Impacts and concerns for vCJD in blood transfusion: current status

The impact of vCJD upon blood transfusion practice hinges on its lymphoreticular involvement. B lymphocytes play a key supporting role for the capture and replication of infectivity by follicular dendritic cells of the lymphoid tissue in animal models of transmissible spongiform encephalopathies (TSE) and tonsils, spleen and appendix in man can harbour vCJD infectivity, a situation not seen with the other human TSEs. Leucodepletion of blood donations in the UK was implemented to reduce possible vCJD transmission and preliminary data suggests that white cell associated infectivity will be effectively removed although plasma infectivity will not. Blood screening assays are under development but none yet are ready for application. The conformation dependant immunoassay, based on differences in secondary and tertiary structure between normal and TSE-associated abnormal prion protein, has a sensitivity now approaching the best bioassay. Even so further development is needed to detect the fg/ml levels likely in the event that vCJD blood does contain abnormal prion, which is as yet unproven. Surrogate assays, such as for erythroid associated factor, may provide additional means of identifying donors harbouring vCJD. Validation of clearance of TSEs from pooled plasma products consistently demonstrates effective removal of the agents in downscaled systems and studies comparing vCJD, BSE and scrapie agents yield similar results. Many approaches to therapy are under investigation, in cell culture and animal models, targeted to normal or abnormal prion metabolism, including chemical and immunological interventions. Efficacy of quinacrine/chlorpromazine and pentosan polysulphate in a clinical setting, and agents yet to be used, will be more accurately known following recent agreement of clinical drug evaluation protocols.     

Iron overload following red blood cell transfusion and its impact on disease severity

Transfusion of red blood cells can be a life-saving therapy both for patients with chronic anemias and for those who are critically ill with acute blood loss. However, transfusion has been associated with significant morbidity. Chronic transfusion results in accumulation of excess iron that surpasses the binding capacity of the major iron transport protein, transferrin. The resulting non-transferrin bound iron (NTBI) can catalyze the production of highly reactive oxygen species (ROS) leading to significant and wide spread injury to the liver, heart, and endocrine organs as well as increases in infection. Acute transfusion of red blood cells in critically ill patients likewise has significant effects including increased mortality, prolonged hospital stays, and elevated risk of nosocomial infection. These effects appear to be more profound with increasing age of stored blood. The progressive release of free iron associated with storage time suggests that morbidity following acute transfusion, like that seen in chronic transfusion, may be due in part to elevated levels of NTBI. It is clear that transfusion is necessary in many instances; however, its risks and benefits must be carefully balanced before proceeding to avoid unnecessary iron toxicity.     

All or none fibrillogenesis of a prion peptide.

Amyloid proteins and peptides comprise a diverse group of molecules that vary both in size and amino-acid sequence, yet assemble into amyloid fibrils that have a common core structure. Kinetic studies of amyloid fibrillogenesis have revealed that certain amyloid proteins form oligomeric intermediates prior to fibril formation. We have investigated fibril formation with a peptide corresponding to residues 195-213 of the human prion protein. Through a combination of kinetic and equilibrium studies, we have found that the fibrillogenesis of this peptide proceeds as an all-or-none reaction where oligomeric intermediates are not stably populated. This variation in whether oligomeric intermediates are stably populated during fibril formation indicates that amyloid proteins assemble into a common fibrillar structure; however, they do so through different pathways.     

Detection of prion protein particles in blood plasma of scrapie infected sheep

Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The agent of the disease is the prion consisting mainly, if not solely, of a misfolded and aggregated isoform of the host-encoded prion protein (PrP). Transmission of prions can occur naturally but also accidentally, e.g. by blood transfusion, which has raised serious concerns about blood product safety and emphasized the need for a reliable diagnostic test. In this report we present a method based on surface-FIDA (fluorescence intensity distribution analysis), that exploits the high state of molecular aggregation of PrP as an unequivocal diagnostic marker of the disease, and show that it can detect infection in blood. To prepare PrP aggregates from blood plasma we introduced a detergent and lipase treatment to separate PrP from blood lipophilic components. Prion protein aggregates were subsequently precipitated by phosphotungstic acid, immobilized on a glass surface by covalently bound capture antibodies, and finally labeled with fluorescent antibody probes. Individual PrP aggregates were visualized by laser scanning microscopy where signal intensity was proportional to aggregate size. After signal processing to remove the background from low fluorescence particles, fluorescence intensities of all remaining PrP particles were summed. We detected PrP aggregates in plasma samples from six out of ten scrapie-positive sheep with no false positives from uninfected sheep. Applying simultaneous intensity and size discrimination, ten out of ten samples from scrapie sheep could be differentiated from uninfected sheep. The implications for ante mortem diagnosis of prion diseases are discussed.     

Fatal prion disease in a mouse model of genetic E200K Creutzfeldt-Jakob disease

Genetic prion diseases are late onset fatal neurodegenerative disorders linked to pathogenic mutations in the prion protein-encoding gene, PRNP. The most prevalent of these is the substitution of Glutamate for Lysine at codon 200 (E200K), causing genetic Creutzfeldt-Jakob disease (gCJD) in several clusters, including Jews of Libyan origin. Investigating the pathogenesis of genetic CJD, as well as developing prophylactic treatments for young asymptomatic carriers of this and other PrP mutations, may well depend upon the availability of appropriate animal models in which long term treatments can be evaluated for efficacy and toxicity. Here we present the first effective mouse model for E200KCJD, which expresses chimeric mouse/human (TgMHu2M) E199KPrP on both a null and a wt PrP background, as is the case for heterozygous patients and carriers. Mice from both lines suffered from distinct neurological symptoms as early as 5-6 month of age and deteriorated to death several months thereafter. Histopathological examination of the brain and spinal cord revealed early gliosis and age-related intraneuronal deposition of disease-associated PrP similarly to human E200K gCJD. Concomitantly we detected aggregated, proteinase K resistant, truncated and oxidized PrP forms on immunoblots. Inoculation of brain extracts from TgMHu2ME199K mice readily induced, the first time for any mutant prion transgenic model, a distinct fatal prion disease in wt mice. We believe that these mice may serve as an ideal platform for the investigation of the pathogenesis of genetic prion disease and thus for the monitoring of anti-prion treatments.     

Copper-associated liver disease: a proteomics study of copper challenge in a sheep model

Sheep display a variant phenotype with respect to their susceptibility to copper and derivative pathology. The North Ronaldsay sheep are acutely sensitive to environmental copper while the Cambridge breed is much more copper-tolerant. A study of protein expression in the liver of the two different breeds of sheep as a result of copper challenge would aid in the understanding of their differing pathophysiologies and contribute to knowledge of copper toxicosis in man. In this initial study, Cambridge breed sheep were challenged with oral copper and liver proteins were analyzed by two-dimensional (2-D) gel electrophoresis. Proteins whose expression pattern was modified by copper exposure were then identified by peptide mass fingerprinting using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. In conclusion, the pattern of changes in protein expression were consistent with an early adaptive response to oxidative challenge. This was followed by evidence of an impaired ability of the liver to compensate as copper loading increased, accompanied by oxidative stress-induced injury.     

Circulating plasma proteins in experimental acute hemorrhage and transfusion of blood components

In the experiments of dogs reactive changes of proteins in circulation in acute blood loss were studied. With the aim of prevention of homeostasis disorders caused by deficit of plasma proteins, blood protein components should be included into transfusion means.     

Tracheal venous blood and lymph collection: a model to study airway injury in sheep

Airway injury is a frequent result of the inhalation or aspiration of toxic material. Although upper airway damage can be identified endoscopically, pathophysiological changes are difficult to evaluate. This paper describes an animal model in which changes in tracheal blood and lymph flow rates, wet-to-dry weight ratios, and lymph-to-plasma protein ratios can be evaluated after injury. In this model, 12 cm of the cervical trachea were isolated using a double-cuffed endotracheal tube and injured with cotton smoke at near room temperature. Injury to the trachea was evaluated in twenty-five anesthetized sheep 4 (n = 3), 8 (n = 3), 24 (n = 3), 48 (n = 3), 96 (n = 3), and 192 (n = 2) h after smoke exposure and compared with sham control animals (n = 8). A significant increase in tracheal venous blood flow from 1.3 +/- 0.4 (SD) ml.min-1.cm-1 for the noninjured trachea to 2.8 +/- 1.2 was noted 24 h after injury (P less than 0.01). Lymph flow significantly increased from 1.3 +/- 0.4 microliters.min-1.cm-1 for the noninjured trachea to 9.8 +/- 3.3 24 h after injury while wet-to-dry weight ratios were elevated from 3.0 +/- 0.2 for noninjured trachea to 4.6 +/- 0.9 from 4 to 24 h after injury (P less than 0.01) and decreased to 3.7 +/- 0.5 by 96 h. Cast material consisting of airway exudate, cellular debris, and intact ciliated epithelial cells was both expectorated and found in the trachea when the animals were killed.(ABSTRACT TRUNCATED AT 250 WORDS)