Analysis of the Electrophoretic Profiles of Prion Protein in Carcinous and Pericarcinous Lysates of Six Different Types of Cancers / 生物医学与环境科学（英文）

Objective@#To find the different electrophoretic profiles of prion protein in carcinous and individual pericarcinous tissues in lysates of gastric, colon, liver, lung, thyroid, and laryngeal cancers.@*Methods@#Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were used to test the amounts and electrophoretic patterns of total PrP and the tolerance of PK (protease K) digestion among six various cancer tissue types.@*Results@#A mass of PrP signals with a large molecular weight were identified in the homogenates of peripheral tissues. The amounts and electrophoretic patterns of total PrP did not differ significantly between carcinous and pericarcinous tissues. PrPs in all types of the tested cancer samples were PK sensitive but showed diversity in the tolerance of PK digestion among various tissue types.@*Conclusions@#The study revealed that the included electrophoretic patterns of carcinous and pericarcinous tissues were almost similar. Unlike PrP-specific immunohistochemical assay, evaluation of PrP electrophoretic patterns in the peripheral organs and tissues by Western blot does not reflect tumor malignancy.

Genetic Prion Disease: Insight from the Features and Experience of China National Surveillance for Creutzfeldt-Jakob Disease / 神经科学通报·英文版

Human genetic prion diseases (gPrDs) are directly associated with mutations and insertions in the PRNP (Prion Protein) gene. We collected and analyzed the data of 218 Chinese gPrD patients identified between Jan 2006 and June 2020. Nineteen different subtypes were identified and gPrDs accounted for 10.9% of all diagnosed PrDs within the same period. Some subtypes of gPrDs showed a degree of geographic association. The age at onset of Chinese gPrDs peaked in the 50-59 year group. Gerstmann-Sträussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI) cases usually displayed clinical symptoms earlier than genetic Creutzfeldt-Jakob disease (gCJD) patients with point mutations. A family history was more frequently recalled in P105L GSS and D178N FFI patients than T188K and E200K patients. None of the E196A gCJD patients reported a family history. The gCJD cases with point mutations always developed clinical manifestations typical of sporadic CJD (sCJD). EEG examination was not sensitive for gPrDs. sCJD-associated abnormalities on MRI were found in high proportions of GSS and gCJD patients. CSF 14-3-3 positivity was frequently detected in gCJD patients. Increased CSF tau was found in more than half of FFI and T188K gCJD cases, and an even higher proportion of E196A and E200K gCJD patients. 63.6% of P105L GSS cases showed a positive reaction in cerebrospinal fluid RT-QuIC. GSS and FFI cases had longer durations than most subtypes of gCJD. This is one of the largest studies of gPrDs in East Asians, and the illness profile of Chinese gPrDs is clearly distinct. Extremely high proportions of T188K and E196A occur among Chinese gPrDs; these mutations are rarely reported in Caucasians and Japanese.

Creutzfeldt-Jakob disease: one hundred years of participation in the design of the transmissible spongiform encephalopathies / Doença de Creutzfeldt-Jakob: cem anos de participação no desenho das encefalopatias espongiformes transmissíveis

Creutzfeldt and Jakob's disease (CJD) has its initial milestone in the publication issued 100 years ago that precipitated its better clinical-pathological and etiological understanding. Now, it is established that it belongs to the group of the prion diseases or transmissible spongiform encephalopathies family. CJD is itself divided into several types, the most common being sporadic that is further subdivided according to the anatomoclinical expression, but mainly due to its aetiology regarding prionic protein or genotype.

A doença de Creutzfeldt e Jakob (CJD) tem seu marco inicial na publicação emitida há 100 anos que precipitou seu melhor entendimento clínico- patológico e etiológico. Agora, está estabelecido que pertence ao grupo da família das doenças de príons ou encefalopatias espongiformes transmissíveis. A própria CJD se divide em vários tipos, sendo o mais comum o esporádico que também se subdivide de acordo com a expressão anatomoclínica, mas principalmente devido à sua etiologia em relação à proteína priônica ou genótipo.

Heidenhain variant of Creutzfeldt-Jakob disease in Brazil: a case report / Variante Heidenhain da Doença de Creutzfeldt-Jakob no Brasil: um relato de caso

Creutzfeldt-Jakob disease (CJD) is a rare spongiform encephalopathy characterized by a rapid neurodegenerative progress, caused by a misfolded variant of the cellular prion protein (PrP) known as PrPSc. The clinical presentation of sCJD includes a wide range of neurological signs of cortical, subcortical, or cerebellar origin, either isolated or in various combinations. Due to this protean clinical presentation form, sCJD must be distinguished from other dementias. In this case report, we discuss the Heidenhain variant of Creutzfeldt-Jakob disease (HvCJD), a rare variant characterized by early visual symptoms and typical findings in imaging scans. Our patient presented rapidly progressive dementia and a history of visual hallucinations. As for other prion diseases, only symptomatic treatment is available for HvCJD. Thirty years of clinical investigation of patients with prion disease have resulted in little progress in either defining or evaluating potential treatments.

A doença de Creutzfeldt-Jakob (DCJ) é uma encefalopatia rara caracterizada por rápida progressão neurodegenerativa, causada pelo enovelamento incorreto da proteína priônica celular (PrP), conhecido como PrPSc. O quadro clínico da DCJ esporádica inclui um amplo espectro de sinais neurológicos de origens cortical, subcortical ou cerebelar, seja de forma isolada, seja combinada. Por causa da sua apresentação clínica variável, a DCJ esporádica deve ser distinguida de outras demências. Neste relato de caso, discutimos a variante Heidenhain da DCJ (vHDCJ), uma variante rara caracterizada por sintomas visuais precoces e características específicas no exame de imagem. Nossa paciente apresentou demência rapidamente progressiva e histórico de alucinações visuais. Assim como para as demais doenças priônicas, apenas o tratamento sintomático está disponível para a vHDCJ. Trinta anos de investigação clínica de pacientes com doença priônica têm resultado em pouco progresso, seja definindo os potenciais tratamentos, seja avaliando-os.

Preparation of PrP-specific Polyclonal Antibody Immunization of -knockout Mice with Recombinant Human PrP Protein / 生物医学与环境科学（英文）

Objective@#The definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrP in the brain tissues of suspected cases. Thus, developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification.@*Methods@#We prepared a PrP-specific polyclonal antibody (pAb P54) in a -knockout mouse model immunization with recombinant full-length human PrP protein residues 23-231. Thereafter, we verified that pAb in Western blot, immunohistochemistry (IHC), and immunofluorescent (IFA) assays.@*Results@#Western blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein, normal brain PrP from healthy rodents and humans, and pathological PrP in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases. The electrophoretic patterns of brain PrP and PrP observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies. Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54. IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139A-infected mice and 263K-infected hamsters. IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei.@*Conclusion@#The newly prepared pAb P54 demonstrated reliable specificity and sensitivity and, thus, may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.

Doença de Creutzfeldt-Jakob: relato de caso / Creutzfeldt-Jakob disease: case report

As doenças priônicas fazem parte do grupo das síndromes de demência rapidamente progressiva com neurodegeneração. Em humanos, a doença de Creutzfeldt-Jakob é a mais prevalente. Atualmente, seu diagnóstico pode ser baseado em uma combinação do quadro clínico, ressonância magnética e eletroencefalograma com alterações típicas, juntamente da detecção de proteína 14- 3-3 no líquido cefalorraquidiano. Este relato descreve o caso de uma paciente de 74 anos, natural de Ubá (MG), admitida em um hospital da mesma cidade com quadro de demência de rápida progressão, com declínio cognitivo, ataxia cerebelar e mioclonias. No contexto clínico, aventou-se a possibilidade de doença de Creutzfeldt-Jakob e, então, foi iniciada investigação para tal, com base nos critérios diagnósticos. Também foram realizados exames para descartar a possibilidade de doenças com sintomas semelhantes. O caso foi diagnosticado como forma esporádica de doença de Creutzfeldt-Jakob. (AU)

Prion diseases are part of the rapidly progressive dementia syndromes with neurodegeneration. In humans, Creutzfeldt-Jakob disease is the most prevalent. Currently, its diagnosis may be based on a combination of clinical picture, magnetic resonance imaging, and electroencephalogram with typical changes, along with the detection of 14-3-3 protein in cerebrospinal fluid. This report describes the case of a 74-year-old woman from the city of Ubá, in the state of Minas Gerais, who was admitted to a hospital in the same city with a rapidly progressive dementia, cognitive decline, cerebellar ataxia and myoclonus. In the clinical context, the possibility of Creutzfeldt-Jakob disease was raised, and then investigation was started for this disease, based on the its diagnostic criteria. Tests have also been conducted to rule out the possibility of diseases with similar symptoms. The case was diagnosed as a sporadic form of Creutzfeldt-Jakob disease. (AU)

Identification of single nucleotide polymorphisms in the prion protein gene in Santa Ines and Dorset sheep / Identificação de polimorfismos de nucleotídeos únicos em ovinos Santa Inês e Dorset através do gene da proteína priônica

Scrapie is a transmissible spongiform encephalopathy (TSE) that affects sheep and goats and results from accumulation of the abnormal isoform of a prion protein in the central nervous system. Resistance or susceptibility to the disease is dependent on several factors, including the strain of infecting agent, the degree of exposure, and the presence of single nucleotide polymorphisms (SNPs) in the prion protein gene. The most important polymorphisms are present in codons 136, 154, and 171. SNPs have also been identified in other codons, such as 118, 127, 141, 142, and 143. The objective of this study was to investigate the genotypic profile of Santa Ines (n=94) and Dorset (n=69) sheep and identify polymorphisms in the prion protein gene using real-time PCR techniques and sequencing. We analyzed SNPs in 10 different codons (127, 136, 138, 140, 141, 142, 143, 154, 171, and 172) in Santa Ines sheep. Classification of the flock into risk groups associated with scrapie revealed that approximately 68% of the Santa Ines herd was considered at moderate risk (group 3), and the most frequent haplotype was ARQ/ARQ (47.8%). For Dorset sheep, 42% of the herd was considered at moderate risk (group 3), 40% at low risk (group 2), and 12% at very low risk (group 1). These findings improve our understanding of the genotype breed and further highlight the importance of genotyping and identification of polymorphisms in Brazilian herds to assess their effects on potential infections upon exposure to the sheep prion.(AU)

Scrapie é uma encefalopatia espongiforme transmissível que afeta ovinos e caprinos, resultante do acúmulo de uma isoforma anormal da proteína priônica no sistema nervoso central. A resistência ou susceptibilidade está relacionada a diversos fatores, tais como, a cepa do agente infectante, o grau de exposição e o polimorfismo de nucleotídeo único (SNPs) do gene da proteína priônica. Os principais polimorfismos estão presentes nos códons 136, 154 e 171. SNPs também são identificadas em outros códons, tais como, 118, 127, 141, 142, e 143. O objetivo do trabalho foi descrever o perfil genotípico de um rebanho da raça Santa Inês (n=94) e um rebanho da raça Dorset (n=89) para identificar potenciais polimorfismos através da técnica de PCR em tempo real e sequenciamento. Os achados no rebanho Santa Inês indicaram a presença de polimorfismos de nucleotídeos únicos em 10 códons diferentes (127, 136, 138, 140, 141, 142, 143, 154, 171 e 172). A classificação do rebanho, quanto aos grupos de risco associados ao scrapie, relevaram que aproximadamente 68% dos ovinos foram considerados do grupo de risco moderado (grupo 3), onde o haplótipo mais frequente foi ARQ/ARQ (47,8%). Para os ovinos da raça Dorset, 42% do rebanho foi considerado do grupo de risco moderado (grupo 3), 40% do grupo de risco baixo (grupo 2) e 12% do grupo de risco muito baixo. Os dados encontrados contribuem para o conhecimento do genótipo das raças, destacando a importância de trabalhos que relatam os polimorfismos genéticos para a identificação de rebanhos brasileiros, bem como o seu impacto a infecções com exposição ao príon ovino.(AU)

Papel da Stress Inducible Phosphoprotein One (STI1) e da proteína prion celular na angiogênese tumoral / The role of Stress Inducible Phosphoprotein One (STI1) and cellular prion protein in tumor angiogenesis

Proteínas multifuncionais podem ser consideradas uma vantagem evolutiva celular, ao passo que uma única proteína divide-se, simultaneamente, entre suas funções intracelulares e a sinalização extracelular para complementar mecanismos de responsividade tecidual. No papel de co-chaperona, a STI1 se complexa com HSP70/90, auxiliando no dobramento de proteínas. Reciprocamente, a secreção não convencional da STI1, em geral associada a vesículas extracelulares, permite a sua integração dinâmica com a demanda fisiológica pelas suas funções neurotróficas, ou até vasculogênicas, pelo recrutamento de precursores endoteliais para a regeneração do tecido cerebral durante a isquemia, sendo ambas mediadas via receptor PrPC (proteína príon). Em glioblastomas, a sinalização via STI1-PrPC também se mostrou determinante para a progressão tumoral. Dessa maneira, a STI1 apresenta um grande potencial de estudo como agente regulador dos processos angiogênicos tumorais em glioblastomas. As funções da STI1 foram avaliadas no modelo de glioma murino GL261 in vitro, ex vivo e in vivo em relação à invasão perivascular e à angiogênese peritumoral. A secreção de STI1 pelas células GL261 ocorre de maneira abundante, em sua maior parte na forma solúvel, não associada a vesículas extracelulares, por mecanismos ainda a serem desvendados, mas não controlados por hipóxia. O papel da STI1 no estímulo à giogênese sobre células endoteliais foi verificado em ensaios de formação de tubos (2D) e microvasos (3D) e em anéis aórticos. Porém, a contaminação por LPS nas preparações bacterianas de STI1 recombinante tornou inválidos nossos esforços e os ensaios foram interrompidos. Os ensaios in vivo foram realizados por meio da injeção intracerebral (ortotópica) de células GL261 em animais tipo-selvagem, haploinsuficientes (STI1+/-) e superexpressores de STI1 (TgA). A sobrevida dos animais STI1+/- e TgA foi estatisticamente superior a dos animais tipo-selvagem. Imunofluorescências com o marcador de células endoteliais CD31 indicaram uma maior vascularização intratumoral nos animais STI1+/-, enquanto animais TgA apresentaram marcação menor que animais tipo-selvagem. Tratamentos com anticorpos neutralizantes para STI1, da mesma forma que implantes ortotópicos de GL261 em animais deficientes para PrPC (PrP0/0), não produziram alterações no crescimento tumoral, que apresentou o mesmo padrão de invasão perivascular e de angiogênese peritumoral. A ausência de resposta também se reproduziu nos ensaios ex vivo dos tumores de GL261, a partir de co-culturas organotípicas com fatias de cérebros de animais STI1+/- e PrP0/0, em relação aos animais tipo-selvagem. Como abordagem final, as células GL261, previamente silenciadas para a expressão de STI1 ou HIF-1α, foram implantadas ortotopicamente no cérebro de animais tipo-selvagem. Apesar de terem sido selecionados os clones com menor nível de expressão de cada proteína, novamente não se verificaram alterações do crescimento tumoral e da angiogênese. Em conjunto, os resultados indicam que a proteína STI1 não possui efeito sobre o crescimento dos tumores de GL261, especialmente no que diz respeito à invasão perivascular e a vascularização intra/peritumoral. Linhagens celulares, porém, como a GL261, podem perder sua assinatura genética pela pressão seletiva a que são submetidas durante o seu período em cultura, o que compromete a sua representatividade in vivo em relação ao fenótipo tumoral infiltrativo próprio dos gliomas. A função extracelular de STI1 como modulador da angiogênese sobre células endoteliais ainda pode ser determinada com a utilização de proteína recombinante livre de LPS, ou sendo esta produzida por sistema heterólogo não-bacteriano

Multifunctional proteins might provide cells with an evolutionary advantage since a sole protein engages in concerted mechanisms of tissue responsiveness by simultaneously acting as a mediator of complementary intracellular and extracellular signaling pathways. Intracellularly, the cochaperone STI1 is an adaptor protein that aids in the folding of client proteins through the HSP90/70 complex. Conversely, the availability of cellular stocks of STI1 enables its dynamic mobilization by unconventional secretion, mostly associated with extracellular vesicles, in order to meet physiological demands extracellularly. Originally described as a neurotrophic growth factor, STI1 also holds vasculogenic functions, given the recruitment of endothelial progenitor cells positive for PrPC (prion protein) receptors from bone marrow to ischemic regions of mice brain, where it prompts vascular and functional recovery. In glioblastomas, signaling via STI1-PrPC axis was also showed to govern tumor progression. Thus, STI1 represents a potential target for modulation of tumor angiogenesis in glioblastomas (GBMs). We used the GL261 murine GBM cell line as an in vitro, ex vivo and in vivo model to evaluate the role of STI1 in certain aspects of perivascular invasion and peritumoral angiogenesis. GL261 cells secrete large amounts of freely soluble STI1, which is not influenced by hypoxia, and a small proportion associated to extracellular vesicles, in agreement with previously studied human GBM cell lines, although the mechanism for this protein segregation is still unknown. Recombinant STI1 apparently stimulated the endothelial sprouting as verified in 2D and 3D in vitro assays (matrigel tube formation and microcarrier-based microvessels formation), besides 3D ex vivo aortic ring assays. However, during these experiments, we found our recombinant protein with a high level of LPS contamination, due to failures in the established purification techniques used to process our bacterial preparations, which prevented the confirmation of STI1 as a pro-angiogenic factor. Orthotopic tumors were obtained by intracerebral injection of GL261 cells in wild-type, superexpressing STI1 (TgA) and haploinsufficient for STI1 (STI1+/-) C57BL/6J mice. Survival of STI1+/-and TgA mice was statistically higher compared to wild-type ones. Immunofluorescence staining of brain tissue sections with the endothelial cell marker CD31 revealed enhanced intratumoral vascularization, related to the wild-type controls, in STI1+/- mice, whereas TgAs presented as the least vascularized. Despite variations in intratumoral vascularization, tumor growth remained encapsulated within the brain parenchyma, without the typical difuse infiltration of GBMs associated with peritumoral angiogenesis. Even when tumors in wild-type mice were treated with neutralizing antibody for STI1, or GL261 cells were orthotopically implanted in PrP0/0 mice brains, tumor histology remained the same, reproducing results from organotypic co-cultures of wild-type, STI1+/-and PrP0/0 mice brain slices with GL261 tumors ex vivo. Unexpectedely, the highest knockdown of STI1 or HIF-1α obtained by lentiviral transduction in selected GL261 cell clones equally produced no effect over orthotopic tumor growth, perivascular invasion, and both intratumoral and peritumoral angiogenesis in wild-type mice brains. Taken together, our results show that STI1 might not affect GL261 tumor growth, and also raises conceptual caveats to the experimental utility of cancer cell lines as an in vivo representative of tumor phenotype. Future experiments might reveal the true angiogenic potential of LPS-free recombinant STI1 produced by a nonbacterial heterologous system

Papel do complexo PrPc-HOP e vesículas extracelulares em câncer colorretal / Role of PrPC-HOP complex and extracellular vesicles in colorectal cancer

O câncer colorretal (CCR) é o terceiro tipo de câncer mais comum no mundo. Apesar dos avanços nos tratamentos convencionais, aproximadamente dois terços dos pacientes com CCR são submetidos à cirurgia potencialmente curativa. Entretanto, grande parte desses pacientes evolui mal, apresentando recidivas e/ou metástases. A busca de novos alvos moleculares para a terapia do CCR revelou a proteína celular Prion (PrPC) como um possível candidato. Trabalhos recentes sugerem participação direta ou indireta de PrPC no crescimento de tumores, na formação de metástases, na composição de complexos multiproteicos e na indução de vias de sinalização envolvidas em diversos processos biológicos, como proliferação. Além disso, PrPC foi descrito como um importante modulador do crescimento de tumor colorretal. Resultados prévios mostraram que a interação da proteína PrPC com a proteína HSP70/HSP90 Organizing Protein (HOP) induz proliferação em glioblastomas. HOP é uma proteína predominantemente citoplasmática, podendo também ser secretada associada às vesículas extracelulares. Assim, o presente estudo objetivou avaliar o papel do complexo PrPC-HOP e das vesículas extracelulares no desenvolvimento e progressão dos tumores colorretais. Os nossos resultados mostram que HOP induziu migração e invasão em linhagens de CCR de maneira dependente de PrPC, uma vez que o uso do peptídeo sem atividade que compete pelo sítio ligação de HOP a PrPC inibiu estes processos. Além disso, nossos dados apontaram que o aumento de migração e invasão das células de CCR induzida pela interação PrPC-HOP é mediada pela ativação da via ERK1/2. Os achados in vitro estimularam a avaliação do perfil de expressão de PrPC e HOP por imuno-histoquímica em tecidos de pacientes com diferentes tipos de tumores colorretais. Nossos resultados sugeriram que essas proteínas são importantes no início do desenvolvimento tumoral e na transição de adenomas para adenocarcinomas, não havendo correlação entre a presença de HOP e/ou PrPC com metástase, linfonodos acometidos, estadiamento, sobrevida ou região tumoral versus tecido normal. Em relação ao papel das vesículas extracelulares na progressão dos tumores colorretais, nossos resultados mostraram que linhagens celulares que apresentam padrões parecidos de agressividade tumoral podem ter perfis de secreção de proteínas e vesículas extracelulares bastante diferentes, induzindo, portanto, processos biológicos com intensidades distintas. O meio condicionado e as vesículas extracelulares da linhagem WiDr apresentaram maior potencial de indução de migração quando comparado com a linhagem HCT8. Além disso, a modulação negativa da proteína VPS4, uma das responsáveis pela formação dos corpos multivesiculares, mostrou-se uma abordagem interessante no estudo da secreção de vesículas por células de CCR, uma vez que o dominante negativo de VPS4 promoveu diminuição do cargo proteico e da secreção de vesículas extracelulares, redução da proliferação celular e do efeito indutor do processo de migração na linhagem WiDr. Assim, em conjunto, o presente trabalho indicou que o complexo PrPC-HOP pode ser um bom alvo terapêutico nos processos de migração e invasão em CCR. Ainda, essas proteínas se mostraram importantes nos estágios iniciais da formação dos tumores. A modulação da secreção de vesículas extracelulares pode contribuir para retardar a progressão dos tumores colorretais

Colorectal cancer (CRC) is the third most common type of cancer in the world. Despite improvements in conventional treatments, approximately two-thirds of CRC patients undergo potentially curative surgery. However, most of these patients evolve poorly, showing recurrence and/or metastasis. Search of new molecular targets for CRC therapy revealed the cellular protein Prion (PrPC) as a putative candidate. Recent studies have shown that PrPC exhibit direct or indirect participation in tumor growth, formation of metastasis, composition of multiprotein complexes and induction of signaling pathways involved in many biological processes such as proliferation. Moreover, PrPC has been described as an important modulator of colorectal tumor growth. Previous findings showed that the interaction between PrPC and its ligand HSP70/90 heat shock organizing protein (HOP) induces gliobastoma proliferation. It is well known that HOP localizes mainly in the cytoplasm but HOP is also secreted associated with extracellular vesicles. In this way, the present study sought to evaluate the role of PrPC-HOP complex and extracellular vesicles in the development and progression of CRC. We demonstrate that HOP induces the migration and invasion of CRC cell lines in a PrPC-dependent manner because the use of HOP peptide, which is able to bind to PrPC, blocking PrPC-HOP complex formation, inhibited the migration and invasion processes. In addition, our data showed that the enhancement of migration and invasion induced by PrPC-HOP interaction is mediated by ERK1/2 pathway activation. These in vitro results lead us to evaluate the PrPC and HOP expression by immunohistochemistry in tissues from patients with different tumor types. Our data showed that these proteins could be important for the initial steps of tumor development, represented by the transition from adenoma to adenocarcinoma. No correlation was found among HOP and/or PrPC expression and metastasis, lymph node involvement, staging, survival or tumor area versus normal tissue. Regarding the role of extracellular vesicles in the progression of colorectal tumors, our results showed that cell lines exhibiting similar aggressive tumor behavior can have a different protein secretion pattern and a distinct profile of extracellular vesicles release, which could induce biological process with different intensities. The conditioned medium and the extracellular vesicles derived from WiDr cell line showed a higher potential to induce migration than HCT8 cell line. Moreover, the negative modulation of VPS4, one of the proteins responsible for multivesicular body formation, showed to be an interesting approach in the study of extracellular vesicles secretion secreted by CRC cells; the negative dominant of VPS4 promoted in the WiDr cell line a reduction in the protein cargo and secretion of the extracellular vesicles, a decrease of cell proliferation and induction of migration process. Therefore, taken together, our data highlights that PrPC-HOP complex can be considered a new therapeutic target in migration and invasion processes of CRC. Moreover, these proteins appeared to be important at onset of tumor formation. The modulation of extracellular vesicles secretion may contribute for delaying the progression of colorectal tumors

Prion protein gene polymorphisms and accumulation of pathogenic prion protein (PrPSc) in a herd with previously confirmed scrapie cases / Polimorfismos do gene da proteína priônica celular (PRNP) e distribuição da proteína priônica alterada (PrPSc) em rebanho com surto anterior de scrapie

Scrapie in sheep is associated with at least three polymorphisms in the prion protein gene (PRNP) on codons 136, 154, and 171. Countries where scrapie is endemic have been using breeding programs based on selection for the most resistant alleles. There are some PRNP genotyping data on sheep in Brazil, and scrapie has sporadically been observed since 1978. Paraná is the Brazilian state where most of the cases of scrapie have been diagnosed. A flock that had three clinical scrapie cases in 2003 and 2004 was genotyped (128 sheep: 53 pure Hampshire Down and 75 crossbred) and slaughtered (111 sheep: 47 pure Hampshire Down and 64 crossbred) in 2006. Samples of lymphoid and central nervous tissues were examined by immunohistochemistry (IHC) for altered prion protein (PrPSc). Six genotypes were detected in the 128 genotyped animals: ARR/ARQ was the most frequent (45.3%), followed by ARQ/ARQ (28.1%), ARR/ARR (14.1%), and ARQ/VRQ (8.6%). ARR/VRQ and ARQ/AHQ showed less than 2.5% genotype frequency. IHC identified 16 positive sheep. Palatine tonsil tissue had the highest percentage of reactive samples: 81.25% of the total positive samples. Of these 16 positive animals, nine (56.25%) had genotype ARR/ARQ, five (31.25%) had genotype ARQ/ARQ, and the remaining two (12.5%) had genotype ARQ/VRQ. All the positive animals were clinically healthy, and therefore represented 14.14% of pre-clinical cases of scrapie in this flock.

Scrapie nos ovinos está associada a pelo menos três polimorfismos do gene da proteína priônica celular (PRNP) nos códons 136, 154 e 171. Países onde o scrapie é endêmico têm utilizado programas de melhoramento, com a seleção para os alelos mais resistentes. Há alguns dados disponíveis de genotipagem do PRNP em ovinos no Brasil, e o scrapie tem sido observado esporadicamente desde 1978. O Paraná é o Estado brasileiro onde a maioria dos casos de scrapie foi diagnosticada. Um rebanho, que teve três casos clínicos de scrapie em 2003 e 2004, foi genotipado (128 ovinos - 53 Hampshire Down e 75 mestiços) e abatido (111 ovinos - 47 Hampshire Down e 64 mestiços) em 2006. Amostras de tecido linfóide e sistema nervoso central foram examinadas por imunohistoquímica (IHQ) para presença de proteína priônica alterada (PrPSc). Seis genótipos foram encontrados nos 128 animais genotipados: ARR/ARQ foi o mais frequente (45,3%), seguido por ARQ/ARQ (28,1%), ARR/ARR (14,1%) e ARQ/VRQ (8,6%). ARR/VRQ e ARQ/AHQ apresentaram menos de 2,5% de freqüência do genótipo. Na IHC, 16 animais com exame positivo para a presença da proteína priônica celular alterada (PrPSc) foram detectados. As tonsilas foram o tecido com a mais alta porcentagem de amostras reativas: 81,25% do total das amostras positivas. Considerando os 16 animais positivos, nove (56,25%) tinham o genótipo ARR/ARQ, seguido pelo genótipo ARQ/ARQ com 31,25% (n = 5) e ARQ/VRQ com 12,5% (n = 2). Todos os animais positivos estavam clinicamente saudáveis, representando, portanto, 14,14% de casos pré-clínicos de scrapie neste rebanho.

Polimorfismo do gene da proteína prion celular (PrPC) e imunohistoquímica de tecido linfóide em ovinos / Polymorphism of cellular prion protein (PrPC) and immunohistochemistry of lymphoid tissue of sheep

O objetivo deste trabalho foi avaliar o polimorfismo do gene da proteína prion celular (PRPN) de ovinos introduzidos numa propriedade onde ocorreu um surto de scrapie, e relacionar com a suscetibilidade à doença por meio da análise da presença da proteína prion celular alterada (PrPSc), utilizando imunohistoquímica (IHQ) de tecido linfóide associado à mucosa reto-anal. Foram avaliados 42 ovinos, mestiços Texel. Eram fêmeas entre um e oito anos de idade, sendo que sete (16,67%) ovelhas foram introduzidas adultas na propriedade em 2006. As demais, 83,33%, eram nascidas na fazenda. A genotipagem do PRPN foi feita pela análise do polimorfismo de comprimento de fragmento de restrição - RFLP ("Restriction Fragment Lenght Polimorphism"). O genótipo ARQ/ARQ foi o mais freqüente, encontrado em 73,81% dos animais, seguido do genótipo ARR/ARQ, com 16,67% e do ARQ/VRQ, com 9,52%. Os alelos ARH e AHQ não foram encontrados nestes animais. O resultado da IHQ foi negativo em todas as amostras. Não foi possível, portanto, estabelecer uma relação entre genótipo e maior susceptibilidade ao scrapie, devido à ausência de PrPSc na amostras examinadas. No Brasil, há poucos dados de genotipagem do gene da proteína prion celular (PRNP) em ovinos e, até o momento, nenhum tipo de controle baseado em cruzamentos direcionados foi implementado.

The aim of this work was to study the polymorphism of the prion protein gene (PRNP) of a sheep flock raised in a farm where a scrapie outbreak had occurred, and to relate to disease susceptibility of possible animals infected with altered prion protein (PrPSc), by immunohistochemical analysis of recto-anal mucosa-associated lymphoid tissue (RAMALT). Forty two sheep, crossbred with Texel, Ile de France, Dorper and Suffolk were used. Females were between one and eight years old, and seven (16.67%) were adult ewes when they entered the flock in 2006. The rest, 83.33% were born in the farm. The PRNP genotyping was performed by RFLP ("restriction fragment length polymorphism") analysis. The most frequent genotype was ARQ/ARQ, found in 73.81% of the animals, followed by ARR/ARQ, with 16.67% and ARQ/VRQ, with 9.52%. The ARH and AHQ alleles were not found. All RAMALT samples were negative in immunohistochemical analysis. It was not possible to establish a relation between PRNP polymorphisms and susceptibility to scrapie, due to the lack of positive samples to PrPSc. In Brazil, there is little available PRNP genotyping data of sheep and, so far, no type of controlled breeding scheme for scrapie has been implemented.

Establishment of hamster- and human-PRNP transgenic mice / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To create transgenic mice expressing hamster- and human-PRNP as a model for understanding the physiological function and pathology of prion protein (PrP), as well as the mechanism of cross-species transmission of transmissible spongiform encephalopathies (TSEs).</p><p><b>METHODS</b>Hamster and human-PRNP transgenic mice were established by conventional methods. The copy number of integrated PRNP in various mouse lines was mapped by real-time PCR. PRNP mRNA and protein levels were determined by semi-quantitative RT-PCR, real-time RT-PCR, and western blot analysis. Histological analyses of transgenic mice were performed by hematoxylin and eosin (H & E) staining and immunohistochemical (IHC) methods.</p><p><b>RESULTS</b>Integrated PRNP copy number in various mouse lines was 53 (Tg-haPrP1), 18 (Tg-huPrP1), 3 (Tg-huPrP2), and 16 (Tg-huPrP5), respectively. Exogenous PrPs were expressed at both the transcriptional and translational level. Histological assays did not detect any abnormalities in brain or other organs.</p><p><b>CONCLUSION</b>We have established one hamster-PRNP transgenic mouse line and three human-PRNP transgenic mouse lines. These four transgenic mouse lines provide ideal models for additional research.</p>

The first Chinese case of Creutzfeldt-Jakob disease with mutation of E200K in PRNP / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate epidemiological, clinical and genetic features of the first Chinese case of Creutzfeldt-Jakob disease (CJD ) with mutation of E200K in PRNP.</p><p><b>METHODS</b>The general epidemiological and clinical data were collected; CSF 14-3-3 protein was analyzed by Western blot; The PRNP was amplified by PCR and analyzed.</p><p><b>RESULTS</b>A missense mutation in codon 200 (E200K) of the PRNP was identified in this patient; CSF 14-3-3 protein was positive; sleep disturbance was the initial sign and the other symptoms gradually appeared, including memory loss, dizziness and ataxia.</p><p><b>CONCLUSION</b>The CJD patient who was first reported in China has a missense mutation in codon 200 (E200K) of the PRNP, and the codon 129 is a methionine homozygous genotype.</p>

Enfermedad de creutzfeldt-jakob esporádico: síntomas neuropsiquíatricos seguidos de daño cognitivo progresivo: reporte de caso / Sporadic creutzfeldt-jakob disease: neuropsychiatric symptoms followed by a progressive congnitive decline: a case report

La enfermedad de Creutzfeldt-Jakob (ECJ) es la enfermedad por priones más común y es la única entre los desórdenes humanos que se puede presentar en forma familiar, esporádica y transmitida. La hipótesis prevalente, basada en numerosos estudios en ratones transgénicos, sugiere que la ECJ y otras enfermedades por priones se inician y propagan por conversión de una proteína prionica normal (PrP) en una isoforma conformacional anormal (PrPreS). Esta última se acumula en el cerebro y es una de las características bioquímicas importantes en ECJ. Se reporta el caso de una mujer de 37 años de edad, quien murió en un hospital de tercer nivel por ECJ esporádico comprobado por autopsia. Este caso muestra la importancia de un examen post-mortem para confirmar el diagnóstico. A la luz de la observación clínica, discutimos el caso, el cual debe ser considerado en pacientes queevolucionan a una demencia rápidamente progresiva.

Creutzfeldt-Jakob disease (CJD) is the most common human prion disease and is unique among all known human disorders in being comprised of familial, sporadic, and transmitted forms. Theprevailing hypothesis, based largely on studies in transgenic animals, suggests that CJD and other prion diseases are initiated and propagated by conversion of normal host-encoded prion protein (PrP) into a conformationally abnormal isoform (PrPreS). The latter has been shown to accumulate in the brain and is the biochemical hallmark of CJD. We report the case of a 37-year-old woman who died in third level hospital of sporadic CJD proven by autopsy. This case illustrates the great importance of apost mortem exam in such context. In light of this clinical observation, we discuss this rare diagnosis which should be considered in patients when confronted with a rapidly progressive dementia.

The construction and identification of the PRNP vectors with ubiquitin or the lysosome-targeting signal / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluate PrP expression characteristic of PRNP nucleic acid vaccine vector with ubiquitin or the lysosome-targeting signal.</p><p><b>METHODS</b>The gene of ubiquitin and lysosome-targeting signal were ligated to PRNP and pcDNA3.1 vector that is, pcDNA3.1-UPrP and pcDNA3.1-PrPL were constructed. The expression characteristics of PrP with two signals were evaluated by Western Blot and the localization was observed by indirect immune fluorescence.</p><p><b>RESULTS</b>The protein expressed by pcDNA3.1-UPrP and pcDNA3.1-PrPL with ubiquitin and lysosome-targeting signal can be recognized by prion-specific antibody. The protein has three glycosylation molecules form as native PrP.PrP with ubiquitin was degraded gradually with time extension,whereas quantity of PrP with lysosome signal reduced in 48 h after transfection. The protein with two location signals can direct fusion proteins to cytoplasm.</p><p><b>CONCLUSION</b>The PRNP vectors with ubiquitin or the lysosome-targeting signal were constructed and expressed in eukaryocyte successfully. There will be one of good foundation on PRNP nucleic acid vaccine.</p>

Study on the characteristic of Surveillance Creutzfeldt-Jakob disease patients from January to August in 2006 in China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To describe the incidence condition and characteristic of Creutzfeldt-Jakob disease (CJD) in China.</p><p><b>METHODS</b>The clinical and epidemical information of patients from China CJD surveillance network was analyzed. Blood and cerebrospinal fluid (CSF) specimens from these patients were differently collected to be used to detect the 14-3-3 protein in the CSF and to analyze the PRNP gene.</p><p><b>RESULTS</b>Ten possible and 8 probable clinically diagnosed CJD patients were found. These patients had sporadic CJD. There were no geographic clustering and occupational risk with these patients. The mean age at onset of disease was approximately 60 years. The male to female ratio was approximately 1:1; rapidly progressive dementia was the main early symptom, which was present in 44% of patients.</p><p><b>CONCLUSION</b>The geographic distribution, occupation, the ratio of male to female and the mean age of onset were consistent with the characteristics of sporadic CJD.</p>

Preliminary analyses for influence of mutant PrPs with different number of octapeptide / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>The present study was conducted to understand the effects of PrP in different octapeptide repeats on proliferation of HeLa cells.</p><p><b>METHODS AND RESULTS</b>Mutant PrPs with octapeptide repeat insertion were transiently expressed in HeLa cells and their results of MTT assay showed stronger cytotoxic effect on the proliferation of cells than wild-type PrP. Annexin V/PI assay also demonstrated that the expression of mutant PrPs was much easier to induce apoptosis than wild-type in HeLa cells. The percentage of both early and late stage apoptosis in mutant groups were significantly higher than that of wild type.</p><p><b>CONCLUSION</b>These data suggest that the expression of mutant PrPs associated with familial CJD is much easier to induce apoptosis in cultured cells than expression of wild type PrP.</p>