De novo prion aggregates trigger autophagy in skeletal muscle.

In certain sporadic, familial, and infectious prion diseases, the prion protein misfolds and aggregates in skeletal muscle in addition to the brain and spinal cord. In myocytes, prion aggregates accumulate intracellularly, yet little is known about clearance pathways. Here we investigated the clearance of prion aggregates in muscle of transgenic mice that develop prion disease de novo. In addition to neurodegeneration, aged mice developed a degenerative myopathy, with scattered myocytes containing ubiquitinated, intracellular prion inclusions that were adjacent to myocytes lacking inclusions. Myocytes also showed elevated levels of the endoplasmic reticulum chaperone Grp78/BiP, suggestive of impaired protein degradation and endoplasmic reticulum stress. Additionally, autophagy was induced, as indicated by increased levels of beclin-1 and LC3-II. In C2C12 myoblasts, inhibition of autophagosome maturation or lysosomal degradation led to enhanced prion aggregation, consistent with a role for autophagy in prion aggregate clearance. Taken together, these findings suggest that the induction of autophagy may be a central strategy for prion aggregate clearance in myocytes. IMPORTANCE In prion diseases, the prion protein misfolds and aggregates in the central nervous system and sometimes in other organs, including muscle, yet the cellular pathways of prion aggregate clearance are unclear. Here we investigated the clearance of prion aggregates in the muscle of a transgenic mouse model that develops profound muscle degeneration. We found that endoplasmic reticulum stress pathways were activated and that autophagy was induced. Blocking of autophagic degradation in cell culture models led to an accumulation of aggregated prion protein. Collectively, these findings suggest that autophagy has an instrumental role in prion protein clearance.

Spongiform encephalopathy in transgenic mice expressing a point mutation in the ß2-α2 loop of the prion protein.

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases attributed to misfolding of the cellular prion protein, PrP(C), into a ß-sheet-rich, aggregated isoform, PrP(Sc). We previously found that expression of mouse PrP with the two amino acid substitutions S170N and N174T, which result in high structural order of the ß2-α2 loop in the NMR structure at pH 4.5 and 20°C, caused transmissible de novo prion disease in transgenic mice. Here we report that expression of mouse PrP with the single-residue substitution D167S, which also results in a structurally well ordered ß2-α2 loop at 20°C, elicits spontaneous PrP aggregation in vivo. Transgenic mice expressing PrP(D167S) developed a progressive encephalopathy characterized by abundant PrP plaque formation, spongiform change, and gliosis. These results add to the evidence that the ß2-α2 loop has an important role in intermolecular interactions, including that it may be a key determinant of prion protein aggregation.

Biological effects and use of PrPSc- and PrP-specific antibodies generated by immunization with purified full-length native mouse prions.

The prion agent is the infectious particle causing spongiform encephalopathies in animals and humans and is thought to consist of an altered conformation (PrP(Sc)) of the normal and ubiquitous prion protein PrP(C). The interaction of the prion agent with the immune system, particularly the humoral immune response, has remained unresolved. Here we investigated the immunogenicity of full-length native and infectious prions, as well as the specific biological effects of the resulting monoclonal antibodies (MAbs) on the binding and clearance of prions in cell culture and in in vivo therapy. Immunization of prion knockout (Prnp(0/0)) mice with phosphotungstic acid-purified mouse prions resulted in PrP-specific monoclonal antibodies with binding specificities selective for PrP(Sc) or for both PrP(C) and PrP(Sc). PrP(Sc)-specific MAb W261, of the IgG1 isotype, reacted with prions from mice, sheep with scrapie, deer with chronic wasting disease (CWD), and humans with sporadic and variant Creutzfeldt-Jakob disease (CJD) in assays including a capture enzyme-linked immunosorbent assay (ELISA) system. This PrP(Sc)-specific antibody was unable to clear prions from mouse neuroblastoma cells (ScN2a) permanently infected with scrapie, whereas the high-affinity MAb W226, recognizing both isoforms, PrP(Sc) and PrP(C), did clear prions from ScN2a cells, as determined by a bioassay. However, an attempt to treat intraperitoneally prion infected mice with full-length W226 or with a recombinant variable-chain fragment (scFv) from W226 could only slightly delay the incubation time. We conclude that (i) native, full-length PrP(Sc) elicits a prion-specific antibody response in PrP knockout mice, (ii) a PrP(Sc)-specific antibody had no prion-clearing effect, and (iii) even a high-affinity MAb that clears prions in vitro (W226) may not necessarily protect against prion infection, contrary to previous reports using different antibodies.

Evaluation of a new commercial enzyme-linked immunosorbent assay for the detection of porcine antibodies against Trichinella spp.

Trichinellosis is a zoonotic disease that is caused by the nematode Trichinella spp. Both European Union regulations and guidelines from the World Organization for Animal Health foresee the possibility of conducting serological surveillance for Trichinella spp. A newly developed commercial enzyme-linked immunosorbent assay (ELISA) was evaluated against 2 existing diagnostic techniques: an in-house ELISA and an in-house Western blot. A total of 875 Trichinella larva-negative samples of pigs and 93 Trichinella larva-positive samples of both naturally and experimentally infected pigs were included in the study. Bayesian modeling techniques were used to correct for the absence of a perfect reference test. The sensitivity and specificity of the commercial ELISA was 97.1-97.8% and 99.5-99.8%, respectively. Sensitivity analysis demonstrated high stability in the models. In a serological surveillance system, ELISA-positive samples should be tested by a confirmatory test. The Western blot is a suitable test for this purpose. With the use of the results of the models, the sensitivity and specificity of a test protocol in both ELISA and Western blot were 95.9% and 99.9%, respectively. The high sensitivity and specificity were achieved with a lower limit of detection than that of the routine artificial digestion test, suggesting that serological surveillance is a valuable alternative in surveillance for Trichinella spp. in pig production.

Assessment of safety and interferon gamma responses of Mycobacterium bovis BCG vaccine in goat kids and milking goats.

Vaccination of domestic animals has emerged as an alternative long-term strategy for the control of tuberculosis (TB). A trial under field conditions was conducted in a TB-free goat herd to assess the safety of the Mycobacterium bovis BCG vaccine. Eleven kids and 10 milking goats were vaccinated with BCG. Bacterial shedding and interferon gamma (IFN-γ) responses were monitored throughout the study. Comprehensive pathological examination and mycobacterial culture of target tissues were performed. BCG vaccine strain was only isolated from the draining lymph node of the injection site of a kid euthanized at week 8 post-vaccination. The remaining animals were euthanized at week 24. Six out of 20 showed small granulomas at the injection site. BCG shedding was not detected in either faeces or in milk throughout the study. All vaccinated kids showed BCG-induced IFN-γ responses at week 8 post-vaccination. BCG vaccination of goats showed no lack of biological safety for the animals, environment and public health, and local adverse reactions were negligible.

Stability and Reproducibility Underscore Utility of RT-QuIC for Diagnosis of Creutzfeldt-Jakob Disease.

Real-time quaking-induced conversion (RT-QuIC) allows the amplification of miniscule amounts of scrapie prion protein (PrP(Sc)). Recent studies applied the RT-QuIC methodology to cerebrospinal fluid (CSF) for diagnosing human prion diseases. However, to date, there has not been a formal multi-centre assessment of the reproducibility, validity and stability of RT-QuIC in this context, an indispensable step for establishment as a diagnostic test in clinical practice. In the present study, we analysed CSF from 110 prion disease patients and 400 control patients using the RT-QuIC method under various conditions. In addition, "blinded" ring trials between different participating sites were performed to estimate reproducibility. Using the previously established cut-off of 10,000 relative fluorescence units (rfu), we obtained a sensitivity of 85% and a specificity of 99%. The multi-centre inter-laboratory reproducibility of RT-QuIC revealed a Fleiss' kappa value of 0.83 (95% CI: 0.40-1.00) indicating an almost perfect agreement. Moreover, we investigated the impact of short-term CSF storage at different temperatures, long-term storage, repeated freezing and thawing cycles and the contamination of CSF with blood on the RT-QuIC seeding response. Our data indicated that the PrP(Sc) seed in CSF is stable to any type of storage condition but sensitive to contaminations with blood (>1250 erythrocytes/µL), which results in a false negative RT-QuIC response. Fresh blood-contaminated samples (3 days) can be rescued by removal of erythrocytes. The present study underlines the reproducibility and high stability of RT-QuIC across various CSF storage conditions with a remarkable sensitivity and specificity, suggesting RT-QuIC as an innovative and robust diagnostic method.

The Priority position paper: Protecting Europe's food chain from prions.

Bovine spongiform encephalopathy (BSE) created a global European crisis in the 1980s and 90s, with very serious health and economic implications. Classical BSE now appears to be under control, to a great extent as a result of a global research effort that identified the sources of prions in meat and bone meal (MBM) and developed new animal-testing tools that guided policy. Priority ( www.prionpriority.eu ) was a European Union (EU) Framework Program 7 (FP7)-funded project through which 21 European research institutions and small and medium enterprises (SMEs) joined efforts between 2009 and 2014, to conduct coordinated basic and applied research on prions and prion diseases. At the end of the project, the Priority consortium drafted a position paper ( www.prionpriority.eu/Priority position paper) with its main conclusions. In the present opinion paper, we summarize these conclusions. With respect to the issue of re-introducing ruminant protein into the feed-chain, our opinion is that sustaining an absolute ban on feeding ruminant protein to ruminants is essential. In particular, the spread and impact of non-classical forms of scrapie and BSE in ruminants is not fully understood and the risks cannot be estimated. Atypical prion agents will probably continue to represent the dominant form of prion diseases in the near future in Europe. Atypical L-type BSE has clear zoonotic potential, as demonstrated in experimental models. Similarly, there are now data indicating that the atypical scrapie agent can cross various species barriers. More epidemiological data from large cohorts are necessary to reach any conclusion on the impact of its transmissibility on public health. Re-evaluations of safety precautions may become necessary depending on the outcome of these studies. Intensified searching for molecular determinants of the species barrier is recommended, since this barrier is key for important policy areas and risk assessment. Understanding the structural basis for strains and the basis for adaptation of a strain to a new host will require continued fundamental research, also needed to understand mechanisms of prion transmission, replication and how they cause nervous system dysfunction and death. Early detection of prion infection, ideally at a preclinical stage, also remains crucial for development of effective treatment strategies.

Characteristic CSF prion seeding efficiency in humans with prion diseases.

The development of in vitro amplification systems allows detecting femtomolar amounts of prion protein scrapie (PrP(Sc)) in human cerebrospinal fluid (CSF). We performed a CSF study to determine the effects of prion disease type, codon 129 genotype, PrP(Sc) type, and other disease-related factors on the real-time quaking-induced conversion (RT-QuIC) response. We analyzed times to 10,000 relative fluorescence units, areas under the curve and the signal maximum of RT-QuIC response as seeding parameters of interest. Interestingly, type of prion disease (sporadic vs. genetic) and the PRNP mutation (E200K vs. V210I and FFI), codon 129 genotype, and PrP(Sc) type affected RT-QuIC response. In genetic forms, type of mutation showed the strongest effect on the observed outcome variables. In sporadic CJD, MM1 patients displayed a higher RT-QuIC signal maximum compared to MV1 and VV1. Age and gender were not associated with RT-QuIC signal, but patients with a short disease course showed a higher seeding efficiency of the RT-QuIC response. This study demonstrated that PrP(Sc) characteristics in the CSF of human prion disease patients are associated with disease subtypes and rate of decline as defined by disease duration.

Current ante-mortem techniques for diagnosis of bovine tuberculosis.

Bovine tuberculosis (TB), mainly caused by Mycobacterium bovis, is a zoonotic disease with implications for Public Health and having an economic impact due to decreased production and limitations to the trade. Bovine TB is subjected to official eradication campaigns mainly based on a test and slaughter policy using diagnostic assays based on the cell-mediated immune response as the intradermal tuberculin test and the gamma-interferon (IFN-γ) assay. Moreover, several diagnostic assays based on the detection of specific antibodies (Abs) have been developed in the last few years with the aim of complementing the current diagnostic techniques in the near future. This review provides an overview of the current ante-mortem diagnostic tools for diagnosis of bovine TB regarding historical background, methodologies and sensitivity (Se) and specificity (Sp) obtained in previous studies under different epidemiological situations.

MAP1272c encodes an NlpC/P60 protein, an antigen detected in cattle with Johne's disease.

The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that contains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne's disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were expressed and evaluated with sera from cattle with Johne's disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a distinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis.

Prion disease blood test using immunoprecipitation and improved quaking-induced conversion.

UNLABELLED: A key challenge in managing transmissible spongiform encephalopathies (TSEs) or prion diseases in medicine, agriculture, and wildlife biology is the development of practical tests for prions that are at or below infectious levels. Of particular interest are tests capable of detecting prions in blood components such as plasma, but blood typically has extremely low prion concentrations and contains inhibitors of the most sensitive prion tests. One of the latter tests is quaking-induced conversion (QuIC), which can be as sensitive as in vivo bioassays, but much more rapid, higher throughput, and less expensive. Now we have integrated antibody 15B3-based immunoprecipitation with QuIC reactions to increase sensitivity and isolate prions from inhibitors such as those in plasma samples. Coupling of immunoprecipitation and an improved real-time QuIC reaction dramatically enhanced detection of variant Creutzfeldt-Jakob disease (vCJD) brain tissue diluted into human plasma. Dilutions of 10(14)-fold, containing ~2 attogram (ag) per ml of proteinase K-resistant prion protein, were readily detected, indicating ~10,000-fold greater sensitivity for vCJD brain than has previously been reported. We also discriminated between plasma and serum samples from scrapie-infected and uninfected hamsters, even in early preclinical stages. This combined assay, which we call "enhanced QuIC" (eQuIC), markedly improves prospects for routine detection of low levels of prions in tissues, fluids, or environmental samples. IMPORTANCE: Transmissible spongiform encephalopathies (TSEs) are largely untreatable and are difficult to diagnose definitively prior to irreversible clinical decline or death. The transmissibility of TSEs within and between species highlights the need for practical tests for even the smallest amounts of infectivity. A few sufficiently sensitive in vitro methods have been reported, but most have major limitations that would preclude their use in routine diagnostic or screening applications. Our new assay improves the outlook for such critical applications. We focused initially on blood plasma because a practical blood test for prions would be especially valuable for TSE diagnostics and risk reduction. Variant Creutzfeldt-Jakob disease (vCJD) in particular has been transmitted between humans via blood transfusions. Enhanced real-time quaking-induced conversion (eRTQ) provides by far the most sensitive detection of vCJD to date. The 15B3 antibody binds prions of multiple species, suggesting that our assay may be useful for clinical and fundamental studies of a variety of TSEs of humans and animals.

Assessment of Mycobacterium tuberculosis OmpATb as a novel antigen for the diagnosis of bovine tuberculosis.

In the search for better tools to control bovine tuberculosis, the development of diagnostic tests with improved specificity and sensitivity has a high priority. We chose to search for novel immunodiagnostic reagents. In this study, Rv0899 (outer membrane protein A of Mycobacterium tuberculosis [OmpATb]) was evaluated as a stimulation antigen in a gamma interferon (IFN-gamma) release assay to diagnose bovine tuberculosis. OmpATb induced IFN-gamma responses in cattle experimentally infected with M. bovis as early and as persistently as ESAT-6 and CFP-10, the current lead diagnostic antigens. In naturally infected cattle, OmpATb stimulated IFN-gamma production in 22 of 26 animals (85%). Importantly, OmpATb detected a portion of M. bovis-infected cattle which did not respond to ESAT-6 and CFP-10 (five of six cattle). The combined diagnostic sensitivity of OmpATb, ESAT-6, and CFP-10 for a preselected group consisting of naturally infected cattle with an overrepresentation of ESAT-6/CFP-10 nonresponders was 96% (25 of 26 animals). The specificity of OmpATb for uninfected cattle was 100% (27 cattle were tested; 12 of them gave false-positive results with tuberculins). In summary, our results indicate that OmpATb has the potential to enhance the sensitivity of previously described diagnostic tests based on ESAT-6 and CFP-10 and that the combined use of OmpATb, ESAT-6, CFP-10, and other proteins may achieve at least equal sensitivity to that obtained with purified protein derivative, but at a higher specificity. Further studies evaluating the diagnostic performance of OmpATb in combination with other proteins are ongoing.

Optimization of a whole-blood gamma interferon assay for detection of Mycobacterium bovis-infected cattle.

Antigens of Mycobacterium bovis elicit a cell-mediated immune response upon intradermal injection in cattle. In vitro, such antigens stimulate the production of gamma interferon (IFN-gamma) by bovine T cells in whole-blood culture (IFN-gamma assay). We have analyzed various parameters of the in vitro IFN-gamma assay, ranging from blood sampling to execution of the IFN-gamma test, in view of potential simplifications of the assay. Here, we show that IFN-gamma responses may be reduced under certain animal handling/holding conditions and that a delayed time from blood collection to culture may lead to a reduced in vitro IFN-gamma response. Delayed initiation of culture in a purified-protein-derivative-based assay (24 h compared to 8 h after blood collection), however, resulted in a significant improvement of specificity (97% compared to 85%), whereas there was only a modest reduction of sensitivity (from 96% to 90%), which was statistically not significant. Furthermore, we show that the stimulation temperature needs to be 33 degrees C or higher; that carbon dioxide is not required for stimulation; and that various plate formats, ranging from 24 to 96 wells per plate, can be utilized. The produced IFN-gamma is stable at 4 degrees C for 28 days as well as after repeated freeze-thaw cycles. Thus, stimulation of samples may be initiated in the field without the need for a carbon dioxide source, and bovine IFN-gamma is stable under various routine laboratory temperature scenarios. These findings demonstrate opportunities for improvements in the bovine IFN-gamma test platform and flexibilities in test application.

Aggravation of ischemic brain injury by prion protein deficiency: role of ERK-1/-2 and STAT-1.

The cellular isoform of prion protein, PrPc, may confer neuroprotection in the brain, according to recent studies. To elucidate the role of PrPc in stroke pathology, we subjected PrPc-knockout (Prnp(0/0)), wild-type and PrPc-transgenic (tga20) mice to 30 min of intraluminal middle cerebral artery occlusion, followed by 3, 24 or 72 h reperfusion, and examined how PrPc levels influence brain injury and cell signaling. In immunohistochemical experiments and Western blots, we show that PrPc expression is absent in the brains of Prnp(0/0) mice, detectable in wild-type controls and approximately 4.0-fold elevated in tga20 mice. We provide evidence that PrPc deficiency increases infarct size by approximately 200%, while transgenic PrPc restores tissue viability, albeit not above levels in wild-type animals. To elucidate the mechanisms underlying Prnp(0/0)-induced injury, we performed Western blots, which revealed increased activities of ERK-1/-2, STAT-1 and caspase-3 in ischemic brains of Prnp(0/0)mice. Our data suggest a role of cytosolic signaling pathways in Prnp(0/0)-induced cell death.

Immunodetection of disease-associated mutant PrP, which accelerates disease in GSS transgenic mice.

The absence of infectivity-associated, protease-resistant prion protein (PrP(Sc)) in the brains of spontaneously sick transgenic (Tg) mice overexpressing PrP linked to Gerstmann-Sträussler Scheinker syndrome, and the failure of gene-targeted mice expressing such PrP to develop disease spontaneously, challenged the concept that mutant PrP expression led to spontaneous prion production. Here, we demonstrate that disease in overexpressor Tg mice is associated with accumulation of protease-sensitive aggregates of mutant PrP that can be immunoprecipitated by the PrP(Sc)-specific monoclonal antibody designated 15B3. Whereas Tg mice expressing multiple transgenes exhibited accelerated disease when inoculated with disease-associated mutant PrP, Tg mice expressing mutant PrP at low levels failed to develop disease either spontaneously or following inoculation. These studies indicate that inoculated mutant PrP from diseased mice promotes the aggregation and accumulation of pre-existing pathological forms of mutant PrP produced as a result of transgene overexpression. Thus, while pathological mutant PrP possesses a subset of PrP(Sc) characteristics, we now show that the attribute of prion transmission suggested by previous studies is more accurately characterized as disease acceleration.

Diagnosis of prion diseases.

Prion diseases are usually diagnosed clinically and confirmed by post-mortem histopathological examination of brain tissue. The only reliable molecular marker for prion diseases is PrP(Sc), the pathological conformer of the prion protein that accumulates in the central nervous system and, to a lesser extent, in lymphoreticular tissues. For BSE, several commercial diagnostic kits based on the post-mortem immunochemical detection of PrP(Sc) in brain tissue are now available. These rapid screening tests have been used in active surveillance of BSE and have greatly improved the detection of infected cattle before their entry into the human food chain. At present, no diagnostic test exists for the detection of prion diseases in live animals or humans. New diagnostic techniques aimed at increasing sensitivity and specificity of PrP(Sc) detection in body fluids and at identifying novel surrogate markers are under development. In this report, we review the classical diagnostic methods as well as present and future tools for the diagnosis of prion diseases.

Soluble dimeric prion protein binds PrP(Sc) in vivo and antagonizes prion disease.

Conversion of cellular prion protein (PrP(C)) into a pathological conformer (PrP(Sc)) is thought to be promoted by PrP(Sc) in a poorly understood process. Here, we report that in wild-type mice, the expression of PrP(C) rendered soluble and dimeric by fusion to immunoglobulin Fcgamma (PrP-Fc(2)) delays PrP(Sc) accumulation, agent replication, and onset of disease following inoculation with infective prions. In infected PrP-expressing brains, PrP-Fc(2) relocates to lipid rafts and associates with PrP(Sc) without acquiring protease resistance, indicating that PrP-Fc(2) resists conversion. Accordingly, mice expressing PrP-Fc(2) but lacking endogenous PrP(C) are resistant to scrapie, do not accumulate PrP-Fc(2)(Sc), and do not transmit disease to others. These results indicate that various PrP isoforms engage in a complex in vivo, whose distortion by PrP-Fc(2) affects prion propagation and scrapie pathogenesis. The unique properties of PrP-Fc(2) suggest that soluble PrP derivatives may represent a new class of prion replication antagonists.