Impact of vCJD on blood supply.

Variant Creutzfeldt-Jakob disease (vCJD) is an at present inevitably lethal neurodegenerative disease which can only be diagnosed definitely post mortem. The majority of the approximately 200 victims to date have resided in the UK where most contaminated beef materials entered the food chain. Three cases in the UK demonstrated that vCJD can be transmitted by blood transfusion. Since BSE and vCJD have spread to several countries outside the UK, it appears advisable that specific risk assessments be carried out in different countries and geographic areas. This review explains the approach adopted by Germany in assessing the risk and considering precautionary measures. A fundamental premise is that the feeding chain of cattle and the food chain have been successfully and permanently cleared from contaminated material. This raises the question of whether transmissions via blood transfusions could have the potential to perpetuate vCJD in mankind. A model calculation based on actual population data showed, however, that this would not be the case. Moreover, an exclusion of transfusion recipients from blood donation would add very little to the safety of blood transfusions, but would have a considerable impact on blood supply. Therefore, an exclusion of transfusion recipients was not recommended in Germany.     

A protocol for TILLING and Ecotilling in plants and animals

We describe Targeting-Induced Local Lesions IN Genomes (TILLING), a reverse-genetic strategy for the discovery and mapping of induced mutations. TILLING is suitable for essentially any organism that can be mutagenized. The TILLING procedure has also been adapted for the discovery and cataloguing of natural polymorphisms, a method called Ecotilling. To discover nucleotide changes within a particular gene, PCR is performed with gene-specific primers that are end-labeled with fluorescent molecules. After PCR, samples are denatured and annealed to form heteroduplexes between polymorphic DNA strands. Mismatched base pairs in these heteroduplexes are cleaved by digestion with a single-strand specific nuclease. The resulting products are size-fractionated using denaturing polyacrylamide gel electrophoresis and visualized by fluorescence detection. The migration of cleaved products indicates the approximate location of nucleotide polymorphisms. Throughput is increased and costs are reduced by sample pooling, multi-well liquid handling and automated gel band mapping. Once genomic DNA samples have been obtained, pooled and arrayed, thousands of samples can be screened daily.     

Molecular genetics of human prion diseases in Germany

Human prion diseases may be acquired as infectious diseases, they may be inherited in an autosomal dominant fashion or occur sporadically. Mutations and polymorphisms in the sequence of the coding region of the prion protein gene (PRNP) have been established as an important factor in all of these three types of prion diseases. Therefore, a total of 578 patients with suspect prion diseases referred to the German Creutzfeldt-Jakob disease (CJD) surveillance unit over a period of 4.5 years have been examined for mutations and polymorphisms in the coding region of PRNP. We found 40 cases with a missense mutation previously reported as pathogenic. Amongst these, the aspartate to asparagine change at codon 178 (D178N) was the most common mutation. All of these cases carried the D178N mutation in coupling with methionine at codon 129, resulting in the typical fatal familial insomnia (FFI) genotype. Most cases with pathogenic mutations were not found in the group of clinically "probable" cases according to established clinical criteria, supporting the notion that inherited prion diseases often exhibit atypical features. Two novel missense mutations (T188R and P238S) and several silent polymorphisms were found, demonstrating the quality of our screening procedure based on a modified version of the single-stranded conformational polymorphism technique. In "definite" CJD cases with no pathogenic mutation, the patients clinically classified as "probable" were mostly homozygous for methionine at the common polymorphism at codon 129, whereas there was a marked over-representation of patients homozygous for valine amongst those clinically classified as "possible". This large study on suspect cases of human prion diseases in Germany clearly shows that PRNP genetics is essential for a comprehensive analysis of prion diseases.     

PrP mRNA and protein expression in brain and PrPc in CSF in Creutzfeldt-Jakob disease MM1 and VV2

Creutzfeldt-Jakob disease (CJD) is a heterogenic neurodegenerative disorder associated with abnormal post-translational processing of cellular prion protein (PrP^c). CJD displays distinctive clinical and pathological features which correlate with the genotype at the codon 129 (methionine or valine: M or V respectively) in the prion protein gene and with size of the protease-resistant core of the abnormal prion protein PrP^sc (type 1: 20/21 kDa and type 2: 19 kDa). MM1 and VV2 are the most common sporadic CJD (sCJD) subtypes. PrP mRNA expression levels in the frontal cortex and cerebellum are reduced in sCJD in a form subtype-dependent. Total PrP protein levels and PrP^sc levels in the frontal cortex and cerebellum accumulate differentially in sCJD MM1 and sCJD VV2 with no relation between PrP^sc deposition and spongiform degeneration and neuron loss, but with microgliosis, and IL6 and TNF-α response. In the CSF, reduced PrP^c, the only form present in this compartment, occurs in sCJD MM1 and VV2. PrP mRNA expression is also reduced in the frontal cortex in advanced stages of Alzheimer disease, Lewy body disease, progressive supranuclear palsy, and frontotemporal lobe degeneration, but PrP^c levels in brain varies from one disease to another. Reduced PrP^c levels in CSF correlate with PrP mRNA expression in brain, which in turn reflects severity of degeneration in sCJD.     

Polymorphisms within the prion-like protein gene (Prnd) and their implications in human prion diseases, Alzheimer's disease and other neurological disorders

Only 10% of human transmissible spongiform encephalopathies (TSEs) are associated with mutations of the Prnp region encoding the prion protein (PrP). Recently, the murine PrP-like protein doppel (Dpl) was described and was shown to be overexpressed in certain strains of PrP knockout mice and to cause neurological diseases such as ataxia and Purkinje cell loss. To answer the question of whether there are any polymorphisms within the PrP-like protein gene (Prnd) that might cause or be involved in the development of TSEs, we investigated the complete open reading frame of the human Prnd gene from 58 patients who had died of genetic or sporadic Creutzfeldt-Jakob disease (CJD), Alzheimer's disease or other neurological disorders and from 111 controls. We found five new polymorphisms and one frame shift mutation. One silent polymorphism, which does not lead to an altered amino acid sequence, was also observed. Statistical analysis revealed a significant difference in the distribution of the Prnd genotype at codon 174 between sporadic CJD patients and healthy controls.     

Enhanced Chemokine Receptor Expression on Leukocytes of Patients with Alzheimer's Disease

Although primarily a neurological complaint, systemic inflammation is present in Alzheimer''s Disease, with higher than normal levels of proinflammatory cytokines and chemokines in the periphery as well as the brain. A gradient of these factors may enhance recruitment of activated immune cells into the brain via chemotaxis. Here, we investigated the phenotypes of circulating immune cells in AD patients with multi-colour flow cytometry to determine whether their expression of chemokine receptors is consistent with this hypothesis. In this study, we confirmed our previously reported data on the shift of early- to late-differentiated CD4+ T-cells in AD patients. The percentage of cells expressing CD25, a marker of acute T-cell activation, was higher in patients than in age-matched controls, and percentages of CCR6+ cells were elevated. This chemokine receptor is primarily expressed on pro-inflammatory memory cells and Th17 cells. The proportion of cells expressing CCR4 (expressed on Th2 cells) and CCR5 (Th1 cells and dendritic cells) was also greater in patients, and was more pronounced on CD4+ than CD8+ T-cells. These findings allow a more detailed insight into the systemic immune status of patients with Alzheimer''s disease and suggest possible novel targets for immune therapy.