Renewed assessment of the risk of emergent advanced cell therapies to transmit neuroproteinopathies.

The inadvertent transmission of long incubating, untreatable and fatal neurodegenerative prionopathies, notably iatrogenic Creutzfeldt-Jakob disease, following transplantation of cadaver-derived corneas, pituitary growth, hormones and dura mater, constitutes a historical precedent which has underpinned the application of precautionary principles to modern day advanced cell therapies. To date these have been reflected by geographic or medical history risk-based deferral of tissue donors. Emergent understanding of other prion-like proteinopathies, their potential independence from prions as a transmissible agent and the variable capability of scalably manufacturable stem cells and derivatives to take up and clear or to propagate prions, substantiate further commitment to qualifying neurodegenerative proteinopathy transmission risks. This is especially so for those involving direct or facilitated access to a recipient's brain or connected visual or nervous system such as for the treatment of stroke, retinal and adult onset neurodegenerative diseases, treatments for which have already commenced. In this review, we assess the prospective global dissemination of advanced cell therapies founded on transplantation or exposure to allogeneic human cells, recap lessons learned from the historical precedents of CJD transmission and review recent advances and current limits in understanding of prion and other neurodegenerative disease prion-like susceptibility and transmission. From these we propose grounds for a reassessment of the risks of emergent advanced cell therapies to transmit neuroproteinopathies and suggestions to ACT developers and regulators for risk mitigation and extension of criteria for deferrals.

Distribution and Quantitative Estimates of Variant Creutzfeldt-Jakob Disease Prions in Tissues of Clinical and Asymptomatic Patients.

In the United-Kingdom, ≈1 of 2,000 persons could be infected with variant Creutzfeldt-Jakob disease (vCJD). Therefore, risk of transmission of vCJD by medical procedures remains a major concern for public health authorities. In this study, we used in vitro amplification of prions by protein misfolding cyclic amplification (PMCA) to estimate distribution and level of the vCJD agent in 21 tissues from 4 patients who died of clinical vCJD and from 1 asymptomatic person with vCJD. PMCA identified major levels of vCJD prions in a range of tissues, including liver, salivary gland, kidney, lung, and bone marrow. Bioassays confirmed that the quantitative estimate of levels of vCJD prion accumulation provided by PMCA are indicative of vCJD infectivity levels in tissues. Findings provide critical data for the design of measures to minimize risk for iatrogenic transmission of vCJD.

Production and characterization of a panel of monoclonal antibodies against native human cellular prion protein.

The human prion diseases, such as variant Creutzfeldt-Jakob disease (vCJD), are characterized by the conversion of the normal cellular prion protein (PrP(C)) into an abnormal disease associated form (PrP(Sc)). Monoclonal antibodies (MAbs) that recognize these different PrP isoforms are valuable reagents both in the diagnosis of these diseases and in prion disease research in general but we know of no attempts to raise MAbs against native human PrP(C). We immunized prion protein gene ablated (PrP(-/-)) mice with native human PrP(C) purified from platelets (pHuPrP) generating a predominantly IgG isotype anti-pHuPrP polyclonal antibody response in all mice. Following fusion of splenocytes from the immunized mice with SP2/0 myeloma cells, we were able to identify single cell clone and cryopreserve 14 stable hybridoma cell lines producing MAbs that reacted with pHuPrP. The properties of these MAbs (such as isotype, binding to native/denatured pHuPrP, and HuPrP epitopes recognized) are described. Furthermore, several of these MAbs showed a selectivity in their ability to immunoprecipitate disease associated PrP(Sc) and its corresponding protease resistant core (PrP(res)).

Application of Atomic Dielectric Resonance Spectroscopy for the screening of blood samples from patients with clinical variant and sporadic CJD.

BACKGROUND: Sub-clinical variant Creutzfeldt-Jakob disease (vCJD) infection and reports of vCJD transmission through blood transfusion emphasise the need for blood screening assays to ensure the safety of blood and transplanted tissues. Most assays aim to detect abnormal prion protein (PrPSc), although achieving required sensitivity is a challenge. METHODS: We have used innovative Atomic Dielectric Resonance Spectroscopy (ADRS), which determines dielectric properties of materials which are established by reflectivity and penetration of radio/micro waves, to analyse blood samples from patients and controls to identify characteristic ADR signatures unique to blood from vCJD and to sCJD patients. Initial sets of blood samples from vCJD, sCJD, non-CJD neurological diseases and normal healthy adults (blood donors) were screened as training samples to determine group-specific ADR characteristics, and provided a basis for classification of blinded sets of samples. RESULTS: Blood sample groups from vCJD, sCJD, non-CJD neurological diseases and normal healthy adults (blood donors) screened by ADRS were classified with 100% specificity and sensitivity, discriminating these by a co-variance expert analysis system. CONCLUSION: ADRS appears capable of recognising and discriminating serum samples from vCJD, sCJD, non-CJD neurological diseases, and normal healthy adults, and might be developed to provide a system for primary screening or confirmatory assay complementary to other screening systems.

Abnormal prion protein in the pituitary in sporadic and variant Creutzfeldt-Jakob disease.

By using high-sensitivity Western blotting and immunohistochemistry, pituitary glands from patients with sporadic and variant Creutzfeldt-Jakob disease (sCJD and vCJD, respectively) were analysed for the presence of the protease-resistant form of the prion protein (PrPres). PrPres was detected in a greater proportion of vCJD pituitaries than sCJD pituitaries and was localized predominantly in the neurohypophysis. PrPres was also detected in a recurrent pituitary adenoma from an sCJD patient. Immunohistochemical analysis showed sparse positive labelling, predominantly in folliculostellate cells, in vCJD and sCJD adenohypophyses. The PrPres glycosylation pattern in the vCJD neurohypophyses showed a predominance of the unglycosylated band, which differed markedly from patterns found in all other vCJD tissues. The presence of PrPres in the pituitary of CJD patients at autopsy suggests that human growth hormone-related iatrogenic CJD may have indeed resulted from infectivity in collected pituitaries rather than necessarily from contamination of pituitary pools by adjacent brain tissue.

Transplantation of ocular tissue from a donor with sporadic Creutzfeldt-Jakob disease.

BACKGROUND: One definite, one probable and several possible transmissions of sporadic Creutzfeldt-Jakob disease (sCJD) have followed corneal transplantation. We report an incident in the UK in 1997 in which both corneas and scleras from a donor, subsequently confirmed to have had sCJD, were transplanted. The final clinical outcome for two surviving recipients is still not yet known. CASE REPORT: In 1997, a 56-year-old woman died from biopsy-proven carcinoma of the bronchus. Both eyes were donated for transplantation. Shortly before she died, she had developed neurological symptoms thought to be due to brain metastases. However, the final result of a neurological post-mortem examination revealed evidence of sCJD. By this time the corneas had been transplanted, one 3 months previously into a 40-year-old man for keratoconus and the other 4 months previously into an 85-year-old woman for Fuchs' dystrophy. In addition, both scleras had been transplanted into a 36-year-old man undergoing oculoplastic reconstructive surgery. The surgeons and patients were informed and removal of tissue was advised but undertaken in only two of the patients. Immunohistochemistry failed to demonstrate the presence of the abnormal form of the prion protein in explanted tissue. CONCLUSIONS: Eight years after the event, two patients remain free of symptoms suggestive of iatrogenic CJD (http://www.cjd.ed.ac.uk/criteria.htm). The third having died aged 92 years, some 7 years after surgery, showing signs of dementia not considered indicative of iatrogenic CJD. Nevertheless this adverse incident attracted substantial publicity. Coupled with continuing concerns in the UK about person-to-person transmission of variant CJD, this has lead to a number of important consequences in donor eye retrieval, ocular tissue banking and transplantation.

Prion protein accumulation in eyes of patients with sporadic and variant Creutzfeldt-Jakob disease.

PURPOSE: Creutzfeldt-Jakob disease (CJD) primarily affects the brain. This study was conducted to assess the possible involvement of the eye in sporadic and variant CJD by testing for the presence of the disease-associated, protease-resistant isoform of the prion protein (PrP(Sc)) in ocular tissue. METHODS: Human eyes from donors with CJD and non-prion neurodegenerative disease control eyes were studied. In situ hybridization and Western blot analysis were used to determine the normal pattern of cellular prion protein (PrP(C)) expression. Western blot analysis and immunohistochemistry were then used to determine the localization, abundance, and isotype of PrP(Sc) in eyes in CJD. RESULTS: PrP(C) was expressed in the nuclear layers of the retina. In both the sporadic and variant forms of CJD, PrP(Sc) accumulated throughout the synaptic layers of the retina. The levels of PrP(Sc) found in the retina were comparable with those found in the brain. Lower levels of PrP(Sc) could be found in the optic nerve, but no PrP(Sc) was detectable in other ocular tissues. The glycoform ratio of PrP(Sc) in the retina did not correspond to that found in the brain. CONCLUSIONS: Presumptive centrifugal spread of PrP(Sc) from the brain through the optic nerve occurs in two major types of CJD. PrP(Sc) is a marker of CJD infectivity. Given that routine decontamination may not remove PrP(Sc) from surgical instruments, a careful risk assessment should be made of possible iatrogenic spread of sporadic and variant CJD after surgery to the retina or optic nerve.