Elias E. Manuelidis

Evidence suggesting that PrP is not the infectious agent in Creutzfeldt-Jakob disease.

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.

Experimental Creutzfeldt-Jakob Disease Transmitted Via the Eye with Infected Cornea

Scrapie of sheep, transmissible mink encephalopathy and Creutzfeldt-Jakob disease and kuru of man constitute the nosologic group of subacute spongiform virus encephalopathies.1 The possible transmi...

Ependymomas of the spinal cord

Many patients with spinal cord ependymomas (SCE) undoubtedly benefit from post-operative radiation therapy; however, because of the wide variability in the total doses given, the optimal post-operative dose for SCE remains unclear. Several recent papers recommend total doses of 4000 rad to 5000 rad in 4 1/2 to 6 weeks. Unfortunately, only a small number of patients reported in the literature have been consistently treated to these high dose recommendations. Nine consecutive adult patients with SCE have been treated in a consistent way at Yale-New Haven Hospital with total doses of approximately 4500 rad to 5000 rad at 180 rad to 200 rad per day. The acute and chronic morbidity from such treatment has been minimal and no patient has had a local recurrence at 8 months to 8 years following treatment.

Transmission of Creutzfeldt-Jakob disease with scrapie-like syndromes to mice.

THE transmission of Creutzfeldt–Jakob disease of man, one of the subacute spongiform virus encephalopathies1, to guinea pigs2,3 and to hamsters4 has been reported from this laboratory; Brownell et al. have also claimed transmission of Creutzfeldt–Jakob disease to mice5,6. Here we present evidence that mice are susceptible to this transmissible encephalopathy and that there are similarities between the clinical and pathological findings in experimental Creutzfeldt–Jakob disease in mice, and those observed in the same host in experimental scrapie7–11.

Ganglioside content and pattern in human gliomas in culture. Correlation of morphological changes with altered gangliosides.

SummaryThe ganglioside level and pattern of human gliomas in monolayer cultures were examined. These gliomas revealed morphological variations that correlated with several features of ganglioside analysis. Glioblastoma lines TC 178 and TC 501 that morphologically had changed during extended subculture revealed reduced amounts and a simplified pattern of gangliosides with almost total loss of the characteristic brain complex gangliosides. In contrast, two glioblastoma lines TC 526 and TC 593, as well as the oligodendroglioma line TC 620 showed brain-like gangliosides and the cells in these cultures had maintained their characteristic morphology observed during early subcultures. The possibility that altered ganglioside levels occur in conjunction with morphological changes after propagation in vitro is discussed.

Creutzfeldt-Jakob Disease

The historical aspects of spongiform encephalopathies, Creutzfeldt-Jakob disease (CJD) and kuru of man, as well as scrapie and transmissible mink encephalopathy, are outlined. Transmissions of these diseases to animal hosts are presented, with emphasis on CJD transmissions to guinea pigs, hamsters, and mice. The relationship of CJD to scrapie with reference to the pathological findings is discussed. In CJD the incubation period is cut in half in guinea pigs and hamsters in the second passage. The spongiform changes occurring in the neuropil are reviewed. These changes are related to the type of inoculum, e.g., there is more vacuolization after inoculation with brain, and less after inoculation with spleen. Spongiform changes are also dependent upon the route of inoculation; these are more severe in intracerebral inoculation compared to intraperitoneal inoculation. Viremia is present. Maternal transmission and lateral transmission are absent. No virus-like particles are detected, and no other organisms are visible by electron microscopy. Isolations of the causative agent and strains of the agent in spongiform encephalopathies remain elusive. The hypotheses concerning the nature of the agent are critically reviewed. Novel data on the production of tumors derived from CJD brains are presented. Tissue culture cells arising from such brains become permanent lines and are similar to neoplastic lines. When such CJD lines are injected subcutaneously into nude mice, malignant neoplasms are formed. No evidence of an infectious etiology in Alzheimers disease exists. Reported similarities between this disease and CJD are reviewed. Animal models of CJD are useful for the investigation of dementias.

An autoradiographic study of the proliferation and differentiation of glial cellsin vitro

SummaryOrganotypic cultures of mammalian embryonic spinal cord-dorsal root ganglia combinations treated with H3 thymidine, were studied with light and electron microscopic autoradiography. Cultures pulsed with H3 thymidine for up to six hours showed 0–2% of labelled neuroepithelial cells when fixed immediately thereafter, and 7 to 15 times as many labelled cells when fixed on subsequent daysin vitro (DIV). Cultures exposed to radioactive material for days, at the initiation of the culture, and prior to myelinationin vitro, were compared. The cumulative evidence showed that the peak of proliferative activity occurred not after explantation, but between DIV 7 and DIV 12, which is after synapse formation and prior to myelination. Another smaller proliferative peak occurred after myelination between DIV 15 and DIV 19.Morphological observations with the light microscope revealed labelling of predominantly small dark cells during the first proliferative peak, that is, prior to myelination. Using electron microscopic criteria for identification, these small dark cells were “large glioblasts”, “large glial precursors” and “young” oligodendrocytes, and transitions could be observed between these cells in that order of differentiation. Oligodendrocytes when closely connected with myelin sheaths did not become labelled.Labelling and proliferation of medium large light cells inconspicuously preceded that of oligodendrocytes and their precursors, and continued modestly throughout myelination, achieving a modest peak and predominance during DIV 15 and DIV 19. Astrocytic features could be demonstrated ultrastructurally in these medium large light cells and mitotic division in this type of cell was observed. The possibility that part of this astrocytic population arose from “large glioblasts” was discussed.No labelled cells were seen ultrastructurally fulfilling criteria of amitotic division.No neurons showed labelling with H3 thymidine.

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

SummaryRepresentative 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 ∼ 7,000-fold specific purification of infectivity with respect of 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 (∼ 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.

Ultrastructural findings in experimental Creutzfeldt-Jakob disease in guinea pigs.

Brains from six clinically ill guinea pigs from various serial passages infected with brain homogenate from a patient with Creutzfeldt-Jakob disease were studied by light and electron microscopy. There were no appreciable morphologic differences among the animals at different passages. Light microscopy revealed clearing, swelling, and vacuolar changes in neurons and astrocytes. These changes occurred in both perikarya and cell processes. Large isolated vacuoles in the neuropil appeared to develop by progressive fusion between swollen cellular processes. By electron microscopy many neuronal processes contained degenerating organelles, osmiophilic bodies, membranous bodies and ˜10 nm filamentous structures. In addition, vacuoles and membranous inclusions were seen in some neuronal nuclei, changes which have not been previously observed in spongiform encephalopathies.

Serial ultrastructural study of experimental Creutzfeldt-Jacob disease in guinea pigs

SummaryGuinea pigs inoculated with brain homogenate from serially passaged Creutzfeldt-Jakob disease (CJD) were killed biweekly starting at week 2 until terminal illness (about 200 days following inoculation). A mild swelling of postsynaptic dendrites and an increase in the number of glial filaments in astrocytic processes was seen at week 4, followed by increased swelling and lucency of axons and dendrites by week 6 post inoculation (p.i.). Severe undulation and focal interruptions of synaptic membranes were also observed both at weeks 4 and 6. By week 8, one could see cystically dilated cellular processes. These sometimes showed continuity with adjacent swollen processes through focally disrupted plasma membranes, and most likely represent a progressive enlargement of vacuoles through fusion and subsequent addition of adjoining processes. The spongiform changes increased mildly between week 8 and week 10 and remained essentially the same in subsequent weeks. After week 24 there was a sharp increase in both the number and size of vacuoles. At week 24 severe structural alterations were present both in the neurons and astrocytes, and numerous intranuclear inclusions were demonstrated in many neuronal nuclei. This study shows that morphological changes in the brain occur considerably earlier than the clinical manifestations of the disease. In the early phase of the disease, there were significant alterations on the dendrites and synapses.