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Integrated OR systems nowadays are closed and proprietary, so that the interconnection of components from third-party vendors is only possible with high time and cost effort. An integrated operating theatre with open interfaces, giving clinical operators the opportunity to choose individual medical devices from different manufacturers, is currently being developed in the framework of the BMBF (Federal Ministry of Education and Research) funded project OR.NET [1]. Actual standards and concepts regarding technical feasibility and accreditation process do not cope with the requirements for modular integration based on an open standard. Therefore, strategies as well as service and device profiles to enable a procedure for risk management and certifiability are in the focus of the project work. Amongst others, a concept for User Interface Profiles (UI-Profiles) has been conceived in order to describe medical device functions and the entire user interface regarding Human-Machine-Interaction (HMI) characteristics with the aim to identify human-induced risks of central user interfaces. The use of standardized device and service profiles shall allow the manufacturers to integrate their medical devices in the OR.NET network, without disclosing the medical devices’ risk analysis and related confidential knowledge or proprietary information.

In our previous study we demonstrated that presenilin 1 (PS1) interacts with cytoplasmic linker protein 170/Restin (CLIP-170/Restin). Herein we show that disruption of the interaction of these proteins within neuronal cell-lines (SY5Y and N2a) can be accomplished by the transfection of vectors that drive the expression of peptide fragments corresponding to their binding domains (BDPs). Interestingly, the disruption of the PS1/CLIP-170 complex is associated with both decreased secretion of endogenous Abeta and decreased uptake of exogenous Abeta from the medium. BDP-expressing cells were also more resistant to surges of Abeta secretion induced by thapsigargin and ionomycin (that elevate intracellular calcium concentrations) and mutations in PS1 linked to familial Alzheimer's disease. Uptake of Abeta by SY5Y cells was amplified when preincubated with ApoE and was mediated through lipoprotein receptor-related protein (LRP). BDP-expressing cells or cells treated with PS1 anti-sense oligonucleotides were less capable of taking up Abeta from the medium compared with controls, indicating that the PS1/CLIP-170 interaction is involved and that PS1 cannot be substituted. In this study, we also mapped the minimum binding domains (mBDPs) of PS1 and CLIP-70 to regions corresponding to the N-terminal end of the large cytoplasmic loop of PS1 and the metal binding motif-containing C-terminal end of CLIP-170. Further, our data obtained from experiments involving in vitro taxol-polymerization of tubulin and confocal immunofluorescence suggest that PS1, via CLIP-170, may serve as an anchor to the microtubules for specific subcellular fractions containing amyloidogenic fragments. Interestingly, Notch is absent from this population of microtubule binding subcellular fractions and its cleavage was unaffected in cells transfected with the PS1-based BDP. This raises the possibility that the interaction of PS1 with CLIP-170 could provide the conceptual basis for anti-amyloidogenic therapeutic strategies with improved specificity. However, this approach may be hampered by a low efficiency, because it may also block Abeta clearance from the interstitial space of the CNS.

Between March 1981 and June 1995, a neurological disease characterized histologically by spongiform encephalopathy was diagnosed in 49 free-ranging cervids from northcentral Colorado (USA). Mule deer (Odocoileus hemionus) were the primary species affected and accounted for 41 (84%) of the 49 cases, but six Rocky Mountain elk (Cervus elaphus nelsoni) and two white-tailed deer (Odocoileus virginianus) were also affected. Clinical signs included emaciation, excessive salivation, behavioral changes, ataxia, and weakness. Emaciation with total loss of subcutaneous and abdominal adipose tissue and serous atrophy of remaining fat depots were the only consistent gross findings. Spongiform encephalopathy characterized by microcavitation of gray matter, intraneuronal vacuolation and neuronal degeneration was observed microscopically in all cases. Scrapie-associated prion protein or an antigenically indistinguishable protein was demonstrated in brains from 26 affected animals, 10 using an immunohistochemical staining procedure, nine using electron microscopy, and seven using Western blot. Clinical signs, gross and microscopic lesions and ancillary test findings in affected deer and elk were indistinguishable from those reported in chronic wasting disease of captive cervids. Prevalence estimates, transmissibility, host range, distribution, origins, and management implications of spongiform encephalopathy in free-ranging deer and elk remain undetermined.

Characterization of eleven monoclonal antibodies (MAbs), raised to isolated sodium dodecyl sulfate (SDS)-treated Alzheimer's neurofibrillary tangles (ANT), has revealed the presence of at least two different epitopes. MAbs were tested for reactivity to ubiquitin and paired helical filaments (PHF) isolated by three different procedures. The effect of protease and/or alkaline phosphatase pretreatment on the reactivity of the MAbs with isolated PHF was also examined. All MAbs that had reacted strongly in the ELISA with sonicated SDS-treated ANT also immune decorated isolated PHF to varying degrees. Two MAbs exhibited a high reactivity to PHF: 3-39 and 5-25. MAb 3-39 was found to recognize a protease sensitive epitope. In contrast MAb 5-25 was found to consistently decorate isolated PHF in all preparations and exhibited a strong reactivity to ubiquitin, and the epitope in isolated PHF was not protease sensitive. Thus structural PHF after protease treatment and detergent treatment contain an antigenic site that is present in ubiquitin.

Scrapie-associated fibrils (SAF) and protease-resistant proteins (PrP) were isolated from spleens and brains of clinical animals (mice and hamsters) from three scrapie agent-host strain combinations, and their concentrations were compared with infectivity levels. The spleens of infected animals contained lower levels of infectivity, PrP, and SAF than did brains. Regardless of the route of infection, both SAF and infectivity were detected in spleen before brain. Infectivity increased in brains and spleens of 139A-infected mice before the detection and increase in SAF, suggesting that the synthesis of SAF and PrP may not be the limiting factor in agent replication. In contrast to those in ME7- and 263K-infected animals, the Western blot profiles for PrP from brain and spleen of 139A-infected mice exhibited distinct differences. Results indicate that SAF and PrP found in the spleens are both organ- and scrapie strain-specific.

We have identified, for the first time, regions of cis-acting RNA elements within the bacteriophage Q beta replicase cistron by analyzing the infectivities of 76 replicase gene mutant phages in the presence of a helper replicase. Two separate classes of mutant Q beta phage genomes (35 different insertion mutants, each containing an insertion of 3 to 15 nucleotides within the replicase gene, and 41 deletion genomes, each having from 15 to 935 nucleotides deleted from different regions of the gene) were constructed, and their corresponding RNAs were tested for the ability to direct the formation of progeny virus particles. Each mutant phage was tested for plaque formation in an Escherichia coli (F+) host strain that supplied helper Q beta replicase in trans from a plasmid DNA. Of the 76 mutant genomes, 34% were able to direct virus production at or close to wild-type levels (with plaque yield ratios of greater than 0.5), another 36% also produced virus particles, but at much lower levels than those of wild-type virus (with plaque yield ratios of less than 0.05), and the remaining 30% produced no virus at all. From these data, we have been able to define regions within the Q beta replicase gene that contain functional cis-acting RNA elements and further correlate them with regions of RNA that are solely required to code for functional RNA polymerase.

Scrapie-associated fibrils, first observed in brains of scrapie-infected mice, were also observed in scrapie-infected hamsters and monkeys, in humans with Creutzfeldt-Jakob disease, and in kuru-infected monkeys. These fibrils were not found in a comprehensive series of control brains from humans and animals affected with central nervous system disorders resulting in histopathologies, ultrastructural features, or disease symptoms similar to those of scrapie, kuru, and Creutzfeldt-Jakob disease. These fibrils are also found in preclinical scrapie and in the spleens of scrapie-infected mice; they are a specific marker for the "unconventional" slow virus diseases, and may be the etiological agent.

The association between scrapie infectivity and scrapie-associated fibrils (SAF) during a partial purification procedure for infectivity was investigated. Scrapie infectivity and SAF can be separated from most membrane components by subcellular fractionation of infected mouse brain to obtain a synaptosomal fraction, followed by detergent treatment and density gradient centrifugation. After different detergent treatments, with either octyl glucoside or sodium N-lauroyl sarcosinate, SAF showed differing sedimentation characteristics but nevertheless cosedimented with scrapie infectivity in both cases. Copurification under two different conditions provides more evidence that SAF may be a form of the infectious agent of scrapie.

Isolated paired helical filaments (PHF) of Alzheimer's disease and senile dementia of the Alzheimer type (AD/SDAT) were studied by negative stain techniques and electron microscopy (EM). The helical structure of the PHF was confirmed by tilt analysis of negatively stained isolated PHF. A substructure of four protofilaments, each 3-5 nm in diameter, was also observed. A comparison of PHF substructure, neurofilaments and microtubules, revealed no similarity among the three structures.

A method of purifying postsynaptic densities (PSD) of Cohen et al. (1977) has been modified, primarily by the substitution of octyl glucoside as the detergent used to solubilize synaptosomal fractions. Subsequent extraction with other detergents resulted in the selective removal of specific polypeptides. In particular sulphobetaine 3-14 removed most of the beta-tubulin but not alpha-tubulin. Sodium N-lauroyl sarcosinate completely destroyed the structural integrity of the PSD when the in vitro formation of intermolecular disulphide bonds was minimized. These results suggest that the structure of PSDs is more labile than previously thought and demonstrate a technique for further examining their composition.

Antisera raised to scrapie-associated fibril (SAF) proteins were used to detect scrapie-specific polypeptides in three different non-purified brain preparations: a synaptosomal-mitochondrial fraction, 20% brain homogenate and 20% brain homogenate extracted with Sarkosyl. The concentration of SAF proteins in the preparations was greater than the quantity of SAF as detected by negative stain electron microscopy. This suggests that not all of the protein exists in the form of SAF. An immunologically reactive 33K to 35K protein was detected in both normal and scrapie brain preparations. This protein was susceptible to complete proteinase K (PK) digestion in normal brain preparations and it is suggested that scrapie infection is responsible for post-translational modifications which confer PK resistance in scrapie preparations. These modifications may also play a role in the antigenic differences seen in a variety of scrapie agents. SAF-specific proteins were also detected in the spinal cords and spleens from scrapie-affected animals. Detergent extraction of material followed by PK treatment and Western blot analysis is a highly specific and sensitive method for the detection of SAF proteins. This procedure could be applied to human neurological diseases of unknown aetiology.

Unconventional slow infections are progressive transmissible degenerative disorders of the central nervous system. The human diseases belonging to this group are Creutzfeld-Jakob disease, kuru, and Gerstmann-Straussler syndrome. Scrapie, transmissible mink encephalopathy, chronic wasting disease of mule deer and elk, and the recently discovered bovine spongiform encephalopathy are similar diseases found in animals. Unusual characteristics of the unconventional slow infections clearly distinguish these disorders from conventional infections. These include: unusually long incubation periods (from months to years); progressive CNS degeneration with characteristic histopathological lesions; the lack of an immune or inflammatory response; unconventional biological and physical properties of the etiologic agents. There has been considerable controversy concerning the nature of the causative agent. The 3 main hypotheses, virus, virino, and modified host protein, are reviewed relative to their ability to explain the properties of the agent and the unusual characteristics of the disease process. The discovery of an abnormal structure, termed scrapie associated fibrils (SAF) and an abnormally modified 33-37 kDa host-encoded glycoprotein unique to unconventional slow infections opened new areas of intense interest and investigation. SAF are abnormal filamentous structures which copurify with infectivity and possess characteristics of "amyloids." The major component of SAF is the host-encoded scrapie-specific protease resistant glyco-protein. Considerable data has accumulated on the biochemistry, immunology and molecular biology of this host coded scrapie protein. The relationship of SAF and the scrapie-specific protein to the infectious agent is discussed in the context of each of the "nature of the agent" hypotheses.

Scrapie-associated fibrils (SAFs) are abnormal filamentous structures that are uniquely associated with unconventional slow virus diseases. The antigenic relationships of SAFs from animals infected with four biologically distinct scrapie strains were investigated by using antisera raised to purified SAF proteins. Rabbit antisera were raised to SAFs isolated from mice infected with the ME7 scrapie strain and to SAFs isolated from hamsters infected with the 263K scrapie strain. A strong antigenic relationship was shown among SAF proteins (PrPs) isolated from all scrapie-infected animals (ME7, 139A, and 87V in mice and 263K in hamsters), and this relationship was demonstrable regardless of which antiserum was used. SAF proteins were antigenically distinct from those of paired helical filaments or amyloid isolated from patients with Alzheimer disease. Distinct Western blot profiles were demonstrated for SAFs isolated from animals infected with each scrapie strain. Differences seen among SAFs were independent, at least in part, of host species or genotype, implying that certain specific structural and molecular properties of SAFs are mediated by the strain of scrapie agent.

Abnormal fibrillary structures, designated "scrapie-associated fibrils" (SAF), have been observed using negative stain techniques in subfractions of brains from scrapie-affected animals. SAF have been observed in all combinations of strain of scrapie agent and strain or species of host examined, regardless of their histopathology, in particular the presence or absence of amyloid plaques. SAF consist either of two or four filaments. They are morphologically dissimilar to the normal brain fibrils--microtubules, neurofilaments, glial filaments, and F actin. However, SAF do bear a resemblance to amyloid.

A method has been developed for the bulk isolation of Alzheimer neurofibrillary tangles (ANT) of paired helical filaments (PHF) from histopathologically confirmed cases of Alzheimer disease/senile dementia of the Alzheimer type (AD/SDAT). The fresh or frozen autopsied cerebral cortex affected with Alzheimer neurofibrillary changes is dissociated by homogenization and sieving through nylon bolting cloth and the ANT are separated by a combination of sucrose discontinuous density gradient centrifugation, glass bead column chromatography, and sodium dodecyl sulfate (SDS) treatment. The isolated ANT produce red-green birefringence when viewed through polarized light after staining with Congo red. Ultrastructurally, the isolated PHF are well preserved and have the dimensions of the PHF seen in situ. Two major Populations of ANT which exist in different proportions in AD/SDAT brains are identified on the basis of their solubility in SDS. The ANT I and the ANT II are soluble and insoluble respectively on treatment with 2% SDS at room temperature for 5 min. Solubilization of the ANT II requires several repeated extractions with a solution containing 10% each of SDS and beta-mercaptoethanol (BME) at 100 degrees C for 10 min. Sonication of the ANT II greatly facilitates their solubilization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated ANT reveals the presence of two major polypeptides with molecular weights (MW) of 62,000 and 57,000, several minor polypeptides with MW below 57,000, and a significant amount of material not entering the stacking and the resolving gels. Re-electrophoresis of polypeptides extracted from various areas of the resolving gel or of the material which does not enter the gel generates the same polypeptide profile as on the first gel, suggesting that the PHF material which does not enter the gel may result from the reaggregation of the polypeptides that enter the resolving gel. None of the polypeptides that enter the resolving gel. None of the polpeptides observed in the isolated PHF comigrate in the SDS-PAGE with any of the neurofilament polypeptides, tubulin, actin, or myosin.

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.

Scrapie of sheep, and Creutzfeldt-Jakob disease (CJD) of man belong to a group of transmissible encephalopathies which have been successfully transmitted to a variety of hosts. In susceptible hosts, these diseases are characterized by progressive degeneration of the central nervous system leading inevitably to death. The agents responsible for these diseases have not yet been identified, but they exhibit similar physicochemical characteristics. Abnormal fibrils designated 'scrapie associated fibrils' (SAF) have been observed in synaptosomal preparations of scrapie infected brain. They have never been observed in various types of control animals. We report here that SAF are present in CJD brain fractions in the experimentally transmitted disease as well as in a few naturally occurring human cases of CJD. SAF are also present in spleen extracts of animals experimentally infected with scrapie or CJD. This close association of SAF with these two diseases and two different tissues (brain and spleen) known to contain titres of infectivity, suggest that the SAF are: (1) a unique pathological response to the disease or (2) the infectious agent of these diseases.

Scrapie-associated fibrils (SAF) were detected by negative-stain electron microscopy in the brains (by two different isolation procedures) and spleens of sheep naturally and experimentally infected with scrapie. Although the numbers of SAF varied from case to case, the yield of SAF from brains of naturally affected sheep was lower than that from experimentally affected sheep. SAF-specific, protease-resistant proteins (PrPs) were detected by silver staining and western blot analysis in most samples of brain from experimentally affected sheep. PrPs, however, could be detected in only a limited number of natural cases of sheep scrapie because of the lower yields of SAF. PrPs from sheep SAF appear biochemically and antigenically similar to PrPs from other species infected with unconventional agents. This study further establishes the unique association of SAF and PrPs with natural or experimentally induced scrapie in its natural host.

The structure of partially purified, CNS amyloid fibrils from three different sources have been compared by negative stain EM. The fibrils isolated from brains with senile dementia of Alzheimer type were 4-8 nm in diameter, narrowing every 30-40 nm and apparently composed of two 2-4 nm filaments. The fibrils from a Gerstmann-Sträussler syndrome brain were 7-9 nm in diameter, narrowing every 70-80 nm and with a suggestion that they are composed of two 3-5 nm filaments. The fibrils isolated from 87V scrapie-affected mouse brains were 4-8 nm in diameter with a twist every 15-25 nm presumably composed of two 2-4 nm filaments. The fibrils from the scrapie brains were usually observed in pairs. The shape of the clusters of the isolated amyloid fibrils observed in each disease was similar in negative stain and thin section EM preparations and was related to the characteristic morphology of the amyloid fibrils in the neuritic and amyloid plaques in situ. The structural differences between the CNS amyloid fibrils from the various diseases studied by us may reflect differences in the polypeptides which comprise the fibril and/or a different pathogenesis in the formation of the amyloid fibrils.

An antibody was raised to a synthetic peptide corresponding to a published sequence for the first 24 residues of a cerebrovascular amyloid peptide (CVAP). Immunohistochemical staining of tissue sections revealed that the antibody bound extensively to cerebrovascular amyloid in Alzheimer disease (AD/SDAT) and Down's syndrome cases. The antibody bound less extensively to neuritic plaques (primitive and mature) and indetectably to neurofibrillary tangles. The antibody did not label scrapie plaques, scrapie-associated fibrils, or Gerstmann-Sträussler syndrome plaques. Immunoblotting experiments showed that the cerebrovascular amyloid peptide epitopes contaminating the neurofibrillary tangle preparations could be extracted with urea, leaving the neurofibrillary tangles intact. These data confirm that the cerebrovascular amyloid peptide is a component of cerebrovascular amyloid, and suggest that its epitopes are also components of neuritic plaque amyloid. The reduced level of immunostaining on amyloid cores in tissue sections suggests that either the cerebrovascular amyloid peptide epitopes are a minor component of amyloid cores, or that their mode of packing or state of processing in amyloid cores renders them relatively inaccessible to the antibody. We also conclude that the cerebrovascular amyloid peptide is not a component of neurofibrillary tangles. The synthetic cerebrovascular amyloid peptide possesses amyloid-like properties: at neutral pH it forms insoluble aggregates consisting of 5-7-nm fibrils, which form red-green birefringent adducts with Congo red and fluoresce with thioflavine S.

Considerable controversy exists concerning the origin and composition of Alzheimer neurofibrillary tangles (ANT) and of paired helical filaments (PHF), the abnormal cytoplasmic fibers which ultrastructurally are the major components of ANT. Thus far, the unusual solubility properties of PHF have hindered the analysis of ANT. A new procedure is presented for isolating purified PHF which are soluble in the presence of sodium dodecyl sulfate. The purification protocol involves differential and rate zonal centrifugation, treatment with the detergents sarcosyl and sulfobetain 3-14, and sonication. The isolated PHF from Alzheimer disease/senile dementia of the Alzheimer type (7 cases) and Down's syndrome (one case) have been characterized structurally by negative-stain electron microscopy, biochemically by PAGE, and immunologically by both the ELISA technique and Western blot analysis using a monoclonal antibody prepared against ANT. Distinct polypeptides were shown to be associated with this structure and not seen in preparations from young and age-matched normal brains.

Brains obtained within 2-4 hours post mortem and histopathologically confirmed for Alzheimer's disease and non-Alzheimer brains from age-matched controls were examined for in-vitro assembly of microtubules and neurofilaments. Microtubule assembly was observed only in control but not in Alzheimer brains, and neurofilaments were obtained from both types of brain. The microtubule-associated protein tau, which stimulates assembly of microtubules from tubulin, was abnormally phosphorylated in Alzheimer but not in control brain microtubule preparations. Alzheimer brains did not show the presence of any inhibitor of microtubule assembly or any abnormality of tubulin. DEAE-dextran, a polycation which mimics tau in stimulating microtubule assembly, induced the assembly of microtubules in Alzheimer brain. Tubulin from both normal and Alzheimer brains was labelled on western blots by a monoclonal antibody to the tyrosinylated carboxy-terminal epitope of alpha tubulin. These studies suggest that in Alzheimer's disease tubulin can be assembled into brain microtubules, but the process is defective, probably because of abnormal phosphorylation of tau. This post-translational alteration of tau might be the cause of the neurofibrillary abnormality in Alzheimer's disease.

Antibody response in mice to scrapie-associated fibril proteins (protease-resistant proteins [PrPs]) was generated to different epitopes depending on the source of antigen. Mice responded differently to PrPs isolated from scrapie-infected animals of homologous (mouse) versus heterologous (hamster) species. An enzyme-linked immunosorbent assay established to monitor this antibody response in mice immunized with PrPs was unable to detect such a response in scrapie-infected mice. A monoclonal antibody (MAb), 263K 3F4, derived from a mouse immunized with hamster 263K PrPs reacted with hamster but not mouse PrPs. MAb 263K 3F4 also recognized normal host protein of 33 to 35 kilodaltons in brain tissue from hamsters and humans but not from bovine, mouse, rat, sheep, or rabbit brains. This is the first demonstration of epitope differences on this host protein in different species. The defining of various epitopes on PrP through the use of MAbs will lead to a better understanding of the relationship of PrPs to their host precursor protein and to the infectious scrapie agent.

Neurofilaments were assembled in vitro from the high speed supernatant of mammalian CNS homogenate in a disassembly buffer containing 4-morpholine-ethane sulfonic acid, MgCl2 and EGTA at 4 degrees C in the presence of 4 M glycerol. The assembled neurofilaments were depolymerized by dialysis against the disassembly buffer and repolymerized by the addition of glycerol to the clarified supernatant obtained afer disassembly. The filament assembly reaction was complete in less than 30 s as measured by turbidimetric changes at 415 nm and did not require any added nucleotide. No assembly of filaments was detected when using frozen tissue. The assembled filaments corresponded to the enrichment of neurofilament triplet, the 210,000, 160,000 and 70,000 dalton polypeptides on SDS-polyacrylamide gels and appeared morphologically and immunochemically identical to neurofilaments isolated by axonal flotation methods. These studies demonstrate in vitro assembly of neurofilaments under native conditions which raises the possibility that like microtubules, neurofilaments or a subpopulation of neurofilaments might be in a dynamic state of assembly--disassembly in situ.

Many years ago, observations of natural scrapie revealed differences in clinical manifestations and in the areas of the brain that showed the most intense histopathological changes. In experimental work with scrapie in mice two fundamental points were established in early studies: (1) A mouse gene, termed Sinc for scrapie incubation, affects the length of the incubation period. (2) In a single strain of mice, incubation periods of different "strains" of scrapie can differ by as much as 3-4 fold, e.g., the incubation period of the ME7 scrapie strain in C57BL mice is approximately 122 days, whereas the incubation period for the 22A strain is 360 days. From this "classic" beginning our knowledge about the parameters influenced by genetic characteristics of both host and agent have expanded. In a number of instances, initial examination of an aspect of host-agent interaction has subsequently revealed surprising influences of informational molecules from both host and agent. A partial list of the parameters under genetic control follows: (1) The extent of vacuolation in various regions of the brain; (2) The quantity of amyloid plaques in the brain; (3) The occurrence of increased weight in the preclinical phase of disease; (4) The development of aberrant glucose tolerance; (5) The area of the brain which yields the shortest incubation period after stereotaxic injection; and (6) The physical-chemical and immunological characteristics of scrapie associated fibrils. In several well documented instances differences between scrapie strains were evident after repeated passages of the strains in the same host strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Scrapie-associated fibrils (SAF) are an infection-specific structure observed in the unconventional-agent diseases. Polyclonal antisera raised to scrapie proteins were used to test the antigenic relationship between purified fibrils and SAF isolated from non-protease-treated synaptosomal-mitochondrial preparations. The experimental design utilized fibrils from scrapie strain 263K-infected hamsters, scrapie strain 139A-infected mice, and scrapie strain ME7-infected mice. Preparations were examined by negative-stain immune electron microscopy and Western blot analysis of the polypeptides. Fibrils and polypeptides from each preparation reacted with a rabbit antiserum raised to each of the following: hamster 263K prion protein (PrP 27-30), hamster 263K SAF protein, and mouse ME7 SAF protein. Immune electron microscopy and Western blot analysis revealed similar antigenic relationships among the three scrapie antisera. Thus, fibrils and polypeptides can be considered to be the same in each preparation. No reactivity of the fibrils was observed with antisera raised to Alzheimer neurofibrillary tangles or a synthetic peptide of cerebrovascular amyloid. Thus, the fibrils observed in purified preparations share structural and antigenic similarities plus biochemically related peptides with SAF present in non-protease-treated preparations.

A decrease in the percentage of polymorphonuclear neutrophils (PMN) in the peripheral blood of mice appeared 3 days after intracerebral (IC) inoculation with scrapie mouse brain homogenate. Mice inoculated IC with normal mouse brain had PMN percentages similar to those found for uninoculated mice. This difference between normal and scrapie-inoculated mice continued throughout the preclinical phase of the disease. In the clinical phase of the disease, the percentage of PMN was either higher or lower than that found in normals. The factor causing the decrease in PMN percentages was found in the filtrates from 220-, 100-, and 50-nm filters, but not in the filtrates from a 25-nm filter. Sodium periodate treatment of the scrapie brain samples eliminated their ability to cause the decrease in PMN percentages, whereas sodium iodate had no effect. In addition to two genetically different scrapie mouse brain isolates, homogenates of mouse spleen, sheep brain, and sheep spleen from scrapie-affected animals caused a decrease in percent PMN, whereas the corresponding normal tissue homogenates did not.

Mice inoculated with brain homogenates from multiple sclerosis (MS) cases showed marked changes in their leukocyte differential counts, with a decrease in per cent polymorphonuclear neutrophils (PMN) and an increase in the per cent lymphocytes. These changes were based upon an absolute decrease in the number of circulating PMN. The decrease in PMN percentages was apparent at 16 hr after infection and persisted for at least 11 months. The factor responsible for the decrease in PMN was (a) recoverable from 12 hr to 8(1/2) months after inoculation, (b) present in human brain homogenate at a concentration of 3 x 10(12), and (c) between 25 and 50 nm in diameter. Inoculation of 100 units of factor into mice and subsequent titration showed that the factor had undergone a net increase in the mouse of at least 10(9)-fold. The factor causing the PMN decrease was found in all MS material thus far tested: three brains, one spleen, three sera, and two cerebrospinal fluid (CSF) from nine cases of MS. The factor was not found in normal human material that included two brains, one spleen, two sera, and two CSF.