Diane Ritchie

Prevalence of lymphoreticular prion protein accumulation in UK tissue samples.

This study aims to provide an estimate of the number of individuals in the UK who may be incubating variant Creutzfeldt‐Jakob disease and at risk of causing iatrogenic spread of the disease. Lymphoreticular accumulation of prion protein is a consistent feature of variant Creutzfeldt‐Jakob at autopsy and has also been demonstrated in the pre‐clinical phase. Immunohistochemical accumulation of prion protein in the lymphoreticular system remains the only technique that has been shown to predict neurological disease reliably in animal prion disorders. In this study, immunohistochemistry was used to demonstrate the presence of prion protein, with monoclonal antibodies KG9 and 3F4, in surgically removed tonsillectomy and appendicectomy specimens. The samples were collected from histopathology departments across the UK and anonymised prior to testing. Samples were tested from 16 703 patients (14 964 appendectomies, 1739 tonsillectomies), approximately 60% of whom were from the age group 20–29 years at operation. Twenty‐five per cent of the samples were excluded from the final analyses because they contained inadequate amounts of lymphoid tissue. Three appendicectomy samples showed lymphoreticular accumulation of prion protein, giving an estimated prevalence of 3/12 674 or 237 per million (95% CI 49–692 per million). The pattern of lymphoreticular accumulation in two of these samples was dissimilar from that seen in known cases of variant Creutzfeldt‐Jakob disease. Although it is uncertain whether immunohistochemical accumulation of prion protein in the lymphoreticular system is specific for variant Creutzfeldt‐Jakob disease, it has not been described in any other disease, including other forms of human prion disease or a range of inflammatory and infective conditions. These findings reinforce the importance of measures taken by the UK Department of Health to reduce the risk of spread of variant Creutzfeldt‐Jakob via blood products and surgical instruments, and of the urgency to proceed with large‐scale screening of fresh tonsil specimens for the presence of prion protein. Copyright

Prion protein accumulation and neuroprotection in hypoxic brain damage

The function of the normal conformational isoform of prion protein, PrP(C), remains unclear although lines of research have suggested a role in the cellular response to oxidative stress. Here we investigate the expression of PrP(C) in hypoxic brain tissues to examine whether PrP(C) is in part regulated by neuronal stress. Cases of adult cerebral ischemia and perinatal hypoxic-ischemic injury in humans were compared with control tissues. PrP(C) immunoreactivity accumulates within neuronal processes in the penumbra of hypoxic damage in adult brain, and within neuronal soma in cases of perinatal hypoxic-ischemic injury, and in situ hybridization analysis suggests an up-regulation of PrP mRNA during hypoxia. Rodents also showed an accumulation of PrP(C) in neuronal soma within the penumbra of ischemic lesions. Furthermore, the infarct size in PrP-null mice was significantly greater than in the wild type, supporting the proposed role for PrP(C) in the neuroprotective adaptive cellular response to hypoxic injury.

Peripheral Tissue Involvement in Sporadic, Iatrogenic, and Variant Creutzfeldt-Jakob Disease An Immunohistochemical, Quantitative, and Biochemical Study

Human prion diseases are rare fatal neurodegenerative conditions that occur as acquired, familial, or idiopathic disorders. A key event in their pathogenesis is the accumulation of an altered form of the prion protein, termed PrP(Sc), in the central nervous system. A novel acquired human prion disease, variant Creutzfeldt-Jakob disease, is thought to result from oral exposure to the bovine spongiform encephalopathy agent. This disease differs from other human prion diseases in its neurological, neuropathological, and biochemical phenotype. We have used immunohistochemistry and Western blot techniques to analyze the tissue distribution and biochemical properties of PrP(Sc) in peripheral tissues in a unique series of nine cases of variant Creutzfeldt-Jakob disease. We have compared this with the distribution and biochemical forms found in all of the major subtypes of sporadic Creutzfeldt-Jakob disease and in a case of iatrogenic Creutzfeldt-Jakob disease associated with growth hormone therapy. The results show that involvement of the lymphoreticular system is a defining feature of variant Creutzfeldt-Jakob disease, but that the biochemical isoform of PrP(Sc) found is influenced by the cell type in which it accumulates.

Variant Creutzfeldt-Jakob disease: prion protein genotype analysis of positive appendix tissue samples from a retrospective prevalence study

Abstract Objective To perform prion protein gene (PRNP) codon 129 analysis in DNA extracted from appendix tissue samples that had tested positive for disease associated prion protein. Design Reanalysis of positive cases identified in a retrospective anonymised unlinked prevalence study of variant Creutzfeldt-Jakob disease (vCJD) in the United Kingdom. Study samples Three positive appendix tissue samples out of 12 674 samples of appendix and tonsil tested for disease associated prion protein. The patients from whom these samples were obtained were aged 20-29 years at the time of surgery, which took place in 1996-9. Setting Pathology departments in two tertiary centres in England and Scotland. Results Adequate DNA was available for analysis in two of the three specimens, both of which were homozygous for valine at codon 129 in the PRNP. Conclusions This is the first indication that the valine homozygous subgroup at codon 129 in the PRNP is susceptible to vCJD infection. All tested clinical cases of vCJD have so far occurred in the methionine homozygous subgroup, and a single case of probable iatrogenic vCJD infection has been identified in one patient who was a methionine/valine heterozygote at this genetic locus. People infected with vCJD with a valine homozygous codon 129 PRNP genotype may have a prolonged incubation period, during which horizontal spread of the infection could occur either from blood donations or from contaminated surgical instruments used on these individuals during the asymptomatic phase of the illness.

Immunocytochemical appearance of cytokines, prostaglandin E2 and lipocortin-1 in the CNS during the incubation period of murine scrapie correlates with progressive PrP accumulations

The appearance of immunoreactive interleukin (IL)-1beta, IL-6 and tumour necrosis factor (TNF)-alpha, prostaglandin (PG) E2 and lipocortin-1 in the central nervous system was investigated during the development of lesions in a 301V/VM murine scrapie model. Focal PrP(Sc) deposition was present after 30 days of the 115-120 day incubation period; this immunoreactivity increased in intensity and distribution thereafter. Staining for IL-1beta and TNF alpha in perivascular macrophages, and PGE2 immunoreactivity in astrocytes, was detected in those areas showing PrP(Sc) deposition from 60 days. Increased GFAP and F4/80 immunoreactivity, indicating activation of astrocytes and microglia, was also evident in these areas from 60 days. Glial cytokine and lipocortin immunoreactivity was detected after 90 days, in the absence of clinical signs. The disease-induced cytokine, PG and lipocortin immunoreactivity occurred only in those brain areas showing PrP(Sc) deposition, glial activation and, in later stages, vacuolation. These findings support the concept that PrP(Sc) deposition induces glial cytokine production. These glial cytokines may contribute to the development of the pathological lesions in scrapie.

Accumulation of prion protein in tonsil and appendix: review of tissue samples

Variant Creutzfeldt-Jakob disease is almost certainly caused by the bovine spongiform encephalopathy agent, and although the disease is rare (115 deaths to date) there is uncertainty about future numbers of cases.1 The lack of a conventional immune response and the inability to detect abnormal prion protein in blood has hampered the development of a blood test.1 Lymphoreticular accumulation of prion protein has been used as a preclinical test for scrapie (the form of the disease in sheep and goats) and is a consistent feature of variant Creutzfeldt-Jakob disease, occurring before the onset of symptoms.2–4 We screened large numbers of specimens from appendicectomies and tonsillectomies for the presence of prion protein in lymphoreticular tissue to determine the number of people with preclinical variant Creutzfeldt-Jakob disease. We retrieved appendix and tonsil samples removed between 1995 and 1999 from patients aged 10-50 from histopathology departments across the United Kingdom. The samples were anonymised before testing. We also examined appendix samples …

Prevalence of disease related prion protein in anonymous tonsil specimens in Britain: cross sectional opportunistic survey

Objective To establish with improved accuracy the prevalence of disease related prion protein (PrPCJD) in the population of Britain and thereby guide a proportionate public health response to limit the threat of healthcare associated transmission of variant Creutzfeldt-Jakob disease (vCJD). Design Cross sectional opportunistic survey. Study samples Anonymised tonsil pairs removed at elective tonsillectomy throughout England and Scotland. Setting National anonymous tissue archive for England and Scotland. Main outcome measure Presence of PrPCJD determined by using two enzyme immunoassays based on different analytical principles, with further investigation by immunohistochemistry or immunoblotting of any samples reactive in either assay. Results Testing of 63 007 samples was completed by the end of September 2008. Of these, 12 753 were from the birth cohort in which most vCJD cases have arisen (1961-85) and 19 908 were from the 1986-95 cohort that would have been also exposed to bovine spongiform encephalopathy through infected meat or meat products. None of the samples tested was unequivocally reactive in both enzyme immunoassays. Only two samples were reactive in one or other enzyme immunoassay and equivocal in the other, and nine samples were equivocally reactive in both enzyme immunoassays. Two hundred and seventy six samples were initially reactive in one or other enzyme immunoassay; the repeat reactivity rate was 15% or less, depending on the enzyme immunoassay and cut-off definition. None of the samples (including all the 276 initially reactive in enzyme immunoassay) that were investigated by immunohistochemistry or immunoblotting was positive for the presence of PrPCJD. Conclusions The observed prevalence of PrPCJD in tonsils from the 1961-95 combined birth cohort was 0/32 661 with a 95% confidence interval of 0 to 113 per million. In the 1961-85 cohort, the prevalence of zero with a 95% confidence interval of 0 to 289 per million was lower than, but still consistent with, a previous survey of appendix tissue that showed a prevalence of 292 per million with a 95% confidence interval of 60 to 853 per million. Continuing to archive and test tonsil specimens, especially in older birth cohorts, and other complementary large scale anonymous tissue surveys, particularly of post-mortem tissues, will further refine the calculated prevalence of PrPCJD.

Specificity of lymphoreticular accumulation of prion protein for variant Creutzfeldt–Jakob disease

Background: Immunocytochemical accumulation of prion protein (PrP) in lymphoid tissues is a feature of variant Creutzfeldt–Jakob disease (vCJD) that has been used both to aid in the diagnosis of patients and as a basis of large scale screening studies to assess the prevalence of preclinical disease in the UK. However, the specificity of this approach is unknown. Aim: To assess the specificity of lymphoreticular accumulation of PrP for vCJD by examining a range of human diseases. Methods: Paraffin wax embedded lymphoreticular tissues from patients with several reactive conditions (58 cases), tumours (27 cases), vCJD (54 cases), and other human prion diseases (56 cases) were assessed. PrP accumulation was assessed by immunocytochemistry using two different monoclonal anti-PrP antibodies and a sensitive detection system. Results: All cases of vCJD showed widespread lymphoreticular accumulation of PrP; however, this was not seen in the other conditions examined. Conclusion: Lymphoreticular accumulation of PrP, as assessed by immunocytochemistry, appears to be a highly specific feature of vCJD.

Phenotypic variability in human prion diseases

Human prion diseases are rare neurodegenerative disorders that can occur as sporadic, familial or acquired disorders. Within each of these categories there is a wide range of phenotypic variation that is not encountered in other neurodegenerative disorders. The identification of the prion protein and its key role in the pathogenesis of this diverse group of diseases has allowed a fuller understanding of factors that influence disease phenotype. In particular, the naturally occurring polymorphism at codon 129 in the prion protein gene has a major influence on the disease phenotype in sporadic, familial and acquired prion diseases, although the underlying mechanisms remain unclear. Recent technical advances have improved our ability to study the isoforms of the abnormal prion protein in the brain and in other tissues. This has lead to the concept of molecular strain typing, in which different isoforms of the prion protein are proposed to correspond to individual strains of the transmissible agent, each with specific biological properties. In sporadic Creutzfeldt‐Jakob disease there are at least six major combinations of codon 129 genotype and prion protein isotype, which appear to relate to distinctive clinical subgroups of this disease. However, these relationships are proving to be more complex than first considered, particularly in cases with more than a single prion protein isotype in the brain. Further work is required to clarify these relationships and to explain the mechanism of neuropathological targeting of specific brain regions, which accounts for the diversity of clinical features within human prion diseases.

Advances in the detection of prion protein in peripheral tissues of variant Creutzfeldt-Jakob disease patients using paraffin-embedded tissue blotting

The accumulation of PrPSc, an abnormal and disease‐associated form of the normal prion protein (PrPc), within the central nervous system (CNS) is a key pathological feature of Creutzfeldt‐Jakob disease (CJD). Following limited proteolytic digestion of PrPSc, the detection of PrPres within lymphoid tissues is a unique characteristic of variant CJD in comparison with other human prion diseases, raising fears of an increased risk of iatrogenic spread. Because levels of PrPres in lymphoid tissues are lower than those found in CNS tissue, there is concern that other peripheral tissues may harbour infectivity at levels that current detection systems cannot demonstrate PrPres. We have modified the paraffin‐embedded tissue blot (PET blot), a technique combining immunohistochemistry (IHC), histoblot and Western blotting, for the detection of PrPres in paraffin sections in peripheral tissues in variant CJD. Five cases of variant CJD were examined, using a panel of anti‐PrP antibodies. In each of these five cases, spleen, tonsil, lymph nodes and dorsal root ganglia showed an increase in the sensitivity and specificity of labelling using the PET blot when compared with optimized PrPres IHC methods. Control cases showed no evidence of PrP accumulation in either peripheral or CNS tissues. Autopsy and biopsy brain material from sporadic CJD cases also showed an increased sensitivity of PrPres detection with the PET blot, confirming its value as an important diagnostic and research tool in human prion diseases.