Evaluation of cold atmospheric plasma for the decontamination of flexible endoscopes.

BACKGROUND: Despite adherence to standard protocols, residues including live micro-organisms may remain on the various surfaces of reprocessed flexible endoscopes. Prions are infectious proteins that are notoriously difficult to eliminate. AIM: To test the potential of cold atmospheric plasma (CAP) for the decontamination of various surfaces of flexible endoscopes, measuring total proteins and prion residual infectivity as indicators of efficacy. METHODS: New PTFE endoscope channels and metal test surfaces spiked with test soil or prion-infected tissues were treated using different CAP-generating prototypes. Surfaces were examined for the presence of residues using very sensitive fluorescence epimicroscopy. Prion residual infectivity was determined using the wire implant animal model and a more sensitive cell infectivity assay. FINDINGS: A CAP jet applied perpendicularly at close range on flat test surfaces removed soil within 3 min, but left microscopic residues and failed to eliminate prion infectivity according to the wire implant animal assay. The longitudinal gas flow from CAP prototypes developed for the treatment of long channels led to the displacement and sedimentation of residual soil towards the distal end, when applied alone. Observations of the plasma inside glass tubes showed temporal and spatial heterogeneity within a limited range. After the standard enzymatic manual pre-wash, 'CAP-activated' gas effluents prevented prion transmission from treated endoscope channels according to the prion infectivity cell assay. CONCLUSION: CAP shows promising results as a final step for decontamination of surgical surfaces. Optimizing CAP delivery could further enhance CAP efficacy, offering a safe, chemical-free alternative for the reprocessing of all luminal flexible endoscope surfaces.

Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein.

The agent responsible for transmissible spongiform encephalopathies (TSEs) is thought to be a malfolded, protease-resistant version (PrPres) of the normal cellular prion protein (PrP). The interspecies transmission of bovine spongiform encephalopathy (BSE) to mice was studied. Although all of the mice injected with homogenate from BSE-infected cattle brain exhibited neurological symptoms and neuronal death, more than 55 percent had no detectable PrPres. During serial passage, PrPres appeared after the agent became adapted to the new host. Thus, PrPres may be involved in species adaptation, but a further unidentified agent may actually transmit BSE.

Prion infection-impaired functional blocks identified by proteomics enlighten the targets and the curing pathways of an anti-prion drug.

Prion-induced neurodegeneration results from multiple cellular alterations among which the accumulation of a modified form of the host protein PrP is but a hallmark. Drug treatments need understanding of underlying mechanisms. Proteomics allows getting a comprehensive view of perturbations leading to neuronal death. Heparan sulfate mimetics has proved to be efficient to clear scrapie protein in cultured cells and in animals. To investigate the mechanisms of drug attack, protein profiles of the neuronal cell line GT1 and its chronically Chandler strain infected counterpart were compared, either in steady state cultures or after a 4-day drug treatment. Differentially expressed proteins were associated into functional blocks relevant to neurodegenerative diseases. Protein structure repair and modification, proteolysis, cell shape and energy/oxidation players were affected by infection, in agreement with prion biology. Unexpectedly, novel affected blocks related to translation, nucleus structure and DNA replication were unravelled displaying commonalities with proliferative processes. The drug had a double action in infected cells by reversing protein levels back to normal in some blocks and by heightening survival functions in others. This study emphasizes the interest of a proteomic approach to unravel novel networks involved in prion infection and curing.

Prion inactivation using a new gaseous hydrogen peroxide sterilisation process.

Prions pose a challenge to decontamination, particularly before the re-use of surgical instruments. They have relatively high resistance to standard decontamination methods and require extreme chemical and/or heat-based treatments for devices used in known or suspected cases of disease. This study investigated the effectiveness of a new gaseous hydrogen peroxide sterilisation process for prions as an alternative low-temperature method. Gaseous peroxide, in addition to known antimicrobial efficacy, was shown to inactivate prions both in in-vitro and in-vivo assays. In contrast to the gas form, liquid peroxide was not effective. The mechanism of action of gaseous peroxide suggested protein unfolding, some protein fragmentation and higher sensitivity to proteolytic digestion. Hydrogen peroxide liquid showed a degree of protein clumping and full resistance to protease degradation. The use of gaseous peroxide in a standard low-temperature sterilisation process may present a useful method for prion inactivation.

Alpha-synuclein accumulates in the brain of scrapie-affected sheep and goats.

Immunohistochemical examination demonstrated widespread granular deposits of alpha-synuclein (alphaSN) in the brains of sheep and goats with natural scrapie, especially in the cornu ammonis and subiculum of the hippocampus; this contrasted with the diffuse and non-granular immunolabelling seen in healthy controls. There was non-regular "co-localization" of PrP(Sc) and alphaSN. The findings resembled those reported in Creutzfeldt-Jakob disease and in experimental prion disease in hamsters and mice. The results suggest that perturbation of alphaSN metabolism plays a role in human and animal prion diseases.

Differential overexpression of SERPINA3 in human prion diseases.

Prion diseases are fatal neurodegenerative disorders with sporadic, genetic or acquired etiologies. The molecular alterations leading to the onset and the spreading of these diseases are still unknown. In a previous work we identified a five-gene signature able to distinguish intracranially BSE-infected macaques from healthy ones, with SERPINA3 showing the most prominent dysregulation. We analyzed 128 suitable frontal cortex samples, from prion-affected patients (variant Creutzfeldt-Jakob disease (vCJD) n = 20, iatrogenic CJD (iCJD) n = 11, sporadic CJD (sCJD) n = 23, familial CJD (gCJD) n = 17, fatal familial insomnia (FFI) n = 9, Gerstmann-Sträussler-Scheinker syndrome (GSS)) n = 4), patients with Alzheimer disease (AD, n = 14) and age-matched controls (n = 30). Real Time-quantitative PCR was performed for SERPINA3 transcript, and ACTB, RPL19, GAPDH and B2M were used as reference genes. We report SERPINA3 to be strongly up-regulated in the brain of all human prion diseases, with only a mild up-regulation in AD. We show that this striking up-regulation, both at the mRNA and at the protein level, is present in all types of human prion diseases analyzed, although to a different extent for each specific disorder. Our data suggest that SERPINA3 may be involved in the pathogenesis and the progression of prion diseases, representing a valid tool for distinguishing different forms of these disorders in humans.

Probing the dynamics of prion diseases with amphotericin B.

Amphotericin B (AmB) is one of the rare drugs that affect the course of experimental prion diseases and modify the kinetics of abnormal prion protein accumulation in the central nervous system. Therefore, AmB could be used as a pharmacological tool to contribute to our understanding of the pathogenic mechanisms involved in these neurodegenerative disorders.

Tissue distribution of bovine spongiform encephalopathy agent in primates after intravenous or oral infection.

BACKGROUND: The disease-associated form of prion protein (PrP(res)) has been noted in lymphoreticular tissues in patients with variant Creutzfeldt-Jakob disease (vCJD). Thus, the disease could be transmitted iatrogenically by surgery or use of blood products. We aimed to assess transmissibility of the bovine spongiform encephalopathy (BSE) agent to primates by the intravenous route and study its tissue distribution compared with infection by the oral route. METHODS: Cynomolgus macaques were infected either intravenously or orally with brain homogenates from first-passage animals with BSE. They were clinically monitored for occurrence of neurological signs and killed humanely at the terminal stage of the disease. Brain, lymphoreticular tissues, digestive tract, and peripheral nerves were obtained and analysed by sandwich ELISA and immunohistochemistry for quantitative and qualitative assessment of their PrP(res) content. FINDINGS: Incubation periods after intravenous transmission of BSE were much shorter than after oral infection. We noted that PrP(res) was present in lymphoreticular tissues such as spleen and tonsils and in the entire gut from the duodenum to the rectum. In the gut, PrP(res) was present in Peyer's patches and in the enteric nervous system and nerve fibres of intestinal mucosa. Furthermore, PrP(res) was found in locomotor peripheral nerves and the autonomic nervous system. Amount of PrP(res) ranged from 0.02% to more than 10% of that recorded in brain. Distribution of PrP(res) was similar in animals infected by the intravenous or oral route. INTERPRETATION: Our findings suggest that the possible risk of vCJD linked to endoscopic procedures might be currently underestimated. Human iatrogenic vCJD cases infected intravenously raise the same public-health concerns as primary cases and need the same precautionary measures with respect to blood and tissue donations and surgical procedures.

Neuropathologic variants of sporadic Creutzfeldt-Jakob disease and codon 129 of PrP gene.

OBJECTIVES: To determine the contribution of methionine/valine (Met/Val) polymorphism at codon 129 of the prion protein (PrP) gene in the neuropathologic pattern and mechanisms of lesion development in sporadic Creutzfeldt-Jakob disease. BACKGROUND: Creutzfeldt-Jakob disease is a transmissible spongiform encephalopathy characterized by a conformational change of PrP and a variety of PrP deposits in the brain, some of which aggregate into amyloid plaques. METHODS: The authors semiquantitatively assessed neuropathologic lesions and performed PrP immunolabeling in 70 patients (39 Met/Met, 11 Met/Val, 20 Val/Val) who had died in France between 1994 and 1998. RESULTS: Met/Met cases (mild lesions mostly involving the occipital areas, low PrP load, few focal PrP nonamyloid deposits, no amyloid plaques) contrasted with Met/Val cases (marked lesions especially in the parahippocampal gyrus, high PrP load, numerous amyloid plaques) and with Val/Val cases (younger patients, longer course of disease: 11.5 +/- 3 months, and distinct neuropathology: severe lesions heavily involving the hippocampal formation and basal ganglia, high PrP load, numerous focal nonamyloid deposits, rare amyloid plaques). The course of Val/Val patients younger than age 55 was particularly long (19.9 +/- 7 months), and the isocortex bore the brunt of the pathology, suggesting a distinct variety. CONCLUSIONS: Polymorphism at codon 129 modulates the phenotype of sporadic Creutzfeldt-Jakob disease. The Val genotype enhances the production of proteinase-resistant PrP, and the Met/Val genotype facilitates its aggregation into amyloid plaques.

Creutzfeldt-Jakob disease from contaminated growth hormone extracts in France.

We diagnosed Cruetzfeldt-Jakob disease in 34 patients (16 definite, 18 probable) who had received human growth hormone extract for various period of time (mean +/- SD, 2.9 years), but particularly during the period between January 1984 and July 1985, a potential high-risk factor. Disease duration for deceased patients (n = 30) was 17 +/- 9 months. The clinical picture was homogeneous, starting with cerebellar ataxia and ocular motor disorders in about 90% of the patients. Neurologic deterioration, including dementia and myoclonic jerks, occurred within months. The high number of cases (1.5% of those treated between 1959 and 1988, 3% of those treated during the putative high-risk period) is still unexplained. We discuss the possibility that new cases will be detected,the risk of contaminating the general public, and the sanitary measures undertaken to prevent this.

Incubation period of Creutzfeldt-Jakob disease in human growth hormone recipients in France.

OBJECTIVE: To estimate the statistical distribution of the incubation period of Creutzfeldt-Jakob disease (CJD) in human growth hormone (hGH) recipients in France. BACKGROUND: Published papers suggest that the median incubation period of hGH-related CJD is approximately 15 years, but there are as yet no statistical data that support this assertion. METHODS: Of the 1,361 hGH recipients who were included in this study, 55 had developed CJD at the time of the study. Individual data on hGH treatment history were available. Different mathematical models were used to estimate the statistical distribution of the incubation period. One main feature of the models was to take into account the occurrence of future CJD cases. RESULTS: Models showed that the mean incubation period was 9 to 10 years, and the 95th percentile of the distribution was 15 to 16 years. Data and models indicated that the incubation period was significantly shorter in homozygotes at codon 129 of the prion protein gene than in heterozygotes. CONCLUSIONS: The short mean incubation period of CJD in French hGH recipients may be due to high infectivity in hormone lots. Estimates of the 95th percentile indicate that the number of hGH-related CJD cases may continue to increase in the coming years.

Dementia with Lewy bodies in a neuropathologic series of suspected Creutzfeldt-Jakob disease.

Discriminating Creutzfeldt-Jakob disease (CJD) from dementia with Lewy bodies (DLB) may be clinically difficult to achieve. The authors describe 10 patients with DLB initially referred to the French Network of Human Spongiform Encephalopathies as having suspected CJD. In a series of 465 autopsied cases, DLB ranked second among degenerative alternative diagnoses to CJD. The authors analyzed the factors that contributed to misleading the diagnosis, and suggest that the detection of 14-3-3 protein in CSF may be useful to distinguish CJD from DLB.

Regulation of the glial fibrillary acidic protein, beta actin and prion protein mRNAs during brain development in mouse.

Developmental regulation in mRNAs of three brain proteins has been investigated by Northern blot evaluation in C57BL/6 mice. The mRNAs of two cytoskeletal components, glial fibrillary acidic protein (GFAP) and beta actin, varied significantly, and differently, during brain development (0-56 days postnatal). The beta actin mRNAs peaked at day 1 after a slight increase, then dropped rapidly during the first 15 days postnatal, and thereafter remained at a level which was strictly maintained throughout development and adulthood. Conversely, the GFAP mRNAs increased during the first two weeks after birth (astroglial proliferation), and then slightly declined until the adult stage (astroglial cell differentiation). The prion protein (PrP) mRNAs were detectable as soon as birth, and increased 4-fold during brain maturation. Then, during the adult life, the GFAP and PrP mRNAs did not change markedly. Nevertheless, slight but significant increases in the mRNA levels of both GFAP and PrP were observed at older stages (360 days). These results are analysed in the light of the implications of PrP and GFAP in scrapie infection models.

Modulation of prion protein gene expression by growth factors in cultured mouse astrocytes and PC-12 cells.

The present study was performed on primary cultures of mouse astrocytes and cultures of rat pheochromocytoma PC-12 in order to investigate the regulation of the prion protein (PrP) gene expression in relation to proliferation and differentiation. Treatment of PC-12 cells with interleukin-6 (IL-6) and beta-nerve growth factor (NGF) resulted in induction of neuronal differentiation. Northern blot analysis demonstrated a 4-fold increase of PrP mRNA in relation to cellular differentiation, after 7 days of treatment with either of the two factors. In astrocytes, PrP and glial fibrillary acidic protein (GFAP) mRNA levels were found to be regulated in a similar manner during development in vitro. A 3-fold increase of their mRNAs was observed from 5 to 14 days of culture (proliferation period). Then, their gene expressions showed a slight decrease from 14 to 28 days (maturation period). Treatment of astrocytes with IL-6, basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) appeared to markedly down-regulate the expression of GFAP mRNAs, which might reflect cell maturation. In contrast, they had no significant effect on the expression of PrP gene. These results suggest that the PrP gene expression is differently regulated in neural cells. In neuronal cells, it is mainly associated with differentiation. On the other hand, in astrocytes, the PrP mRNA level seems to be not only related to the proliferation and differentiation stages.

Specific determination of the proteinase K-resistant form of the prion protein using two-site immunometric assays. Application to the post-mortem diagnosis of BSE.

The aim of this work was to establish an immunological test suitable for specifically detecting PrPres in tissues from animals or humans developing TSEs. We chose to use as detection method a conventional two-site immunometric assay (sandwich immunoassay) because over the last 20 years this technique has clearly been shown to be more sensitive and specific than other tests. We have established numerous two-site immunometric assays based on the use of monoclonal antibodies and suitable for measurement of PrPsen in various mammalian species (human, bovine, ovine, mouse and hamster). A detection limit below 100 pg/ml was estimated from standard curves established using ovine recombinant PrP. PrPres was selectively detected by processing samples (currently brain homogenates) to enable specific purification and concentration of PrPres, which was finally solubilized by a strong denaturing treatment. This sample-processing procedure can be achieved within 30 minutes. The capacity of this test to detect bovine PrPres was estimated in the framework of an evaluation study organized by the Directorate-General XXIV of the European Commission during May 1999. On this occasion, a blind test on 1400 brain stem samples taken from either healthy (1000) or BSE-infected (300) cows demonstrated 100% sensitivity and specificity. In addition, dilution experiments showed that the test can significantly detect PrPres in homogenates diluted 1/300 and was at least as sensitive as a conventional bioassay performed on mice.

Microglial cells respond to amyloidogenic PrP peptide by the production of inflammatory cytokines.

The scrapie isoform of the prion protein (PrPres) induces neurodegeneration and gliosis in the central nervous system. These features may be reproduced in vitro on exposure of neuronal and glial cultures to PrPres and the peptide HuPr P106-126. In the present study, we investigated the role of microglial cells and astrocytes in the pathological process by studying their molecular response to PrP 106-126 exposure. PrP 106-126 elicited a specific overproduction of pro-inflammatory cytokines IL1beta and IL6 in microglial cells (but not increased expression of TNFalpha, IL10, and TGFbeta1) and over-expression of GFAP in astrocytes. These effects were strictly dependent on the ability of the peptide to form amyloid fibrils. These data strongly suggest that microglial cells contribute to prion-related neurodegenerative processes by producing proinflammatory cytokines in the brain areas of amyloid PrP deposition.

A simple and rapid method for sequencing the entire coding region of the human prion protein (PrP) gene.

The accumulation in brain of the 'prion protein' (PrP), a host-encoded sialoglycoprotein, is the unique specific molecular marker of subacute spongiform transmissible encephalopathies (SSTE). Furthermore, the primary sequence of the PrP gene (PRNP) seems to contain some genetic determinants of great importance in the development of SSTE. Here we present a simple and rapid polymerase chain reaction (PCR)-based method for direct sequencing of the entire coding sequence of the PrP gene, PRNP, in patients. The ability to determine sequences of both alleles of the PRNP gene is demonstrated in the analysis of 3 patients previously established as codon 129 heterozygotes by the use allele-specific oligonucleotide hybridization method.

Astrocyte gene expression in experimental mouse scrapie.

The biological hallmark of transmissible spongiform encephalopathies is a significant accumulation, in brain, of the scrapie prion protein (PrPsc), often associated with an increased glial fibrillary acidic protein (GFAP) expression. This study was focused on astrocyte gene expression during scrapie development over a period of 172 days in intracerebrally inoculated newborn mice. The levels of expression of PrP and two specific astrocyte proteins, -GFAP and glutamine synthetase (GS)-, were investigated by Western and Northern blots. In brain, a 10-fold increased expression of GFAP mRNAS was demonstrated from 112 days post-inoculation to 172 days, whereas the "upregulation" of GS mRNAs was two-fold. GFAP was observed to increase 10- to 20-fold in scrapie-infected brain from day 112 to day 172, while PrP showed a three- to four-fold elevation. Both proteins were found in greater amount in the frontal cortex and cerebellum of animals with clinical scrapie than in those given an injection of normal brain. PrPsc was detected in scrapie brain from day 84 after inoculation, and thereafter increased about 20-fold until day 172. On the other hand, the concentration of glutamine synthetase remained constant in brain throughout the scrapie disease. To conclude, these results show that GFAP and GS mRNAs are differently upregulated in brain in the scrapie mouse model.

MS-8209, an amphotericin B analogue, delays the appearance of spongiosis, astrogliosis and PrPres accumulation in the brain of scrapie-infected hamsters.

The histopathological response of scrapie-infected hamsters treated at the late stage of the infection with an "anti-scrapie" drug, a polyene macrolide antibiotic designated MS-8209, was evaluated in the brain. The results showed that (1) MS-8209 prolonged significantly the incubation time of the experimental disease, (2) MS-8209 delayed the appearance of spongiosis and astrogliosis in the brain, (3) immunodetection of abnormal prion protein and glial fibrillary acidic protein was significantly reduced in the central nervous system. In addition, this report indicates that polyene antibiotics markedly delay the development of the classical brain lesions that result from scrapie infection.

[Animal models of transmissible subacute spongiform encephalopathies]. / Modèles animaux d'encéphalopathies subaiguës spongiformes transmissibles.

The lack of suitable in vitro test explains the major importance of animal models in the study of Transmissible Spongiform Encephalopathies (TSE). Models using big animals are closer to natural situations, while solely rodent adapted models can be used currently in laboratories. Different scientific approaches based on the utilization of these models permitted to evidence the multifactorial character of TSE, with factors linked to the host and others linked to the TSE agents, also called prions. Prion protein (PrP) is the major host factor as it has been recently demonstrated by transgenetic studies. Among agent related factors, the notion of independant strains of TSE agents is now well established in inbred mice even if molecular studies have hitherto not been able to precise their exact nature. Although these models constitute powerful tools for the study of TSE, it has to be reminded that they can be far from natural situations.