Training and accreditation standards for pathologists undertaking clinical trial work.

Clinical trials rely on multidisciplinary teams for successful delivery. Pathologists should be involved in clinical trial design from the outset to ensure that protocols are optimised to deliver maximum data collection and translational research opportunities. Clinical trials must be performed according to the principles of Good Clinical Practice (GCP) and the trial sponsor has an obligation to ensure that all of the personnel involved in the trial have undergone training relevant to their role. Pathologists who are involved in the delivery of clinical trials are often required to undergo formal GCP training and may additionally undergo Good Clinical Laboratory Practice training if they are involved in the laboratory analysis of trials samples. Further training can be provided via trial-specific investigator meetings, which may be either multidisciplinary or discipline-specific events. Pathologists should also ensure that they undertake External Quality Assurance schemes relevant to the area of diagnostic practice required in the trial. The level of engagement of pathologists in academia and clinical trials research has declined in the United Kingdom over recent years. This paper recommends the optimal training and accreditation for pathologists undertaking clinical trials activities with the aim of facilitating increased engagement. Clinical trials training should ideally be provided to all pathologists through centrally organised educational events, with additional training provided to pathologists in training through local postgraduate teaching. Pathologists in training should also be strongly encouraged to undertake GCP training. It is hoped that these recommendations will increase the number of pathologists who take part in clinical trials research in order to ensure a high level and standard of data collection and to maximise the translational research opportunities.

Identification of clinical target areas in the brainstem of prion-infected mice.

AIMS: While prion infection ultimately involves the entire brain, it has long been thought that the abrupt clinical onset and rapid neurological decline in laboratory rodents relates to involvement of specific critical neuroanatomical target areas. The severity and type of clinical signs, together with the rapid progression, suggest the brainstem as a candidate location for such critical areas. In this study we aimed to correlate prion pathology with clinical phenotype in order to identify clinical target areas. METHOD: We conducted a comprehensive survey of brainstem pathology in mice infected with two distinct prion strains, which produce different patterns of pathology, in mice overexpressing prion protein (with accelerated clinical onset) and in mice in which neuronal expression was reduced by gene targeting (which greatly delays clinical onset). RESULTS: We identified specific brainstem areas that are affected by prion pathology during the progression of the disease. In the early phase of disease the locus coeruleus, the nucleus of the solitary tract, and the pre-Bötzinger complex were affected by prion protein deposition. This was followed by involvement of the motor and autonomic centres of the brainstem. CONCLUSIONS: Neurodegeneration in the locus coeruleus, the nucleus of the solitary tract and the pre-Bötzinger complex predominated and corresponded to the manifestation of the clinical phenotype. Because of their fundamental role in controlling autonomic function and the overlap with clinical signs in sporadic Creutzfeldt-Jakob disease, we suggest that these nuclei represent key clinical target areas in prion diseases.

Filamentous white matter prion protein deposition is a distinctive feature of multiple inherited prion diseases.

BACKGROUND: Sporadic, inherited and acquired prion diseases show distinct histological patterns of abnormal prion protein (PrP) deposits. Many of the inherited prion diseases show striking histological patterns, which often associate with specific mutations. Most reports have focused on the pattern of PrP deposition in the cortical or cerebellar grey matter. RESULTS: We observed that the subcortical white matter in inherited prion diseases frequently contained filamentous depositions of abnormal PrP, and we have analysed by immunohistochemistry, immunofluorescence and electron microscopy 35 cases of inherited prion disease seen at the UK National Prion Clinic. We report here that filamentous PrP is abundantly deposited in myelinated fibres in inherited prion diseases, in particular in those with N-terminal mutations. CONCLUSIONS: It is possible that the presence of filamentous PrP is related to the pathogenesis of inherited forms, which is different from those sporadic and acquired forms.

Development of antibody fragments for immunotherapy of prion diseases.

Prions are infectious proteins responsible for a group of fatal neurodegenerative diseases called TSEs (transmissible spongiform encephalopathies) or prion diseases. In mammals, prions reproduce themselves by recruiting the normal cellular protein PrP(C) and inducing its conversion into the disease-causing isoform denominated PrP(Sc). Recently, anti-prion antibodies have been shown to permanently cure prion-infected cells. However, the inability of full-length antibodies and proteins to cross the BBB (blood-brain barrier) hampers their use in the therapy of TSEs in vivo. Alternatively, brain delivery of prion-specific scFv (single-chain variable fragment) by AAV (adeno-associated virus) transfer delays the onset of the disease in infected mice, although protection is not complete. We investigated the anti-prion effects of a recombinant anti-PrP (D18) scFv by direct addition to scrapie-infected cell cultures or by infection with both lentivirus and AAV-transducing vectors. We show that recombinant anti-PrP scFv is able to reduce proteinase K-resistant PrP content in infected cells. In addition, we demonstrate that lentiviruses are more efficient than AAV in gene transfer of the anti-PrP scFv gene and in reducing PrP(Sc) content in infected neuronal cell lines. Finally, we have used a bioinformatic approach to construct a structural model of the D18scFv-PrP(C) complex. Interestingly, according to the docking results, Arg(PrP)(151) (Arg(151) from prion protein) is the key residue for the interactions with D18scFv, anchoring the PrP(C) to the cavity of the antibody. Taken together, these results indicate that combined passive and active immunotherapy targeting PrP might be promising strategies for therapeutic intervention in prion diseases.

Single treatment with RNAi against prion protein rescues early neuronal dysfunction and prolongs survival in mice with prion disease.

Prion diseases are fatal neurodegenerative conditions for which there is no effective treatment. Prion propagation involves the conversion of cellular prion protein, PrP(C), to its conformational isomer, PrP(Sc), which accumulates in disease. Here, we show effective therapeutic knockdown of PrP(C) expression using RNAi in mice with established prion disease. A single administration of lentivirus expressing a shRNA targeting PrP into each hippocampus of mice with established prion disease significantly prolonged survival time. Treated animals lived 19% and 24% longer than mice given an "empty" lentivirus, or not treated, respectively. Lentivirally mediated RNAi of PrP also prevented the onset of behavioral deficits associated with early prion disease, reduced spongiform degeneration, and protected against neuronal loss. In contrast, mice receiving empty virus or no treatment developed early cognitive impairment and showed severe spongiosis and neuronal loss. The focal use of RNAi therapeutically in prion disease further supports strategies depleting PrP(C), which we previously established to be a valid target for prion-based treatments. This approach can now be used to define the temporal, quantitative, and regional requirements for PrP knockdown for effective treatment of prion disease and to explore mechanisms involved in predegenerative neuronal dysfunction and its rescue.