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It has been suggested that the infectious agents of scrapie and Creutzfeldt‐Jakob disease (CJD) are ‘prions’ constituted by a protease resistant glycopeptide, PrP. To analyze the role of PrP in CJD infectivity we re‐evaluated the biochemical characteristics of infectivity. First, when the infectious agent is not aggregated, infectivity is exquisitely sensitive to proteinase K treatment, and therefore a proteinase‐K‐resistant molecule (e.g. PrP) is unlikely to contain information essential for agent replication. Second, removal of sugar residues from Gp34 (the major precursor of the proteolyzed PrP band) failed to reduce infectivity. Third, one‐half of the PrP peptides could be separated from significant infectivity using nondenaturing conditions with practical quantitative recovery of infectivity. These studies suggest that PrP in itself is unlikely to be the replicating component of the infectious agent. We suggest that these as yet undefined agents may consist of core protein and nucleic acid that are incompletely assembled in, and protected by, cell membranes. This hypothesis would explain the absence of conventional viral particles in these diseases, account for observed membrane pathology including altered behavior of endogenous membrane proteins, and would be consistent with the replication and transforming properties of CJD that indicate there is an agent specific nucleic acid.

Summary A biotinylated Po cDNA was hybridizedin situ to aldehyde-fixed vibratome sections of trigeminal ganglia from day 2, day 7, day 15, day 30 and adult rats. Nickel-enhanced horseradish peroxidase (HRP) was used in an antibody sandwich method to detect hybridization.

The transmission and serial propagation of Creutzfeldt-Jakob disease from man to guinea pigs are reported. The latency, symptomatology, and morphology of the infection during the first four passages are presented. The incubation period between the first and subsequent passages was halved. One hundred percent take, morbidity, and mortality were achieved in all inoculated animals. All guinea pigs developed a subacute spongiform virus encephalopathy with marked neuronal destruction in the cerebral cortex and subcortical grey structures. The neuronal loss resulted in cerebral atrophy and hydrocephalus ex vacuo.

Paraformaldehyde-fixed tissue from mouse cerebellum was hybridized with biotin-labeled satellite DNA for identification of centromeres. By using avidin-peroxidase conjugates, it was possible to define the nuclear position of centromeres at the ultrastructural level. Three-dimensional analysis of well-resolved centromere arrays were aided by computer reconstruction of serial sections. Different cell types displayed distinct, nonrandom centromere locations. In Purkinje neurons, the majority of detected sequences were clustered together around the central nucleolus, whereas in granule neurons, more numerous, dispersed centromere clusters were associated with the nuclear membrane. In Purkinje cells, peroxidase-labeled regions corresponded to dense heterochromatic aggregates were detected in Purkinje cells of several different species. These observations suggest that in these highly differentiated cells, the nuclear position of centromeres is maintained in evolution despite species differences in centromeric DNA sequence. Such defined ordering of centromeres may be integral to specific functional capacities.

Small amounts of brain tissue (2 g) infected with Creutzfeldt-Jakob disease (CJD) can be fractionated by using a simple 1-day method that includes lysis with N-lauroylsarcosine. Unique fibrils have been identified previously in scrapie- and CJD-infected tissue. These fibrils were abundant in final fractions. Preparations from human CJD autopsy material and from experimental hamster and guinea pig CJD all displayed readily identifiable fibrils that were not seen in control preparations. Thus, these methods appear to be of value in biopsy diagnosis of suspected human cases of CJD. Lysis with N-lauroylsarcosine quantitatively solubilized infectivity from membrane-rich fractions. Significant infectivity was recovered in microfractionations. After proteinase K digestion, a diffuse band at 29 kDa was detectable on NaDodSO4/PAGE. This 29-kDa material was not present in uninfected control brain and was similar to that seen in scrapie. Protein blots of human, guinea pig, and hamster CJD fractions were tested with an antibody raised against a 29-kDa band from mouse scrapie; 29-kDa proteins were labeled in all CJD and scrapie fractions but not in controls. These results indicate that specific proteins in both these diseases share common antigenic determinants. Ricin and wheat germ agglutinin, but not concanavalin A, also labeled a portion of the 29-kDa band from hamster CJD and hamster scrapie fractions, but they did not label any bands in normal hamster fractions at the same gel protein loads. When proteinase K treatment was omitted, specific bands of approximately equal to 35 kDa were detected in CJD samples. These results are consistent with the idea that some CJD- and scrapie-specific proteins are glycoproteins or sialoglycoproteins that can reside in or possibly derive from cell membranes.

Gliosis (hyperplasia and hypertrophy of astrocytes), the fundamental response of the central nervous system to tissue destruction, typically becomes apparent only several weeks after injury. The biochemical hallmark of this response is a marked accumulation of the specific astrocyte intermediate filament glial fibrillary acidic protein (GFAP). To date despite its importance, the mechanisms of GFAP gene regulation have not been studied in any developmental or pathological system to our knowledge, and the molecular signals for GFAP mRNA and protein accumulation are not defined. In Creutzfeldt-Jakob disease, a progressive dementing illness caused by an "unconventional agent," we find steadily increasing elevations of GFAP mRNA throughout the later stages of disease, using two independent GFAP cDNA clones, representing the entire insert or the 3'-noncoding region (pScr-1). The accumulation of GFAP, assessed immunocytochemically, follows GFAP mRNA elevation. A 5-fold stimulation of GFAP gene expression precedes the development of florid histologic lesions in the cerebrum, and in the cerebellum 5- to 6-fold increases occurred with no detectable spongiform changes at any time during disease. Therefore, these GFAP changes cannot be simply a response to neuronal damage. These effects are directly or indirectly caused by high local concentrations of agent and possibly involve a humoral factor.

Experimental Creutzfeldt-Jakob disease was serially transmitted from guinea pigs to Syrian hamsters with 100% incidence, morbidity, and mortality. All animals developed a subacute spongiform virus encephalopathy with neuronal destruction and concomitant astrocytic changes. In the first passage three different clinical syndromes were recorded, each with widely variant incubation times; these results suggested there may be different strains of the Creutzfeldt-Jakob agent, some of which may be partially separated when the agent is passaged from one species to another. Accumulations of neurofilaments in neuronal perikarya and processes and increased lipofuscin were suggestive of changes seen in senility and aging.

The etiology of Alzheimer disease (AD) is unknown. To investigate the transmissibility of AD, the buffy coat of the blood from 11 relatives of AD patients, including 2 with suspicious or early signs of AD, was inoculated intracerebrally into hamsters. In these pilot experiments, 5 individuals produced histologically documented spongiform encephalopathy on primary passage in recipient hamsters. Material from 3 of these positives was serially transmitted in a second passage. The histological alterations observed in the brains of positive hamsters were similar to those seen in experimental Creutzfeldt-Jakob disease (CJD). These transmission results raise the intriguing possibility that CJD-like agents may be involved in at least some forms of AD.

Isolates from six patients with Creutzfeldt-Jakob disease (CJD) were injected into various strains of hamsters and mice, and the infective agent was propagated. Serially passaged cultures were established from these CJD agent-infected brains and from uninfected control brains. All healthy cultures (21 out of 21) from CJD agent-infected brains became immortal and/or transformed. In contrast only 3 out of 13 normal brain cultures became immortal, and the rest died out with serial propagation in vitro. The fact that permanent cell lines were readily derived from multiple rodent strains and all CJD isolates tested suggests that a transforming capability is an intrinsic property of CJD agents. This conclusion is supported by demonstrations of in vitro cell transformation by CJD infectious brain fractions. Although the molecular mechanism of transformation events associated with the CJD agent is not presently known, a provocative possibility is that the CJD agent has a direct effect on the host genome by mechanisms analogous to those known for slowly oncogenic retroviruses.

In this report, we present the first physical characterization of the Creutzfeld-Jakob disease agent. Preparations with high yields of infectivity (assayed infectious units) were obtained by a novel, gentle procedure in which initially sedimenting Gp34 ("prion" protein) was disaggregated by a variety of criteria with no subsequent loss of infectivity. Studies with this preparation indicate that most of the Creutzfeld-Jakob disease agent has both a viruslike size and density. In velocity sedimentation and isopycnic sucrose gradients, infectivity comigrated with nucleic acid-protein complexes of appreciable size.

Creutzfeldt-Jakob disease (CJD), a progressive dementia of humans, is caused by an infectious agent that is closely related to the scrapie agent of sheep. Although the molecular nature of these "unconventional" agents is still a matter of speculation and controversy, even less is known concerning the mechanism(s) of their effects on the central nervous system. To gain insight into the cellular effects of these agents, we have examined a series of cell lines derived directly from CJD-infected hamster brain or produced from nontransformed rodent lines by exposure to CJD infectious fractions in vitro. These cell lines appear transformed by a variety of criteria and secrete growth factors into the culture medium. All CJD lines produce a factor that is like alpha-transforming growth factor (alpha-TGF). Conditioned medium from these CJD lines also stimulates the synthesis of glial fibrillary acidic protein in normal astrocytic cells in vitro. This effect is mimicked by purified alpha-TGF and platelet-derived growth factors. Further study of CJD-induced growth factor production may elucidate fundamental properties of these unconventional agents.

Summary A biotinylated P0 glycoprotein cDNA was hybridized in situ to aldehyde-fixed vibratome sections and to aldehyde-fixed thin sections of Lowicryl-embedded trigeminal ganglia of 15 day old rats. Alkaline phosphatase and peroxidase detectors were used for light microscopic (LM) studies and peroxidase or colloidal gold were employed for electron microscopic (EM) detection. In both LM and EM sections, probe was found in cytoplasmic areas of myelinforming Schwann cells that were enriched in granular endoplasmic reticulum, demonstrating that these regions contain P0 mRNA. Interestingly, P0 mRNA tended to cluster in regions close to the developing myelin sheath. Relatively simple methods are here described for EM detection of mRNA with reasonable tissue preservation and high resolution. These methods may be useful for developmental and disease-related studies of specific mRNAs in mammalian tissues.

Plasmid clones containing up to 94 kilobases of single-copy DNA from band q22.3 of chromosome 21 and a complete pool of insert DNA from a chromosome 21 recombinant library have been used to rapidly detect numerical and structural aberrations of chromosome 21 by in situ hybridization in both metaphase and interphase cells. A trisomic karyotype, diagnostic of Down syndrome, is readily detected in nonmitotic cells because the majority of their nuclei exhibit three discrete foci of hybridization, in contrast to normal diploid cells, which show two foci. Chromosomal translocations involving chromosome 21 sequences were also detected with these probes, and the intranuclear location of 21q22.3 DNA sequences in "normal" human brain neurons was established with the plasmid DNA probe set. These results suggest that chromosome 21-specific probes may have utility in clinical diagnostics, especially by facilitating the direct analysis of interphase cells.