Heino Diringer

Early Spread of Scrapie from the Gastrointestinal Tract to the Central Nervous System Involves Autonomic Fibers of the Splanchnic and Vagus Nerves

ABSTRACT Although the ultimate target of infection is the central nervous system (CNS), there is evidence that the enteric nervous system (ENS) and the peripheral nervous system (PNS) are involved in the pathogenesis of orally communicated transmissible spongiform encephalopathies. In several peripherally challenged rodent models of scrapie, spread of infectious agent to the brain and spinal cord shows a pattern consistent with propagation along nerves supplying the viscera. We used immunocytochemistry (ICC) and paraffin-embedded tissue (PET) blotting to identify the location and temporal sequence of pathological accumulation of a host protein, PrP, in the CNS, PNS, and ENS of hamsters orally infected with the 263K scrapie strain. Enteric ganglia and components of splanchnic and vagus nerve circuitry were examined along with the brain and spinal cord. Bioassays were carried out with selected PNS constituents. Deposition of pathological PrP detected by ICC was consistent with immunostaining of a partially protease-resistant form of PrP (PrPSc) in PET blots. PrPSc could be observed from approximately one-third of the way through the incubation period in enteric ganglia and autonomic ganglia of splanchnic or vagus circuitry prior to sensory ganglia. PrPSc accumulated, in a defined temporal sequence, in sites that accurately reflected known autonomic and sensory relays. Scrapie agent infectivity was present in the PNS at low or moderate levels. The data suggest that, in this scrapie model, the infectious agent primarily uses synaptically linked autonomic ganglia and efferent fibers of the vagus and splanchnic nerves to invade initial target sites in the brain and spinal cord.

Dextran sulphate 500 delays and prevents mouse scrapie by impairment of agent replication in spleen.

Treatment of scrapie-infected mice with dextran sulphate (DS) 500 resulted in considerably reduced spleen titres over a long period of time. Subsequently, the central nervous system disease was delayed or even prevented during the 350-day period of observation. Both effects increased after multiple injections of the compound. The potency of DS 500 to protect against scrapie was greatest when treatment and infection were carried out simultaneously. Under these conditions the lethality of 500 to 1000 LD50 was reduced to almost zero. Treatment as early as 10 weeks before infection still prolonged the incubation periods. Of several other polyions tested, dextran sulphate 5 and pentosan polysulphate also impaired scrapie pathogenesis.

Sequential appearance and accumulation of pathognomonic markers in the central nervous system of hamsters orally infected with scrapie

Both infectivity and TSE-specific amyloid protein (also referred to as protease resistant- or prion protein, PrP) are pathognomonic markers for transmissible spongiform encephalopathies (TSE). This paper presents a new densitometric method for the quantification of TSE-specific amyloid protein and its application to studying the pathogenesis of scrapie in Syrian hamsters after infection with scrapie strain 263K. A first study established a close correlation between infectivity and TSE-specific amyloid protein with a doubling time of 2-2.6 days in the brain and cervical spinal cord for both markers. The ratio of infectivity and TSE-specific amyloid protein was relatively constant at a mean value of about 10(6) protein molecules per infectious unit during the course of infection. A subsequent study addressed the temporal-spatial spread of infection in the central nervous system by tracing the accumulation of the pathological protein. The pathogenetic process was first detected in the spinal cord between vertebrae T4 and T9, and then showed an anterograde and retrograde spread with a rate of 0.8-1.0 mm/day. There were also some indications for a possible alternative route of spread of infection from the periphery to the brain, other than via the spinal cord. Involvement of the spleen did not appear essential for the early pathogenesis in hamsters orally infected with the 263K strain of scrapie.

The Original Gerstmann-Sträussler-Scheinker Family of Austria: Divergent Clinicopathological Phenotypes but Constant PrP Genotype

We present new data on the original Austrian kindred with Gerstmann‐Sträussler‐Scheinker disease (GSS) which encompasses currently 221 members in 9 generations. The mode of inheritance is autosomal dominant. Predominant clinical features are slowly progressive ataxia and late impairment of higher cerebral functins. In contrast, a recent case with proven P102L mutation of the PRNP gene had rapidly developing dementia and severe cortical damage indistinguishable from the clinicopathological phenotype of Creutzfeldt‐Jakob disease (CJD). PRNP codon 129 was homozygous for methionine in both the historic and recent cases. Neuropathology confirms spongiosis of variable degree and numerous protease resistant / prion protein (PrP) amyloid plaques scattered throughout most of the brain as constant features in this family. Some amyloid deposits are surrounded by dystrophic neurites with accumulation of phosphorylated neurofilaments and abnormal organelles, reminiscent of Alzheimer‐type plaques. Severe telencephalic damage and a synaptic‐type fine granular immunoreactivity in laminar distribution in the cortex with anti‐PrP after hydrated autoclaving of sections were seen only in the recent patient. In conclusion, factors in addition to the PRNP genotype at codons 102 and 129 must play a role in determining clinicopathological characteristics of this inherited brain amyloidosis.

Chemoprophylaxis of scrapie in mice

Three applications of the polyanion pentosanpolysulphate about 2 months before infection of mice with scrapie completely protected animals infected with up to 100 LD50, and considerably prolonged the lifespan of those infected with 100 to 10,000 LD50. The clinical diagnosis was confirmed by immunoblot analysis for the protein of scrapie-associated fibrils.

A rapid and efficient method to enrich SAF-protein from scrapie brains of hamsters

Scrapie hamster brains contain at least 5–10 μg of scrapie-associated fibrils (SAF) per brain as estimated by the amount of its major constituent, a protein of about 26 000 daltons (SAF-protein). It can be extracted efficiently by a 10% solution of sarkosyl and can be enriched by differentia] centrifugation and buffer extraction. Scrapie infectivity, SAF, and SAF-protein copurify.

Evidence for an alternative direct route of access for the scrapie agent to the brain bypassing the spinal cord.

Scrapie is a disease which occurs naturally in sheep and goats and belongs to a group of neurodegenerative disorders known as transmissible spongiform encephalopathies, or TSEs. There is currently no cure for TSEs, and the causative agent has not yet been identified. Numerous experiments, however, have addressed the pathogenetic process following a TSE infection. In this paper we present a study of the spread of the scrapie agent after intraperitoneal infection of hamsters. The accumulation of TSE-specific amyloid protein, TSE-AP (also known as PrP), was used as a marker for infectivity. The data suggested three points of agent entry into the spinal cord: the most important one between thoracic vertebrae T7-9, and two minor ones in the lower cervical spinal cord and between vertebrae T13-L2. Further, strong evidence was found for the existence of a direct route of access to the brain which bypasses the spinal cord and most likely terminates in the medulla oblongata. The indication of an alternative pathway to the brain was confirmed by the data from orally infected hamsters. The spleen appeared to play a potential, but non-essential role in pathogenesis after intraperitoneal infection in our animal model.

Biochemical Differences among Scrapie-associated Fibrils Support the Biological Diversity of Scrapie Agents

Scrapie-associated fibrils (SAF) were isolated and purified from animals infected with three different scrapie agents: ME7 and 139A in mice, and 263K in hamsters. Mouse ME7 and 139A SAF differed from hamster 263K SAF in morphology, sedimentation rate and protein composition. SAF from the three scrapie agents were distinguishable from each other by their sensitivity to proteinase K digestion. SAF copurified with infectivity in both the hamster and mouse systems. SAF appear to be a unique class of structures which are related but specific for each individual scrapie agent. These properties may correlate with the biological and pathological differences seen among these agents.

Pathogenesis of Scrapie: Study of the Temporal Development of Clinical Symptoms, of Infectivity Titres and Scrapie-associated Fibrils in Brains of Hamsters Infected Intraperitoneally

After an intraperitoneal infection of hamsters with scrapie agent, early low and constant titres of about 100 LD50/brain between days 10 to 50 were followed by a dramatic increase to maximum levels of 3 X 10(9) LD50/brain within about 15 days. The plateau of maximum infectivity remained unchanged from day 70 to the time of the first and final signs of disease at 95 and 123 days post-infection, respectively. Scrapie-associated fibrils (SAF) as measured by immunoblotting of SAF protein could not be detected before 79 days post-infection even when a total brain was used for analysis. Subsequently, the concentration of SAF increased gradually by about 100,000-fold until the time of clinical disease. The kinetics suggest a virus-induced amyloidosis of the brain as the cause of disease.

Sustained viremia in experimental hamster scrapie

SummaryAfter an intraperitoneal injection of scrapie agent into hamsters, a viremic phase is maintained from day 10 to at least day 40 post infection. Infectivity can be detected in the brain as early as 5–10 days after inoculation.