Early detection of chronic wasting disease prions in urine of pre-symptomatic deer by real-time quaking-induced conversion assay.

Chronic wasting disease (CWD) is a prion disease of captive and free-ranging deer (Odocoileus spp), elk (Cervus elaphus nelsonii) and moose (Alces alces shirasi). Unlike in most other prion diseases, in CWD prions are shed in urine and feces, which most likely contributes to the horizontal transmission within and between cervid species. To date, CWD ante-mortem diagnosis is only possible by immunohistochemical detection of protease resistant prion protein (PrP (Sc) ) in tonsil or recto-anal mucosa-associated lymphoid tissue (RAMALT) biopsies, which requires anesthesia of animals. We report on detection of CWD prions in urine collected from pre-symptomatic deer and in fecal extracts by using real time quaking-induced conversion (RT-QuIC). This assay can be useful for non-invasive pre-symptomatic diagnosis and surveillance of CWD.

Aptamer-based spectrofluorometry for cellular prion protein using N,N'-bis[3,3'-(dimethylamino)propylamine]-3,4,9,10-perylenetetracarboxylic diimide.

A new spectrofluorometric method for cellular prion protein (PrP(C)) was developed based on the regulation of N,N'-bis[3,3'-(dimethylamino)propylamine]-3,4,9,10-perylenetetracarboxylic diimide (DAPER) fluorescence. As a perylene derivative, DAPER emits strong fluorescence in the form of free monomer in aqueous medium, but not in the form of aggregates. In this contribution, we found that the aptamer of PrP(C) could induce the aggregation of DAPER, and the bright fluorescence of DAPER was completely quenched. The quenched fluorescence, however, was recovered if PrP(C) was further added, which was ascribed to the specific binding of PrP(C) to its aptamer and the releasing of free DAPER monomers. This signalling mechanism makes it possible to detect PrP(C) by fluorescence spectroscopy. The assay allows the selective determination of PrP(C) in aqueous solution with high sensitivity and exhibits a good linear range from 0.4 to 1.6 nmol L(-1). Moreover, this probe can be applied to monitor the level of PrP(C) in human urine samples with satisfactory results.

Iatrogenic prion diseases in humans: an update.

Although Creutzfeldt-Jakob disease (CJD) was first identified in 1920, prevention of transmission raised particular concern all over the world when a new variant of the disease was first described in 1996. There is good evidence of iatrogenic transmission of this new variant among human beings through blood, blood components, tissues and growth hormone. Furthermore, four cases of iatrogenic transmission of CJD through fertility treatment with human pituitary-derived gonadotrophins have been reported. It is important to distinguish the categories of infectivity and categories of risk, which require consideration not only of the level of infectivity of a given tissue or fluid, but also the amount of tissue/fluid to which a person is exposed, the duration of exposure and the route by which infection is transmitted. The potential presence and infectivity of prion proteins in human urinary gonadotrophin preparations is a matter of debate. Differences in the sensitivity of bioassay methods are of paramount importance when considering the infectivity of a tissue. Some new methods might detect small amounts of agent in some tissues currently thought to be free of infectivity. No cases of human prion disease due to the use of urinary gonadotrophins have been recognized to date. However, the detection of prions in the urine of experimental animals and in some urine-based preparations, and the young age of fertility drug recipients, require the application of the precautionary principle to urinary preparations.

Assessing prion infectivity of human urine in sporadic Creutzfeldt-Jakob disease.

Prion diseases are neurodegenerative conditions associated with a misfolded and infectious protein, scrapie prion protein (PrP(Sc)). PrP(Sc) propagate prion diseases within and between species and thus pose risks to public health. Prion infectivity or PrP(Sc) presence has been demonstrated in urine of experimentally infected animals, but there are no recent studies of urine from patients with Creutzfeldt-Jakob disease (CJD). We performed bioassays in transgenic mice expressing human PrP to assess prion infectivity in urine from patients affected by a common subtype of sporadic CJD, sCJDMM1. We tested raw urine and 100-fold concentrated and dialyzed urine and assessed the sensitivity of the bioassay along with the effect of concentration and dialysis on prion infectivity. Intracerebral inoculation of transgenic mice with urine from 3 sCJDMM1 patients failed to demonstrate prion disease transmission, indicating that prion infectivity in urine from sCJDMM1 patients is either not present or is <0.38 infectious units/mL.

Detection of prion protein in urine-derived injectable fertility products by a targeted proteomic approach.

BACKGROUND: Iatrogenic transmission of human prion disease can occur through medical or surgical procedures, including injection of hormones such as gonadotropins extracted from cadaver pituitaries. Annually, more than 300,000 women in the United States and Canada are prescribed urine-derived gonadotropins for infertility. Although menopausal urine donors are screened for symptomatic neurological disease, incubation of Creutzfeldt-Jakob disease (CJD) is impossible to exclude by non-invasive testing. Risk of carrier status of variant CJD (vCJD), a disease associated with decades-long peripheral incubation, is estimated to be on the order of 100 per million population in the United Kingdom. Studies showing infectious prions in the urine of experimental animals with and without renal disease suggest that prions could be present in asymptomatic urine donors. Several human fertility products are derived from donated urine; recently prion protein has been detected in preparations of human menopausal gonadotropin (hMG). METHODOLOGY/PRINCIPAL FINDINGS: Using a classical proteomic approach, 33 and 34 non-gonadotropin proteins were identified in urinary human chorionic gonadotropin (u-hCG) and highly-purified urinary human menopausal gonadotropin (hMG-HP) products, respectively. Prion protein was identified as a major contaminant in u-hCG preparations for the first time. An advanced prion protein targeted proteomic approach was subsequently used to conduct a survey of gonadotropin products; this approach detected human prion protein peptides in urine-derived injectable fertility products containing hCG, hMG and hMG-HP, but not in recombinant products. CONCLUSIONS/SIGNIFICANCE: The presence of protease-sensitive prion protein in urinary-derived injectable fertility products containing hCG, hMG, and hMG-HP suggests that prions may co-purify in these products. Intramuscular injection is a relatively efficient route of transmission of human prion disease, and young women exposed to prions can be expected to survive an incubation period associated with a minimal inoculum. The risks of urine-derived fertility products could now outweigh their benefits, particularly considering the availability of recombinant products.

Characterization of the prion protein in human urine.

The presence of the prion protein (PrP) in normal human urine is controversial and currently inconclusive. This issue has taken a special relevance because prion infectivity has been demonstrated in urine of animals carrying experimental or naturally occurring prion diseases, but the actual presence and tissue origin of the infectious prion have not been determined. We used immunoprecipitation, one- and two-dimensional electrophoresis, and mass spectrometry to prove definitely the presence of PrP in human urine and its post-translational modifications. We show that urinary PrP (uPrP) is truncated mainly at residue 112 but also at other residues up to 122. This truncation makes uPrP undetectable with some commonly used antibodies to PrP. uPrP is glycosylated and carries an anchor which, at variance with that of cellular PrP, lacks the inositol-associated phospholipid moiety, indicating that uPrP is probably shed from the cell surface. The detailed characterization of uPrP reported here definitely proves the presence of PrP in human urine and will help determine the origin of prion infectivity in urine.

Detection of proteinase K resistant proteins in the urine of patients with Creutzfeldt-Jakob and other neurodegenerative diseases.

Recent concern about the possible secondary spread of vCJD through blood transfusion and blood products has highlighted the need for a sensitive test for the identification of PrP(TSE/res) in clinical specimens collected in a non-invasive way. In addition, a more accurate estimate of the prevalence of pre-clinical vCJD in the population may be possible if there were a test that could be applied to easily available material such as urine. As a step towards this goal,the detection of putative PrP(TSE/res) in the urine of CJD patients has been improved, based on Proteinase K digestion of samples and western blotting. The modified western blot uses concentrated urine as a starting material. After proteolytic treatment followed by electrophoresis and western blotting, membranes are incubated with an anti-PrP antibody conjugated directly with horseradish peroxidase. This study was conducted on urine samples of CJD and other neurodegenerative disease affected individuals. Proteinase K resistant high molecular weight proteins were detected, which are suggested to be a complex of urinary PrP and immunoglobulin proteins. Whether urine can be used as a diagnostic tool for the detection of PrP could not be answered in this study.

Urinary excretion and blood level of prions in scrapie-infected hamsters.

Prions, infectious agents causing transmissible spongiform encephalopathy (TSE), are composed primarily of the pathogenic form (PrP(Sc)) of the host-encoded prion protein. Although very low levels of infectivity have been detected in urine from scrapie-infected rodents, no reports of urinary PrP(Sc) have been substantiated. Studies on the dynamics of urinary PrP(Sc) during infection are needed to ensure the safety of urine-derived biopharmaceuticals and to assess the possible horizontal transmission of prion diseases. Using the protein misfolding cyclic amplification technique, a time-course study of urinary excretion and blood levels of PrP(Sc) was performed in Sc237-infected hamsters and a high rate of PrP(Sc) excretion was found during the terminal stage of the disease. Following oral administration, PrP(Sc) was present in all buffy coat samples examined; it was also present in most of the plasma samples obtained from hamsters in the symptomatic stage. PrP(Sc) was excreted in urine for a few days after oral administration; subsequently, urinary PrP(Sc) was not detected until the terminal disease stage. These results represent the first biochemical detection of PrP(Sc) in urine from TSE-infected animals.

Infectious prions in the saliva and blood of deer with chronic wasting disease.

A critical concern in the transmission of prion diseases, including chronic wasting disease (CWD) of cervids, is the potential presence of prions in body fluids. To address this issue directly, we exposed cohorts of CWD-naïve deer to saliva, blood, or urine and feces from CWD-positive deer. We found infectious prions capable of transmitting CWD in saliva (by the oral route) and in blood (by transfusion). The results help to explain the facile transmission of CWD among cervids and prompt caution concerning contact with body fluids in prion infections.

Prion urine comprises a glycosaminoglycan-light chain IgG complex that can be stained by Congo red.

Light chain IgG, a known amyloidotic protein, is present in the urine of prion disease affected individuals in a protease resistant form. In addition, it was shown recently that prion urine samples comprise a significant excess of glycosaminoglycans. Since amyloidotic proteins and glycosaminoglycans are the major components of amyloid aggregates, a Congo red dot blot assay was developed for detection of Creutzfeldt-Jacob disease (CJD) in urine. This assay was also positive for about 10% of patients suffering from diseases such as Alzheimer disease, cerebrovascular attacks and multiple sclerosis, but negative for healthy controls. Both glycosaminoglycans and proteins such as light chain IgG were required for the binding of Congo red to the urine fractions, as shown by the fact that Proteinase K digestion of the samples either after guanidine or after choindrotinase abolished the Congo red signal from the CJD samples.

Consensus statement on the bio-safety of urinary-derived gonadotrophins with respect to Creutzfeldt-Jakob disease.

Human transmissible spongiform encephalopathies (TSE) encompass a group of rare neurodegenerative diseases. In April 2004, a group of international experts and regulators met in Buenos Aires, Argentina, to review the safety and to reach consensus on the use of urinary-derived gonadotrophins with respect to TSE. Iatrogenic transmission of Creutzfeldt-Jakob Disease (CJD) from pituitary-derived gonadotrophins has been reported, no infectivity in urine has been demonstrated, and no definite cases of transmission via urine have been reported. It is currently not possible to monitor donor urine or finished product for the presence of prions. Therefore the assessment of risk has to be based on the likelihood of infection in urine, the source of the urine, and the capacity of the manufacturing process to remove any adventitious infection. Urine for the production of medicinal products should be obtained from sources that minimize the possible presence of materials derived from subjects suffering from human TSE. As no strong evidence for TSE infectivity in urine exists, it can be concluded that the risk of disease-generating prions and TSE infectivity being present in donor urine is low. Current evidence indicates that, with respect to the risk of TSE infection, urinary-derived gonadotrophins appear to be safe.

Prion biology relevant to bovine spongiform encephalopathy.

Bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD) of deer and elk are a threat to agriculture and natural resources, as well as a human health concern. Both diseases are transmissible spongiform encephalopathies (TSE), or prion diseases, caused by autocatalytic conversion of endogenously encoded prion protein (PrP) to an abnormal, neurotoxic conformation designated PrPsc. Most mammalian species are susceptible to TSE, which, despite a range of species-linked names, is caused by a single highly conserved protein, with no apparent normal function. In the simplest sense, TSE transmission can occur because PrPsc is resistant to both endogenous and environmental proteinases, although many details remain unclear. Questions about the transmission of TSE are central to practical issues such as livestock testing, access to international livestock markets, and wildlife management strategies, as well as intangible issues such as consumer confidence in the safety of the meat supply. The majority of BSE cases seem to have been transmitted by feed containing meat and bone meal from infected animals. In the United Kingdom, there was a dramatic decrease in BSE cases after neural tissue and, later, all ruminant tissues were banned from ruminant feed. However, probably because of heightened awareness and widespread testing, there is growing evidence that new variants of BSE are arising "spontaneously," suggesting ongoing surveillance will continue to find infected animals. Interspecies transmission is inefficient and depends on exposure, sequence homology, TSE donor strain, genetic polymorphism of the host, and architecture of the visceral nerves if exposure is by an oral route. Considering the low probability of interspecies transmission, the low efficiency of oral transmission, and the low prion levels in nonnervous tissues, consumption of conventional animal products represents minimal risk. However, detection of rare events is challenging, and TSE literature is characterized by subsequently unsupported claims of species barriers or absolute tissue safety. This review presents an overview of TSE and summarizes recent research on pathogenesis and transmission.

Prion diseases: recent developments toward diagnostic tests.

Concerns about the large-scale European crisis of bovine spongiform encephalopathy, the finding of 1 infected cow in Alberta, Canada, in 2003, and the theoretical concern of prion transmissibility in blood have renewed interest in the development of rapid, minimally invasive diagnostic assays for prion diseases. Herein we review the pathologic features, clinical manifestations, diagnostic criteria, and recent developments that may lead to new diagnostic screening assays for prion diseases.

Prion transmission in blood and urine: what are the implications for recombinant and urinary-derived gonadotrophins?

Evidence is emerging that suggests that the protease-resistant isoform (PrP(sc)) of the normal cellular prion protein (PrP(c)) can be detected in the blood and urine of animals and humans with transmissible spongiform encephalopathies (TSEs). The production of the human menopausal and recombinant gonadotrophin preparations for use in ovarian stimulation protocols in fertility treatment is one area where the pharmaceutical industry needs to be vigilant and take appropriate steps to ensure that the safety of such drugs remains as high as ever. The recombinant preparations utilize fetal calf serum or other animal sera or proteins as part of a culture medium during production. Human urinary-derived menotrophin preparations are exposed to the theoretical risk of infection from menopausal donors of urine. Nevertheless, the failure to demonstrate irrefutably infectivity following intracerebral inoculation with urine from TSE-infected hosts suggests that the risk associated with products derived from urine is merely theoretical. Despite the paucity of evidence to date and its relevance to the infectious spread of TSEs, it is important that robust measures are implemented to either remove or inactivate PrP(sc) in order to minimize contamination. Validation of each production process is required to assess the likelihood of contamination.

The use of urinary gonadotrophins should be discouraged.

In view of concerns regarding the potential presence and infectivity of prion proteins in human urinary gonadotrophin preparations, together with the availability of both recombinant FSH and recombinant LH, it is argued that the use of urinary gonadotrophins should be discouraged.