A transmissible Creutzfeldt-Jakob disease-like agent is prevalent in the human population.

The etiology of most human dementias is unknown. Creutzfeldt-Jakob disease (CJD), a relatively uncommon human dementia, is caused by a transmissible virus-like agent. Molecular markers that are specific for the agent have not yet been defined. However, the infectious disease can be transmitted to rodents from both brain and infected buffy coat (blood) samples. To determine whether human CJD infections are more widespread than is apparent from the low incidence of neurological disease, we attempted to transmit CJD from buffy coat samples of 30 healthy volunteers who had no family history of dementing illness. Primary transmissions from 26 of 30 individuals produced CJD-like spongiform changes in the brains of recipient hamsters at 200-500 days postinoculation. This positive evidence of viremia was found for individuals in all age groups (20-30, 40-50, and 61-71 years old), whereas 12 negatively scored brain samples failed to produce similar changes in hamsters observed for > 900 days in the same setting. We suggest that a CJD agent endemically infects humans but only infrequently produces an infectious dementia. Disease expression is likely to be influenced by several host factors in combination with viral variants that have altered neurovirulence.

Nucleic acid binding proteins in highly purified Creutzfeldt-Jakob disease preparations.

The nature of the infectious agent causing human Creutzfeldt-Jakob disease (CJD), a slowly progressive dementia, is controversial. As in scrapie, no agent-specific proteins or nucleic acids have been identified. However, biological features of exponential replication and agent strain variation, as well as physical size and density data, are most consistent with a viral structure--i.e., a nucleic acid-protein complex. It is often assumed that nuclease treatment, which does not reduce infectious titer, leaves no nucleic acids of > 50 bp. However, nucleic acids of 500-6000 bp can be extracted from highly purified infectious complexes with a mass of approximately 1.5 x 10(7) daltons. It was therefore germane to search for nucleic acid binding proteins that might protect an agent genome. We here use Northwestern blotting to show that there are low levels of nonhistone nucleic acid binding proteins in highly purified infectious 120S gradient fractions. Several nucleic acid binding proteins were clearly host encoded, whereas others were apparent only in CJD, but not in parallel preparations from uninfected brain. Small amounts of residual host Gp34 (prion protein) did not bind any 32P-labeled nucleic acid probes. Most of the minor "CJD-specific" proteins had an acidic pI, a characteristic of many viral core proteins. Such proteins deserve further study, as they probably contribute to unique properties of resistance described for these agents. It remains to be seen if any of these proteins are agent encoded.

Protected endogenous retroviral sequences copurify with infectivity in experimental Creutzfeldt-Jakob disease.

Scrapie and Creutzfeldt-Jakob disease (CJD) are caused by infectious agents that are defined phenomenologically. No agent-specific molecules or particles have been identified. Biological properties, such as exponential agent replication and strain variation, as well as physical characteristics of infectivity indicate a protected viral structure. A host membrane glycoprotein of 34 kDa ("prion" protein) that aggregates at end stages of disease is clearly important in pathology and susceptibility to infection, but has no demonstrable infectivity in any purified or recombinant form. Thus a characterization of more viral-like molecules is important. In order to identify viral-like nucleic acids we previously developed methods to substantially purify the human CJD agent from experimentally infected hamster brains, and demonstrated selected retroviral-like LTR bands at pg levels that were insufficient for sequencing. To further define these and other viral-like sequences we cloned nucleic acids from highly infectious CJD fractions, and tested the efficacy of our methods using a selected retroviral probe. RNA extracted from an infectious 120 S Gaussian peak, which is reproducibly purified approximately 100,000 fold with respect to starting nucleic acids, and contains approximately 20% of the initial brain infectivity, was used to generate a cDNA library in a sequence independent amplification strategy for low levels of RNA (< 6 ng). Reconstituted strong stop experiments using several retroviral tRNA primers had indicated that Syrian hamster IAP (SHIAP) sequences should be present in both CJD and uninfected control fractions. Because SHIAP particles are extremely resistant to denaturation, their representation in a cDNA library would imply adequate extraction of other protected RNAs of viral origin. At least 900 bases of the Syrian hamster retroviral IAP genome were unambiguously identified in the cDNA library, and in independent PCR walks with selected primers, all of which were based on our cloned sequences. Sequencing confirmed the presence of protected LTR and adjacent retroviral motifs. Because these sequences were also present in control preparations they may represent normal endogenous viral contaminants that cosediment with infectivity in size and density gradients. On the other hand, LTRs can drive the expression of many diverse sequences, and it remains to be seen if CJD specific sequences are either transduced, or copackaged with, protected IAP complexes. The effective extraction and amplification of highly protected SHIAP nucleic acids of significant length sets the stage for identifying additional protected viral elements that may specify the CJD agent.

Nuclease-resistant polyadenylated RNAs of significant size are detected by PCR in highly purified Creutzfeldt-Jakob disease preparations.

The molecular nature of the 'unconventional viruses' that cause slow, progressive brain deterioration is still poorly understood. As part of a reinvestigation of potential agent-specific nucleic acids, we developed a protocol for enriching agent-specific sequences. This protocol uses extensive micrococcal nuclease digestion followed by rate zonal sucrose sedimentation. Most of the infectivity in the gradient (84%) had a characteristic mean size of approximately 120S, and was resolved from 70% of a host glycoprotein (PrP) that can cosediment with infectivity. In infectious size fractions, nucleic acids were reduced approximately one million-fold with respect to starting brain homogenate, and specific purification of infectivity was approximately 100,000-fold with respect to nucleic acid. Using a novel polymerase chain reaction strategy, we were able to amplify RNA species in these fractions. Remarkably, host polyadenylated sequences of 1 to over 4 kb were detected in the nuclease-protected infectious fractions. These strategies set the stage for the identification of similar nucleic acids that may be specific for the CJD agent.

Potential retroviral RNAs in Creutzfeldt-Jakob disease.

The molecular nature of the related infectious agents that cause Creutzfeldt-Jakob disease (CJD) and scrapie is poorly understood, and an agent-specific nucleic acid genome has not yet been identified. Several biological manifestations of these agents resemble those seen in retrovirus-induced diseases. We therefore attempted to identify an agent-specific retrovirus-like RNA transcript in CJD infectious fractions. A series of synthetic oligonucleotides complementary to known mammalian retroviral primer binding sites were used in a primer extension assay. Substrate nucleic acids isolated from partially purified hamster brain CJD infectious fractions and from parallel normal brain fractions were compared with total starting brain RNA. This sensitive exogenous strong-stop reaction revealed that CJD infectious fractions contained a series of potential retroviral RNAs including apparent transcripts of endogenous hamster IAP genes. Most transcripts selectively recovered in the fractions were substantially protected from micrococcal nuclease digestion, and at least one substrate RNA, consistent with an intracisternal A particle, was packaged in a form that had the same buoyant density as CJD infectivity. Although a completely CJD-specific transcript was not identified, the copurification of potential retroviral transcripts with CJD infectivity suggests that models of disease involving retrovirus-like nucleic acid elements deserve further consideration.

Nuclease treatment results in high specific purification of Creutzfeldt-Jakob disease infectivity with a density characteristic of nucleic acid-protein complexes.

Representative preparations of partially purified Creutzfeldt-Jakob disease (CJD), including disaggregated density gradient fractions, were treated with a variety of nucleases. RNases as well as exhaustive digestions with micrococcal nuclease did not significantly diminish infectivity, but resulted in an approximately 7,000-fold specific purification of infectivity with respect to nucleic acid. Protected nucleic acids included species of up to 2,000 bases in length. After nuclease treatment, infectivity co-migrated with nucleic acid-protein complexes at a density of 1.27 g/cm3 in sucrose. Substantial specific protein purification were also achieved in the gradient step (approximately 11,000-fold), where 70% the host Gp34 ("prion protein") as well as other free proteins separated from infectivity. These CJD purifications are better than those previously attained in scrapie, and may be useful for further studies of non-host protein and nucleic acid species. The data are consistent with the hypothesis that CJD-like agents are composed of nucleic acid-protein complexes.

Transmission of Alpers' disease (chronic progressive encephalopathy) produces experimental Creutzfeldt-Jakob disease in hamsters.

We successfully and serially transmitted to outbred and inbred strains of hamsters the brain tissue of a 2 1/2-year-old girl with a chronic progressive encephalopathy (Alpers' disease) characterized postmortem as a spongiform encephalopathy. In all hamster strains we produced a spongiform encephalopathy. The light and ultrastructural changes in the brain of hamsters, as well as the clinical signs of experimental disease, are identical to those obtained in transmission experiments of human Creutzfeldt-Jakob disease (CJD). CJD infection may be more widespread than previously recognized and can be manifested in infancy.

Physical properties of the Creutzfeldt-Jakob disease agent.

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.

Immunohistochemistry of capillary hemangioblastoma. Immunoperoxidase-labeled antibody staining resolves the differential diagnosis with metastatic renal cell carcinoma, but does not explain the histogenesis of the capillary hemangioblastoma.

We used a battery of antigens to determine whether immunohistochemistry can (a) contribute to resolving the histogenesis of the stromal component of the capillary hemangioblastoma, and (b) answer cases of difficult pathologic differential diagnosis with metastatic clear cell carcinoma. The stromal cells of the capillary hemangioblastoma are antigenically polymorphous and may express immunoreactive erythropoietin, renin, keratin, Leu M1, Leu 7, actin, neuron-specific enolase, S100 protein, and glial fibrillary acidic protein. However, the use of epithelial membrane antigen allows certain histopathologic distinction between capillary hemangioblastoma and metastatic clear cell carcinoma.

Neuronal alterations in experimental Creutzfeldt-Jakob disease: a Golgi study.

Neuronal alterations in the cortex of hamsters with experimental Creutzfeldt-Jakob disease (CJD) were studied utilizing the rapid Golgi and Golgi-Hortega methods. In terminally ill hamsters many pyramidal neurons showed a considerable decrease in the number of dendritic spines often with moderate irregularities in size. The shafts of apical dendrites in some cortical pyramidal neurons were tortuous. Occasional neuronal cell bodies were irregular in contour and showed hole-like empty spaces. Two types of focal swellings, which involved both axons and dendrites, were observed; focal swellings were either semitranslucent or darkly impregnated. Possible mechanisms for the pathogenesis of the semitranslucent and dark swellings are discussed. The neuronal changes showed no preponderance for a particular neuronal group or for selective segments of individual neurons.

Suggested links between different types of dementias: Creutzfeldt-Jakob disease, Alzheimer disease, and retroviral CNS infections.

Several lines of investigation identify common features of Creutzfeldt-Jakob disease, Alzheimer disease, and retroviral CNS infections. We discuss salient neuropathological, genetic, transmission, and transformation properties that suggest there may be common pathways in the pathogenesis of dementias. We also briefly present potential retroviral mechanisms that may be implicated in all of these dementias.

Malignant glioma of the optic chiasm eight years after radiotherapy for prolactinoma.

A 41-year-old man had rapidly progressive visual loss caused by a malignant glioma that developed in the optic chiasm eight years after radiation therapy for a recurrent prolactinoma. Radiation-induced glioma should be considered as a cause of progressive visual loss in patients who have received irradiation in the region of the sella turcica.

Renin in glioblastoma multiforme and its role in neovascularization.

Significant proliferation of capillaries with hyperplastic vascular endothelium is one of the characteristic histologic features of glioblastoma multiforme (GBM). It has been shown that the renin-angiotensin II cascade stimulates new vessel formation. The presence of renin in several types of highly vascularized neoplasm suggests that it may also be implicated in the mechanism of tumor angiogenesis. In order to study the possible relationship of renin to GBM, immunohistochemical search for human renin was carried out in ten instances of such a tumor. Eight of these cases demonstrated renin-containing neoplastic astrocytes, whereas seven cases of reactive gliosis and six cases of low-grade astrocytoma revealed no renin-containing cells. The immunostaining was not present after preabsorption of the renin antiserum with pure human renin or substitution of preimmune serum for the specific renin antiserum. Because it has also been demonstrated that a product of renin, angiotensin II, has angiogenic properties, it seems reasonable to postulate that renin, through angiotensin II, may play a role in the mechanism of GBM-associated neovascularization.

Growth factor production by Creutzfeldt-Jakob disease cell lines.

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.

Transmission studies from blood of Alzheimer disease patients and healthy relatives.

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.

Cerebral glycosidases in experimental Creutzfeldt-Jakob disease.

In Creutzfeldt-Jakob disease (CJD), there are prominent ultrastructural alterations of the plasma membrane, which contains many glycolipids and glycoproteins. Glycosidases can degrade glycolipids and glycoproteins. Gangliosides, a subset of glycolipids, are decreased in amount at the terminal stages of CJD, and CJD infectivity is closely associated with membrane rich fractions. We therefore studied 10 glycosidases, and found a statistically significant increase in beta-xylosidase, beta-glucuronidase, N-acetyl-beta-D-glucosaminidase and N-acetyl-beta-D-galactosaminidase activities in CJD. In contrast, alpha-glucosidase, beta-glucosidase, alpha-galactosidase, alpha-mannosidase, alpha-fucosidase, and beta-galactosidase were not significantly changed. The above results are consistent with degenerative membrane changes observed morphologically, and with increased degradation of sugar residues on lipids and/or proteins. These changes may be effected by the accumulation of the CJD agent in cell membranes. We suggest that the higher activities of these enzymes in CJD may be partially responsible for some of the structural and biochemical alterations in CJD infected brains.

Potential involvement of retroviral elements in human dementias.

Creutzfeldt-Jakob disease (CJD) is a dementia of humans caused by a class of infectious agents with several biological properties similar to those of conventional viruses. The molecular nature of this group of agents is enigmatic, for neither an agent-specific nucleic acid nor a non-host protein has yet been identified. Recent transmissions of familial CJD dementias to rodents suggest that this class of agent can be integrated into the germline. Furthermore, tissue culture studies indicate that CJD causes transformation of cells in a manner reminiscent of slowly oncogenic retroviruses. Currently characterized retroviral-like elements include many forms that do not have 'typical' retroviral ultrastructural morphology; several forms are also known to be resistant to various types of standard physicochemical inactivation. We suggest that CJD agents are either constituted by retroviral-like nucleic acids or interact with endogenous retroviral sequences to elicit a slowly progressive disease of the central nervous system. Several overlapping properties between infectious CJD and 'non-infectious' dementias, such as Alzheimer's disease, implicate potential common pathogenic mechanisms.

Creutzfeldt-Jakob disease with intranuclear vacuolar inclusions: a biopsy case of negative light microscopic findings and successful animal transmission.

In a 53-year-old man with a progressive mental deterioration and myoclonic jerks, brain biopsy failed to show any significant light microscopical findings. Electron microscopy revealed membrane-bound vacuolar inclusions in many neuronal nuclei as the only prominent finding. Hamsters intracerebrally inoculated with the biopsy material demonstrated typical spongiform changes in the gray structures of the brain when sacrificed on the 309th and 332nd days post inoculation, characteristic of experimental Creutzfeldt-Jakob disease (CJD). These intranuclear vacuolar inclusions, originally reported in experimental Creutzfeldt-Jakob disease in this laboratory, may be a valuable electron microscopic feature in some CJD cases and may play an important role in supporting the diagnosis of CJD.