Linda McCardle

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

Variant CJD infection in the spleen of a neurologically asymptomatic UK adult patient with haemophilia

Summary.  All UK patients with bleeding disorders treated with any UK‐sourced pooled factor concentrates between 1980 and 2001 have been informed that they may be at an increased risk of infection with variant Creutzfeldt–Jakob disease (vCJD). We describe a study to detect disease‐associated, protease‐resistant prion protein (PrPres) in 17 neurologically aysmptomatic patients with haemophilia considered to be at increased risk of vCJD. Materials from 11 autopsy and seven biopsy cases were analysed for PrPres. The tissues available from each case were variable, ranging from a single biopsy sample to a wide range of autopsy tissues. A single specimen from the spleen of one autopsy case gave a strong positive result on repeated testing for PrPres by Western blot analysis. This tissue came from a 73‐year‐old male patient with no history of neurological disease, who was heterozygous (methionine/valine) at codon 129 in the prion protein gene. He had received over 9000 units of factor VIII concentrate prepared from plasma pools known to include donations from a vCJD‐infected donor, and some 400 000 units not known to include donations from vCJD‐infected donors. He had also received 14 units of red blood cells and had undergone several surgical and invasive endoscopic procedures. Estimates of the relative risks of exposure through diet, surgery, endoscopy, blood transfusion and receipt of UK‐sourced plasma products suggest that by far the most likely route of infection in this patient was receipt of UK plasma products.

Laboratory diagnosis of variant Creutzfeldt–Jakob disease

The neuropathological and biochemical features of 33 cases of variant Creutzfeldt–Jakob disease (vCJD) diagnosed up to the end of 1998 are analysed in relation to the 646 cases of suspected CJD referred to the CJD Surveillance Unit laboratory from 1990 to 1998. Morphological studies of the central nervous system, lymphoid tissues and other organs were accompanied by immunocytochemistry; Western blot analysis of PrPRES was performed on frozen brain tissue. The findings were analysed in relation to clinical and genetic data. The pathology of vCJD showed morphological and immunocytochemical characteristics distinct from other cases of CJD. PrP accumulation was widespread in lymphoid tissues in vCJD, but was not identified in other non‐neural tissues. PrPRES accumulation in vCJD brain tissue showed a uniform glycotype pattern distinct from sporadic CJD. All analysed cases of vCJD were methionine homozygotes at codon 129 of the PrP gene.  No evidence currently exists to suggest that cases of CJD diagnosed in individuals who are MV or VV at codon 129 of the PrP gene represent ‘human bovine spongiform encaphalopathy (BSE)’. Continued surveillance is required to further investigate this possibility, with the need to investigate autopsy tissues from suspected cases by histological and biochemical techniques.

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.

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 …

Prion protein heterogeneity in sporadic but not variant Creutzfeldt–Jakob disease: U.K. cases 1991–2002

Human prion diseases can occur as an idiopathic disorder (sporadic Creutzfeldt–Jakob disease) or can be acquired, as is the case for variant Creutzfeldt–Jakob disease. These disorders are characterized by the accumulation of a protease‐resistant form of the host‐encoded prion protein termed PrPSc in the brains of affected individuals. PrPSc has been proposed to be the principal, if not sole, component of the infectious agent, with its accumulation in the central nervous system the primary event leading to neurodegeneration. A major question remains as to whether self‐propagating structural differences in PrPSc might account for the clinicopathological diversity evident in Creutzfeldt–Jakob disease and whether different prion protein types underlie the existence of different strains of causative agent. Here, we describe the results of a large‐scale biochemical study of PrPSc from autopsy‐proved cases of variant Creutzfeldt–Jakob disease (n = 59) and compare these with cases of sporadic Creutzfeldt–Jakob disease (n = 170) in the United Kingdom over the period 1991 to 2002. The results show PrPSc in variant Creutzfeldt–Jakob disease to be remarkably stereotyped. In contrast, considerable heterogeneity in PrPSc exists both between and within cases of sporadic Creutzfeldt–Jakob disease. Ann Neurol 2004;55:851–859

Immunohistochemistry for the Prion Protein: Comparison of Different Monoclonal Antibodies in Human Prion Disease Subtypes

Demonstration of the abnormal form of the prion protein (PrP) in the brain confirms the diagnosis of human prion disease (PrD). Using immunohistochemistry, we have compared ten monoclonal antibodies in PrD subtypes including sporadic and variant Creutzfeldt‐Jakob disease (CJD), fatal familial insomnia, Alzheimers disease (AD), and control brains. CJD subgroups were determined using Western blot analysis for the protease‐resistant PrP type in combination with sequencing to determine the genotype at the methionine/valine polymorphism at codon 129 of the prion protein gene. None of the antibodies labeled given subgroups exclusively, but the intensity of immunoreactivity varied among morphologically distinct types of deposit. Fine granular or synaptic PrP deposits stained weakly or not at all with antibodies against the N‐terminus of PrP, and were visible in one case only with 12F10 and SAF54. Coarser and plaque type deposits were immunolabeled with all antibodies. The immunostaining patterns appear characteristic for the disease subgroups. Labeling of certain neurons in all cases irrespective of disease, and staining at the periphery and/or throughout the senile plaques of AD patients were also noted. Antibodies such as 6H4 and 12F10 failed to give this type of labeling and are therefore less likely to recognise non‐pathological PrP material in immunohistochemistry.

Retrospective study of prion-protein accumulation in tonsil and appendix tissues

To identify individuals who could be at high risk of developing vCJD, a sensitive immunohistochemical technique was used to detect prion protein in a retrospective series of over 3000 tonsil and appendix specimens. No positives were detected but further studies are required to help reduce uncertainties about possible future numbers of vCJD cases in the UK.

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.