Young mice administered adult doses of AAV5-hFVIII-SQ achieve therapeutic factor VIII expression into adulthood.

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) gene transfer provided reduced bleeding for adult clinical trial participants with severe hemophilia A. However, pediatric outcomes are unknown. Using a mouse model of hemophilia A, we investigated the effect of vector dose and age at treatment on transgene production and persistence. We dosed AAV5-hFVIII-SQ to neonatal and adult mice based on body weight or at a fixed dose and assessed human factor VIII-SQ variant (hFVIII-SQ) expression through 16 weeks. AAV5-hFVIII-SQ dosed per body weight in neonatal mice did not result in meaningful plasma hFVIII-SQ protein levels in adulthood. When treated with the same total vector genomes per mouse as adult mice, neonates maintained hFVIII-SQ expression into adulthood, although plasma levels were 3- to 4-fold lower versus mice dosed as adults. Mice <1 week old initially exhibited high hFVIII-SQ plasma levels and maintained meaningful levels into adulthood, despite a partial decline potentially due to age-related body mass and blood volume increases. Spatial transduction patterns differed between mice dosed as neonates versus adults. No features of hepatotoxicity or endoplasmic reticulum stress were observed with dosing at any age. These data suggest that young mice require the same total vector genomes as adult mice to sustain hFVIII-SQ plasma levels.

Case Report: Neuroblastoma-Like Schwannoma in a Domestic Short-Haired Cat.

An axillary mass was detected in a 6-year-old, neutered, male, domestic short-haired cat during a wellness exam. Gross examination following surgical removal revealed a discrete, deep subcutaneous, discoid mass that was between 0.5- and 0.7-cm-in-diameter and diffusely firm and white. Histologically, the mass was well-demarcated, partially encapsulated, and expanded the panniculus carnosus. It was composed of tightly packed, giant rosettes of radially arranged fusiform cells stacked in one to 10 layers with peripherally palisading nuclei and with centrally oriented, fibrillary, cytoplasmic processes, and collagenous fibers. Laminin immunoreactivity and ultrastructural examination highlighted a continuous basal lamina outside the plasma membrane of each neoplastic cell. Neoplastic cells were immunoreactive for GFAP, S100, periaxin, and Sox-10 and were immunonegative for synaptophysin, smooth muscle actin, and pancytokeratin. Collective findings were consistent with a diagnosis of neuroblastoma-like schwannoma. This is the first veterinary report of this rare variant of benign schwannoma.

Society of Toxicologic Pathology Neuropathology Interest Group Article: Neuropathologic Findings in Nonhuman Primates Associated With Administration of Biomolecule-Based Test Articles.

The increasing specificity of novel druggable targets coupled with the complexity of emerging therapeutic modalities for treating human diseases has created a growing need for nonhuman primates (NHPs) as models for translational drug discovery and nonclinical safety assessment. In particular, NHPs are critical for investigating potential unexpected/undesired on-target and off-target liabilities associated with administration of candidate biotherapeutics (nucleic acids, proteins, viral gene therapy vectors, etc.) to treat nervous system disorders. Nervous system findings unique to or overrepresented in NHPs administered biomolecule-based ("biologic") test articles include mononuclear cell infiltration in most neural tissues for all biomolecule classes as well as neuronal necrosis with glial cell proliferation in sensory ganglia for certain viral vectors. Such test article-related findings in NHPs often must be differentiated from procedural effects (e.g., local parenchymal or meningeal reactions associated with an injection site or implanted catheter to administer a test article directly into the central nervous system) or spontaneous background findings (e.g., neuronal autophagy in sensory ganglia).

Exosome-mediated genetic reprogramming of tumor-associated macrophages by exoASO-STAT6 leads to potent monotherapy antitumor activity.

Effectiveness of checkpoint immunotherapy in cancer can be undermined by immunosuppressive tumor-associated macrophages (TAMs) with an M2 phenotype. Reprogramming TAMs toward a proinflammatory M1 phenotype is a novel approach to induce antitumor immunity. The M2 phenotype is controlled by key transcription factors such as signal transducer and activator of transcription 6 (STAT6), which have been "undruggable" selectively in TAMs. We describe an engineered exosome therapeutic candidate delivering an antisense oligonucleotide (ASO) targeting STAT6 (exoASO-STAT6), which selectively silences STAT6 expression in TAMs. In syngeneic models of colorectal cancer and hepatocellular carcinoma, exoASO-STAT6 monotherapy results in >90% tumor growth inhibition and 50 to 80% complete remissions. Administration of exoASO-STAT6 leads to induction of nitric oxide synthase 2 (NOS2), an M1 macrophage marker, resulting in remodeling of the tumor microenvironment and generation of a CD8 T cell-mediated adaptive immune response. Collectively, exoASO-STAT6 represents the first platform targeting transcription factors in TAMs in a highly selective manner.

Nerve Fiber Immunohistochemical Panel Discriminates between Nerve Sheath and Perivascular Wall Tumors.

Benign and malignant nerve sheath tumors (NST) pose a major challenge in routine diagnostic anatomic pathology because of shared histomorphological features with other soft-tissue tumors (STT). As a result, NST are often diagnosed as STT, a broad category that encompasses various entities including perivascular wall tumors (PWT) and that represents approximately 15% of all skin tumors in dogs. Immunohistochemistry (IHC) can assist the identification of histologic subtypes of STT. This IHC pilot study applies various markers largely expressed by peripheral nerves to twelve benign and six malignant NST and determines the intratumoral protein expression of laminin, periaxin-1, Sox-10 and S-100 in the NST subtypes. Furthermore, this study assesses the usefulness of peripheral nerve markers applied to diagnostic work cases and demonstrates the relevance of laminin expression patterns, periaxin-1 and Sox-10 in assisting the differentiation of NST from other STT, in particular from PWT.

Quantitative Assessment of Neuroinflammation, Myelinogenesis, Demyelination, and Nerve Fiber Regeneration in Immunostained Sciatic Nerves From Twitcher Mice With a Tissue Image Analysis Platform.

Scoring demyelination and regeneration in hematoxylin and eosin-stained nerves poses a challenge even for the trained pathologist. This article demonstrates how combinatorial multiplex immunohistochemistry (IHC) and quantitative digital pathology bring new insights into the peripheral neuropathogenesis of the Twitcher mouse, a model of Krabbe disease. The goal of this investigational study was to integrate modern pathology tools to traditional anatomic pathology microscopy workflows, in order to generate quantitative data in a large number of samples, and aid the understanding of complex disease pathomechanisms. We developed a novel IHC toolkit using a combination of CD68, periaxin-1, phosphorylated neurofilaments and SOX-10 to interrogate inflammation, myelination, axonal size, and Schwann cell counts in sciatic nerves from 17-, 21-, 25-, and 35-day-old wild-type and Twitcher mice using self-customized digital image algorithms. Our quantitative analyses highlight that nerve macrophage infiltration and interstitial expansion are the earliest detectable changes in Twitcher nerves. By 17 days of age, while the diameter of axons is small, the number of myelinated axons is still normal. However, from 21 days onward Twitcher nerves contain 75% of wild-type myelinated nerve fiber numbers despite containing 3 times more Schwann cells. In 35-day-old Twitcher mice when demyelination is detectable, nerve myelination drops to 50%.

Prednisolone Does Not Regulate Factor VIII Expression in Mice Receiving AAV5-hFVIII-SQ: Valoctocogene Roxaparvovec.

AAV5-hFVIII-SQ (valoctocogene roxaparvovec) is an adeno-associated virus (AAV)-mediated gene therapy vector containing a B-domain-deleted human factor VIII (hFVIII-SQ) transgene. In a phase 1/2 clinical study of AAV5-hFVIII-SQ for severe hemophilia A (FVIII < 1 IU/dL), participants received prednisolone to mitigate potential immune-mediated reactions to the gene therapy and demonstrated concomitant elevations in plasma FVIII levels, following a single administration of AAV5-hFVIII-SQ. To assess whether prednisolone is capable of directly modulating transgene expression or levels of circulating hepatic enzymes, C57BL/6 mice were given intravenous vehicle, 2 × 1013 vector genomes (vg)/kg AAV5-hFVIII-SQ, or 6 × 1013 vg/kg AAV5-hFVIII-SQ, followed by either daily oral prednisolone or water. Mice were euthanized 4 or 13 weeks after vector administration. Hepatic hFVIII-SQ DNA, RNA, and protein (immunostaining), plasma hFVIII-SQ protein and FVIII activity, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured. Liver hFVIII-SQ DNA, RNA, and plasma hFVIII-SQ protein and activity increased in a dose-dependent manner, with or without prednisolone. In summary, chronic prednisolone treatment in mice treated with AAV5-hFVIII-SQ did not modulate levels of liver hFVIII-SQ DNA, RNA, or the percentage and distribution of hFVIII-SQ-positive hepatocytes, nor did it regulate levels of plasma hFVIII-SQ protein or activity, or affect levels of plasma AST or ALT.

TSPO PET Using [18F]PBR111 Reveals Persistent Neuroinflammation Following Acute Diisopropylfluorophosphate Intoxication in the Rat.

Acute intoxication with organophosphates (OPs) can trigger status epilepticus followed by persistent cognitive impairment and/or electroencephalographic abnormalities. Neuroinflammation is widely posited to influence these persistent neurological consequences. However, testing this hypothesis has been challenging, in part because traditional biometrics preclude longitudinal measures of neuroinflammation within the same animal. Therefore, we evaluated the performance of noninvasive positron emission tomography (PET), using the translocator protein (TSPO) radioligand [18F]PBR111 against classic histopathologic measures of neuroinflammation in a preclinical model of acute intoxication with the OP diisopropylfluorophosphate (DFP). Adult male Sprague Dawley rats administered pyridostigmine bromide (0.1 mg/kg, im) 30 min prior to administration of DFP (4 mg/kg, sc), atropine sulfate (2 mg/kg, im) and 2-pralidoxime (25 mg/kg, im) exhibited moderate-to-severe seizure behavior. TSPO PET performed prior to DFP exposure and at 3, 7, 14, 21, and 28 days postexposure revealed distinct lesions, as defined by increased standardized uptake values (SUV). Increased SUV showed high spatial correspondence to immunohistochemical evidence of neuroinflammation, which was corroborated by cytokine gene and protein expression. Regional SUV metrics varied spatiotemporally with days postexposure and correlated with the degree of neuroinflammation detected immunohistochemically. Furthermore, SUV metrics were highly correlated with seizure severity, suggesting that early termination of OP-induced seizures may be critical for attenuating subsequent neuroinflammatory responses. Normalization of SUV values to a cerebellar reference region improved correlations to all outcome measures and seizure severity. Collectively, these results establish TSPO PET using [18F]PBR111 as a robust, noninvasive tool for longitudinal monitoring of neuroinflammation following acute OP intoxication.

Glomeruloid Microvascular Proliferation, Desmoplasia, and High Proliferative Index as Potential Indicators of High Grade Canine Choroid Plexus Tumors.

Choroid plexus tumors (CPT) are intraventricular neoplasms accounting for 10% of all primary central nervous system tumors in dogs. They are frequently classified according to the human WHO classification into choroid plexus papilloma (CPP, grade I), atypical CPP (aCPP, grade II), and choroid plexus carcinoma (CPC, grade III). Histological features observed in canine CPT such as increased vascular density (IVD) and glomeruloid microvascular proliferation (GMVP) are not part of the WHO classification. This multi-centric study aimed to investigate tumor-associated vascular hyperplasia in dogs by determining the prevalence of GMVP and IVD in 52 canine CPT and their association with tumor grade. In addition, the expression of angiogenic factors was assessed by immunohistochemistry in 25 tumors to investigate the pathogenesis of tumor-associated vascular hyperplasia. Based on the classical histological hallmarks, this study of 52 CPT identified 22 (42%) CPP (grade I) and 30 of (58%) CPC (grade III). GMVP was more prevalent in CPC (13/30; 43%) than CPP (1/22; 4%), whereas IVD occurred to a similar extent in CPP and CPC. Desmoplasia was more common in CPC (19/30; 63%) than CPP (2/22; 9%), and similarly, the proliferative index (PI) of neoplastic epithelium was significantly higher in CPC (5.14%) than CPP (0.94%). The majority of CPT expressed platelet-derived growth factor (PDGF), PDGFRα, PDGFRß, and vascular endothelial growth factor (VEGF) irrespective of tumor grade or tumor-associated vascular hyperplasia. These results suggest that tumor-associated GMVP, desmoplasia, and PI may serve as histological indicators of malignancy in CPT.

From the Cover: MagneticResonance Imaging Reveals Progressive Brain Injury in Rats Acutely Intoxicated With Diisopropylfluorophosphate.

Acute intoxication with organophosphates (OPs) can trigger seizures that progress to status epilepticus, and survivors often exhibit chronic neuropathology, cognitive impairment, affective disorders, and/or electroencephalographic abnormalities. Understanding how acute injury transitions to persistent neurological sequelae is critical to developing medical countermeasures for mitigating damage following OP-induced seizures. Here, we used in vivo magnetic resonance imaging (MRI) to monitor the spatiotemporal patterns of neuropathology for 1 month after acute intoxication with diisopropylfluorophosphate (DFP). Adult male Sprague Dawley rats administered pyridostigmine bromide (0.1 mg/kg, im) 30 min prior to successive administration of DFP (4 mg/kg, sc), atropine sulfate (2 mg/kg, im), and 2-pralidoxime (25 mg/kg, im) exhibited moderate-to-severe seizure behavior. T2-weighted and diffusion-weighted MR imaging prior to DFP exposure and at 3, 7, 14, 21, or 28 days postexposure revealed prominent lesions, tissue atrophy, and ventricular enlargement in discrete brain regions. Lesions varied in intensity and/or extent over time, with the overall magnitude of injury strongly influenced by seizure severity. Importantly, lesions detected by MRI correlated spatially and temporally with histological evidence of brain pathology. Analysis of histogram parameters extracted from frequency distributions of regional apparent diffusion coefficient (ADC) values identified the standard deviation and 90th percentile of the ADC as robust metrics for quantifying persistent and progressive neuropathological changes. The interanimal and interregional variations observed in lesion severity and progression, coupled with potential reinjury following spontaneous recurrent seizures, underscore the advantages of using in vivo imaging to longitudinally monitor neuropathology and, ultimately, therapeutic response, following acute OP intoxication.

Editor's Highlight: Spatiotemporal Progression and Remission of Lesions in the Rat Brain Following Acute Intoxication With Diisopropylfluorophosphate.

Similar to organophosphate (OP) nerve agents, diisopropylfluorophosphate (DFP) rapidly and irreversibly inhibits acetylcholinesterase, leading to convulsions that can progress to status epilepticus (SE). However, in contrast to the OP nerve agents, the long-term consequences of DFP-induced SE are not well known. Thus, we characterized the spatiotemporal profile of neuropathology during the first 2 months following acute DFP intoxication. Adult, male Sprague Dawley rats administered pyridostigmine bromide (0.1 mg/kg, im) 30 min prior to successive administration of DFP (4 mg/kg, sc), atropine sulfate (2 mg/kg, im), and 2-pralidoxime (25 mg/kg, im), exhibited moderate-to-severe seizure behavior, yet survived until euthanized at 0.5 to 60 days post exposure. Analyses of brains and hearts stained with hematoxylin-eosin, or of brains immunostained for neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), or ionized binding adapter molecule 1 (IBA1), revealed progressive neuronal cell death, neuroinflammation, and tissue remodeling across limbic brain regions and the cerebral cortex, with no detectable pathology in the cerebellum or the heart. The lesion type and progression varied according to brain region and time after exposure. Across multiple brain regions, neuronal necrosis peaked after the first week, and neuroinflammation persisted at least 2 months after intoxication. Notably, mineralization was observed at later times in the thalamus, and to a more limited extent, in the hippocampus. Lesion severity was influenced by the initial seizure severity, and spontaneous recurrent seizures were associated with more severe brain damage. These findings parallel descriptions of neuropathology in preclinical models of acute intoxication with OP nerve agents, and other seizurogenic chemicals, suggesting conserved mechanisms of pathology downstream of chemical-induced SE.

Sarcocystis fayeri in skeletal muscle of horses with neuromuscular disease.

Recent reports of Sarcocystis fayeri-induced toxicity in people consuming horse meat warrant investigation on the prevalence and molecular characterization of Sarcocystis spp. infection in horses. Sarcocysts in skeletal muscle of horses have been commonly regarded as an incidental finding. In this study, we investigated the prevalence of sarcocysts in skeletal muscle of horses with neuromuscular disease. Our findings indicated that S. fayeri infection was common in young mature horses with neuromuscular disease and could be associated with myopathic and neurogenic processes. The number of infected muscles and number of sarcocysts per muscle were significantly higher in diseased than in control horses. S. fayeri was predominantly found in low oxidative highly glycolytic myofibers. This pathogen had a high glycolytic metabolism. Common clinical signs of disease included muscle atrophy, weakness with or without apparent muscle pain, gait deficits, and dysphagia in horses with involvement of the tongue and esophagus. Horses with myositis were lethargic, apparently painful, stiff, and reluctant to move. Similar to humans, sarcocystosis and cardiomyopathy can occur in horses. This study did not establish causality but supported a possible association (8.9% of cases) with disease. The assumption of Sarcocysts spp. being an incidental finding in every case might be inaccurate.

Stability of murine scrapie strain 87V after passage in sheep and comparison with the CH1641 ovine strain.

Breed- and prion protein (PRNP) genotype-related disease phenotype variability has been observed in sheep infected with the 87V murine scrapie strain. Therefore, the stability of this strain was tested by inoculating sheep-derived 87V brain material back into VM mice. As some sheep-adapted 87V disease phenotypes were reminiscent of CH1641 scrapie, transgenic mice (Tg338) expressing ovine prion protein (PrP) were inoculated with the same sheep-derived 87V sources and with CH1641. Although at first passage in VM mice the sheep-derived 87V sources showed some divergence from the murine 87V control, all the characteristics of murine 87V infection were recovered at second passage from all sheep sources. These included 100 % attack rates and indistinguishable survival times, lesion profiles, immunohistochemical features of disease-associated PrP accumulation in the brain and PrP biochemical properties. All sheep-derived 87V sources, as well as CH1641, were transmitted to Tg338 mice with identical clinical, pathological, immunohistochemical and biochemical features. While this might potentially indicate that sheep-adapted 87V and CH1641 are the same strain, profound divergences were evident, as murine 87V was unable to infect Tg338 mice but was lethal for VM mice, while the reverse was true for CH1641. These combined data suggest that: (i) murine 87V is stable and retains its properties after passage in sheep; (ii) it can be isolated from sheep showing a CH1641-like or a more conventional scrapie phenotype; and (iii) sheep-adapted 87V scrapie, with conventional or CH1641-like phenotype, is biologically distinct from experimental CH1641 scrapie, despite the fact that they behave identically in a single transgenic mouse line.

Feasibility Study of Canine Epidermal Neural Crest Stem Cell Transplantation in the Spinal Cords of Dogs.

UNLABELLED: This pilot feasibility study aimed to determine the outcome of canine epidermal neural crest stem cell (cEPI-NCSC) grafts in the normal spinal cords of healthy bred-for-research dogs. This included developing novel protocols for (a) the ex vivo expansion of cEPI-NCSCs, (b) the delivery of cEPI-NCSCs into the spinal cord, and (c) the labeling of the cells and subsequent tracing of the graft in the live animal by magnetic resonance imaging. A total of four million cEPI-NCSCs were injected into the spinal cord divided in two locations. Differences in locomotion at baseline and post-treatment were evaluated by gait analysis and compared with neurological outcome and behavioral exams. Histopathological analyses of the spinal cords and cEPI-NCSC grafts were performed at 3 weeks post-transplantation. Neurological and gait parameters were minimally affected by the stem cell injection. cEPI-NCSCs survived in the canine spinal cord for the entire period of investigation and did not migrate or proliferate. Subsets of cEPI-NCSCs expressed the neural crest stem cell marker Sox10. There was no detectable expression of markers for glial cells or neurons. The tissue reaction to the cell graft was predominantly vascular in addition to a degree of reactive astrogliosis and microglial activation. In the present study, we demonstrated that cEPI-NCSC grafts survive in the spinal cords of healthy dogs without major adverse effects. They persist locally in the normal spinal cord, may promote angiogenesis and tissue remodeling, and elicit a tissue response that may be beneficial in patients with spinal cord injury. SIGNIFICANCE: It has been established that mouse and human epidermal neural crest stem cells are somatic multipotent stem cells with proved innovative potential in a mouse model of spinal cord injury (SCI) offering promise of a valid treatment for SCI. Traumatic SCI is a common neurological problem in dogs with marked similarities, clinically and pathologically, to the syndrome in people. For this reason, dogs provide a readily accessible, clinically realistic, spontaneous model for evaluation of epidermal neural crest stem cells therapeutic intervention. The results of this study are expected to give the baseline data for a future clinical trial in dogs with traumatic SCI.

Correlation between infectivity and disease associated prion protein in the nervous system and selected edible tissues of naturally affected scrapie sheep.

The transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative disorders characterised by the accumulation of a pathological form of a host protein known as prion protein (PrP). The validation of abnormal PrP detection techniques is fundamental to allow the use of high-throughput laboratory based tests, avoiding the limitations of bioassays. We used scrapie, a prototype TSE, to examine the relationship between infectivity and laboratory based diagnostic tools. The data may help to optimise strategies to prevent exposure of humans to small ruminant TSE material via the food chain. Abnormal PrP distribution/accumulation was assessed by immunohistochemistry (IHC), Western blot (WB) and ELISA in samples from four animals. In addition, infectivity was detected using a sensitive bank vole bioassay with selected samples from two of the four sheep and protein misfolding cyclic amplification using bank vole brain as substrate (vPMCA) was also carried out in selected samples from one animal. Lymph nodes, oculomotor muscles, sciatic nerve and kidney were positive by IHC, WB and ELISA, although at levels 100-1000 fold lower than the brain, and contained detectable infectivity by bioassay. Tissues not infectious by bioassay were also negative by all laboratory tests including PMCA. Although discrepancies were observed in tissues with very low levels of abnormal PrP, there was an overall good correlation between IHC, WB, ELISA and bioassay results. Most importantly, there was a good correlation between the detection of abnormal PrP in tissues using laboratory tests and the levels of infectivity even when the titre was low. These findings provide useful information for risk modellers and represent a first step toward the validation of laboratory tests used to quantify prion infectivity, which would greatly aid TSE risk assessment policies.

Susceptibility of European red deer (Cervus elaphus elaphus) to alimentary challenge with bovine spongiform encephalopathy.

European red deer (Cervus elaphus elaphus) are susceptible to the agent of bovine spongiform encephalopathy, one of the transmissible spongiform encephalopathies, when challenged intracerebrally but their susceptibility to alimentary challenge, the presumed natural route of transmission, is unknown. To determine this, eighteen deer were challenged via stomach tube with a large dose of the bovine spongiform encephalopathy agent and clinical signs, gross and histological lesions, presence and distribution of abnormal prion protein and the attack rate recorded. Only a single animal developed clinical disease, and this was acute with both neurological and respiratory signs, at 1726 days post challenge although there was significant (27.6%) weight loss in the preceding 141 days. The clinically affected animal had histological lesions of vacuolation in the neuronal perikaryon and neuropil, typical of transmissible spongiform encephalopathies. Abnormal prion protein, the diagnostic marker of transmissible encephalopathies, was primarily restricted to the central and peripheral nervous systems although a very small amount was present in tingible body macrophages in the lymphoid patches of the caecum and colon. Serial protein misfolding cyclical amplification, an in vitro ultra-sensitive diagnostic technique, was positive for neurological tissue from the single clinically diseased deer. All other alimentary challenged deer failed to develop clinical disease and were negative for all other investigations. These findings show that transmission of bovine spongiform encephalopathy to European red deer via the alimentary route is possible but the transmission rate is low. Additionally, when deer carcases are subjected to the same regulations that ruminants in Europe with respect to the removal of specified offal from the human food chain, the zoonotic risk of bovine spongiform encephalopathy, the cause of variant Creutzfeldt-Jakob disease, from consumption of venison is probably very low.

Susceptibility to scrapie and disease phenotype in sheep: cross-PRNP genotype experimental transmissions with natural sources.

It has long been established that the sheep Prnp genotype influences the susceptibility to scrapie, and some studies suggest that it can also determine several aspects of the disease phenotype. Other studies, however, indicate that the source of infection may also play a role in such phenotype. To address this question an experiment was set up in which either of two different natural scrapie sources, AAS from AA136 Suffolk and VVC from VV136 Cheviot sheep, were inoculated into AA136, VA136 and VV136 sheep recipients (n = 52). The immunohistochemical (IHC) profile of disease-associated PrP (PrPd) accumulation in the brain of recipient sheep was highly consistent upon codon 136 homologous and semi-homologous transmission, but could be either similar to or different from those of the inoculum donors. In contrast, the IHC profiles were highly variable upon heterologous transmission (VVC to AA136 and AAS to VV136). Furthermore, sheep of the same Prnp genotype could exhibit different survival times and PrPd profiles depending on the source of infection, and a correlation was observed between IHC and Western blot profiles. It was found that additional polymorphisms at codons 112 or 141 of AA136 recipients resulted in a delayed appearance of clinical disease or even in protection from infection. The results of this study strongly suggest that the scrapie phenotype in sheep results from a complex interaction between source, donor and recipient factors, and that the Prnp genotype of the recipient sheep does not explain the variability observed upon codon 136 heterologous transmissions, arguing for other genetic factors to be involved.

Disease phenotype in sheep after infection with cloned murine scrapie strains.

Prion diseases exhibit different disease phenotypes in their natural hosts and when transmitted to rodents, and this variability is regarded as indicative of prion strain diversity. Phenotypic characterization of scrapie strains in sheep can be attempted by histological, immunohistochemical and biochemical approaches, but it is widely considered that strain confirmation and characterization requires rodent bioassay. Examples of scrapie strains obtained from original sheep isolates by serial passage in mice include ME7, 79A, 22A and 87V. In order to address aspects of prion strain stability across the species barrier, we transmitted the above murine strains to sheep of different breeds and susceptible Prnp genotypes. The experiment included 40 sheep dosed by the oral route alone and 36 sheep challenged by combined subcutaneous and intracerebral routes. Overall, the combined route produced higher attack rates (~100%) than the oral route (~50%) and 2-4 times shorter incubation periods. Uniquely, 87V given orally was unable to infect any sheep. Overall, scrapie strains adapted and cloned in mice produce distinct but variable disease phenotypes in sheep depending on breed or Prnp genotype. Further re-isolation experiments in mice are in progress in order to determine whether the original cloned murine disease phenotype will reemerge.

Factors influencing temporal variation of scrapie incidence within a closed Suffolk sheep flock.

Several studies have shown that transmission of natural scrapie can occur vertically and horizontally, and that variations in scrapie incidence between and within infected flocks are mostly due to differences in the proportion of sheep with susceptible and resistant PRNP genotypes. This report presents the results of a 12-year period of scrapie monitoring in a closed flock of Suffolk sheep, in which only animals of the ARQ/ARQ genotype developed disease. Among a total of 120 of these, scrapie attack rates varied between birth cohorts from 62.5 % (5/8) to 100 % (9/9), and the incidence of clinical disease among infected sheep from 88.9 % (8/9) to 100 % (in five birth cohorts). Susceptible sheep born to scrapie-infected ewes showed a slightly higher risk of becoming infected (97.2 %), produced earlier biopsy-positive results (mean 354 days) and developed disease at a younger age (median 736 days) than those born to non-infected dams (80.3 %, 451 and 782 days, respectively). Taken together, this was interpreted as evidence of maternal transmission. However, it was also observed that, for the birth cohorts with the highest incidence of scrapie (90-100 %), sheep born to infected and non-infected dams had a similar risk of developing scrapie (97.1 and 95.3 %, respectively). Compared with moderate-attack-rate cohorts (62.5-66.7 %), high-incidence cohorts had greater numbers of susceptible lambs born to infected ewes, suggesting that increased rates of horizontal transmission in these cohorts could have been due to high levels of environmental contamination caused by infected placentas.

Cellular and sub-cellular pathology of animal prion diseases: relationship between morphological changes, accumulation of abnormal prion protein and clinical disease.

The transmissible spongiform encephalopathies (TSEs) or prion diseases of animals are characterised by CNS spongiform change, gliosis and the accumulation of disease-associated forms of prion protein (PrP(d)). Particularly in ruminant prion diseases, a wide range of morphological types of PrP(d) depositions are found in association with neurons and glia. When light microscopic patterns of PrP(d) accumulations are correlated with sub-cellular structure, intracellular PrP(d) co-localises with lysosomes while non-intracellular PrP(d) accumulation co-localises with cell membranes and the extracellular space. Intracellular lysosomal PrP(d) is N-terminally truncated, but the site at which the PrP(d) molecule is cleaved depends on strain and cell type. Different PrP(d) cleavage sites are found for different cells infected with the same agent indicating that not all PrP(d) conformers code for different prion strains. Non-intracellular PrP(d) is full-length and is mainly found on plasma-lemmas of neuronal perikarya and dendrites and glia where it may be associated with scrapie-specific membrane pathology. These membrane changes appear to involve a redirection of the predominant axonal trafficking of normal cellular PrP and an altered endocytosis of PrP(d). PrP(d) is poorly excised from membranes, probably due to increased stabilisation on the membrane of PrP(d) complexed with other membrane ligands. PrP(d) on plasma-lemmas may also be transferred to other cells or released to the extracellular space. It is widely assumed that PrP(d) accumulations cause neurodegenerative changes that lead to clinical disease. However, when different animal prion diseases are considered, neurological deficits do not correlate well with any morphological type of PrP(d) accumulation or perturbation of PrP(d) trafficking. Non-PrP(d)-associated neurodegenerative changes in TSEs include vacuolation, tubulovesicular bodies and terminal axonal degeneration. The last of these correlates well with early neurological disease in mice, but such changes are absent from large animal prion disease. Thus, the proximate cause of clinical disease in animal prion disease is uncertain, but may not involve PrP(d).