Enrichment of prion protein in exosomes derived from ovine cerebral spinal fluid

Prion diseases are transmissible neurodegenerative disorders affecting humans and a wide variety of animal species including sheep and cattle. The transmissible agent, the prion, is an abnormally folded form (PrP(Sc)) of the host encoded cellular prion protein (PrP(C)). Distribution of the prion protein in the fluids of species susceptible to these diseases is of importance to human health and the iatrogenic spread of prion disease. Aside from blood which is confirmed to be a source of prion infectivity, it is currently unclear which other body fluids harbor a significant transmission risk. In the current study we examined two ovine fluids; pseudo-afferent lymph and cerebral spinal fluid (CSF), for the presence of exosomes and concurrent enrichment of the normal, cellular form of the prion protein (PrP(C)). Here we demonstrate the existence of exosomes in both pseudo-afferent lymph and CSF isolated from sheep. In the CSF derived exosomes we were able to show an enrichment of PrP(C) over unfractionated CSF. This experimental approach suggests that CSF derived exosomes could be used as a novel means of detecting abnormal forms of the prion protein and provide an in vivo link between these vesicles and prion disease pathogenesis.     

Physiological role of the cellular prion protein

The prion protein (PrP) plays a key role in the pathogenesis of prion diseases. However, the normal function of the protein remains unclear. The cellular isoform (PrP(C)) is expressed most abundantly in the brain, but has also been detected in other non-neuronal tissues as diverse as lymphoid cells, lung, heart, kidney, gastrointestinal tract, muscle, and mammary glands. Cell biological studies of PrP contribute to our understanding of PrP(C) function. Like other membrane proteins, PrP(C) is post-translationally processed in the endoplasmic reticulum and Golgi on its way to the cell surface after synthesis. Cell surface PrP(C) constitutively cycles between the plasma membrane and early endosomes via a clathrin-dependent mechanism, a pathway consistent with a suggested role for PrP(C) in cellular trafficking of copper ions. Although PrP(-/-) mice have been reported to have only minor alterations in immune function, PrP(C) is up-regulated in T cell activation and may be expressed at higher levels by specialized classes of lymphocytes. Furthermore, antibody cross-linking of surface PrP(C) modulates T cell activation and leads to rearrangements of lipid raft constituents and increased phosphorylation of signaling proteins. These findings appear to indicate an important but, as yet, ill-defined role in T cell function. Recent work has suggested that PrP(C) is required for self-renewal of haematopoietic stem cells. PrP(C) is highly expressed in the central nervous system, and since this is the major site of prion pathology, most interest has focused on defining the role of PrP(C) in neurones. Although PrP(-/-) mice have a grossly normal neurological phenotype, even when neuronal PrP(C) is knocked out postnatally, they do have subtle abnormalities in synaptic transmission, hippocampal morphology, circadian rhythms, and cognition and seizure threshold. Other postulated neuronal roles for PrP(C) include copper-binding, as an anti- and conversely, pro-apoptotic protein, as a signaling molecule, and in supporting neuronal morphology and adhesion. The prion protein may also function as a metal binding protein such as copper, yielding cellular antioxidant capacity suggesting a role in the oxidative stress homeostasis. Finally, recent observations on the role of PrP(C) in long-term memory open a challenging field.     

Cloning and characterization of ovine immunoglobulin G Fc receptor III (FcγRIII)

Receptors for the Fc regions of immunoglobin G (IgG) play a critical role in immunoregulation and immune defenses against pathogens. In this study, we describe the cloning, eukaryotic expression and IgG subclass specificity of ovine Fc gamma receptor III (FcγRIII). The newly cloned ovine FcγRIII cDNA contains a 940 bp open-reading frame (ORF), and is predicted to encode a 250 amino acid transmembrane glycoprotein composed of two immunoglobulin-like extracellular domains, a transmembrane region and a short cytoplasmic tail. The overall identity of the ovine FcγRIII amino acid sequence to its cattle, pig and human counterparts was 83.2%, 62.0%, 60.7%, respectively. Overlapping PCR was performed with the extracellular domain of ovine FcγRIII and the transmembrane and intracellular region of ovine Fc gamma chain to construct a chimeric receptor. Rosetting analysis showed that transfected COS-7 cells required Fc receptor gamma chain for the expression of FcγRIII on the surface. COS-7 cells expressing FcγRIII were able to bind chicken erythrocytes sensitized with ovine IgG1, but not IgG2. Identification of ovine FcγRIII will further our understanding of the ovine immune system.     

Molecular cloning and characterization of equine Toll-like receptor 9

Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells and recognizes unmethylated bacterial or viral cytosine-phosphate-guanine DNA (CpG-DNA). To investigate innate immune responses through TLR9 signaling pathway in horses, we cloned and characterized equine TLR9. Protein sequence analysis shows that equine TLR9 has a typically conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, three leucine-rich repeat (LRR) motifs, with greater than 82% identity to human, monkey, bovine, canine, feline, porcine and ovine orthologs. Equine TLR9 mRNA expression was characterized for spleen, lymph node, and peripheral blood leukocyte samples. Flow cytometric analysis of equine TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in PMNs, CD4(+) and CD8(+) T-lymphocytes as well as other leukocytes; similar to human TLR9 expression. The conservation of equine TLR9 and high expression profile in leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity.     

Generation of genuine prion infectivity by serial PMCA

Prions are the causative infectious agents of transmissible spongiform encephalopathies (TSEs). They are thought to arise from misfolding and aggregation of the prion protein (PrP). In serial transmission protein misfolding cyclic amplification (sPMCA) experiments, newly formed misfolded and proteinase K-resistant PrP (PrPres) catalysed the structural conversion of cellular prion protein (PrP(C)) as efficiently as PrP(Sc) from the brain of scrapie-infected (263K) hamsters confirming an autocatalytic misfolding cascade as postulated by the prion hypothesis. However, the fact that PrPres generated in vitro was associated with approximately 10 times less infectivity than an equivalent quantity of brain-derived PrP(Sc) casts doubt on the "protein-only" hypothesis of prion propagation and backs theories that suggest there are additional molecular species of infectious PrP or other agent-associated factors. By combining sPMCA with prion delivery on suitable carrier particles we were able to resolve the apparent discrepancy between the amount of PrPres and infectivity which we were then able to relate to differences in the size distribution of PrP aggregates and consecutive differences in regard to biological clearance. These findings demonstrate that we have designed an experimental set-up yielding in vitro generated prions that are indistinguishable from prions isolated from scrapie-infected hamster brain in terms of proteinase K resistance, autocatalytic conversion activity, and - most notably - specific biological infectivity.     

Strategies for eliminating PrP(c) as substrate for prion conversion and for enhancing PrP(Sc) degradation

Prion diseases are fatal neurodegenerative infectious disorders for which no therapeutic or prophylactic regimens exist. Our work aims to eliminate PrP(c) as substrate for the conversion into PrP(Sc) and to increase the cellular clearance capacity of PrP(Sc). In order to achieve the first objective, we used chemical compounds which interfere with the subcellular trafficking of PrP(c), e.g. by intracellular re-routing. Recently, we found that PrP(c) requires cholesterol for cell surface localisation. Treatment with mevinolin significantly reduced the amount of cell surface PrP(c) and led to its accumulation in the Golgi compartment. These data show that cholesterol is essential for the cell surface localisation of PrP(c), which is in turn known to be necessary for the formation of PrP(Sc). Another anti-prion strategy uses RNA and peptide aptamers directed against PrP(c). We have selected peptide aptamers using a constrained peptide library presented on the active site loop of the Escherichia coli protein TrxA in a Y2H screen. Several peptides reproducibly binding to PrP(c) in several assays were identified. Preliminary data indicate that selected peptide aptamers are able to interfere with prion propagation in prion-infected cells. To obtain additive effects we have tried to clarify cellular mechanisms that enable cells to clear prion infectivity. This goal was achieved by selective interference in intracellular signalling pathways which apparently also increase the cellular autophagy machinery. Finally, we have tried to establish an active auto-vaccination approach directed against PrP, which gave some positive preliminary results in the mouse system. This might open the door to classical immunological interference techniques.     

Immunobiotic lactic acid bacteria beneficially regulate immune response triggered by poly(I:C) in porcine intestinal epithelial cells

ABSTRACT: This study analyzed the functional expression of TLR3 in various gastrointestinal tissues from adult swine and shows that TLR3 is expressed preferentially in intestinal epithelial cells (IEC), CD172a+CD11R1high and CD4+ cells from ileal Peyer's patches. We characterized the inflammatory immune response triggered by TLR3 activation in a clonal porcine intestinal epitheliocyte cell line (PIE cells) and in PIE-immune cell co-cultures, and demonstrated that these systems are valuable tools to study in vitro the immune response triggered by TLR3 on IEC and the interaction between IEC and immune cells. In addition, we selected an immunobiotic lactic acid bacteria strain, Lactobacillus casei MEP221106, able to beneficially regulate the anti-viral immune response triggered by poly(I:C) stimulation in PIE cells. Moreover, we deepened our understanding of the possible mechanisms of immunobiotic action by demonstrating that L. casei MEP221106 modulates the interaction between IEC and immune cells during the generation of a TLR3-mediated immune response.     

Immunisation strategies against prion diseases: prime-boost immunisation with a PrP DNA vaccine containing foreign helper T-cell epitopes does not prevent mouse scrapie

Vaccination against prion diseases constitutes a promising approach for the treatment and prevention of the disease. Passive immunisation with antibodies binding to the cellular prion protein (PrP(C)) can protect against prion disease. However, immunotherapeutic strategies with active immunisation are limited due to the immune tolerance against the self-antigen. In order to develop an anti-prion vaccine, we designed a novel DNA fusion vaccine composed of mouse PrP and immune stimulatory helper T-cell epitopes of the tetanus toxin that have previously been reported to break tolerance to other self-antigens. This approach provoked a strong PrP(C)-specific humoral and cellular immune response in PrP null mice, but only low antibody titres were found in vaccinated wild-type mice. Furthermore, prime-boost immunisation with the DNA vaccine and recombinant PrP protein increased antibody titres in PrP null mice, but failed to protect wild-type mice from mouse scrapie.     

Scrapie resistance alleles are not associated with lower prolificity in Rasa Aragonesa sheep

Scrapie is a prion disease characterised by the accumulation of the pathological associated form of cellular prion protein (PrP(SC)) in the central nervous system. Susceptibility to scrapie is associated with polymorphism in the ovine prion protein (PrP) gene. The European Union has implemented scrapie control programs, relying on selective breeding for scrapie resistance; the use of ARR-carrier and the exclusion of VRQ-carrier were recommended. In this study, 4323 individuals from Rasa Aragonesa Sheep breed were genotyped for the PrP gene and the individual estimated breeding values (EBV) for prolificity were calculated. Most represented PrP alleles do not work against prolificity. Only a significant association between VRQ/VRQ genotype and a lower EBV was observed (p = 0.027, eta2 = 0.002). Therefore, avoiding reproduction of VRQ/VRQ individuals would not cause negative effect regarding prolificity.     

Prion protein in sheep urine.

The misfolded form of cellular prion protein (PrP(C)) is the main component of the infectious agent of transmissible spongiform encephalopathies and the validated biomarker for these diseases. The expression of PrP(C) is highest in the central nervous system and has been found in peripheral tissues. Soluble PrP(C) has been detected in cerebrospinal fluid, urine, serum, milk, and seminal plasma. In this study, attempts were made to characterize prion protein in urine samples from normal and scrapie-infected sheep. Urine samples from scrapie-infected sheep and age-matched healthy sheep were collected and analyzed by Western blot following concentration. A protease K-sensitive protein band with a molecular weight of approximately 27-30 kDa was visualized after immunoblotting with anti-PrP monoclonal antibodies to a C-terminal part of PrP(C), but not after immunoblotting with monoclonal antibodies to an N-terminal epitope of PrP(C) or with secondary antibodies only. The amount of PrP(C) in the urine of 49 animals (control group: n = 16; naturally scrapie-infected group: n = 33) was estimated by comparison with known amounts of ovine recombinant PrP in the immunoblot. Background concentration of PrP(C) in urine was found to be 0-0.16 ng/ml (adjusted to the initial nonconcentrated volume of the urine samples). Seven out of 33 naturally scrapie-infected animals had an elevated level (0.3-4.7 ng/ml) of PrP(C) in urine. The origin of PrP(C) in urine and the reason for the increased level of PrP(C) in scrapie-infected sheep urine has yet to be explored.     

Prion agent diversity and species barrier

Mammalian prions are the infectious agents responsible for transmissible spongiform encephalopathies (TSE), a group of fatal, neurodegenerative diseases, affecting both domestic animals and humans. The most widely accepted view to date is that these agents lack a nucleic acid genome and consist primarily of PrP(Sc), a misfolded, aggregated form of the host-encoded cellular prion protein (PrP(C)) that propagates by autocatalytic conversion and accumulates mainly in the brain. The BSE epizooty, allied with the emergence of its human counterpart, variant CJD, has focused much attention on two characteristics that prions share with conventional infectious agents. First, the existence of multiple prion strains that impose, after inoculation in the same host, specific and stable phenotypic traits such as incubation period, molecular pattern of PrP(Sc) and neuropathology. Prion strains are thought to be enciphered within distinct PrP(Sc) conformers. Second, a transmission barrier exists that restricts the propagation of prions between different species. Here we discuss the possible situations resulting from the confrontation between species barrier and prion strain diversity, the molecular mechanisms involved and the potential of interspecies transmission of animal prions, including recently discovered forms of TSE in ruminants.     

An overview of transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders of humans and animals associated with an accumulation of abnormal isoforms of prion protein (PrP) in nerve cells. The pathogenesis of TSEs involves conformational conversions of normal cellular PrP (PrP(c)) to abnormal isoforms of PrP (PrP(Sc)). While the protein-only hypothesis has been widely accepted as a causal mechanism of prion diseases, evidence from more recent research suggests a possible involvement of other cellular component(s) or as yet undefined infectious agent(s) in PrP pathogenesis. Although the underlying mechanisms of PrP strain variation and the determinants of interspecies transmissibility have not been fully elucidated, biochemical and molecular findings indicate that bovine spongiform encephalopathy in cattle and new-variant Creutzfeldt-Jakob disease in humans are caused by indistinguishable etiological agent(s). Cumulative evidence suggests that there may be risks of humans acquiring TSEs via a variety of exposures to infected material. The development of highly precise ligands is warranted to detect and differentiate strains, allelic variants and infectious isoforms of these PrPs. This article describes the general features of TSEs and PrP, the current understanding of their pathogenesis, recent advances in prion disease diagnostics, and PrP inactivation.     

Cloning and characterization of ovine immunoglobulin G Fc receptor II (FcγRII)

Immunoglobulin G (IgG) Fc receptors (FcγRs) bind to immune complexes through interactions with the Fc region of IgG to initiate or inhibit the defense mechanism of the leukocytes on which they are expressed. In this study, we describe the cloning, sequencing and characterization of ovine FcγRII. By screening a translated expression sequence tag (EST) database with the protein sequence of bovine IgG Fc receptor II, we identified a putative ovine homologue. Using rapid amplification of cDNA ends (RACE), we isolated the cDNA encoding ovine FcγRII from peripheral blood leucocyte RNA. The ovine FcγRII cDNA contains an 894 bp open-reading frame, encoding a 297 amino acid transmembrane glycoprotein composed of two immunoglobulin-like extracellular domains, a transmembrane region and a cytoplasmic tail with an immunoreceptor tyrosine-based inhibitory motif (ITIM). The glycoprotein encoded by the cloned cDNA was then expressed on the surface of COS-7 cells and immunoglobulin-binding assays show that it binds ovine IgG1, but not IgG2. Identification of the ovine FcγRII will aid in the understanding of the molecular basis of IgG–FcγR interaction.     

Identification of heterologous monoclonal antibodies that cross-react with cervine leukocyte subpopulations

The degree to which cross-reactivity between monoclonal antibodies developed against cells of the human, mouse, bovine and ovine immune systems, and cells of the cervine immune system occurs was investigated. It was found that within the ruminants a considerable degree of cross-reactivity does exist while there is virtually none between the cervine and murine or human systems. The highest incidence of cross-reactivity was found between ovine monoclonals and cervine leukocytes (46% cross-reactive) with 25% of bovine monoclonal antibodies cross-reacting with deer leukocytes. Ovine monoclonals were found to be the most useful in identifying a wide range of cervine leukocyte subpopulations. Bioassays showed that ovine anti-class I and II monoclonals detected molecules on cervine leukocytes that are functionally similar to MHC antigens. The possibility that cross-reactive monoclonals detect similar subpopulations in both the homologous and heterologous species is discussed.     

Immunophenotypic and functional effects of bunker C fuel oil on the immune system of American mink (Mustela vison)

The relationship between exposure to environmental contaminants and immunotoxicity in vulnerable marine species is unknown. In this study, we used American mink (Mustela vision) as a surrogate species for the sea otter to examine the immunotoxic effects of chronic exposure to a low concentration of bunker C fuel oil (500 ppm admixed in the feed for 113-118 days). The mink immune system was monitored over time by flow cytometric analysis for alterations in the immunophenotype of blood lymphocytes and monocytes and by mitogen-stimulated proliferation assays for changes in peripheral blood mononuclear cell function. Fuel oil exposure caused a mild, yet significant (P < 0.05) increase in the absolute numbers of specific peripheral blood lymphocyte subsets (CD3+T cells) and monocytes, an increase in the level of expression of functionally significant cell surface proteins (MHC II, CD18), and an increase in mitogen-induced mononuclear cell proliferative responses. This heightened state of cellular activation along with the increase in specific cell surface protein expression on both the innate and adaptive immune cells is similar to the pro-inflammatory or "adjuvant-like" effect described in laboratory models of polycyclic aromatic hydrocarbon exposure in other species. These results show the benefits of using a controlled laboratory model for detecting and characterizing subtle petroleum oil-induced perturbations in immune responses. In addition this study establishes a framework for studying the effects of environmental petroleum oil exposure on the immune system of free-ranging marine mammals. Expansion of these studies to address biolgical significance is warranted.     

Characterisation of antibodies to bovine Toll-like receptor (TLR)-2 and cross-reactivity with ovine TLR2

Host recognition of conserved pathogen-associated molecular patterns (PAMPs) and their interactions with pattern-recognition receptors, including the Toll-like receptors (TLR) is essential for innate immune response induction. The TLR1 family (TLR1, 2, 6 and 10) is involved in the recognition of gram-positive and gram-negative bacteria and heterodimers of TLR1 or TLR6 with TLR2 are crucial for the identification of several PAMPs. Studies on cell surface expression of TLR in ruminants are hampered by the lack of specific antibodies and no convincingly cross-reactive anti-human antibodies have been described so far. We describe herein four antibodies which recognise bovine TLR2. Differences in TLR2 expression were evident on bovine antigen presenting cells with high level expression on peripheral blood monocytes and monocyte-derived macrophages. Lower levels of expression were evident on dendritic cell populations derived in vitro and ex vivo, and on alveolar macrophages. One of the antibodies recognised TLR2 expression on ovine peripheral blood monocytes. The identification of antibodies specific for bovine and ovine TLR2 will facilitate studies of the role of this important PRR in the initiation of immune responses to important pathogens.     

Jaagsiekte sheep retrovirus found in milk macrophages but not in milk lymphocytes or mammary gland epithelia of naturally infected sheep

Jaagsiekte sheep retrovirus (JSRV) causes ovine pulmonary adenocarcinoma. JSRV can be transmitted via infected colostrum or milk, which contain somatic cells (SCs) harboring JSRV provirus. Nevertheless, the cell types involved in this form of transmission and the involvement of the mammary gland remain unknown. We separated adherent cells (macrophages and monocytes) by plastic adherence, and lymphocytes (CD4+ and CD8+ T cells, and B cells) by flow cytometry, from SCs in milk samples from 12 naturally infected, PCR blood test JSRV–positive, subclinical ewes. These cell populations were tested by PCR to detect JSRV provirus. The ewes were euthanized, and mammary gland samples were analyzed immunohistochemically to detect JSRV surface protein. We did not detect JSRV provirus in any milk lymphocyte population, but milk adherent cells were positive in 3 of 12 sheep, suggesting a potential major role of this population in the lactogenic transmission of JSRV. Immunohistochemistry did not reveal positive results in mammary epithelial cells, pointing to a lack of participation of the mammary gland in the biological cycle of JSRV and reducing the probability of excretion of free viral particles in colostrum or milk.     

Detergents modify proteinase K resistance of PrP Sc in different transmissible spongiform encephalopathies (TSEs)

Prion diseases are diagnosed by the detection of their proteinase K-resistant prion protein fragment (PrP(Sc)). Various biochemical protocols use different detergents for the tissue preparation. We found that the resistance of PrP(Sc) against proteinase K may vary strongly with the detergent used. In our study, we investigated the influence of the most commonly used detergents on eight different TSE agents derived from different species and distinct prion disease forms. For a high throughput we used a membrane adsorption assay to detect small amounts of prion aggregates, as well as Western blotting. Tissue lysates were prepared using DOC, SLS, SDS or Triton X-100 in different concentrations and these were digested with various amounts of proteinase K. Detergents are able to enhance or diminish the detectability of PrP(Sc) after proteinase K digestion. Depending on the kind of detergent, its concentration - but also on the host species that developed the TSE and the disease form or prion type - the detectability of PrP(Sc) can be very different. The results obtained here may be helpful during the development or improvement of a PrP(Sc) detection method and they point towards a detergent effect that can be additionally used for decontamination purposes. A plausible explanation for the detergent effects described in this article could be an interaction with the lipids associated with PrP(Sc) that may stabilize the aggregates.     

The first Canadian indigenous case of bovine spongiform encephalopathy (BSE) has molecular characteristics for prion protein that are similar to those of BSE in the United Kingdom but differ from those of chronic wasting disease in captive elk and deer

Brain tissue from a case of bovine spongiform encephalopathy (BSE) from Alberta was subjected to a Western immunoblotting technique to ascertain the molecular profile of any disease-specific, abnormal prion protein, that is, prion protein that is protease-resistant (PrP(res)). This technique can discriminate between isolates from BSE, ovine scrapie, and sheep experimentally infected with BSE. Isolates of brain tissue from the BSE case in Alberta, 3 farmed elk with chronic wasting disease (CWD) from different parts of Saskatchewan, and 1 farmed white-tailed deer with CWD from Edmonton, Alberta, were examined alongside isolates of brain tissue from BSE, ovine scrapie, and sheep experimentally infected with BSE from the United Kingdom (UK). The molecular weights of PrP(res) and the cross reactions to 2 specific monoclonal antibodies (mAbs) were determined for each sample. The BSE isolates from Canada and the UK had very similar PrP(res) molecular weights and reacted with only 1 of the 2 mAbs. The PrP(res) isolated from both elk and white-tailed deer with CWD had a higher molecular weight profile than did the corresponding PrP(res) from the scrapie and BSE isolates. The PrP(res) from CWD cases cross reacted with both mAbs, a property shared with PrP(res) in isolates from scrapie but not with PrP(res) isolates from BSE or sheep experimentally infected with BSE. The results from this study seem to confirm that the PrP(res) isolated from the BSE case in Alberta has similar molecular properties to the PrP(res) isolated from a BSE case in the UK, and that it differs in its molecular and immunological characteristics from the CWD and scrapie cases studied.