All clinically-relevant blood components transmit prion disease following a single blood transfusion: a sheep model of vCJD

Variant CJD (vCJD) is an incurable, infectious human disease, likely arising from the consumption of BSE-contaminated meat products. Whilst the epidemic appears to be waning, there is much concern that vCJD infection may be perpetuated in humans by the transfusion of contaminated blood products. Since 2004, several cases of transfusion-associated vCJD transmission have been reported and linked to blood collected from pre-clinically affected donors. Using an animal model in which the disease manifested resembles that of humans affected with vCJD, we examined which blood components used in human medicine are likely to pose the greatest risk of transmitting vCJD via transfusion. We collected two full units of blood from BSE-infected donor animals during the pre-clinical phase of infection. Using methods employed by transfusion services we prepared red cell concentrates, plasma and platelets units (including leucoreduced equivalents). Following transfusion, we showed that all components contain sufficient levels of infectivity to cause disease following only a single transfusion and also that leucoreduction did not prevent disease transmission. These data suggest that all blood components are vectors for prion disease transmission, and highlight the importance of multiple control measures to minimise the risk of human to human transmission of vCJD by blood transfusion

PRNP variation in UK sporadic and variant Creutzfeldt Jakob disease highlights genetic risk factors and a novel non-synonymous polymorphism

<p>Abstract</p> <p>Background</p> <p>Genetic analysis of the human prion protein gene (<it>PRNP</it>) in suspect cases of Creutzfeldt-Jakob disease (CJD) is necessary for accurate diagnosis and case classification. Previous publications on the genetic variation at the <it>PRNP </it>locus have highlighted the presence of numerous polymorphisms, in addition to the well recognised one at codon 129, with significant variability between geographically distinct populations. It is therefore of interest to consider their influence on susceptibility or the clinico-pathological disease phenotype. This study aimed to characterise the frequency and effect of <it>PRNP </it>open reading frame polymorphisms other than codon 129 in both disease and control samples sourced from the United Kingdom population.</p> <p>Methods</p> <p>DNA was extracted from blood samples and genetic data obtained by full sequence analysis of the prion protein gene or by restriction fragment length polymorphism analysis using restriction enzymes specific to the gene polymorphism under investigation.</p> <p>Results</p> <p>147 of 166 confirmed cases of variant CJD (vCJD) in the UK have had <it>PRNP </it>codon 129 genotyping and all are methionine homozygous at codon 129; 118 have had full <it>PRNP </it>gene sequencing. Of the latter, 5 cases have shown other polymorphic loci: at codon 219 (2, 1.69%), at codon 202 (2, 1.69%), and a 24 bp deletion in the octapeptide repeat region (1, 0.85%). E219K and D202D were not found in sporadic CJD (sCJD) cases and therefore may represent genetic risk factors for vCJD.</p> <p>Genetic analysis of 309 confirmed UK sCJD patients showed codon 129 genotype frequencies of MM: 59.5% (n = 184), MV: 21.4% (n = 66), and VV: 19.1% (n = 59). Thirteen (4.2%) had the A117A polymorphism, one of which also had the P68P polymorphism, four (1.3%) had a 24 bp deletion, and a single patient had a novel missense variation at codon 167. As the phenotype of this latter case is similar to sCJD and in the absence of a family history of CJD, it is unknown whether this is a form of genetic CJD, or simply a neutral polymorphism.</p> <p>Conclusions</p> <p>This analysis of <it>PRNP </it>genetic variation in UK CJD patients is the first to show a comprehensive comparison with healthy individuals (n = 970) from the same population, who were genotyped for the three most common variations (codon 129, codon 117, and 24 bp deletion). These latter two genetic variations were equally frequent in UK sCJD or vCJD cases and a normal (healthy blood donor) UK population.</p

No Major Change in vCJD Agent Strain after Secondary Transmission via Blood Transfusion

The identification of transmission of variant Creutzfeldt-Jakob disease (vCJD) by blood transfusion has prompted investigation to establish whether there has been any alteration in the vCJD agent following this route of secondary transmission. Any increase in virulence or host adaptation would require a reassessment of the risk analyses relating to the possibility of a significant secondary outbreak of vCJD. Since there are likely to be carriers of the vCJD agent in the general population, there is a potential for further infection by routes such as blood transfusion or contaminated surgical instruments.We inoculated both wild-type and transgenic mice with material from the first case of transfusion associated vCJD infection.The strain transmission properties of blood transfusion associated vCJD infection show remarkable similarities to the strain of vCJD associated with transmission from bovine spongiform encephalopathy (BSE).Although it has been hypothesized that adaptation of the BSE agent through secondary passage in humans may result in a greater risk of onward transmission due to an increased virulence of the agent for humans, our data presented here in two murine models suggest no significant alterations to transmission efficiency of the agent following human-to-human transmission of vCJD

vCJD risk in the Republic of Ireland

BACKGROUND: The Republic of Ireland has the second highest incidence of BSE worldwide. Only a single case of vCJD has been identified to date. METHODS: We estimate the total future number of clinical cases of vCJD using an established mathematical model, and based on infectivity of bovine tissue calculated from UK data and on the relative exposure to BSE contaminated meat. RESULTS: We estimate 1 future clinical case (95% CI 0 – 15) of vCJD in the Republic of Ireland. Irish exposure is from BSE infected indigenous beef products and from imported UK beef products. Additionally, 2.5% of the Irish population was exposed to UK beef through residing in the UK during the 'at-risk' period. The relative proportion of risk attributable to each of these three exposures individually is 2:2:1 respectively. CONCLUSIONS: The low numbers of future vCJD cases estimated in this study is reassuring for the Irish population and for other countries with a similar level of BSE exposure

Risk behaviors in a rural community with a known point-source exposure to chronic wasting disease

<p>Abstract</p> <p>Background</p> <p>The emergence and continuing spread of Chronic Wasting Disease (CWD) in cervids has now reached 14 U.S. states, two Canadian provinces, and South Korea, producing a potential for transmission of CWD prions to humans and other animals globally. In 2005, CWD spread for the first time from the Midwest to more densely populated regions of the East Coast. As a result, a large cohort of individuals attending a wild game feast in upstate New York were exposed to a deer that was subsequently confirmed positive for CWD.</p> <p>Methods</p> <p>Eighty-one participants who ingested or otherwise were exposed to a deer with chronic wasting disease at a local New York State sportsman's feast were recruited for this study. Participants were administered an exposure questionnaire and agreed to follow-up health evaluations longitudinally over the next six years.</p> <p>Results</p> <p>Our results indicate two types of risks for those who attended the feast, a <it>Feast Risk </it>and a G<it>eneral Risk</it>. The larger the number of risk factors, the greater the risk to human health if CWD is transmissible to humans. Long-term surveillance of feast participants exposed to CWD is ongoing.</p> <p>Conclusion</p> <p>The risk data from this study provide a relative scale for cumulative exposure to CWD-infected tissues and surfaces, and those in the upper tiers of cumulative risk may be most at risk if CWD is transmissible to humans.</p

Transmission characteristics of heterozygous cases of Creutzfeldt-Jakob disease with variable abnormal prion protein allotypes

In the human prion disease Creutzfeldt-Jakob disease (CJD), different CJD neuropathological subtypes are defined by the presence in normal prion protein (PrPC) of a methionine or valine at residue 129, by the molecular mass of the infectious prion protein PrPSc, by the pattern of PrPSc deposition, and by the distribution of spongiform change in the brain. Heterozygous cases of CJD potentially add another layer of complexity to defining CJD subtypes since PrPSc can have either a methionine (PrPSc-M129) or valine (PrPSc-V129) at residue 129. We have recently demonstrated that the relative amount of PrPSc-M129 versus PrPSc-V129, i.e. the PrPSc allotype ratio, varies between heterozygous CJD cases. In order to determine if differences in PrPSc allotype correlated with different disease phenotypes, we have inoculated 10 cases of heterozygous CJD (7 sporadic and 3 iatrogenic) into two transgenic mouse lines overexpressing PrPC with a methionine at codon 129. In one case, brain-region specific differences in PrPSc allotype appeared to correlate with differences in prion disease transmission and phenotype. In the other 9 cases inoculated, the presence of PrPSc-V129 was associated with plaque formation but differences in PrPSc allotype did not consistently correlate with disease incubation time or neuropathology. Thus, while the PrPSc allotype ratio may contribute to diverse prion phenotypes within a single brain, it does not appear to be a primary determinative factor of disease phenotype

Paracrine Diffusion of PrPC and Propagation of Prion Infectivity by Plasma Membrane-Derived Microvesicles

Cellular prion protein (PrPc) is a physiological constituent of eukaryotic cells. The cellular pathways underlying prions spread from the sites of prions infection/peripheral replication to the central nervous system are still not elucidated. Membrane-derived microvesicles (MVs) are submicron (0.1–1 µm) particles, that are released by cells during plasma membrane shedding processes. They are usually liberated from different cell types, mainly upon activation as well as apoptosis, in this case, one of their hallmarks is the exposure of phosphatidylserine in the outer leaflet of the membrane. MVs are also characterized by the presence of adhesion molecules, MHC I molecules, as well as of membrane antigens typical of their cell of origin. Evidence exists that MVs shedding provide vehicles to transfer molecules among cells, and that MVs are important modulators of cell-to-cell communication. In this study we therefore analyzed the potential role of membrane-derived MVs in the mechanism(s) of PrPC diffusion and prion infectivity transmission. We first identified PrPC in association with the lipid raft components Fyn, flotillin-2, GM1 and GM3 in MVs from plasma of healthy human donors. Similar findings were found in MVs from cell culture supernatants of murine neuronal cells. Furthermore we demonstrated that PrPSc is released from infected murine neuronal cells in association with plasma membrane-derived MVs and that PrPSc-bearing MVs are infectious both in vitro and in vivo. The data suggest that MVs may contribute both to the intercellular mechanism(s) of PrPC diffusion and signaling as well as to the process of prion spread and neuroinvasion

Use of different RT-QuIC substrates for detecting CWD prions in the brain of Norwegian cervids

Chronic wasting disease (CWD) is a highly contagious prion disease affecting captive and free-ranging cervid populations. CWD has been detected in United States, Canada, South Korea and, most recently, in Europe (Norway, Finland and Sweden). Animals with CWD release infectious prions in the environment through saliva, urine and feces sustaining disease spreading between cervids but also potentially to other non-cervids ruminants (e.g. sheep, goats and cattle). In the light of these considerations and due to CWD unknown zoonotic potential, it is of utmost importance to follow specific surveillance programs useful to minimize disease spreading and transmission. The European community has already in place specific surveillance measures, but the traditional diagnostic tests performed on nervous or lymphoid tissues lack sensitivity. We have optimized a Real-Time Quaking-Induced Conversion (RT-QuIC) assay for detecting CWD prions with high sensitivity and specificity to try to overcome this problem. In this work, we show that bank vole prion protein (PrP) is an excellent substrate for RT-QuIC reactions, enabling the detection of trace-amounts of CWD prions, regardless of prion strain and cervid species. Beside supporting the traditional diagnostic tests, this technology could be exploited for detecting prions in peripheral tissues from live animals, possibly even at preclinical stages of the disease

Plasmacytoid Dendritic Cells Sequester High Prion Titres at Early Stages of Prion Infection

In most transmissible spongiform encephalopathies prions accumulate in the lymphoreticular system (LRS) long before they are detectable in the central nervous system. While a considerable body of evidence showed that B lymphocytes and follicular dendritic cells play a major role in prion colonization of lymphoid organs, the contribution of various other cell types, including antigen-presenting cells, to the accumulation and the spread of prions in the LRS are not well understood. A comprehensive study to compare prion titers of candidate cell types has not been performed to date, mainly due to limitations in the scope of animal bioassays where prohibitively large numbers of mice would be required to obtain sufficiently accurate data. By taking advantage of quantitative in vitro prion determination and magnetic-activated cell sorting, we studied the kinetics of prion accumulation in various splenic cell types at early stages of prion infection. Robust estimates for infectious titers were obtained by statistical modelling using a generalized linear model. Whilst prions were detectable in B and T lymphocytes and in antigen-presenting cells like dendritic cells and macrophages, highest infectious titers were determined in two cell types that have previously not been associated with prion pathogenesis, plasmacytoid dendritic (pDC) and natural killer (NK) cells. At 30 days after infection, NK cells were more than twice, and pDCs about seven-fold, as infectious as lymphocytes respectively. This result was unexpected since, in accordance to previous reports prion protein, an obligate requirement for prion replication, was undetectable in pDCs. This underscores the importance of prion sequestration and dissemination by antigen-presenting cells which are among the first cells of the immune system to encounter pathogens. We furthermore report the first evidence for a release of prions from lymphocytes and DCs of scrapie-infected mice ex vivo, a process that is associated with a release of exosome-like membrane vesicles

Extended Thromboprophylaxis with Betrixaban in Acutely Ill Medical Patients

Background Patients with acute medical illnesses are at prolonged risk for venous thrombosis. However, the appropriate duration of thromboprophylaxis remains unknown. Methods Patients who were hospitalized for acute medical illnesses were randomly assigned to receive subcutaneous enoxaparin (at a dose of 40 mg once daily) for 10±4 days plus oral betrixaban placebo for 35 to 42 days or subcutaneous enoxaparin placebo for 10±4 days plus oral betrixaban (at a dose of 80 mg once daily) for 35 to 42 days. We performed sequential analyses in three prespecified, progressively inclusive cohorts: patients with an elevated d-dimer level (cohort 1), patients with an elevated d-dimer level or an age of at least 75 years (cohort 2), and all the enrolled patients (overall population cohort). The statistical analysis plan specified that if the between-group difference in any analysis in this sequence was not significant, the other analyses would be considered exploratory. The primary efficacy outcome was a composite of asymptomatic proximal deep-vein thrombosis and symptomatic venous thromboembolism. The principal safety outcome was major bleeding. Results A total of 7513 patients underwent randomization. In cohort 1, the primary efficacy outcome occurred in 6.9% of patients receiving betrixaban and 8.5% receiving enoxaparin (relative risk in the betrixaban group, 0.81; 95% confidence interval [CI], 0.65 to 1.00; P=0.054). The rates were 5.6% and 7.1%, respectively (relative risk, 0.80; 95% CI, 0.66 to 0.98; P=0.03) in cohort 2 and 5.3% and 7.0% (relative risk, 0.76; 95% CI, 0.63 to 0.92; P=0.006) in the overall population. (The last two analyses were considered to be exploratory owing to the result in cohort 1.) In the overall population, major bleeding occurred in 0.7% of the betrixaban group and 0.6% of the enoxaparin group (relative risk, 1.19; 95% CI, 0.67 to 2.12; P=0.55). Conclusions Among acutely ill medical patients with an elevated d-dimer level, there was no significant difference between extended-duration betrixaban and a standard regimen of enoxaparin in the prespecified primary efficacy outcome. However, prespecified exploratory analyses provided evidence suggesting a benefit for betrixaban in the two larger cohorts. (Funded by Portola Pharmaceuticals; APEX ClinicalTrials.gov number, NCT01583218. opens in new tab.