Nanofiltration as a robust method contributing to viral safety of plasma-derived therapeutics: 20 years' experience of the plasma protein manufacturers.

Background: Nanofiltration entails the filtering of protein solutions through membranes with pores of nanometric sizes that have the capability to effectively retain a wide range of viruses.

Study Design And Methods: Data were collected from 754 virus validation studies (individual data points) by Plasma Protein Therapeutics Association member companies and analyzed for the capacity of a range of nanofilters to remove viruses with different physicochemical properties and sizes. Different plasma product intermediates were spiked with viruses and filtered through nanofilters with different pore sizes using either tangential or dead-end mode under constant pressure or constant flow.

Prion removal capacity of plasma protein manufacturing processes: a data collection from PPTA member companies.

Background: The variant Creutzfeldt-Jakob disease incidence peaked a decade ago and has since declined. Based on epidemiologic evidence, the causative agent, pathogenic prion, has not constituted a tangible contamination threat to large-scale manufacturing of human plasma-derived proteins. Nonetheless, manufacturers have studied the prion removal capabilities of various manufacturing steps to better understand product safety.

Effective virus inactivation and removal by steps of Biotest Pharmaceuticals IGIV production process.

The virus validation of three steps of Biotest Pharmaceuticals IGIV production process is described here. The steps validated are precipitation and removal of fraction III of the cold ethanol fractionation process, solvent/detergent treatment and 35 nm virus filtration. Virus validation was performed considering combined worst case conditions.

Contribution to safety of immunoglobulin and albumin from virus partitioning and inactivation by cold ethanol fractionation: a data collection from Plasma Protein Therapeutics Association member companies.

Background: Virus removal by partitioning into different fractions during cold ethanol fractionation has been described by several authors, demonstrating that cold ethanol fractionation can provide significant contribution to virus removal, even in those cases where virus removal is limited and must be supported by additional measures for virus inactivation during further processing.

Study Design And Methods: Plasma Protein Therapeutics Association (PPTA) member companies collected and evaluated 615 studies on virus removal by the steps of the cold ethanol fractionation process. The studies describe the precipitation and separation of Fraction (F)III or FI/III in the immunoglobulin fractionation process and precipitation and separation of FII/III, FI/II/III, and FIV/IV in the albumin fractionation process.

Impaired axonal transport in motor neurons correlates with clinical prion disease.

Prion diseases are fatal neurodegenerative disorders causing motor dysfunctions, dementia and neuropathological changes such as spongiosis, astroglyosis and neuronal loss. The chain of events leading to the clinical disease and the role of distinct brain areas are still poorly understood. The role of nervous system integrity and axonal properties in prion pathology are still elusive.

Ultramicroscopy reveals axonal transport impairments in cortical motor neurons at prion disease.

The functional imaging of neuronal circuits of the central nervous system is crucial for phenotype screenings or investigations of defects in neurodegenerative disorders. Current techniques yield either low penetration depth, yield poor resolution, or are restricted by the age of the animals. Here, we present a novel ultramicroscopy protocol for fluorescence imaging and three-dimensional reconstruction in the central nervous system of adult mice.

Alteration of B-cell subsets enhances neuroinvasion in mouse scrapie infection.

Acquired forms of prion diseases or transmissible spongiform encephalopathies are believed to occur following peripheral exposure. Prions initially accumulate in the lymphoid system before spreading to the nervous system, but the underlying mechanisms for prion transfer between the two systems are still elusive. Here we show that ablation of the B-cell-specific transmembrane protein CD19, a coreceptor of the complement system, results in an acceleration of prion neuroinvasion.

The tyrosine kinase inhibitor imatinib mesylate delays prion neuroinvasion by inhibiting prion propagation in the periphery.

Prion diseases are fatal neurodegenerative disorders with no effective therapy. A hallmark of prion disease is the conversion of the normal cellular form of prion protein PrP(C) into a disease-associated isoform PrP(Sc). The authors recently have shown that a tyrosine kinase inhibitor, imatinib mesylate, induces clearance of PrP(Sc) via specific inhibition of c-Abl in prion-infected cell culture models.

Immunisation strategies against prion diseases: prime-boost immunisation with a PrP DNA vaccine containing foreign helper T-cell epitopes does not prevent mouse scrapie.

Vaccination against prion diseases constitutes a promising approach for the treatment and prevention of the disease. Passive immunisation with antibodies binding to the cellular prion protein (PrP(C)) can protect against prion disease. However, immunotherapeutic strategies with active immunisation are limited due to the immune tolerance against the self-antigen.

An enzyme-detergent method for effective prion decontamination of surgical steel.

Prions, transmissible agents that cause Creutzfeldt-Jakob disease (CJD) and other prion diseases, are known to resist conventional sterilization procedures. Iatrogenic transmission of classical CJD via neurosurgical instruments is well documented and the involvement of lymphoreticular tissues in variant CJD (vCJD), together with the unknown population prevalence of asymptomatic vCJD infection, has led to concerns about transmission from a wide range of surgical procedures. To address this problem, conditions were sought that destroy PrP(Sc) from vCJD-infected human tissue and eradicate RML prion infectivity adsorbed onto surgical steel.

The role of PrP in health and disease.

Transmissible spongiform encephalopathies (TSEs) such as scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle or Creutzfeldt-Jacob disease (CJD) and Gerstmann-Sträussler-Scheinker syndrome (GSS) in humans, are caused by an infectious agent designated prion. The "protein only" hypothesis states that the prion consists partly or entirely of a conformational isoform of the normal host protein PrPc and that the abnormal conformer, when introduced into the organism, causes the conversion of PrPc into a likeness of itself. Since the proposal of the "protein only" hypothesis more than three decades ago, cloning of the PrP gene, studies on PrP knockout mice and on mice transgenic for mutant PrP genes allowed deep insights into prion biology.

The tyrosine kinase inhibitor STI571 induces cellular clearance of PrPSc in prion-infected cells.

The conversion of the cellular prion protein (PrP(c)) into pathologic PrP(Sc) and the accumulation of aggregated PrP(Sc) are hallmarks of prion diseases. A variety of experimental approaches to interfere with prion conversion have been reported. Our interest was whether interference with intracellular signaling events has an impact on this conversion process.

PrP knock-out and PrP transgenic mice in prion research.

Spongiform encephalopathies such as scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle or Creutzfeldt-Jacob disease (CJD) and Gerstmann-Sträussler-Scheinker syndrome (GSS) in humans is caused by a transmissible agent designated prion. The 'protein only' hypothesis proposes that the prion consists partly or entirely of a conformational isoform of the normal host protein PrP(C), designated PrP(*)(1) and that the abnormal conformer, when introduced into the organism, causes the conversion of PrP(C) into a likeness of itself. PrP(*) may be congruent with PrP(Sc), a protease-resistant, aggregated conformer of PrP that accumulates mainly in brain of almost all prion-infected organisms.

A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions.

Prions are usually quantified by bioassays based on intracerebral inoculation of mice that are slow, imprecise, and costly. We have isolated neuroblastoma N2a sublines highly susceptible to mouse prions, as evidenced by accumulation of infectivity and the scrapie form of prion protein (PrPSc), and developed quantitative in vitro assays for prion infectivity. In the scrapie cell (SC) assay, susceptible N2a cells are exposed to prion-containing samples for 3 days, grown to confluence, and split 1:10 three times, and the proportion of PrPSc-containing cells is determined with automated counting equipment.

Expression of truncated PrP targeted to Purkinje cells of PrP knockout mice causes Purkinje cell death and ataxia.

PrP knockout mice with disruption of only the PrP-encoding region (Zürich I-type) remain healthy, whereas mice with deletions extending upstream of the PrP-encoding exon (Nagasaki-type) suffer Purkinje cell loss and ataxia, associated with ectopic expression of Doppel in brain, particularly in Purkinje cells. The phenotype is abrogated by co-expression of full-length PrP. Doppel is 25% similar to PrP, has the same globular fold, but lacks the flexible N-terminal tail.

Transmission of prions.

The "protein only" hypothesis holds that the infectious agent causing transmissible spongiform encephalopathies is a conformational isomer of PrP, a host protein that is predominantly expressed in the brain. This hypothesis is strongly supported by many lines of evidence. To date, prion diseases are unique among conformational diseases in that they are transmissible-experimentally and by natural routes (mainly by ingestion).

Molecular biology of prions.

The "protein only" hypothesis holds that the infectious agent causing transmissible spongiform encephalopathies is a conformational isomer of PrP, a host protein predominantly expressed in brain and is strongly supported by many lines of evidence. Prion diseases are so far unique among conformational diseases in that they are transmissible, not only experimentally but also by natural routes, mainly by ingestion. The pathway of prions to the brain has been elucidated in outline.

Transmission of prions.

The "protein only" hypothesis states that the infectious agent causing transmissible spongiform encephalopathies is a conformational isomer of PrP, a host protein predominantly expressed in brain, and is strongly supported by many lines of evidence. Prion diseases are so far unique among conformational diseases in that they are transmissible, not only experimentally but also by natural routes, mainly by ingestion. A striking feature of prions is their extraordinary resistance to conventional sterilization procedures, and their capacity to bind to surfaces of metal and plastic without losing infectivity.

Transmission of scrapie by steel-surface-bound prions.

Background: Prions are unusually resistant to conventional disinfection procedures. An electrode used intracerebrally on a Creutzfeldt-Jakob disease (CJD) patient transmitted the disease to two patients in succession and finally to a chimpanzee, despite attempted disinfection. Concerns that surgical instruments may transmit variant CJD have been raised by the finding of PrP(Sc), a surrogate marker for infectivity, in various tissues other than brain.

Similar turnover and shedding of the cellular prion protein in primary lymphoid and neuronal cells.

The cellular prion protein (PrP(C)) is essential for pathogenesis and transmission of prion diseases. Although prion replication in the brain is accompanied by neurodegeneration, prions multiply efficiently in the lymphoreticular system without any detectable pathology. We have used pulse-chase metabolic radiolabeling experiments to investigate the turnover and processing of PrP(C) in primary cell cultures derived from lymphoid and nervous tissues.

Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody.

Exposure of susceptible neuroblastoma N2a cells to mouse scrapie prions leads to infection, as evidenced by the continued presence of the scrapie form of the prion protein (PrP(Sc)) and infectivity after 300 or more cell doublings. We find that exposure to phosphatidylinositol-specific phospholipase C (PIPLC) or to the monoclonal anti-prion protein (PrP) antibody 6H4 not only prevents infection of susceptible N2a cells but also cures chronically scrapie-infected cultures, as judged by the long-term abrogation of PrP(Sc) accumulation after cessation of treatment. A nonpassaged, stationary infected culture rapidly loses PrP(Sc) when exposed to the antibody or PIPLC, indicating that the PrP(Sc) level is determined by steady state equilibrium between formation and degradation, and that depletion of the cellular form of PrP can interrupt the propagation of PrP(Sc).

B lymphocyte-restricted expression of prion protein does not enable prion replication in prion protein knockout mice.

Prion replication in spleen and neuroinvasion after i.p. inoculation of mice is impaired in forms of immunodeficiency where mature B lymphocytes are lacking.

Onset of ataxia and Purkinje cell loss in PrP null mice inversely correlated with Dpl level in brain.

PrP knockout mice in which only the open reading frame was disrupted ('Zürich I') remained healthy. However, more extensive deletions resulted in ataxia, Purkinje cell loss and ectopic expression in brain of Doppel (Dpl), encoded by the downstream gene, PRND: A new PrP knockout line, 'Zürich II', with a 2.9 kb PRNP: deletion, developed this phenotype at approximately 10 months (50% morbidity).

Prion protein devoid of the octapeptide repeat region restores susceptibility to scrapie in PrP knockout mice.

Mice devoid of PrP are resistant to scrapie and fail to replicate the agent. Introduction of transgenes expressing PrP into such mice restores susceptibility to scrapie. We find that truncated PrP devoid of the five copper binding octarepeats still sustains scrapie infection; however, incubation times are longer and prion titers and protease-resistant PrP are about 30-fold lower than in wild-type mice.

Adenoviral and adeno-associated viral transfer of genes to the peripheral nervous system.

Targeted expression of foreign genes to the peripheral nervous system is interesting for many applications, including gene therapy of neuromuscular diseases, neuroanatomical studies, and elucidation of mechanisms of axonal flow. Here we describe a microneurosurgical technique for injection of replication-defective viral vectors into dorsal root ganglia (DRG). Adenovirus- and adeno-associated virus-based vectors with transcriptional competence for DRG neurons led to expression of the gene of interest throughout the first neuron of the sensory system, from the distal portions of the respective sensory nerve to the ipsilateral nucleus gracilis and cuneatus, which contains the synapses to the spinothalamic tracts.