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1 Department of Veterinary Sciences, Michale E. Keeling Center for Comparative Medicine and Research, University of Texas MD Anderson Cancer Center, Bastrop, TX, USA
2 Bethesda, MD, USA
3 National CJD Surveillance Unit, Western General Hospital, Edinburgh, UK
4 Department of Comparative Medicine, College of Medicine, University of South Alabama, Mobile, AL, USA
5 Global Pathogen Safety, Baxter Bioscience, Vienna, Austria
Correspondence
Lawrence Williams
lewillia{at}mdanderson.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Sporadic Creutzfeldt–Jakob disease (sCJD) is by far the most common form of this disease, occurring with no apparent environmental or genetic cause at a worldwide rate of about one case per million population per year. Kuru, the prototype human TSE, occurred only among the Fore linguistic group in the interior of Papua New Guinea as a result of ritualistic cannibalism, and is now virtually extinct. Familial forms of TSE account for about 10 % of all cases and are described as familial CJD, Gerstmann–Sträussler–Scheinker disease (GSS) and fatal familial insomnia (FFI), depending on the causative mutations and their phenotypic expression (Kovacs et al., 2002). Environmentally acquired disease has mostly followed iatrogenic exposures (contaminated pituitary hormones, dura mater grafts or neurosurgical instruments) or oral infections from bovine spongiform encephalopathy (BSE)-contaminated foodstuffs [variant CJD (vCJD)] (Will et al., 1996; Brown et al., 2000). These different forms of human disease tend to show different and sometimes quite distinctive clinical and neuropathological features. sCJD patients typically present with memory loss or some other manifestation of mental deterioration (including behavioural changes) coupled with physical signs of incoordination; the mean duration of illness is about 7 months. vCJD patients typically present with behavioural and sensory symptoms such as leg pain or paraesthesias, and the mean duration of illness is 14 months. The unique pathological hallmark of vCJD is the so-called ‘florid’ plaque, a core of amyloid surrounded by a halo of spongiform change. Cases of vCJD also have a pattern of pathologically misfolded ‘prion’ protein (PrPTSE) in the brain recognizably different from that seen in cases of sCJD. Familial forms of disease can show features similar to those of sCJD or be so different as to have merited different names (GSS and FFI); in general, the duration of illness in most familial disease is longer than in either sCJD or vCJD and often extends to years, rather than a few months.
We here report the first systematic, cliniconeuropathological study of different forms of CJD passaged in the squirrel monkey, a highly susceptible non-human primate species.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
sCJD.
Brain tissue was pooled from two UK patients.
RU 98-88.
An 86-year-old female presented with cognitive difficulties, confusion and ataxia and progressed rapidly to a state of akinetic mutism, dying several months after onset of illness. Methionine was homozygous at codon 129, yielding a PrPTSE type 1 pattern on Western blot analysis. There was widespread spongiform change throughout the cerebral cortex (most prominent in frontal, temporal and occipital lobes), basal ganglia and thalamus. No plaques were present on routine stains. PrPTSE was deposited in a predominantly synaptic pattern in the cerebral cortex, together with occasional areas of perivascular accumulation. PrP immunohistochemistry did not reveal any plaques or plaque-like structures.
RU 98-123.
A 62-year-old female presented with slowly progressive cerebellar syndrome and cognitive impairment and later developed abnormal movements and mutism, dying 3 years after onset of illness. Methionine/valine were heterozygous at codon 129, yielding a PrPTSE type 2A pattern on Western blot analysis. There was widespread spongiform change throughout the cerebral cortex (most prominent in the parietal region), basal ganglia and cerebellum, with milder changes in the thalamus. There were occasional small, kuru-type amyloid plaques in the cerebral cortex and molecular layer of the cerebellum, none of which had a florid appearance. There was also a strong synaptic pattern of PrPTSE deposition throughout the cerebrum, basal ganglia and cerebellar molecular layer, with PrPTSE-positive plaques with some perineuronal staining in the cerebral cortex.
vCJD.
Brain tissue was pooled from three UK patients.
RU 96-110.
A 35-year-old female presented with agitation, social withdrawal and personality change, progressing to ataxia with myoclonus and, finally, akinetic mutism, dying 14 months after onset of illness. Methionine was homozygous at codon 129, yielding a PrPTSE type 2B pattern on Western blot analysis. There was widespread spongiform change in the cerebral and cerebellar cortices and in the caudate nucleus, with numerous non-florid and florid plaques especially prominent in the occipital cortex; milder changes were present in the basal ganglia and thalamus. There was extensive PrPTSE deposition around cells and vessels and within plaques.
RU 97-110.
A 21-year-old female presented with irritability, aggression and social withdrawal, then developed persistent double vision, progressive incoordination, and, finally, akinetic mutism, dying 20 months after onset of illness. Methionine was homozygous at codon 129, yielding a PrPTSE type 2B pattern on Western blot analysis.
RU 97-49.
An 18-year-old male presented with social withdrawal and leg pain, progressing to dysarthria, ataxia, dementia and myoclonus, dying 14 months after onset of illness. Methionine was homozygous at codon 129, yielding a PrPTSE type 2B pattern on Western blot analysis. There was patchy spongiform change in the cerebral cortex, most prominent in the occipital region, accompanied by numerous florid plaques; similar changes in the basal ganglia, thalamus and molecular layer of the cerebellum and widespread perineuronal and perivascular deposition of PrPTSE, with a strong staining of plaques, were seen.
GSS.
The patient was a 35-year-old male presenting with difficulty walking and deteriorating job performance, who then had a fairly rapid progression of mental and behavioural deterioration and ataxia, exaggerated reflexes and myoclonus associated with periodic electroencephalogram activity, dying 3 months after onset of symptoms. There was widespread spongiform encephalopathy with prominent, multifocal PrPTSE amyloid plaques in the cerebellum. PRNP gene sequencing revealed a 192 bp insertion (Western blot for PrPTSE was not performed). Although the patient's illness was characteristic of CJD, other family members with the same mutation had clinical and neuropathological features much more typical of GSS.
Brain tissue was passaged successively in two chimpanzees by intracerebral inoculation of a 10 % homogenate in saline, and leukocytes from the second-passage chimpanzee were inoculated intracerebrally into two squirrel monkeys (Saimiri sciureus), one of which, after an incubation period of 36 months, developed the disease described in the present study.
Inoculated primates.
As part of a large, experimental study of blood infectivity in human subjects with either sCJD or vCJD, in which squirrel monkeys were used to bioassay blood components, a number of positive-control animals were inoculated with brain tissue from the same human subjects. The monkeys were imported and held for a minimum of 6 months prior to inoculation. All animals were adults at the time of inoculation. The inocula were homogenized and diluted in saline, and animals were inoculated with 0.1 ml volumes into the left frontal lobe (Table 1). The 16 animals in the 10–1 and 10–3 dilution sCJD and vCJD groups (four animals per group) were kept under continuous clinical surveillance and were subjected to detailed behavioural testing at regular intervals throughout the preclinical (incubation period) and clinical stages of disease until they met the ethical criteria for euthanasia. One monkey was inoculated with leukocytes from a chimpanzee-passaged case of GSS; 0.1 ml leukocytes in the frontal cortex and 0.5 ml leukocytes diluted 1 : 1 in normal saline intravenously. Squirrel monkeys are uniformly homozygous for methionine at codon 129, and all of the inoculated monkeys showed a type 1 PrPTSE glycoform pattern.
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Housing.
Subjects were housed four or five to a cage. Subjects with similar dilutions of each CJD inoculum where housed together in stainless-steel cages that measured 1.5 m (wide) by 0.75 m (tall) by 0.75 m (deep). Control animals were housed together in a separate cage. Commercial monkey chow and water were provided ad libitum, with fresh fruits and vegetables provided several days per week. A 12 h/12 h light/dark cycle was used.
Behavioural observation and analysis.
Qualitative observations were made by trained observers. They included daily, in-depth observations for abnormal posture, movements and the willingness and ability to accept a food reward.
Quantitative observations were made by using a focal animal method. During a 10 min time block, all behaviour involving the focal animal was recorded. Table 2 provides the operational definitions for the behavioural responses analysed as part of this study. The data generated were converted into estimates of hourly rates and durations for the behaviour recorded. Data were recorded directly onto a hand-held computer and transferred to a desktop computer for analysis using the Noldus Observer program (Azzolin et al., 1998). Up to 24 focal sessions of 10 min were collected during a 3-month study block. For the purposes of this paper, the data were summarized into 30-day blocks by using the date that the subject was removed from the study as time zero (0) and looking backwards to 1 month (0–30 days) and 3 months (60–90 days) prior to removal. Data for the control group were summarized across data-collection blocks in which inoculated subjects exited the study. One-way analysis of variance procedures were used to investigate differences between the three study groups at both time points, with statistical significance set at a probability of 0.05.
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Following dissection of the formalin-fixed tissues from the brain, spleen, lymph nodes, tonsil and small intestine, the tissue blocks were immersed in 96 % formic acid for 1 h prior to routine processing and embedding in paraffin wax. Sections (5 µm) were cut for haematoxylin and eosin (H&E) staining, immunohistochemistry and paraffin-embedded tissue (PET) blotting for pathological prion protein (PrPTSE). Antibody 6H4 (Prionics) was used for immunohistochemistry in combination with a catalysed signal-amplification technique (Dako) (Ironside et al., 2000). For PET blotting, antibody 3F4 (Dako) was used (Ritchie et al., 2004).
Sample preparation and Western blot analysis of PrPres.
Biochemical analysis of PrPres was carried out on frozen brain tissue from four of the six squirrel monkey cases that had undergone pathological investigation. Western blot analysis was performed on extracts of samples (100 mg) of frontal cortex. Samples were digested with proteinase K (VWR International Ltd) and analysed by Western blotting using an established method for the biochemical diagnosis of human CJD brain (Head et al., 2004). Bis–Tris NuPage gels (10 %), buffers and molecular mass markers (MagicMark; Invitrogen Life Technologies) were used in accordance with the manufacturers' instructions. Proteinase K digestion was carried out at a concentration of 50 µg ml–1 for 1 h. The monoclonal anti-PrP antibody 3F4 (Dako) was used at a concentration of 50 ng ml–1 for 1 h. Blotting membranes (Hybond-P), horseradish peroxidase-conjugated anti-mouse secondary antibody, chemiluminescent reagent (ECL+) and X-ray film (Hyperfilm) were all obtained from GE Healthcare. The squirrel-monkey samples under investigation were run between proteinase K-treated samples of cerebral cortex from standard control cases of sCJD (type 1 PrPres) and vCJD (type 2B PrPres).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In the group that received a 10–3 dilution of sCJD brain homogenate, the time to initial clinical signs ranged from 22 to 23 months. The duration of clinical signs ranged from 2 to 7 months. Clinical progression of the disease was similar to the 10–1 group; signs started with mild tremors and ataxia that became progressively worse and the animals developed abnormal postures and intention tremors.
vCJD.
In the 10–1 inoculum-dilution group, clinical signs developed 21–30 months post-inoculation and lasted from 2.5 to 6.0 months. Monkeys had mild gait ataxia and tremors. Toward the end of the illness, subjects had difficulty maintaining upright posture and prehending food. All animals had progressive weight loss. Unlike the sCJD-inoculated monkeys, there were neither myoclonus nor proprioceptive deficits. The subjects remained alert and active and showed no evidence of increased aggression or irritability.
In the 10–3 inoculum-dilution group, clinical signs developed 29–41 months post-inoculation, with signs lasting 3–7 months. As with the sCJD groups, clinical signs in the 10–3 dilution group were similar to those seen in the 10–1 dilution group, only progressing more slowly. The disease started with a mild unsteady gait and ataxia and, as the disease progressed, the animals moved with greater deliberation. All animals showed progressive weight loss.
GSS.
The monkey inoculated with a leukocyte homogenate from a chimpanzee-passaged case of GSS developed clinical signs that were strikingly different from those seen in either the sCJD- or vCJD-inoculated squirrel monkeys. There was no evidence of tremor (either resting or intentional), no myoclonus and none of the exaggerated gait ataxias seen in the other groups. While walking, the monkey moved slowly and carefully; it stayed off perches and narrow walkways, crouched with limbs spread to provide a wider base and would occasionally stumble and fall. When reaching for food, the monkey would grab the bowl to steady itself and would appear to have difficulty targeting an individual food item. The hand would sweep back and forth over a food item before grabbing it. In addition, the animal displayed an exaggerated undulation of the whole body. When sitting, the monkey's head would bob or wiggle up and down or from side to side, causing the animal to lose its balance with a sudden jerk. While standing, the whole body would undulate in a wave-like motion, starting rostrally and moving caudally; at times, it would progress with chameleon-like back-and-forth oscillations.
Quantitative behavioural descriptions
Focal animal observations on the sCJD-inoculated, vCJD-inoculated and control animals were analysed 3 months and 1 month prior to exit from the study, at which time the animals were in an advanced stage of illness. These results are summarized in Table 3. At the earlier clinical stages, there were no significant differences between sCJD- and vCJD-inoculated animals in the duration of non-social object manipulation, time playing, time resting, time eating or in aggression or dominance displays (genital display and mounting).
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At both time points, sCJD-inoculated and vCJD-inoculated animals scratched more than the control animals. However, the sCJD-inoculated animals scratched more at 3 months, but not at 1 month, prior to study exit than the vCJD-inoculated animals. This follows a decrease in all coordinated activity seen in sCJD-inoculated animals later in the study.
At 3 months prior to exit from the study, time spent sitting or self-scratching was significantly higher in sCJD-inoculated than in vCJD-inoculated or control animals. Time spent drinking was significantly higher in vCJD-inoculated compared with the control and sCJD-inoculated animals, but this difference diminished by 1 month prior to study exit.
At 1 month prior to study exit, time spent travelling was significantly higher in vCJD-inoculated compared with sCJD-inoculated animals; conversely, time spent huddled with another animal was significantly higher in sCJD-inoculated than in vCJD-inoculated animals (in fact, no vCJD-inoculated animal was observed to huddle with a social partner during the month prior to study exit).
Neuropathology and immunohistochemistry
H&E-stained sections showed spongiform change within the brain and brainstem in the three sCJD and the three vCJD cases, but not in the control case. Spongiform change was generally most prominent within the cerebral cortex and basal ganglia, with the thalamus, hippocampus, brainstem and cerebellum less affected. Different patterns of lesions were evident in the sCJD-inoculated and vCJD-inoculated animals.
The three sCJD-inoculated monkeys showed comparatively subtle microvacuolar spongiform change, most prominent in the deeper layers of the frontal and temporal lobes (Fig. 1a) and in the basal ganglia. In these cases, immunohistochemistry showed sparse PrPTSE deposition (Fig. 1c), predominantly in a synaptic pattern with perineuronal deposition around occasional large pyramidal cells, and PET blot labelling also showed a widespread synaptic distribution of PrPTSE, similar to that seen in human cases of sCJD. However, these patterns of PrPTSE accumulation were not identical to those seen most frequently in the type 1 subgroup sCJD, where perineuronal accumulation is relatively uncommon (Parchi et al., 1999). Equally, kuru-type amyloid plaques and plaque-like deposits of PrPTSE (which are common in cases of the type 2 subgroup of sCJD) were not observed in any of these cases.
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). Patchy vacuolation was also identified in the thalamus, accompanied by neuronal loss and gliosis. Immunohistochemistry and PET blot labelling in these cases showed widespread and heavy deposition of PrPTSE within the grey matter, in a diffuse and pericellular pattern similar to that observed in human vCJD cases (Fig. 1d). However, the amyloid plaques and plaque-like deposits of PrPTSE that are a prominent feature of vCJD in humans were not observed in any of the three cases. Although the pattern of vacuolation in the cortex and the thalamic pathology in these monkeys were similar to those seen on experimental transmission of vCJD to macaques (Lasmézas et al., 2001), no florid plaques were identified. No lesions or PrPTSE were detected in the brain of the control monkey. No PrPTSE was present in peripheral lymphoid tissues of any of the infected or control animals.
Western blot analysis of protease-resistant PrP (PrPres) in frozen tissue samples from the frontal cortex of the sCJD and vCJD transmissions (Fig. 2) showed that the sCJD transmissions contained a single PrPres isoform that resembled the human sCJD type 1 PrPres isoform, with no type 2 PrPres detected. In contrast, the PrPres isoform in the vCJD transmissions closely resembled the human vCJD PrPres isoform in terms of molecular mass, mobility and glycosylation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), but has never been the subject of systematic clinical, neuropathological or immunohistochemical evaluation. The recent addition of a distinctively different variant form of CJD (vCJD) to the spectrum of human disease has raised the interesting question of whether the strain of TSE responsible for this form of disease is maintained in passage to non-human primates and can be distinguished clinically and neuropathologically from strains causing sCJD.
In view of studies in humans showing different phenotypes in sCJD patients with various combinations of codon 129 genotypes and protein-glycoform patterns (Parchi et al., 1999), and the fact that squirrel monkeys (in common with other non-human primates) are uniformly homozygous for methionine at codon 129, it may be asked whether the disease phenotypes exhibited by the sCJD monkeys might have been influenced by pooling two sCJD samples with different genotypes and glycoform patterns. The question cannot be answered with confidence short of studying large groups of monkeys inoculated with each of the six possible combinations, a prohibitively expensive and probably not very fruitful experiment, given the still-preliminary nature of the classification of human sCJD PrPTSE isoforms.
However, it is worth noting that, despite their differing molecular biology, both sCJD patients presented with cognitive and cerebellar deficits, in sharp contrast to the psychiatric/sensory presentations of all three patients with vCJD. The pooling should therefore not have obscured any clinical differences between the monkeys inoculated with sCJD and vCJD.
Both the mean incubation period and duration of illness were somewhat longer in the vCJD- than sCJD-inoculated groups. The longer vCJD incubation period cannot be interpreted because, although the mean human vCJD incubation period is thought to be 10–15 years, the incubation period of sCJD is unknown. However, the longer duration of illness is consistent with human disease, in that vCJD has a mean duration of 14 months, compared with 7 months for sCJD (Will et al., 2000).
The sCJD squirrel monkeys had more prominent gait ataxia and tremors, as well as myoclonus, and in consequence were unable to move about the cage to associate themselves with potential social partners. They spent more time within one body length of another animal (social proximity). Animals in the vCJD groups, with less-severe clinical signs (mild ataxia, reduced tremors and no myoclonus), generally kept up their usual activities, eating and drinking, but at a slower pace. These observations differ from those reported in vCJD-inoculated cynomolgus macaque monkeys, which were not, however, studied systematically or with standardized behavioural tests (Lasmézas et al., 2001). Unlike macaques, for which an increase in aggression has been reported with vCJD inoculation, there was no increase in aggressive behaviour in vCJD-inoculated squirrel monkeys.
The extraordinary incoordination syndrome shown by the single animal inoculated with a chimpanzee-passaged strain of human GSS was different in both its severity and clinical manifestations from the illnesses seen in all other inoculated animals. This difference may have been due to the strain of TSE or to its passage through a chimpanzee before inoculation into monkeys.
The purpose of our study was to investigate whether the behaviour of CJD-inoculated squirrel monkeys would allow for a differential diagnosis between vCJD- and sCJD-infected animals, and to establish clinicopathological baselines for eventual therapeutic studies. On the basis of clinical observation, it was not possible to say that any given monkey had been infected with one or the other strain of human TSE, although, as a group, the sCJD- and vCJD-inoculated monkeys were recognizably different (the same can be said about human cases of these two forms of disease). However, the distribution of histopathological changes and PrPTSE immunostaining, even in the absence of plaques (thus not exactly duplicating the pathology of human disease) (Ironside et al., 2000), allowed a clear distinction between individual sCJD- and vCJD-inoculated animals, and differences in the nature, severity and distribution of the pathological lesions and PrPTSE in the brain are likely to have influenced the observed clinical manifestations. The pathological features of infection by BSE-related agents differ substantially according to the host species. Thus, BSE infection in cattle results in no florid plaques in the brain and very little PrPTSE deposition in peripheral tissues, in contrast to vCJD in humans. Amyloid plaques are not a feature of BSE infection in mice or cats and, even in primates, the nature, extent and characteristics of BSE-related pathology are variable (Baker et al., 1998; Lasmézas et al., 2001).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 16 February 2006;
accepted 3 October 2006.
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