Littérature scientifique sur le sujet « Encefalopatia spongiforme »

Transmisivne spongiformne encefalopatije su grupa rijetkih, progresivnih neurodegenerativnih bolesti od kojih obolijevaju ljudi i veći broj životinjskih vrsta, a prouzroči ih prion. Infekcija je dugi vremenski period asimptomatska, a bolest je progresivna i neminovno letalnog ishoda. U skupinu transmisivnih spongiformnih encefalopatija spadaju: klasična i atipična goveđa spongiformna encefalopatija, klasični i atipični grebež u ovaca i koza, spongiformna encefalopatija jelena, transmisivna encefalopatija američke vidrice, mačja spongiformna encefalopatija, Creutzfeldt- Jakobova bolest, varijanta Creutzfeldt- Jakobove bolesti, Gerstmann-Sträussler- Scheinker sindrom, familijarna fatalna insomnija i kuru. Za postojanje ove bolesti na ovcama zna se već gotovo tri stoljeća. Predmetne bolesti su se izučavale tijekom 20. stoljeća, ali su se tek pojavom epizootije goveđe spongiformne encefalopatije 80-ih godina prošlog stoljeća u Ujedinjenom Kraljevstvu (UK) te otkrivanjem novih dijagnostičkih metoda, istraživanja intenzivirala. Otkriven je i uzročnik bolesti - prion (proteinska zarazna čestica). Prion je normalni stanični protein pronađen prije svega na površini neurona, ali i drugih stanica u organizmu, koji se zbog još uvijek neobjašnjivih razloga u biokemijskim procesima sinteze proteina promijeni i postaje uzročnik encefalopatije. U ovome članku prikazani su rezultati praćenja goveđe spongiformne encefalopatije i grebeža ovaca u Republici Hrvatskoj od 2001. do kraja 2021. godine. Goveđa spongiformna encefalopatija se sistematski nadzire od 2001., dok se grebež ovaca nadzire od 2002. godine. Do kraja 2021. godine ukupno je pretraženo 454.822 uzoraka moždanog tkiva goveda, a tijekom navedenog perioda nije utvrđen niti jedan pozitivan uzorak. Na grebež ovaca pretraženo je ukupno 25.332 uzoraka moždanog tkiva koza i ovaca. Laboratorijskim pretraživanjem nije utvrđen niti jedan pozitivan uzorak na klasični grebež ovaca, ali su potvrđena četiri slučaja atipičnog grebeža u ovaca. Na temelju svih provedenih aktivnosti i nadziranja goveđe spongiformne encefalopatije u Republici Hrvatskoj je od strane Svjetske organizacije za zdravlje životinja 2014. godine dodijeljen status zemlje sa zanemarivim rizikom u odnosu na goveđu spongiformnu encefalopatiju, što je ujedno i najviši zdravstveni status u odnosu na ovu bolest.

Le Encefalopatie Spongiformi Trasmissibili (TSE) o Malattie da Prioni, costituiscono un gruppo di malattie neurodegenerative fatali, ad eziologia sporadica, genetica o infettiva che colpiscono l’uomo e diverse specie animali. Il meccanismo patogenetico responsabile delle TSE consiste nella conversione conformazionale della normale proteina prionica cellulare (PrPC), con una struttura prevalentemente ad α-elica, in una forma aberrante, ricca in β-sheet, parzialmente insolubile in detergenti non ionici e resistente alla digestione con proteasi, denominata PrPSc, che rappresenta il marcatore diagnostico di queste malattie. Nei bovini, sono due le forme di TSE conosciute: l’encefalopatia spongiforme bovina (BSE) e l’encefalopatia spongiforme amiloidotica bovina (BASE). Della prima sono ampiamente note sia la manifestazione fenotipica sia la caratterizzazione biochimica del ceppo proteico grazie al fiorire di studi in seguito all’epidemia che a partire dal 1985 ha colpito molti allevamenti bovini della Gran Bretagna. Della seconda, invece, non esiste ancora una completa caratterizzazione a causa della sua recente identificazione. Infatti solo nei primi anni del 2000 sono state riconosciute due nuove forme atipiche di BSE, chiamate high type (BSE-H) and low type (BSE-L) in base alla differente migrazione elettroforetica dell’isoforma non glicosilata della PrPSc bovina. Anche la BASE è stata definita una forma atipica di BSE per le notevoli differenze molecolari e neuropatologiche presentate dalla proteina prionica. Infatti la PrPSc associata alla BASE è risultata paragonabile a quella della BSE-L, per il peso e il profilo di glicosilazione delle isoforme, e distinguibile dalla BSE per la presenza di depositi ricchi di amiloide, chiamati placche kuru, a livello cerebrale. E’ stata così suggerita l’ipotesi dell’esistenza di un ceppo di prione della BASE diverso rispetto a quello della BSE, ma uguale a quello della BSE-L. Anche recenti studi di trasmissione in topi PrP-bovinizzati hanno contribuito a confermare tali dati, dimostrando come la BASE sia una patologia più aggressiva e più facilmente trasmissibile della BSE. Mancando sia le conferme a queste preliminari sperimentazioni sia l’identificazione del fenotipo clinico-patologico associato alla BASE, abbiamo progettato uno studio di trasmissione intraspecie di ceppi di BSE e BASE in bovini di razza Frisona e Bruna Alpina. Utilizzando la stessa specie di quella che ha contratto in origine l’infezione, si sono raggiunti due obiettivi: ridurre il periodo di incubazione, evitando il fenomeno dell’adattamento e tutti i processi atti a superare la barriera di specie, e delineare il quadro fenotipico della BASE. Gli animali inoculati con BASE hanno manifestato un comportamento affine alla depressione diverso da quello più irrequieto degli animali infettati con BSE, associato ad una progressiva amiotrofia. Inoltre i bovini hanno presentato profili molecolari e neuropatologici affini alle caratteristiche del donatore, confermando la diversità dei due strains proteici. Lo studio si è avvalso anche di un’analisi ultrastrutturale, che, oltre a confermare la diversità del pattern di deposizione della PrPSc nelle due forme di TSE, ha rilevato significative differenze nella localizzazione subcellulare della proteina prionica. Questa sperimentazione ha permesso di valutare tutti gli aspetti clinici, biochimici, istologici, immunoistochimici e ultrastrutturali associati alla PrPSc ed ottenere quindi una completa caratterizzazione della BASE, confermando i dati già presenti in letteratura sulla diversità del ceppo proteico rispetto quello della BSE e riproducendo per la prima volta la manifestazione fenotipica.
Transmissible Spongiform Encephalopathies (TSE) or Prion diseases are a group of rare, fatal and transmissible neurodegenerative disorders that affect both humans and animals. Clinically these diseases exist in sporadic, genetic and acquired forms, and present with a variety of neurological signs. TSE diseases are characterized by accumulation, primarily in the brain, of an abnormal isoform of the normal host-encoded prion protein (PrPC), named PrPSc that is considered the disease-associated agent. The central event of the TSE is a post-translational conformational change of the PrPC, a plasma membrane glycoprotein, rich in α-helix, into the PrPSc that has a higher β-sheet content. In bovine there are two forms of TSE: Bovine Spongiform Encephalopathy (BSE) and Bovine Amyloidotic Spongiform Encephalopathy (BASE). After the epidemy in cattle in UK in 1985, many researches characterized the phenotype and the biochemistry of PrPSc in BSE. Recently new atypical forms of BSE are discovered and called BSE-H (higher) and BSE-L (lower) for the different molecular mass of unglycosilated isoform of PrPSc. Also BASE is classified as atypical BSE with a prion protein similar to BSE-L and it is distinguishable from BSE for differences in pattern of deposition, consistent in the presence of amyloid plaques, in distinct molecular masses and in glycosilation profile of prion isoforms. These observations suggest that cattle may have two distinct prion strains, refered to two forms of TSE and that prion protein of BASE could be the same of BSE-L. Also recent studies on PrP-bovinized transgenic mice have showed that BASE is a more aggressive disease than BSE, characterized by shorter period of incubation. To investigate the characteristics of prion strains responsible of BSE and BASE, we carried out transmission by inoculation of BSE and BASE isolates in Fresian and Alpine brown cattle. Intraspecies transmission permitted firstly to reduce the period of incubation, overcoming the species barrier, and secondly to identify the phenotype of BASE. BASE inoculated cattle showed a progressive muscle atrophy and a dull behaviour, in contrast with aggressiveness and hypersensitivity of BSE-infected animals. The prion strains of inoculi preserved their different biochemical and neuropathological properties also after transmission. Finally, a more detailed ultrastructural analysis confirmed the distinction in patterns of PrPSc deposition and it showed different subcellular localization of prion protein between BSE and BASE infected bovine. By evaluation of biochemical, immunoistochemical and ultrastructural results, our study gave an improvement in the characterization of BASE prion strain, describing the clinical phenotype.

Transmissible spongiform encephalopathies (TSE) are neurodegenerative diseases caused by the conversion of the host-encoded cellular protein (PrPC) to a disease-associated isoform (PrPSc). The agent responsible for prion diseases may exist as different strains with specific biological and biochemical properties. According to the protein-only hypothesis, prion strain diversity is enciphered in PrPSc conformation. Molecular strain typing methods are based on the electrophoretic mobility of protease resistant core of PrPSc, on the susceptibility to protease digestion, on the glycosylation profile of PrPres and on the conformational stability of PrPSc. In this study a new conformational stability assay was developed based on the differential solubility of PrPC and PrPSc: CSSA (conformational stability and solubility assay). The conformational stability assay was performed by measuring PrPSc solubility in homogenates treated with increasing concentrations of GdnHCl, in the absence of proteinase K. Indeed, dose-response curves allowed estimation of the concentration of GdnHCl able to solubilise 50% of PrPSc. The results showed that this method is valuable for the biochemical typing of strains in bank voles and it is also a promising tool for molecular analysis of natural prion isolates. CSSA also revealed strain-specific PrPSc conformational stabilities of ovine natural isolates so that this feature, combined with the N-terminal PrPSc cleavage, allowed differentiation of classical scrapie, including CH1641-like, from natural goat BSE and experimental sheep BSE. In view of the implications concerning strain similarity between animal and human TSEs, the physico-chemical properties of the Nor98 with two human prion diseases (VPSPr and GSS) were compared in order to investigate the extent of the similarity between animal and human prion strains. The results showed an unexpected heterogeneity of the molecular features among human and sheep TSEs associated with internal PrPres fragments with the possible exception of Nor98 and a case of GSS P102L. These similarities and differences need further investigation by N- and C-terminal sequencing and biological characterization.

Transmissible spongiform encephalopathies (TSE) are neurodegenerative diseases caused by the conversion of the host-encoded cellular protein (PrPC) to a disease-associated isoform (PrPSc). The agent responsible for prion diseases may exist as different strains with specific biological and biochemical properties. According to the protein-only hypothesis, prion strain diversity is enciphered in PrPSc conformation. Molecular strain typing methods are based on the electrophoretic mobility of protease resistant core of PrPSc, on the susceptibility to protease digestion, on the glycosylation profile of PrPres and on the conformational stability of PrPSc. In this study a new conformational stability assay was developed based on the differential solubility of PrPC and PrPSc: CSSA (conformational stability and solubility assay). The conformational stability assay was performed by measuring PrPSc solubility in homogenates treated with increasing concentrations of GdnHCl, in the absence of proteinase K. Indeed, dose-response curves allowed estimation of the concentration of GdnHCl able to solubilise 50% of PrPSc. The results showed that this method is valuable for the biochemical typing of strains in bank voles and it is also a promising tool for molecular analysis of natural prion isolates. CSSA also revealed strain-specific PrPSc conformational stabilities of ovine natural isolates so that this feature, combined with the N-terminal PrPSc cleavage, allowed differentiation of classical scrapie, including CH1641-like, from natural goat BSE and experimental sheep BSE. In view of the implications concerning strain similarity between animal and human TSEs, the physico-chemical properties of the Nor98 with two human prion diseases (VPSPr and GSS) were compared in order to investigate the extent of the similarity between animal and human prion strains. The results showed an unexpected heterogeneity of the molecular features among human and sheep TSEs associated with internal PrPres fragments with the possible exception of Nor98 and a case of GSS P102L. These similarities and differences need further investigation by N- and C-terminal sequencing and biological characterization.

Prion diseases, or trasmissible spongiform encephalopathies (TSEs), are a group of fatal neurodegenerative diseases that affect humans and other mammalians. All these pathologies have long incubation periods followed by a crhonic neurological disease with fatal outcomes and have similar pathology limited to the central nervous system. TSEs are characterized hystopathologically by spongiosis and neural loss. The infectious agent in prion diseases is believed to be represented by a single protein, named prion protein (PrPC). The “Protein Only Hypothesis” [1] suggests that TSE are caused by a disease-associated and improperly folded form of PrPC, termed PrPSc. The nature of prions and the mechanism by which they propagate still poses fascinating basic questions. Human TSEs are biologically unique in that the disease process can be triggered by different phenomena: i) infection with prion infected tissues, such as kuru and the new variant of Creuzfeldt-Jacob disease (vCJD); ii) a sporadic event that generates PrPSc, for example in CJD, and iii) inherited mutations in the human prion protein encoding gene PRNP, which cause fatal familial insomnia (FFI), Gerstmann-Straussler-Scheinker disease (GSS) and familial CJD. The extraordinary structural and functional conservation of genes and pathways observed in distantly related organisms, from yeast to primates, has made possible to use model organisms to further our understanding of human biology. Several studies performed using transgenic animal models have yeld a wealth of new knowledge about infectious, genetic and sporadic prion diseases. The aim of this work is to investigate human inherited prion diseases (IPDs) thus helping to advance our knowledge of molecular mechanism responsible of these pathologies that are presently not understood, using a Drosophila melanogaster animal model. Among invertebrate model organisms, Drosophila presents the highest degree of gene conservation to humans and, importantly, it has an high evolved nervous system. The fly brain is estimated to have more than 300,000 neurons and, similarly to mammals, is organised into areas with separated specialised functions. In addition, the fruit fly offers a number of experimental advantages, including short lifespan, well characterized development and anatomy, sequenced genome and simple transgenic analysis. Several studies indicate that not only basic cell biology but also higher-order events such as organs construction and function have been conserved between human and flies throughout the evolution. Moreover, an unparallel advantage of invertebrates is the ability to carry out large-scale genetic screens rapidly. Finally, Drosophila has an unrivalled battery of genetic tools including rapidly expanding collections of mutants, trasposon-based methods for gene manipulation, systems that allow controlled ectopic gene expression and balancer chromosomes. These attributes, along with the rapid generation time, inexpensive culture requirements and large progeny numbers produced in a single cross, make Drosophila an incisive tool for the analysis of human diseases. Therefore, we generated transgenic fruit flies for constructs carryng either wild type or mutated forms of human PrP (H-PrP). In particular, the mutations were: P102L, associated to GSS syndrome; D178N/129M associated to FFI; D178N/129V and E200K both associated to fCJD. With standard genetic crosses, the different human PrP forms were actively expressed in all tissues, in the entire nervous system, or in motor neurons. Flies expressing H-PrP exhibited severe age-dependent locomotor dysfunction and premature death. These flies showed a progressive loss of the ability to fly, followed by a progressive loss of the capability to walk on the tubes where they are collected: they did not fly, walked slowly, had difficulty righting themselves when they are knocked down. Moreover, they lost the control of their leg movements: leg quivered and the flies often fell on a side, like they missed the support of the legs. At the same time, they showed an altered movement of wings and antennae as they continuously groomed themselves. Locomotor activities were analyzed by specific behavioural tests and evaluating possible alteration in neuromuscolar junctions. Moreover, in order to evaluate the presence of tissue vacuolization/spongiosis and/or PrPSc deposition, adult flies brains were examined by standard hematoxiyin/eosin stained sections at different time points. In particular, we analized brain sections when flies display severe age-dependent behavioural alterations. Our findings indicate that Drosophila could be a powerful tool to study human familial TSEs. In particular, H-PrP expression in transgenic flies gives rise to gross behavioural phenotypes that closely remind the neurodegeneration associated with altered function of prion diseases in humans. While knowledge of the precise ways in which evolutionary conserved processes operate in humans will only come from the investigation of human themselves, studies in Drosophila will result in a better undesrtanding of the gene networks involved, and how their perturbations in humans can lead to disease.
Le malattie da prioni, dette anche encefalopatie spongiformi trasmissibili (TSE), comprendono un gruppo di malattie neurodegenerative che colpiscono l’uomo e altre specie di mammiferi. Tutte queste patologie hanno la caratteristica comune di presentare lunghi periodi di incubazione seguiti da una patologia cronica a carico del sistema nervoso che ha sempre esito fatale. A tutt’oggi, la teoria più accreditata per spiegare le TSE è la “Protein Only Hypothesis” [1], secondo la quale l’agente responsabile di queste malattie è una proteina in grado di replicarsi autonomamente, il prione (PrPSc). Il prione rappresenterebbe un’isoforma patologica di una proteina normalmente presente nelle cellule, la proteina prionica (PrPC), e che risulta particolarmente concentrata a livello delle cellule nervose. Nell’uomo, le malattie da prioni sono presenti in tre forme: i) infettiva, dovuta al meccanismo di conversione della proteina prionica nella sua isoforma patologica mediata dalla presenza di PrPSc, come nel caso del kuru e della variante della malattia di Creuzfeldt-Jacobs (vCJD); ii) sporadica, in seguito al cambiamento spontaneo della forma normale in PrPSc con un meccanismo non ancora noto, come, ad esempio nel caso della CJD, e infine una forma iii) ereditaria, trasmessa come carattere autosomico dominante associato a mutazioni a carico del gene PRNP codificante la proteina prionica. E’ questo il caso di malattie quali l’insonnia familiare fatale (IFF), la malattia di Gerstmann-Straussler-Scheinker (GSS) e la forma familiare della CJD (fCJD). La straordinaria conservazione strutturale e funzionale dei geni in organismi diversi ha permesso di utilizzare modelli animali, dal lievito ai primati, per accrescere la nostra conoscenza della biologia dell’uomo. Per poter contribuire al chiarimento della biologia dei prioni, del meccanismo patogenetico e del ruolo svolto dalle diverse mutazioni, abbiamo analizzato la possibilità di impiegare come organismo modello Drosophila melanogaster, già ampiamente utilizzata con successo per lo studio di altre malattie neurodegenerative umane. Gli invertebrati, a differenza dei mammiferi, risultano particolarmente utili nello studio di geni la cui funzione è sconosciuta in quanto permettono di applicare l’analisi genetica in modo sistematico. La scelta del modello Drosophila si basa sul fatto che questo organismo presenta, tra gli invertebrati, la massima omologia con il genoma umano, un sistema nervoso particolarmente evoluto e un tempo di generazione breve. Inoltre, data l’ampia varietà di tecniche molecolari applicabili, permette un’analisi sistematica offrendo la possiblità di chiarire la funzione di geni in modo relativamente rapido ed efficiente rispetto ad altri modelli animali. Ci siamo quindi proposti da un lato di creare un modello animale per lo studio della funzione della PrP umana normale e mutata che permetta di approfondire i processi neurodegenerativi alla base delle TSE, e dall’altro di utilizzare il moscerino della frutta per effettuare un rapido screening genetico di alcune tra le mutazioni note, in modo da poter determinare quelle che svolgono un ruolo importante nella patogenesi di queste malattie. Tra tutte le mutazioni finora identificate ne sono state scelte tre perchè rappresentative delle tre forme di TSE ereditarie nell’uomo: P102L, associata alla GSS, D178N/129M, associata alla IFF, D178N/129V e E200K associate alla fCJD. Mediante microiniezione degli embrioni di Drosophila, sono state ottenute diverse linee di moscerini transgenici per costrutti codificanti la proteina prionica umana (H-PrP) wild type oppure recante le mutazioni indicate. Avvalendosi del sistema di espressione binario UAS/GAL4, sono stati allestiti gli incroci per promuovere l’espressione tessuto-specifica dei diversi transgeni, in particolare a livello ubiquitario, dell’intero sistema nervoso e nei motoneuroni. E’ stato così possibile osservare che l’espressione delle diverse forme di H-PrP non interferisce con le fasi dello sviluppo del moscerino mentre nell’adulto causa un’alterazione del fenotipo comportamentale che si aggrava progressivamente in relazione all’età degli individui. Con il trascorrere dei giorni, i moscerini esprimenti H-PrP manifestano una progressiva perdita del controllo dei movimenti delle zampe, tremori e perdita dell’equilibrio, ai quali si accompagna una sempre più evidente lentezza dei movimenti, fino alla pressoché totale immobilità e alla morte precoce. I fenotipi motori sono stati analizzati utilizzando specifici test comportamentali. Inoltre, per valutare se i fenotipi patologici osservati fossero imputabili, a livello cerebrale, alla comparsa di vacuolizzazione/spongiosi e/o all’accumulo di PrPSc, sono state osservate le sezioni istologiche ottenute dall’inclusione delle sole teste di moscerini, collezionate a tempi di invecchiamento diversi, in modo da monitorare l’eventuale comparsa di fenomeni degenerativi in dipendenza dell’età degli individui. Nel loro complesso, i risultati ottenuti sembrano indicare che Drosophila melanogaster possa rappresentare un valido modello animale da utilizzare nello studio delle malattie da prioni ereditarie. In particolare, la progressione temporale dei sintomi e il tipo di sintomatologia osservata sembrano riprodurre l’evoluzione delle TSE nei mammiferi, supportando così la validità del modello animale qui proposto. Mentre le conoscenze dei processi che operano nell’uomo potranno essere chiariti solamente studiando l’uomo stesso, gli studi condotti su Drosophila permettono di approfondire quali mutazioni sono coinvolte e come la loro espressione possa portare allo sviluppo della malattia.

Le encefalopatie spongiformi trasmissibili (EST), o malattie da prioni, sono malattie neurodegenerative che colpiscono l'uomo e gli animali. Le più note tra le EST animali sono l’encefalopatia spongiforme bovina (BSE) e la scrapie della pecora e della capra; le forme umane le più note sono la malattia di Creutzfeldt-Jakob (MCJ) e la variante della MCJ (vMCJ), legata all'epidemia di BSE. Le EST sono caratterizzate da tempi di incubazione estremamente lunghi ed esito invariabilmente fatale. Da un punto di vista patologico le lesioni, confinate al sistema nervoso centrale (SNC), consistono in spongiosi del neuropilo, vacuolizzazione e perdita neuronale, iperplasia ed ipertrofia delle cellule gliali. Il momento patogenetico centrale comune a tutte le EST è rappresentato dalla modificazione conformazionale di una proteina cellulare denominata PrPC (proteina prionica cellulare) in una isoforma patologica denominata PrPSc. La PrPSc, insolubile e caratterizzata da una parziale resistenza alle proteasi, si forma in seguito ad alterazioni post-translazionali dell’isoforma cellulare e tende a depositarsi sotto forma di fibrille amiloidee nel SNC dei soggetti colpiti. Nonostante possiedano i caratteri delle malattie neurodegenerative, le EST mostrano la sorprendente proprietà di essere trasmissibili. Obiettivo di questa tesi è stato quello di indagare i meccanismi che governano la trasmissione interspecifica delle malattie da prioni mediante l’utilizzo dell’arvicola rossastra o Myodes glareolus, un nuovo modello animale altamente suscettibile a diversi ceppi di EST. In particolare, la ricerca si è posta l’obiettivo di indagare: 1) se tale suscettibilità fosse da attribuire alla sequenza della PrP dell’arvicola; 2) qualora così fosse, a quali specifici aminoacidi; 3) se tale suscettibilità si esprimesse nei confronti di tutte le malattie da prioni o fosse ceppo-dipendente. Al fine di studiare il ruolo della PrP dell’arvicola in modo indipendente rispetto ad altri possibili fattori cellulari, sono stati condotti studi di trasmissione di diversi ceppi di prione in topi transgenici esprimenti la PrP di arvicola su un background knock out. I tempi di sopravvivenza sono stati analizzati per definire le caratteristiche di trasmissione nel modello transgenico e i risultati messi a confronto con quelli ottenuti inoculando gruppi di controllo di topi wild-type e di arvicola rossastra. Lo studio ha dimostrato che i topi transgenici acquisiscono caratteristiche di suscettibilità simili a quelle dell’arvicola, suggerendo che è la sequenza della PrP il maggiore determinante di suscettibilità dell’arvicola e confermando l’assenza o l’azione minoritaria di fattori specie-specifici diversi dalla PrP. Successivamente, al fine di verificare l’eventuale ruolo prevalente di specifici residui aminoacidici della PrP nel modulare la suscettibilità, sono state inoculate, con differenti ceppi di prione, sei diverse specie di roditori che mostrano diversi gradi di affinità filogenetica e specifici polimorfismi nella sequenza della PrP. L’analisi dei risultati suggerisce che alcuni siti polimorfici della PrP sono in grado di modulare la suscettibilità delle diverse specie ai prioni, facilitando o ostacolando la trasmissione e condizionando - nella trasmissione interspecifica - l’ampiezza della barriera di trasmissione. In particolare, le posizioni Y154N e S169N sembrano essere i principali determinanti della peculiare suscettibilità dell’arvicola e di altri roditori che condividono con essa le stesse caratteristiche nella sequenza della PrP. Infine, i dati di trasmissione hanno indicato come una determinata sequenza della PrP non conferisce elevata o ridotta suscettibilità in termini generali. Alcuni ceppi infatti mostrano di essere trasmissibili in maniera più efficiente alla specie portatrice di una determinata sequenza della PrP piuttosto che ad un’altra, ma il contrario può accadere impiegando altri ceppi. I risultati di questo studio hanno importanti implicazioni nella comprensione dei meccanismi molecolari alla base della trasmissione delle malattie da prioni e dimostrano che la barriera di trasmissione è un fenomeno cui partecipano due componenti intimamente correlate tra loro: la sequenza della PrP della specie infettata (e, in particolare, alcune posizioni “critiche”) ed il ceppo di agente. Grazie alla elevata suscettibilità, l’arvicola rossastra (Myodes glareolus) si dimostra un modello di grande interesse per lo studio delle basi molecolari della trasmissione interspecifica delle EST e per “modellare” il rischio di trasmissione dei prioni da una specie animale ad un’altra
Transmissible spongiform encephalopathies (TSEs) or prion diseases, are a group of fatal neurodegenerative diseases of humans and animals. They include bovine spongiform encephalopathy (BSE) in cattle and scrapie in sheep and goat. The most known human TSEs are Creutzfeldt-Jakob disease (CJD) and variant CJD (vCJD), that is linked to the BSE epidemic. TSEs are characterized by very long incubation periods and invariably fatal outcome. Neurodegenerative changes confined to the central nervous system (CNS) and consisting of neuronal loss, vacuolation of neurons and neuropil, and hyperplasia and hypertrophy of glial cells are the pathological characteristics of these diseases.The conformational modification of the cellular prion protein, named PrPC, into an abnormal protease-resistant isoform, named PrPSc is the key event in TSEs pathogenesis. PrPSc is insoluble, partially resistant to protease and it tends to aggregate into amiloyd fibrils in the CNS of affected subjects. Although they have the characteristic of the neurodegenerative diseases, TSEs show the surprising property of being transmissible. Main objective of the present thesis was to study the mechanisms of interspecies transmission of prion diseases in bank vole or Myodes glareolus, a new animal model highly susceptible to a variety of TSE strains from animals and humans. The study had three different specific aims: 1) investigate whether the susceptibility of vole is imputable to its PrP sequence 2) if so, to identify amino acids residues of PrP which are potentially critical in influencing this susceptibility, 3) whether the susceptibility related to a given PrP sequence is applies to all prion diseases or it is strain-dependent. In order to study the role of bank vole PrP amino acids sequence independently from other cellular factors, the transmission studies were conducted into transgenic mice expressing the PrP of bank vole in a PrP knock-out background. The survival times observed in transgenic animals were compared with those obtained from control groups of voles and wild-type mice. The results showed that transgenic mice have a similar susceptibility to voles and clearly different from wild-type mice, suggesting that the PrP sequence is the principal determinant of susceptibility of voles. This confirmed the minor or absent role of factors different from PrP. Subsequently, with the aim to verify the role of specific amino acid variations on the PrP sequence in modulating the susceptibility of voles, we inoculated a panel of six rodent species showing various degrees of phylogenetic affinity and specific PrP sequence variations, with different TSEs. The results of this studies suggested that specific substitutions modulate the susceptibility of rodents to prion disease, also influencing the dimension of the barrier in interspecies transmission. In comparison to mice, the susceptibility of voles is influenced by the Y154N and S169N variations on the PrP sequence.. Finally, overall results of transmission studies showed that a particular PrP sequence may confer high or reduced susceptibility depending on the prion strains involved. Some strains are transmitted more efficiently to the species with determinate PrP sequence of rather than another, but the opposite may happen using other strains. . In particular, we showed that the Y154N–S169N exchanges, which appeared to confer in vole species a high susceptibility to a given strain, had the opposite effect with another one. These results improve the understanding of the molecular mechanisms underlying the transmission of prion diseases. Here we demonstrate that the transmission barrier is a phenomenon involving two component closely linked each other: the PrP sequence of the infected species (and, in particular, some amino acids residues) and the prion strain. Due to its high susceptibility, the bank vole (Myodes glareolus) is a model of great interest for studing the molecular basis of interspecies transmission of TSEs and helpful for assessing the risk of transmission of prions from one species to another

Dissertação de Mestrado em Segurança Alimentar
A primeira grande crise alimentar da história contemporânea, ocorreu quando em 1986 se provou a relação entre o consumo de produtos alimentares com origem em bovinos infectados com a Encefalopatia Espongiforme Bovina (EEB) e uma nova variante da Doença de Creutzfeld-Jakob, uma doença neurodegenerativa e fatal que afecta o Homem. Na sequência desta descoberta, a União Europeia (U.E) impõe uma série de medidas: Planos de Vigilância, Controlo e Erradicação da EEB; retirada de certos produtos da cadeia alimentar humana e animal (MRE); restrições à utilização de certas proteínas de origem animal na alimentação de animais de exploração (Feed Ban). Portugal revelou-se um dos países da União Europeia com maior incidência de EEB. Neste contexto foi alvo da imposição, por parte da U.E, de um embargo à exportação de produtos de origem bovina e de bovinos vivos. Neste trabalho procurou-se caracterizar, para o intervalo temporal entre 2002 e 2009, a evolução da epidemia de EEB em Portugal, os factores de risco, a eficácia das medidas preventivas e, em simultâneo, extrapolar possíveis futuros cenários caso a U.E, baseada nos dados obtidos nos diferentes países e num aconselhamento científico sólido, decida pela flexibilização de algumas medidas preventivas. Constatou-se que a EEB continua maioritariamente localizada nas Regiões Norte e Centro do país sendo que o número de casos positivos por milhão de bovinos adultos diminuiu de 137, em 2003, para 9 em 2009. A média etária dos animais positivos é de 104 meses e 98,8% desses bovinos nasceram antes de 2000, sendo 1997 o ano de nascimento do maior número de casos de EEB.
ABSTRACT - BSE IN PORTUGAL IN THE PERIOD 2002-2009 – EPIDEMIOLOGICAL EVOLUTION AND FUTURE CONSIDERATIONS - The major food safety crisis of contemporary history occurred in 1986 when an association was established between the consumption of products of bovine origin from BSE infected cattle and a new variant of Creutzfeld-Jacob Disease, a fatal human neurodegenerative condition. Following the finding, the European Union (EU) made compulsive a set of measures: surveillance plans; control and eradication programmes; removal of specified risk materials (MRE) from the food chain; feed ban of meat and bone meal (MBM) from the diet of food production animal species. Portugal became one of the EU Member States with high BSE incidence. Due to this fact, an embargo on the export of Portuguese live cattle and bovine products was put into force by the EU. In this study the evolution of the BSE epidemic in Portugal is described for the period 2002- 2009. Risk factors, the efficacy of preventive measures and future surveillance plans are discussed by the light of recent scientific findings and the industry pressure to make flexible the preventive measures. The BSE incidence was reduced from 137 cases per million of adult bovines in 2003 to 9 cases per million of adult bovines in 2009. BSE cases continue to be mainly located at the North and Centre Regions of Portugal. The average age of infected animals was 104 months. 98.8% of the BSE cases were born before 2000. 1997 being the birth year of the largest number of BSE cases.

En aquesta tesi doctoral, s’ha desenvolupat metodologia analítica per a la purificació, separació i caracterització de prió cel•lular (PrPC) i superòxid dismutasa (SOD-1), dues proteïnes relacionades amb les Encefalopaties Espongiformes Transmissibles (TSEs) i l’Esclerosi Lateral Amiotròfica (ALS), respectivament. Les TSEs es caracteritzen per l’acumulació de la forma patològica del PrPC (PrPSc) al cervell dels animals afectats, mentre que a l’ALS s’observa la formació d’agregats de SOD-1. Avui dia, encara es desconeixen els factors que inicien i regulen les interaccions que condueixen a la formació d’agregats proteics en moltes malalties neurodegeneratives. Alguns autors suggereixen mecanismes basats en els canvis estructurals que s’observen entre la proteïna nativa i la patològica, que estan relacionats amb la conformació, la seqüència d’aminoàcids, els metalls o les modificacions post-transduccionals. En proteïnes oligomèriques com la SOD-1, també s’ha considerat la dissociació dels oligòmers fins a monòmers abans de l’agregació. És doncs necessari millorar el coneixement de l’estructura d’aquestes proteïnes i aprofundir en els mecanismes que governen l’agregació. En aquest treball es proposa una estratègia de purificació per recuperar de manera eficient PrPC de cervell boví emprant mètodes de purificació convencionals no immuniquímics i western blot (WB) per detectar la presència PrPC en les diferents etapes. Tot seguit s’estudia exhaustivament la separació i caracterització de la SOD-1 mitjançant electroforesi capil•lar acoblada a l’espectrometria de masses amb analitzadors de trampa iònica i de temps de vol (CE-IT-MS i CE-TOF-MS), espectrometria de masses amb font d’ionització per desorció amb làser assistida per una matriu i analitzador de temps de vol (MALDI-TOF-MS) i espectrometria de masses de mobilitat iònica amb font d’ionització per nano-electrosprai (n-ESI-IM-MS). Els estudis amb SOD-1 purificada a partir de mostres de sang d’individus sans i pacients amb ALS han permès obtenir algunes conclusions preliminars interessants sobre els canvis estructurals en la proteïna associats a la malaltia.
In this thesis, we developed an analytical method for the purification, separation and characterization of cellular prion (PrPC) and superoxide dismutase (SOD-1), two proteins related to Transmissible Spongiform Encephalopathies (TSEs) and the Amyotrophic Lateral Sclerosis (ALS), respectively. The TSEs are characterized by the accumulation of the pathological form of PrPC (PrPSc) in the brain of affected animals, whereas in ALS it is observed the formation of aggregates of SOD-1. Today, factors that initiate and regulate the interactions that lead to the formation of protein aggregates in many neurodegenerative diseases are still unknown. Some authors suggest mechanisms based on the structural changes observed between the native and the pathology protein which cold be related with the conformation, the amino acid sequence, metals or post-translational modifications. In oligomeric proteins such as SOD-1, the dissociation of oligomers to monomers before aggregation it is also considered. So, it is crucial to increase the knowledge of the structure of these proteins and the mechanisms that govern its aggregation for understanding the disease development. This paper proposes a strategy for having an efficient recovery in the purification of bovine brain PrPC using conventional purification methods that not involves immunochemical procedures. The presence of PrPC was checked at different stages by western blot (WB). Then, the separation and characterization of the SOD-1 by capillary electrophoresis coupled to mass spectrometry with ion trap and time of flight analyzers (CE-IT-MS and CE-TOF-MS), matrix-assisted laser desorption/ionization with a time of flight mass analyzer (MALDI-TOF-MS) and ion mobility mass spectrometry with power nano-electrospray ionization source (n-ESI-IM-MS) was studied. The comparison of purified SOD-1 from blood samples of healthy individuals and patients with ALS have yielded some preliminary interesting conclusions about structural changes in the protein associated with cold be related with the disease.

Assessment of occurrence and diagnostic methods of viral diseases in cattle – viral diarrhoea (BVD), infectious bovine rhinotracheitis (IBR), rabies, enzootic bovine leukosis (EBL), and spongiform encephalopathy (BSE) – was carried out for the first time in Lithuania. It was established that viruses of rabies, infectious rhinotraheitis and viral diarrhoea are most widespread in the country. It was determined that occurrence of rabies in cattle is parallel with the infection of wildlife with rabies virus. Analysis of eradication programme of enzootic bovine leucosis was done. It revealed that only combined application of diagnostic and preventive measures allowed reducing the cattle infection up to 0.2%. Though bovine spongiform encephalopathy has not been recorded in Lithuania, it is feasible to implement its diagnostic and prevention programme. An overall financial analysis of expenditures on BSE and viral diseases diagnostics and control was for the first time done in Lithuania. It showed that BSE and EBL occupied the leading positions in the structure of expenditures on viral diseases. In 2001, expenditures on BSE investigations accounted for 76.68% and in 2004 for 86.74% of the total. Expenditures on EBL investigations relatively reduced from 86.98% in 2000 to 8.47% in 2004. During the time under consideration, expenditures on investigations of other viral diseases changed but little. It was determined that consistent and wide-scale preventive vaccination created... [to full text]

Prion diseases are fatal transmisible neurodegenerative and infectious disorders (TSEs) of humans and animals, characterized by structural transition of the host-encoded cellular prion protein (PrPC) into the aberrantly folded pathologic isoform PrPSc. The main aim of this work is to summarize present information about prion diseases and their possibilities of determination pointed to electrochemical techniques. For this purpose cyclic voltammetry (CV), differential pulse voltammetry, differential pulse voltammetry Brdicka reaction and chronopotentiometric stripping analysis (CPSA) were used. The estimated detection limits were 32 ug/ml by CV, 16 ug/ml by DPV, 16 ug/ml by DPV -- Brdicka reaction and 8 ug/ml by CPSA. Subsequently, the influence of heat denaturation was observed. It clearly follows from the obtained results that signals of prion decreased linearly depending on the duration of the heat treatment at 99°C for various time intervals 0, 15, 30, 45, and 60 min. Moreover, we aimed our attention on studying of prion protein interaction with CdTe quantum dots (QDs) using electrochemistry. Primarily, we characterized electrochemical properties of QDs and the detection limit at 100 fg/ml was estimated. Further, electrochemical study of prion and QD interactions was carried out to find the most suitable conditions for sensitive detection of prion proteins. Detection limit (3 S/N) was estimated as 1 fg in 5 ul. This makes labeling of proteins with QDs of great importance due to easy applicability and possibility to use in miniaturized devices, which can be used in situ. This should open new possibilities how to determine the presence of these proteins on surgical equipment and other types of materials, which could be contagious.

Transmissive spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrPTSE ) in brain. PrPTSE is at present the only specific biochemical marker of human and animal TSEs. Diagnostic tests are based on the detection of PrPres after proteinase K digestion of brain homogenate using Western blot or on the immunohistochemistry of fixed brain tissue, which are both difficult and time consuming. In this work we focused on development of a new type of tests based on PrP detection without need of proteinase K digestion. As deposits of PrPTSE remain in the body for a long time, there is a substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrPTSE may have a potential to serve as a diagnostic marker. We prepared monoclonal antibodies specific for carboxymethyl lysine/arginine modified prion protein. Bacterially expressed and purified recombinant human prion protein (rhPrP) was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of laboratory mice and hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of 4...