Pall leukotrap affinity prion-reduction filter removes exogenous infectious prions and endogenous infectivity from red cell concentrates.

BACKGROUND AND OBJECTIVES: Three recent probable cases of transmission of a variant of human Creutzfeldt-Jakob disease (vCJD) through blood transfusion suggest that the disease can be transmitted through transfusion of blood components from presymptomatic blood donors. In this study, we investigated the performance of a new filter for reducing the levels of infectious prions (PrP(Sc)) from red cell concentrates (RCC). MATERIALS AND METHODS: Endogenous Infectivity: A pool of 500 ml of whole blood was collected from 263K-strain scrapie-infected hamsters into an anticoagulant, processed into non-leucoreduced RCC (NL-RCC), and then passed through a prion-reduction filter. Pre- and postfiltration samples were tested for PrP(Sc) by Western blot and infectivity by inoculation of healthy hamsters. Results of the endogenous infectivity study after 200 days post-inoculation are discussed. Exogenous (Spiking) Study: Scrapie-infected hamster brain homogenates containing PrP(Sc) were added to human RCC and then filtered. Levels of PrP(Sc) were determined by Western blot assay. The effect of prior leucodepletion of 'spiked' RCC on PrP(Sc) removal by the prion-removal filter was also assessed. RESULTS: In the endogenous infectivity study, at 200-day observation time, the prefiltered RCC transmitted disease to six of the 187 hamsters, whereas the filtered RCC did not transmit disease to any of 413 animals, P = 0.001. The prion filter also significantly reduced the concentration of leucocytes in the RCC by about 4 logs, P < 0.05. In the exogenous (spiking) study, the level of PrP(res) was significantly reduced in RCC P < 0.05. Prior leucodepletion of the RCC with a leucoreduction filter did not significantly reduce the concentration of exogenously spiked PrP(Sc), P > 0.05. CONCLUSION: The use of this new prion-reduction filter should reduce the risk of vCJD transmission through transfusion of RCC, the most widely transfused blood component.

Characteristics of scrapie isolates derived from hay mites.

Previous epidemiological evidence suggested that in some instances a vector and/or reservoir is involved in the occurrence and spread of transmissible spongiform encephalopathies (TSEs). In a preliminary study, hay mite preparations from five Icelandic farms with a history of scrapie were injected into mice, and some of these mice became sick after long incubation periods. To confirm that the disease was scrapie, subsequent passages in mice were performed. In addition, the characteristics of the disease process in these passages were assessed and the results compared to those findings with standard scrapie strains. As expected for scrapie, subsequent passages in the same host led to shortened incubation periods compared to those in primary isolate mice, and all mice had spongiform changes in brain. Results were similar for three of four isolates with regard to clinical manifestations, the incubation periods in mice of the three scrapie incubation-period genotypes (s7s7, s7p7, p7p7), and the PrPSc Western blot (WB) pattern. The characteristics of the fourth isolate were markedly different from the other three isolates with regard to these parameters. Comparison of the characteristics of standard mouse-adapted scrapie strains and the four isolates revealed differences; these differences were particularly pronounced for the fourth isolate.

Reversion of prion protein conformational changes by synthetic beta-sheet breaker peptides.

BACKGROUND: Transmissible spongiform encephalopathies are associated with a structural transition in the prion protein that results in the conversion of the physiological PrPc to pathological PrP(Sc). We investigated whether this conformational transition can be inhibited and reversed by peptides homologous to the PrP fragments implicated in the abnormal folding, which contain specific residues acting as beta-sheet blockers (beta-sheet breaker peptides). METHODS: We studied the effect of a 13-residue beta-sheet breaker peptide (iPrP13) on the reversion of the abnormal structure and properties of PrP(Sc) purified from the brains of mice with experimental scrapie and from human beings affected by sporadic and variant Creutzfeldt-Jakob disease. In a cellular model of familial prion disease, we studied the effect of the peptide in the production of the abnormal form of PrP in intact cells. The influence of the peptide on prion infectivity was studied in vivo by incubation time assays in mice with experimental scrapie. FINDINGS: The beta-sheet breaker peptide partly reversed in-vitro PrP(Sc) to a biochemical and structural state similar to that of PrPc. The effect of the peptide was also detected in intact cells. Treatment of prion infectious material with iPrP13 delayed the appearance of clinical symptoms and decreased infectivity by 90-95% in mice with experimental scrapie. INTERPRETATION: Beta-sheet breaker peptides reverse PrP conformational changes implicated in the pathogenesis of spongiform encephalopathies. These peptides or their derivatives provide a useful tool to study the role of PrP conformation and might represent a novel therapeutic approach for prion-related disorders.

Biochemical and conformational variability of human prion strains in sporadic Creutzfeldt-Jakob disease.

The pathogenesis of prion (PrP) diseases is thought to be related to conformational changes of a normal cellular protein, PrPC, into a protease resistant protein called PrPSc, which is infectious by itself. A difficulty with this 'protein only' hypothesis is the existence of numerous PrP strains, that require PrPSc to have multiple conformations. Sporadic Creutzfeldt-Jakob disease (CJD), which accounts for nearly 80% of human prionoses, was reported to include at least two 'strains' termed types 1 and 2 which differ by electrophoretic patterns of their proteinase K (PK)-resistant fragments (PrP27-30). We have analyzed the biochemical and structural properties of PrPSc and PrP27-30 isolates from six sporadic CJD patients. Fourier transform-infra-red spectroscopy, PrP27-30 glycosylation patterns and studies of PK sensitivity revealed a striking heterogeneity. Furthermore, one isolate yielded a PrP27-30 fragment with a lower mobility clearly different from previously described sporadic CJD types. Although the average beta-sheet content was higher among type 1 isolates, there was overlap between the two types. Our study suggests that human sporadic CJD-related prions display a significant heterogeneity.

Immune surveillance and antigen conformation determines humoral immune response to the prion protein immunogen.

Transmissible spongiform encephalopathies (TSE) are progressive degenerative disorders of the central nervous system. PrP(Sc) is a TSE-specific marker derived from the host-encoded glycoprotein, PrPc. The generation of antibodies to PrP plays an important role in the diagnosis of these diseases. In this study the role of the PrP immunogen and the species being immunized was examined in relation to specific epitopes. Various mammals (mice, hamsters, rabbits and PrP null mice) were immunized with formic acid-treated PrP(Sc) isolated from mice, hamsters and sheep. Both the species being immunized and the source of immunogen played an important role in the antibody response. Response to a limited number of linear epitopes was seen among the various immunized animals. One region in the C-terminal portion of PrP appeared highly immunogenic in all species. Comparison of immunoreactivity and the pepscan-defined linear epitope sites suggests both linear and conformational directed responses in many of the animals. Information on the forces directing immune responses to PrP will lead to a better understanding of host-PrP interactions. It will also assist in the development of new strategies for generating additional tools for immunodiagnosis.

Biondi ring tangles in the choroid plexus of Alzheimer's disease and normal aging brains: a quantitative study.

The choroid plexus (CP) performs the vital function of producing up to 90% (450-1000 ml/day) of cerebrospinal fluid (CSF) to nourish and to protect the brain in the CSF suspension. The CP also acts as a selective barrier between blood and CSF to regulate ions and other essential molecules. However, the accumulation of intracellular inclusions called Biondi ring tangles (BRTs) in CP cells of Alzheimer's disease (AD)/aging brains may affect these vital functions of the CP. Statistical analysis of quantitative data on the numbers of CP cells containing BRTs from 54 brains (29 AD and 25 normal control), age range 1-100 years, indicated a significant difference (p<0.00004) between AD and control brains, using analysis of covariance (ANCOVA) with age as covariate. This study compiled the first set of archives to reveal the distribution pattern of BRTs in the CP of AD brains at various ages. Electron microscopy of negatively stained isolated BRTs revealed that these tangles are made of tightly packed bundles of long filaments with diameter around 10 nm that are morphologically distinct from the more loosely packed/shorter bundles of 6-8 nm amyloid fibrils of neuritic plaques (NPs) and from the 24 nm paired helical filaments of neurofibrillary tangles (NFTs) in AD brain. These data suggest that BRTs may represent a significant and measurable biomarker for AD in addition to NPs and NFTs.

Cell-lysate conversion of prion protein into its protease-resistant isoform suggests the participation of a cellular chaperone.

A conformational transition between the normal cellular prion protein (PrPC) and the beta-sheet-rich pathological isoform (PrPSc) is a central event in the pathogenesis of spongiform encephalopathies. The prion infectious agent seems to contain mainly, if not exclusively, PrPSc, which has the ability to propagate its abnormal conformation by transforming the host PrPC into the pathological isoform. We have developed an in vitro system to induce the PrPC --> PrPSc conversion by incubating a cell-lysate containing mouse PrPC with partially purified mouse PrPSc. After 48 h of incubation with a 10-fold molar excess of PrPSc, the cellular protein acquired PK-resistance resembling a PrPSc-like state. Time course experiments suggest that the conversion follows a stepwise mechanism involving kinetic intermediates. The conversion was induced by PrPSc extracted from mice infected with two different prion strains, each propagating its characteristic Western blot profile. The latter results and the fact that all the cellular components are present in the conversion reaction suggest that PrPC-PrPSc interaction is highly specific and required for the conversion. No transformation was observed under the same conditions using purified proteins without cell-lysate. However, when PrPC-depleted cell-lysate was added to the purified proteins the conversion was recovered. These findings provide direct evidence for the participation of a chaperone-like activity involved in catalyzing the conversion of PrPC into PrPSc.

Astrocytosis and amyloid deposition in scrapie-infected hamsters.

In scrapie infection, prion protein (PrPSc) is localized in areas where there is neurodegeneration and astrocytosis. It is thought that PrPSc is toxic to neurons and trophic for astrocytes. In our study, paraffin sections from scrapie infected (263K and 139H) and control hamsters were examined with histological and immunocytochemical staining. We found that PrPSc was present in the ependymal cells of both 263K- and 139H-infected hamsters. In 139H-infected hamsters, PrPSc was found in the cytoplasm of neurons in cerebral cortex and in hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. In contrast, neuronal cytoplasm and nuclei, were positive for PrPSc in most areas such as cortex, hippocampus, and thalamus in 263K-infected hamsters. Many aggregations of PrPSc could be seen in the cortex, hippocampus, substantia nigra and around the Pia mater, corpus callosum, fimbria, ventricles, and blood vessels in sections from 139H- and/or 263K-positive animals. Furthermore, PrPSc was also co-localized with glial fibrillary acidic protein (GFAP) in many reactive astrocytes (approximately 90%) in certain areas such as the hippocampus in 263K-infected hamsters, but not 139H-infected hamsters. The patterns of astrocytosis and PrPSc formation were different between 139H- and 263K-infected hamsters, which may be used for a diagnosis purpose. Our results suggest a hypothesis that multiple cell-types are capable of PrPSc production. Our results also confirm that reactive astrocytes can produce and/or accumulate PrPSc during some scrapie strain infections. The findings suggest a 'snowball effect', that is: astrocytosis might play an important role in amyloidosis, while amyloidosis may induce further astrocytosis at least in 263K-infected hamsters.

Astrocytosis and proliferating cell nuclear antigen expression in brains of scrapie-infected hamsters.

Scrapie is a neurodegenerative disease in sheep and goats. Neuropathological examination shows astrocytosis. One issue is whether the astrocytosis seen in scrapie is a function of an increase in reactivity of individual cells, or whether there is actual replication of astrocytes. We used double-label immunohistochemistry for proliferating cell nuclear antigen (PCNA) and for glial fibrillary acidic protein (GFAP) to determine the mitotic state of cells and to confirm their identity as astrocytes. Brain sections from hamsters (strain LVG/LAK) infected with 139H or 263K scrapie isolates were examined. GFAP immunostaining was increased in astrocytes in most regions of the brains of scrapie-infected hamsters. These qualitative observations were confirmed by computerized image analysis quantification. A proportion of the hypertrophic astrocytes (0.5-10.8%, depending on specific location) were PCNA immunoreactive. The PCNA-immunopositive astrocytes were most frequently found in cerebral cortex, corpus callosum, subependymal areas, fimbria, caudate, thalamus, hypothalamus, hippocampus, and dentate gyrus. Our results suggest that the astrocytosis seen in scrapie-infected animals is, at least in part, owing to actual replication of astrocytes in these animals. We hypothesize that the astrocytes may be an important locus for the disease process.

Immunodiagnosis of prion disease.

The immunodiagnosis of prion diseases is of critical importance due to the transmissibility of these conditions and their fatal prognosis. A panel of monoclonal and polyclonal antibodies have been generated for use in the study and diagnosis of these diseases. This manuscript describes the generation and characterization of these antibodies as well as their diagnostic application.

Histopathological changes in the islets of Langerhans in scrapie 139H-affected hamsters.

Previous studies showed that the 139H strain of scrapie injected into hamsters caused obesity, a marked hypertrophy of the islets of Langerhans, generalized endocrinopathy and marked hypoglycaemia-hyperinsulinaemia. In the current study, female weanling Syrian hamsters (LVG/LAK strain) were inoculated intracerebrally with scrapie strain 139H or 263K, or with normal hamster brain. Sections of the pancreas stained with haematoxylin and eosin or Gomori's one-step trichrome were examined by light microscopy. The 139H-affected hamsters showed extensive vacuolization, cellular hypertrophy, cellular atrophy, cytoplasmic vesicles and nuclear pathological changes in the islets of Langerhans. Also observed were abnormal structures, termed blood vessel cores, in the islets of 139H-affected hamsters. These structures were almost always centrally located within islets and were surrounded by B cells, some of which were abnormally elongated. None of these pathological changes were seen in the islets of Langerhans in control or 263K-affected hamsters. The level of scrapie-specific protease resistant protein (PrPSc) in pancreas was much lower than that in brain, a finding consistent with previous data showing low scrapie infectivity titres in pancreas.

The role of antibodies to PrP in the diagnosis of transmissible spongiform encephalopathies.

Transmissible spongiform encephalopathies (TSE) are progressive degenerative disorders of the central nervous system. Efficient and accurate identification of these disorders is necessitated by their transmissibility and fatal prognosis. The availability of polyclonal and monoclonal antibodies to a TSE disease-specific protein marker PrPSC affords the sensitivity and specificity for immuno-diagnostic assays. The majority of PrPSC antigenic sites are species-directed, involve non-self sites and are common to both the normal host precursor (PrPC) and the modified disease form. The availability of these antigenic sites is highly restricted by conformational influences resulting in epitope-dependent restrictions on antibody binding. Diagnostic immunoassays for TSE have relied largely on immunocytochemistry and immunoblotting. Restrictions on epitope availability have lead to the formulation by several laboratories of a variety of techniques to unmask PrP specific epitopes. In addition, diagnosis requires the ability to detect PrPSC specifically in tissue which can also contain immuno-reactive PrPC. Immuno-detection techniques are discussed relative to their range of application, ease of interpretation, specificity and sensitivity.

Brain regional distribution of prion protein PrP27-30 in mice stereotaxically microinjected with different strains of scrapie.

Stereotaxic inoculation was used to examine the role of scrapie agent strain, inoculum, and injection site on the brain regional distribution of the prion protein, PrP27-30. Neither the type of inoculum nor the injection site influenced the distribution of PrP27-30 in brains of mice. Among the parameters examined, only the strain of agent affected the pattern of distribution and the yield of PrP27-30. Although mice injected into the cerebellum had the shortest incubation period, the cerebellum gave the lowest yield of the PrP27-30 among the seven brain regions examined. The positive correlation between PrP27-30 regional distribution and lesion profile (degree of vacuolation) reinforces the role of the PrPSC protein in scrapie pathogenesis.

Demonstration of scrapie strain diversity in infected PC12 cells.

Scrapie strain replication in the nerve growth factor-induced, differentiated PC12 cell culture system was examined. Differences in replication between mouse-derived agents were demonstrated, with the 139A scrapie strain yielding 100- to 1000-fold higher levels of infectivity than the ME7 scrapie strain. Replication was not detected in PC12 cells infected with either the hamster-derived 263K or rat-derived 139R scrapie strains. Studies on the neurotransmitters in infected PC12 cells demonstrated that the adrenergic pathway was unchanged but the cholinergic pathway was altered. Furthermore, the degree of alteration correlated with the level of scrapie strain replication. Comparison of infectivity titres and enzymatic changes in ME7-infected PC12 cells with those in Chandler agent-infected mouse neuroblastoma cells suggests that the significant changes in neurotransmitter levels in cultures exhibiting low titres of infectivity involve factors in addition to strain replication. The variation in the range of scrapie strain replication in PC12 cells is discussed in relationship to species barrier, cell targeting, genetic susceptibility and species strain specificity. These studies further emphasize the value of the PC12 cell model system in examining the scrapie strain-host cell interaction and in addition support the concept of variation among scrapie strains.

Nearly ubiquitous tissue distribution of the scrapie agent precursor protein.

The "modified host protein" model of scrapie proposes that the transmissible agent is composed of the degradation-resistant protein, Sp33-37, and that clinical and pathologic signs result from neurotoxic accumulations of this protein. Sp33-37 is an abnormal, amyloidogenic isoform of the normally occurring cellular protein Cp33-37. This study investigated the tissue distribution of Cp33-37 in hamster. In brain, Cp33-37 was most concentrated in the hippocampal formation. Immunohistochemical studies localized Cp33-37 to neurons and surrounding neuropil in hippocampus; septal, caudate, and thalamic nuclei; dorsal root ganglia cells; and large-diameter dorsal root axons. In non-neuronal hamster tissues, Cp33-37 was detected in circulating leukocytes, heart, skeletal muscle, lung, intestinal tract, spleen, testis, ovary, and some other organs. The presence of Cp33-37 in extracerebral tissues indicates that its function is not unique to brain. These results indicate that the molecular substrate for the production of Sp33-37, the scrapie agent, and scrapie amyloid is present in a variety of cerebral and extracerebral sites.

Scrapie-infected spleens: analysis of infectivity, scrapie-associated fibrils, and protease-resistant proteins.

Scrapie-associated fibrils (SAF) and protease-resistant proteins (PrP) were isolated from spleens and brains of clinical animals (mice and hamsters) from three scrapie agent-host strain combinations, and their concentrations were compared with infectivity levels. The spleens of infected animals contained lower levels of infectivity, PrP, and SAF than did brains. Regardless of the route of infection, both SAF and infectivity were detected in spleen before brain. Infectivity increased in brains and spleens of 139A-infected mice before the detection and increase in SAF, suggesting that the synthesis of SAF and PrP may not be the limiting factor in agent replication. In contrast to those in ME7- and 263K-infected animals, the Western blot profiles for PrP from brain and spleen of 139A-infected mice exhibited distinct differences. Results indicate that SAF and PrP found in the spleens are both organ- and scrapie strain-specific.