was tested for its ability to remove transmissible spongiform encephalopathies prion protein from an immune globulin preparation during ultrafiltration., Study Design and Methods: Hamster-adapted 263K scrapie brain homogenate (SBH) was spiked into Rh0(D) immune globulin (human) at 1 in 300 and 1 in 1000 dilutions. Before spiking, the SBH was treated with detergent, sonicated, and filtered through serial 0.45-, 0.22-, and 0.1-microm filters to present a rigorous filter challenge. Process variables were monitored throughout the ultrafiltration to ensure that the spiked material did not compromise the membrane flux. Removal of scrapie prion protein (PrP(Sc)) material was determined by use of a sensitive Western blot assay., Results: The turbid SBH became completely translucent after sonication and passage through the 0.45-, 0.22-, and 0.1-microm filters. The filtration of the immune globulin containing PrP(Sc) material was more difficult to perform than was filtration of immune globulin spiked with the normal cellular isoform. Even during tangential flow filtration, the fibril material prevented the PrP(Sc)-spiked immune globulin from passing as readily through the filter. Western blot results indicated a removal of greater than or equal to 2.5 log PrP(Sc), while remaining within the normal filtration limits., Conclusions: The composition, physical condition, and the amount of SBH introduced have significant effects on the filtration of the immune globulin and the log removal values obtained. By use of a detergent-treated, sonicated, and filtered preparation of SBH, it was demonstrated that the Viresolve 180 effectively removes PrP(Sc) from the immune globulin.

Any biopharmaceutical product that has involved the use of animal-derived material during the manufacturing process has the potential to be contaminated with animal viruses. To ensure safety of these products, extensive testing is performed on the starting materials, such as the cell banks, and on the raw materials used in manufacture. Additional testing is also performed at various stages of production and, in some cases, on the final product as well. Because of inherent limitations in direct testing methods, the capacity of the downstream purification process to remove/inactivate potential viral contaminants is also studied to give an extra degree of assurance that the final product will be free of infectious viruses.

The prion diseases or transmissible spongiform encephalopathies (TSE) constitute a group of hereditary or acquired neurodegenerative disorders. The Authors evaluate the etiopathogenetic background, the clinical features and the current concerns with special attention to bovine spongiform encephalopathy and new variant Creutzfeldt Jacob disease.

A template-assisted conformational change of the cellular prion protein (PrP(C)) from a predominantly helical structure to an amyloid-type structure with a higher proportion of beta-sheet is thought to be the causative factor in prion diseases. Since flexibility of the polypeptide is likely to contribute to the ability of PrP(C) to undergo the conformational change that leads to the infective state, we have undertaken a comprehensive examination of the dynamics of two recombinant Syrian hamster PrP fragments, PrP(29-231) and PrP(90-231), using (15)N NMR relaxation measurements. The molecular motions of these PrP fragments have been studied in solution using (15)N longitudinal (T(1)) and transverse relaxation (T(2)) measurements as well as [(1)H]-(15)N nuclear Overhauser effects (NOE). These data have been analyzed using both reduced spectral density mapping and the Lipari-Szabo model free formalism. The relaxation properties of the common regions of PrP(29-231) and PrP(90-231) are very similar; both have a relatively inflexible globular domain (residues 128-227) with a highly flexible and largely unstructured N-terminal domain. Residues 29-89 of PrP(29-231), which include the copper-binding octarepeat sequences, are also highly flexible. Analysis of the spectral densities at each residue indicates that even within the structured core of PrP(C), a markedly diverse range of motions is observed, consistent with the inherent plasticity of the protein. The central portions of helices B and C form a relatively rigid core, which is stabilized by the presence of an interhelix disulfide bond. Of the remainder of the globular domain, the parts that are not in direct contact with the rigid region, including helix A, are more flexible. Most significantly, slow conformational fluctuations on a millisecond to microsecond time scale are observed for the small beta-sheet. These results are consistent with the hypothesis that the infectious, scrapie form of the protein PrP(Sc) could contain a helical core consisting of helices B and C, similar in structure to the cellular form PrP(C). Our results indicate that residues 90-140, which are required for prion infectivity, are relatively flexible in PrP(C), consistent with a lowered thermodynamic barrier to a template-assisted conformational change to the infectious beta-sheet-rich scrapie isoform.

The term 'prion diseases' refers to a group of neurodegenerative disorders thought to be caused by prions, pathogenic agents with novel modes of replication and transmission. Prion diseases are characterized by long incubation periods ranging from months to years and are invariably fatal once clinical symptoms have appeared. They are also called transmissible spongiform encephalopathies (TSE), on account of the predominant neuropathological change observed in the central nervous system. The most important members of this group are Creutzfeldt-Jakob disease (CJD) of man displaying sporadic, inherited and infectious forms, bovine spongiform encephalopathy (BSE, 'mad cow disease') of cattle, and scrapie of sheep. Despite their rarity, human prion diseases have recently been covered extensively in the media because of the likely connection between a new variant of human CJD (vCJD) and BSE and the possibility of contamination of human blood and blood products by the vCJD agent. This short review discusses the basic biological properties of prions, followed by a presentation of the clinical and pathological features of the most important human prion diseases.

We present a theoretical framework that enables us to dissect out the parametric dependencies of the pathogenesis of prion diseases. We are able to determine the influence of both host-dependent factors (connectivity, cell density, protein synthesis rate, and cell death) and strain-dependent factors (cell tropism, virulence, and replication rate). We use a model based on a linked system of differential equations on a lattice to explore how the regional distribution of central nervous system pathology in Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome, and fatal familial insomnia relates to each of these factors. The model then is used to make qualitative predictions about the pathology for two possible hypothetical triggers of neuronal loss in prion diseases. Pathological progression in overexpressing mouse models has been shown to depend on the site of initial infection. The model allows us to compare the pathologies resulting from different inoculation routes.

Prions are very resistant to inactivation, and accidental transmission has occurred through the use of inadequate decontamination procedures. Strong sodium hypochiorite solutions achieve inactivation but other chlorine-releasing compounds are less effective. 2M sodium hydroxide leads to substantial but incomplete inactivation; other chemical procedures such as the use of proprietary phenolic disinfectants are much less less effective. Infectivity can survive autoclaving at 132-138 degrees C, and under certain conditions the effectiveness of autoclaving actually declines as the temperature is increased. The small resistant subpopulations that survive autoclaving are not inactivated by simply re-autoclaving, and they acquire biological characteristics that differentiate them from the main population. Despite the limitations of autoclaving, combining autoclaving (even at 121 degrees C) with a sodium hydroxide treatment is extremely effective. Protein-fixation (e.g., by ethanol or formalin) substantially enhances the thermostability of these agents. This suggests that future successful inactivation strategies might best be developed by studying procedures that avoid protein-fixation.

The word "prion" was created in 1982 to name the etiological agent of the transmissible spongiform encephalopathies (TSE), a group of degenerative diseases affecting central nervous system of man and animals, including bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD). Prions present two isoforms: PrPC, cellular or normal, which exists in all vertebrates and is sensitive to detergents and proteases, and PrPSc, disease associated, partially resistant. The molecular weight of both PrPC and PrPSc is 30-35 kD; after treatment with detergents and proteases PrPSc originates PrP27-30 (27-30 kD). PrPC is also denominated PrPsens, and PrPSc is PrPres. PrPSc and PrP27-30 cause disease. PrPC presents polymorphisms specifically associated with some TSE. The "prion hypothesis" says that PrPSc transmits its characteristic resistance to PrPC through conformational changes, and accumulation of the protein, without involvement of nucleic acids, causes disease. Most of the hypothesis has been demonstrated with transgenic mice, computer models and recombinant proteins, but the existence of strains of the TSE agents has not been explained. The description of similar mechanisms of propagation of protein conformational properties in Saccharomyces cereviseae has extended the meaning of the prion definition. Although the transmission of conformational changes between PrPC and PrPSc was experimentally shown, the pathogenesis of the TSE remains unknown. The relationship between BSE and vCJD is mentioned.

PrP is a host-encoded glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs) or 'prion' diseases. The normal form of the protein (PrP(C)) is heavily but incompletely glycosylated; it shows structural diversity in three neuroanatomically distinct regions of the brain. No effect of TSE infection on PrP(C) glycosylation has been detected. TSE-specific forms of PrP (PrP(Sc)) vary in their degree of glycosylation according to strain of TSE infectious agent. PrP(Sc) also varies independently in the amount and pattern of glycosylation according to brain region. This diversity shows that the glycosylation of PrP is under both host- and TSE agent-specified control, probably within the biosynthetic pathway for protein N-glycosylation. These findings challenge assumptions that PrP(Sc) is formed from the normal, mature form of PrP(Sc) but are compatible with a model in which the glycosylation phenotype of PrP(Sc) is under the control of both host cellular factors and TSE agent-specified information.

An infectious illness, attributed to atypically structured cytopathic "stealth" viruses, occurred in 1996 in the Mohave Valley region of the United States. A stealth virus-infected child from this region has developed a severe noninflammatory, vacuolating (spongiform) en cephalopathy. The illness initially presented as a behavioral problem without overt neurological signs. Extensive investigations, including repeated magnetic resonance imaging, two brain biopsies, and stealth virus cultures, have helped define the disease process occurring in this child. Significant clinical benefit with apparent retardation of disease progression occurred during a 6-week course of ganciclovir therapy. The potential contributing role of stealth virus infections in children presenting with behavioral problems needs to be addressed., (Copyright 1999 Academic Press.)

A retroviral etiology might explain why amyloid plaque and/or spongiosis are or are not associated with neuronal death in prion diseases. While retroviral genes themselves may be responsible for neuronal death, a retrovirus may also cause mutations in cellular genes. Hence, the prion gene may be altered by a retrovirus in the same way as a cellular proto-oncogene is altered to produce an oncogene, either by transduction or by integration of the provirus in its vicinity. In both cases, the resulting abnormal prion protein, acting as a catalyst, may induce the formation of amyloid plaques. In addition, a wild type retrovirus may recombine to the vesicular stomatitis virus (VSV) to give rise to a pseudotyped retrovirus able to induce spongiosis. It is reported here that in scrapie, a blood monocytoid cell proliferates in vitro. If confirmed in other species, this raises the question of the potential link between prion disease and leukemia. Indeed neurovirulent strains of murine leukemia virus, a slow acting retrovirus, are known to induce spongiform encephalopathies. A preliminary attempt to purify reverse transcriptase by chromatography, using the classical protocol, failed because of the presence of a prion-like protein secreted by the blood mononuclear cells which stuck to the phosphocellulose column. Therefore, if a retrovirus is present in prion diseases, it would be evidenced only in animals developing the disease in the absence of prion protein. From this point of view, mice obtained in 1997 by the group of D. Dormont in France, offer a unique opportunity to test the retroviral hypothesis.

Amphotropic Moloney-murine leukemia virus recombinants (Mo-AmphoV) induce a severe spongiform encephalomyelopathy in newborn mice. We show here that a coisogenic recombinant with a 10A1-MuLV host range (Mo-10A1V) also induces a neurodegenerative disease, clinically characterized by mild tremor and ataxia. Spongiform lesions are most severe in the metencephalon and mesencephalon but extend into the prosencephalon and spinal cord. Significantly, the quality of histopathology was indistinguishable between Mo-AmphoV and Mo-10A1V, probably reflecting a final common pathogenic pathway. Common receptor use thus may be an important determinant in the pathogenicity of these viruses. These results have implications for the clinical use of retroviral pseudotypes that use phosphate transporters for cell entry., (Copyright 1998 Academic Press.)