30 research outputs found
Inhibition of Plasmodium liver infection by Ivermectin
Copyright © 2017 Mendes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.Avermectins are powerful endectocides with an established potential to reduce the incidence of vector-borne diseases. Here, we show that several avermectins inhibit the hepatic stage of Plasmodium infection in vitro Notably, ivermectin potently inhibits liver infection in vivo by impairing parasite development inside hepatocytes. This impairment has a clear impact on the ensuing blood stage parasitemia, reducing disease severity and enhancing host survival. Ivermectin has been proposed as a tool to control malaria transmission because of its effects on the mosquito vector. Our study extends the effect of ivermectin to the early stages of mammalian host infection and supports the inclusion of this multipurpose drug in malaria control strategies.A.M.M., P.M., and M.P. acknowledge the Fundação para a Ciência e Tecnologia, Portugal, for grants SFRH/BPD/80693/2011, SFRH/BD/71098/2010, and Investigador FCT (2013), respectively. This work was supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) grant PTDC/SAU-MIC/117060/2010.info:eu-repo/semantics/publishedVersio
A systematic review of peptide-based serological tests for the diagnosis of leishmaniasis
Serological methods should meet the needs of leishmaniasis diagnosis due to their high sensitivity and specificity, economical and adaptable rapid diagnostic test format, and ease of use. Currently, the performances of serological diagnostic tests, despite improvements with recombinant proteins, vary greatly depending on the clinical form of leishmaniasis and the endemic area. Peptide-based serological tests are promising as they could compensate for antigenic variability and improve performance, independently of Leishmania species and subspecies circulating in the endemic areas. The objective of this systematic review was to inventory all studies published from 2002 to 2022 that evaluate synthetic peptides for serological diagnosis of human leishmaniases and also to highlight the performance (e.g., sensitivity and specificity) of each peptide reported in these studies. All clinical forms of leishmaniasis, visceral and tegumentary, and all Leishmania species responsible for these diseases were considered. Following PRISMA statement recommendations, 1,405 studies were identified but only 22 articles met the selection criteria and were included in this systematic review. These original research articles described 77 different peptides, of which several have promising performance for visceral or tegumentary leishmaniasis diagnosis. This review highlights the importance of and growing interest in synthetic peptides used for serological diagnosis of leishmaniases, and their performances compared to some widely used tests with recombinant proteins
Propriedades métricas do Oxford Knee Score em pessoas com osteoartrite após artroplastia do joelho: Revisão Sistemática da Literatura
Objective: To evaluate the metric properties of the Oxford Knee Score applied in people with osteoarthritis after knee arthroplasty.
Method: Systematic review of the literature. The EBSCOhost platform was used to access the MEDLINE and LILACS database and the SCiELO platform. The descriptors were validated on the DeCS and MESH platforms, except for "Oxford Knee Score" and "responsiveness". We selected articles published in the last five years, available in Portuguese, English and Spanish.
Results: There were eight articles that revealed that OKS is a valid, reliable and reproducible instrument. Responsiveness was the least studied metric property.
Conclusion: The Oxford Knee Scale is adequate to evaluate the functionality and the impact of pain in people with Osteoarthritis after knee arthroplasty.Objetivo: Evaluar las propiedades métricas de la Oxford Knee Score aplicada en personas con osteoartritis después de la artroplastia de la rodilla.
Método: Revisión sistemática de la literatura. Se recurrió a la plataforma EBSCOhost que permitió acceder a la base de datos MEDLINE y LILACS y plataforma SCiELO. Los descriptores fueron validados en las plataformas DeCS y MESH, con excepción de "Oxford Knee Score" y "responsividad". Se seleccionaron los artículos publicados en los últimos cinco años, disponibles en Portugués, Inglés y Español.
Resultados: Se han obtenido ocho artículos que revelaron que Oxford Knee Score es un instrumento válido, fiable y reproductivo. La responsividad fue la propiedad métrica menos estudiada.
Conclusión: El Oxford Knee Score es adecuado para evaluar la funcionalidad y el impacto del dolor en personas con Osteoartritis después de la artroplastia de la rodilla.Objetivo: Avaliar as propriedades métricas da Oxford Knee Score aplicada em pessoas com osteoartrite após artroplastia do joelho.
Método: Revisão sistemática da literatura. Recorreu-se à plataforma EBSCOhost que permitiu aceder à base de dados MEDLINE e LILACS e plataforma SCiELO. Os descritores foram validados nas plataformas DeCS e MESH, à exceção de “Oxford Knee Score” e “responsividade”. Foram selecionados artigos publicados nos últimos cinco anos, disponíveis em português, inglês e espanhol.
Resultados: Obtiveram-se oito artigos que revelaram que o Oxford Knee Score é um instrumento válido, fiável e reprodutivo. A responsividade foi a propriedade métrica menos estuda.
Conclusão: O Oxford Knee Score é adequado para avaliar a funcionalidade e o impato da dor em pessoas com Osteoartrite após atroplastia do joelho
Evidence for the first multi-species shark nursery area in Atlantic Africa (Boa Vista Island, Cabo Verde)
Funding: This research is part of project NGANDU (The Importance of Shark Populations and Sustainable Ocean Use for Human Well-being in Cabo Verde and São Tomé and Príncipe, West Africa) funded by the Portuguese Foundation for Science and Technology (FCT) and the Aga Khan Development Network (AKDN) under grant agreement FCT AGA-KHAN/541746579/2019. All authors acknowledge funding from FCT under the strategic project UIDB/04292/2020 granted to MARE and project LA/P/0069/2020 granted to the Associate Laboratory ARNET. TM acknowledges funding from the strategic project UIDB/00006/2020 granted to CEAUL. CF acknowledges funding from FCT research contract 2020.03704.CEECIND and FCT grant agreement PTDC/CTA-AMB/30226/2017. VP acknowledges funding from FCT PhD grant 2020.05435.BD. CS acknowledges funding from FCT PhD grant SFRH/BD/117890/2016, FCT research grants under PTDC/CTA-AMB/30226/2017 through FCiencias.ID and AGA-KHAN/541746579/2019 through Nova School of Business and Economics. EN acknowledges funding from FCT PhD grant SFRH/BD/135438/2017. JRP acknowledges funding from FCT research contract 2021.01030.CEECIND. JV acknowledges funding from the Intergovernmental Panel on Climate Change (PhD grant, IPCC Scholarship Programme–Prince Albert II of Monaco Foundation) and the Camões–Instituto da Cooperação e da Língua, I.P. (Scholarship Programme).This study describes the first potential multi-species shark nursery area in Atlantic Africa (Sal Rei Bay – SRB, Boa Vista Island, Cabo Verde). From August 2016 to September 2019, 6162 neonates and juveniles of 5 different shark species were observed in SRB using beach gillnet-based bycatch surveys, namely milk (Rhizoprionodon acutus; n= 4908), scalloped hammerhead (Sphyrna lewini; n= 1035), blacktip (Carcharhinus limbatus; n=115), Atlantic weasel (Paragaleus pectoralis; n= 93) and nurse (Ginglymostoma cirratum; n= 12) sharks. Except for nurse sharks, significant seasonal variations in shark relative abundance were observed, with higher levels being recorded during summer and autumn. These findings, together with local knowledge (interviews to fishermen), denote the consistent use of SRB by juvenile sharks and its preference relative to other areas in the region. Ensuring the protection and conservation of SRB nursery area is especially relevant as, according to IUCN, all identified shark species are threatened with extinction over the near-future – in particular, scalloped hammerheads (critically endangered) and Atlantic weasel sharks (endangered). The effective protection of SRB will not only support the conservation of shark populations, but also of other charismatic fauna (e.g., loggerhead turtles) and broader benthic and pelagic ecosystems.Publisher PDFPeer reviewe
Developpement d'un vaccin peptidique multi-epitope contre les leishmanioses humaines
Leishmaniasis is a vector-borne neglected tropical disease endemic to 98 countries worldwide. Twenty Leishmania species are capable of establishing intracellular infection within human macrophages, causing different clinical presentations. Vaccine development against leishmaniases is supported by evidence of natural immunity against infection, mediated by a dominant cellular Th1 response and production of IFN-γ, IL-2 and TNF-α by polyfunctional TCD4+ and TCD8+ cells, ultimately leading to macrophage activation and parasite killing.Excreted-secreted proteins are important virulence factors present throughout Leishmania life stages and are able to induce durable protection in dogs, a good model for human infection. We aim to develop a second generation vaccine from the Leishmania secretome, with the potential for large scale dissemination in a cost-effective, reproducible approach.The secretome of six main pathogenic species (plus L. tarentolae) was analysed by Mass-Spectrometry and conserved candidate antigens were searched in the complete dataset. A total of 52 vaccine antigen candidates were selected, including 28 previously described vaccine candidates, and an additional 24 new candidates discovered through a reverse vaccinology approach.In silico HLA-I and –II epitope binding prediction analysis was performed on all selected vaccine antigens, with world coverage regarding HLA restriction. To select the best epitopes, an automated R script was developed in-house, according to strict rational criteria. From thousands of potential epitopes, the automated script, in combination with optimal IC50, homology to host and solubility properties, allowed us to select 50 class I and 24 class II epitopes, synthesized as individual peptides. In vitro toxicity assays showed these selected peptides are non-toxic to cells.The peptides’ immunogenicity was evaluated using immunoscreening assays with immune cells from human donors, allowing for the validation of in silico epitope predictions and selection, and the assessment of the peptide’s immunogenicity and prophylactic potential. Healed individuals, which had active infection and received treatment, possess Leishmania-specific memory responses and are resistant to reinfection, being considered the gold standard of protective immunity. On the other hand, the naive population is extremely important to include in the experimental validation step since it is the target population to vaccinate with a prophylactic vaccine. Importantly, a minimum specific T-cell precursor frequency is needed to induce long-lasting memory protective responses. Furthermore, there is also a positive correlation between immunodominant epitopes and a high frequency of specific T-cell precursors. Peptides able to induce Th1 and/or cytotoxic immune responses in both background are promising candidates for a vaccine formulation. Altogether,experimental validation exclusively in human samples will provide us a very strong base for a vaccine formulation and allow to accelerate translation to the field.Results show Leishmania-specific peptides successfully induce IFN-γ production by total PBMC from healed donors, and by specific T cells amplified from the naïve repertoire. Preliminary evidence exists for peptides which are immunogenic in both immune backgrounds (eight HLA-class I 9-mer peptides and five class II 15-mer peptides) which are, for now, the most promising candidates to advance for the multi-epitope peptide design.Through the combination of proteomic analysis and in silico tools, promising peptide candidates were swiftly identified and the secretome was further established as an optimal starting point for vaccine development. The proposed vaccine preclinical development pipeline delivered a rapid selection of immunogenic peptides, providing a powerful approach to fast-track the deployment of an effective pan-specific vaccine against Leishmaniases.La leishmaniose est une maladie tropicale négligée à transmission vectorielle qui est endémique dans 98 pays dont les plus pauvres. Vingt espèces de Leishmania sont capables d’établir une infection intracellulaire au sein des macrophages humains, provoquant différentes manifestations cliniques. Le développement d'un vaccin contre les leishmanioses est étayé par des preuves d'immunité naturelle contre l'infection, induite par une réponse à médiation cellulaire de type Th1 dominante associée à la production d'IFN-γ, d'IL-2 et de TNF-α par des cellules T polyfonctionnelles TCD4+ et TCD8+, conduisant à l'activation classique des macrophages entrainant la destruction des parasites. Induire une protection robuste et durable et déterminer les épitopes immunodominants responsables de la protection naturelle représente un véritable défi.Les protéines sécrétées sont des facteurs de virulence jouant un rôle important dans le cycle de vie des leishmanies et sont capables d’induire une protection durable chez le chien, un bon modèle pour l’infection humaine. Notre objectif est de développer, à partir du sécrétome de Leishmania, un vaccin de seconde génération reproductible et facile à produire à bas prix dans les zones d’endémie, avec des rendements de production rendant possible son utilisation à grande échelle.Les sécrétomes des six espèces les plus pathogènes de leishmanie (plus L. tarentolae) ont été analysés et comparées par spectrométrie de masse. Les antigènes candidats ont été recherchés dans l'ensemble des données disponibles (analyses protéomiques, littérature…). 52 antigènes candidats vaccin ont ainsi été sélectionnés, dont 28 avaient déjà été décrits et 24 sont nouveaux et découverts grâce à une approche de vaccinologie réverse.Une analyse de la prédiction de liaison des épitopes in silico HLA-I et –II a été réalisée sur tous les antigènes candidats vaccin, prenant ainsi en compte le polymorphisme HLA de la population mondiale. Pour sélectionner les meilleurs épitopes parmi des milliers d’épitopes potentiels, un script R automatisé a été développé en interne, selon des critères rationnels stricts. Ainsi, 50 épitopes de classe I et 24 épitopes de classe II ont été sélectionnés et synthétisés sous forme de peptides individuels. Des essais de toxicité in vitro ont montré l’absence de toxicité cellulaire de ces peptides.Les individus guéris par chimiothérapie généralement développent des réponses immunitaires protectrices à Leishmania. Des tests de stimulation des PBMC ont donc été réalisés avec des échantillons biologiques provenant de donneurs guéris de Tunisie et la production d'IFN-γ a été évaluée par ELISpot. De plus, il était important d'inclure dans l'étape de validation expérimentale des peptides des échantillons provenant d’individus naïfs, population cible à vacciner avec un vaccin prophylactique. Les résultats montrent que des peptides spécifiques de Leishmania induisent avec succès la production d'IFN-γ par les PBMC totaux provenant de donneurs guéris et par les lymphocytes T spécifiques amplifiés à partir du répertoire naïf.Globalement, la validation expérimentale des peptides réalisée exclusivement sur des échantillons humains nous fournira une base préclinique très solide pour développer un vaccin efficace capable de protéger les populations touchées par ces maladies. Elle constituera un moyen sûr et rentable de mieux sélectionner les candidats retenus pour le vaccin et d'éliminer ceux qui présentent un risque d'échec élevé au tout début du processus de développement du vaccin.Grâce à la combinaison de l'analyse protéomique et d'outils in silico, des candidats peptidiques prometteurs ont été rapidement identifiés pour le développement d'un vaccin. Le « pipeline » de développement préclinique du vaccin proposé fournit une sélection rapide de peptides immunogènes, offrant une approche puissante pour accélérer le déploiement d'un vaccin pan-spécifique efficace contre les leishmanioses
Developpement d'un vaccin peptidique multi-epitope contre les leishmanioses humaines
La leishmaniose est une maladie tropicale négligée à transmission vectorielle qui est endémique dans 98 pays dont les plus pauvres. Vingt espèces de Leishmania sont capables d’établir une infection intracellulaire au sein des macrophages humains, provoquant différentes manifestations cliniques. Le développement d'un vaccin contre les leishmanioses est étayé par des preuves d'immunité naturelle contre l'infection, induite par une réponse à médiation cellulaire de type Th1 dominante associée à la production d'IFN-γ, d'IL-2 et de TNF-α par des cellules T polyfonctionnelles TCD4+ et TCD8+, conduisant à l'activation classique des macrophages entrainant la destruction des parasites. Induire une protection robuste et durable et déterminer les épitopes immunodominants responsables de la protection naturelle représente un véritable défi.Les protéines sécrétées sont des facteurs de virulence jouant un rôle important dans le cycle de vie des leishmanies et sont capables d’induire une protection durable chez le chien, un bon modèle pour l’infection humaine. Notre objectif est de développer, à partir du sécrétome de Leishmania, un vaccin de seconde génération reproductible et facile à produire à bas prix dans les zones d’endémie, avec des rendements de production rendant possible son utilisation à grande échelle.Les sécrétomes des six espèces les plus pathogènes de leishmanie (plus L. tarentolae) ont été analysés et comparées par spectrométrie de masse. Les antigènes candidats ont été recherchés dans l'ensemble des données disponibles (analyses protéomiques, littérature…). 52 antigènes candidats vaccin ont ainsi été sélectionnés, dont 28 avaient déjà été décrits et 24 sont nouveaux et découverts grâce à une approche de vaccinologie réverse.Une analyse de la prédiction de liaison des épitopes in silico HLA-I et –II a été réalisée sur tous les antigènes candidats vaccin, prenant ainsi en compte le polymorphisme HLA de la population mondiale. Pour sélectionner les meilleurs épitopes parmi des milliers d’épitopes potentiels, un script R automatisé a été développé en interne, selon des critères rationnels stricts. Ainsi, 50 épitopes de classe I et 24 épitopes de classe II ont été sélectionnés et synthétisés sous forme de peptides individuels. Des essais de toxicité in vitro ont montré l’absence de toxicité cellulaire de ces peptides.Les individus guéris par chimiothérapie généralement développent des réponses immunitaires protectrices à Leishmania. Des tests de stimulation des PBMC ont donc été réalisés avec des échantillons biologiques provenant de donneurs guéris de Tunisie et la production d'IFN-γ a été évaluée par ELISpot. De plus, il était important d'inclure dans l'étape de validation expérimentale des peptides des échantillons provenant d’individus naïfs, population cible à vacciner avec un vaccin prophylactique. Les résultats montrent que des peptides spécifiques de Leishmania induisent avec succès la production d'IFN-γ par les PBMC totaux provenant de donneurs guéris et par les lymphocytes T spécifiques amplifiés à partir du répertoire naïf.Globalement, la validation expérimentale des peptides réalisée exclusivement sur des échantillons humains nous fournira une base préclinique très solide pour développer un vaccin efficace capable de protéger les populations touchées par ces maladies. Elle constituera un moyen sûr et rentable de mieux sélectionner les candidats retenus pour le vaccin et d'éliminer ceux qui présentent un risque d'échec élevé au tout début du processus de développement du vaccin.Grâce à la combinaison de l'analyse protéomique et d'outils in silico, des candidats peptidiques prometteurs ont été rapidement identifiés pour le développement d'un vaccin. Le « pipeline » de développement préclinique du vaccin proposé fournit une sélection rapide de peptides immunogènes, offrant une approche puissante pour accélérer le déploiement d'un vaccin pan-spécifique efficace contre les leishmanioses.Leishmaniasis is a vector-borne neglected tropical disease endemic to 98 countries worldwide. Twenty Leishmania species are capable of establishing intracellular infection within human macrophages, causing different clinical presentations. Vaccine development against leishmaniases is supported by evidence of natural immunity against infection, mediated by a dominant cellular Th1 response and production of IFN-γ, IL-2 and TNF-α by polyfunctional TCD4+ and TCD8+ cells, ultimately leading to macrophage activation and parasite killing.Excreted-secreted proteins are important virulence factors present throughout Leishmania life stages and are able to induce durable protection in dogs, a good model for human infection. We aim to develop a second generation vaccine from the Leishmania secretome, with the potential for large scale dissemination in a cost-effective, reproducible approach.The secretome of six main pathogenic species (plus L. tarentolae) was analysed by Mass-Spectrometry and conserved candidate antigens were searched in the complete dataset. A total of 52 vaccine antigen candidates were selected, including 28 previously described vaccine candidates, and an additional 24 new candidates discovered through a reverse vaccinology approach.In silico HLA-I and –II epitope binding prediction analysis was performed on all selected vaccine antigens, with world coverage regarding HLA restriction. To select the best epitopes, an automated R script was developed in-house, according to strict rational criteria. From thousands of potential epitopes, the automated script, in combination with optimal IC50, homology to host and solubility properties, allowed us to select 50 class I and 24 class II epitopes, synthesized as individual peptides. In vitro toxicity assays showed these selected peptides are non-toxic to cells.The peptides’ immunogenicity was evaluated using immunoscreening assays with immune cells from human donors, allowing for the validation of in silico epitope predictions and selection, and the assessment of the peptide’s immunogenicity and prophylactic potential. Healed individuals, which had active infection and received treatment, possess Leishmania-specific memory responses and are resistant to reinfection, being considered the gold standard of protective immunity. On the other hand, the naive population is extremely important to include in the experimental validation step since it is the target population to vaccinate with a prophylactic vaccine. Importantly, a minimum specific T-cell precursor frequency is needed to induce long-lasting memory protective responses. Furthermore, there is also a positive correlation between immunodominant epitopes and a high frequency of specific T-cell precursors. Peptides able to induce Th1 and/or cytotoxic immune responses in both background are promising candidates for a vaccine formulation. Altogether,experimental validation exclusively in human samples will provide us a very strong base for a vaccine formulation and allow to accelerate translation to the field.Results show Leishmania-specific peptides successfully induce IFN-γ production by total PBMC from healed donors, and by specific T cells amplified from the naïve repertoire. Preliminary evidence exists for peptides which are immunogenic in both immune backgrounds (eight HLA-class I 9-mer peptides and five class II 15-mer peptides) which are, for now, the most promising candidates to advance for the multi-epitope peptide design.Through the combination of proteomic analysis and in silico tools, promising peptide candidates were swiftly identified and the secretome was further established as an optimal starting point for vaccine development. The proposed vaccine preclinical development pipeline delivered a rapid selection of immunogenic peptides, providing a powerful approach to fast-track the deployment of an effective pan-specific vaccine against Leishmaniases
Development of a new and rapid method to introduce heterologous DNA sequences into the Plasmodium genome
Tese de mestrado. Biologia (Microbiologia Aplicada). Universidade de Lisboa, Faculdade de Ciências, 2010Malaria is one of the most important parasitic diseases nowadays with a reported 243 million cases each year and almost half of the world’s population at risk of contracting the disease. The development of new and more effective drugs as well as a prophylactic vaccine is imperative and even though, in the recent years, several advances have been made with some vaccine candidates there is still no vaccine available. Genome manipulation and reverse genetics constitute powerful tools that contributed to these advances. Together, these techniques allow a better understanding of parasite-host interactions as well as new insight into gene function and expression. With the help of cloning and transfection technologies we aim to develop the proposed Swift Knock-in Markerfree (SKIM) method, a more efficient way to knock-in and/or knock-out genes of interest that overcomes some of the limitations of previously described transfection technologies namely, the time needed to obtain transfectants, low transfection efficiencies and the recycling of the drug resistance markers. The technique is based on a motherline expressing a fused positive/negative selectable marker and, through homologous recombination by double crossover, a heterologous DNA sequence replaces the selectable marker cassette and parasites can be selected by negative selection. We tested the viability of the technique and optimized the negative selection of the parasites with a fluorescent construct – our results show that not only through the SKIM method successful integration is achieved but also the transfection efficiency is high – 93% efficiency with 4 consecutive days of negative selection. Also, we use the technique to express transmission-blocking antigens – P. falciparum and P. berghei P25 and P48/45 – in parallel to the traditional positive selection transfection, with which we were able to introduce most of the antigens.A malária ou paludismo é actualmente uma das doenças parasitárias de maior impacto global com 243 milhões de casos reportados anualmente e com quase metade da população em risco de contrair a doença; é prevalente sobretudo em países em desenvolvimento, sendo que 85% dos casos reportados se encontram em África. Estima-se que haja cerca de 1 milhão de mortes por ano sobretudo em crianças com idade inferior a 5 anos, o que constitui um enorme fardo para as populações onde a malária é endémica. É uma doença causada por parasitas protozoários da espécie Plasmodium. Existem cinco espécies capazes de causar infecção no homem – P. falciparum, P. vivax, P. ovale, P. malariae e P. knowlesi, inicialmente um parasita de primatas. P. falciparum é o patogéneo mais importante sendo responsável por um maior número de casos e complicações graves, nomeadamente malária cerebral e placentária. Os parasitas diferem nas características microscópicas, distribuição geográfica e alguns aspectos do quadro clínico, mas partilham o mesmo ciclo de vida. O parasita possui como hospedeiros o humano e os mosquitos do género Anopheles. De forma resumida, o parasita reproduz-se sexuamente e assexuadamente no interior do mosquito e este, durante a picada, injecta esporozoítos que migram até ao fígado onde se reproduzem assexuadamente sem causar sintomatologia (fase hepática); em seguida, os merozoítos libertam-se dos hepatócitos, entram na corrente sanguínea onde invadem os eritrócitos. Assim tem início a fase sanguínea e o quadro clínico associado à malária – febre alta e com periodicidade, hepatoesplenomegalia, mal-estar, cefaleias e suores. Quando o mosquito fêmea volta a alimentar-se de sangue humano ingere eritrócitos infectados e o ciclo de vida do parasita prossegue. É um ciclo de vida complexo que dificulta o controlo e eventual eliminação do parasita. Desde a descoberta do parasita já foram adquiridos conhecimentos significativos e desenvolvidos fármacos capazes de eliminar o parasita, no entanto, ainda não está disponível nenhuma vacina profiláctica, essencial ao objectivo de erradicação. Já existem alguns candidatos que alcançaram a fase de ensaios clínicos e outros continuam a surgir com a ajuda da biologia molecular e outros instrumentos de investigação. A ideia de produzir uma vacina whole-organism é promissora e o seu potencial já foi provado em 1967 com imunização com esporozoítos inactivados por irradiação. É possível também produzir parasitas geneticamente atenuados, capazes de parar o seu desenvolvimento na fase hepática ou sanguínea, estimulando o sistema imune e assim actuar como vacina. Actualmente, existe um interesse crescente em desenvolver vacinas transmission-blocking, que terão um papel preponderante na eliminação do parasita da malária, juntamente com outras medidas de prevenção, uma vez que neutralizam o parasita no interior do mosquito de modo a que o seu ciclo de vida termine nesta fase e o mosquito deixe de contribuir para a transmissão da doença. As técnicas de modificação genética disponíveis em Plasmodium como a transfecção, mutação aleatória e silenciamento de genes, que contribuíram para a aquisição de conhecimento ao nível da biologia e patogénese do parasita e sobre função de proteínas e expressão génica, são então também essenciais ao desenvolvimento de uma vacina profiláctica contra a malária. A transfecção permite introduzir ADN directamente no genoma e assim fazer o knock-in (expressão de transgenes) ou knock-out (disrupção) de genes. Estes sistemas de transfecção já foram desenvolvidos quer para o parasita humano P. falciparum quer para parasitas murinos (P. berghei, P. yoellii, P. chabaudi) que oferecem a vantagem de estudar interacções parasita-hospedeiro in vivo enquanto o parasita humano está limitado a cultura in vitro. Os sistemas de transfecção actuais envolvem a introdução de um marcador que atribui uma característica fenotípica específica ao parasita, passível de ser seleccionada – geralmente, resistência a drogas. Existem sistemas de selecção positiva, sendo o mais utilizado a resistência à pirimetamina com a introdução do gene dhfr-ts (dihidrofolato redutase-timidilato sintase) proveniente de P. falciparum, de P. berghei (o modelo murino) ou de Toxoplasma gondii (outro parasita Apicomplexa); e existem também sistemas de selecção negativa nos quais os parasitas são naturalmente resistentes à droga, mas com a introdução de genes como yfcu (que codifica a proteína de fusão yCD::yUPRT – uracilfosforibosiltransferase) ou timidina cinase os parasitas tornam-se susceptíveis a fármacos como a 5-fluorocitosina. Estes marcadores encontram-se num vector de ADN que, após transfecção, pode ser integrado no genoma por crossover simples ou duplo, ou permanecer epissomal. Estes marcadores são suficientes para estudos de expressão de transgenes ou de disrupção de genes mas se se desejar obter parasitas livres de marcador ou modificar, eliminar e/ou introduzir outros genes são necessários marcadores adicionais ou outras técnicas. A técnica proposta SKIM (Swift Knock-in Markerfree) baseia-se numa linha de parasitas que expressa um marcador de selecção positiva e negativa, isto é, um gene de fusão entre o gene dhfr humano e o gene yfcu, integrado no genoma num locus não-essencial ao parasita, como o 230p; esta linha de parasitas é positivamente seleccionada (com pirimetamina) e é utilizada em transfecções subsequentes para introduzir a cassette de expressão do gene de interesse. Constrói-se um vector que apenas contêm o gene de interesse com o respectivo promotor e as sequências de integração no locus não-essencial 230p, idênticas às usadas previamente para inserir os marcadores de selecção. Deste modo, por recombinação homóloga entre as sequências de integração, ocorre a substituição de uma cassette por outra – os parasitas que sofreram integração expressam o gene de interesse, e serão resistentes à selecção negativa; os parasitas não transfectados, ainda com o marcador de selecção negativa, não sobreviverão ao tratamento. Assim, obtemos uma linha pura de parasitas transgénicos livre de marcador de selecção que pode ser sujeita a modificações genómicas adicionais. Esta técnica permite a utilização de qualquer locus para integração desde que as sequências de integração utilizadas sejam idênticas quer para o vector do marcador de selecção positiva e negativa quer para o vector que contém o gene de interesse. Este novo método baseia-se no facto de a integração em Plasmodium ser quase exclusivamente por recombinação homóloga que, aliada à selecção positiva e selecção negativa, oferece várias vantagens em relação a técnicas estabelecidas previamente nomeadamente: uma maior facilidade na realização de ensaios de complementação ou de introdução de transgenes, uma redução no tempo para obter parasitas integrantes e uma maior simplicidade de construção dos vectores, vectores estes de menores dimensões e que, por não terem marcador de selecção, não se mantêm epissomais. Após a construção da linha original que expressa o marcador de selecção positiva e negativa, integrado no locus 230p (1596cl1), testou-se a técnica com vectores de fluorescência (mCherry) e testou-se a eficiência de transfecção em experiências independentes que variam quanto à administração de 5-fluorocitosina, o fármaco de selecção negativa. Por análise microscópica e por PCR (Polymerase chain reaction) confirmou-se a correcta integração da cassette de fluorescência e, por análise de citometria de fluxo, observou-se uma eficiência de transfecção de 93% para a linha 1645, sujeita a 4 dias consecutivos de selecção negativa. Para além do desenvolvimento e optimização da técnica proposta, um dos objectivos é também a expressão dos antigénios transmission-blocking P25 e P48/45 de P. berghei e P. falciparum sob o controlo dos promotores ef1αa que assegura a expressão dos antigénios durante todo o ciclo de vida do parasita, e CS (promotor do gene que codifica a circumsporozoite protein) responsável pela expressão génica no esporozoíto e em todas as formas de diferenciação do parasita, na fase sexuada no interior do mosquito. Utilizou-se a transfecção por selecção positiva (vectores com as cassettes do gene de interesse e do marcador de selecção positiva TgDHFR) e a transfecção utilizando o método SKIM (vectores apenas com a cassette do gene de interesse introduzidos na linha de parasitas que expressa o marcador de selecção positiva e negativa) para a expressão dos antigénios seleccionados. Através da transfecção por selecção positiva podemos obter informações em relação à expressão e localização dos antigénios introduzidos. Após confirmação da correcta integração, por PCR e FIGE (Field-inversion Gel Electrophoresis), algumas linhas foram clonadas de modo a obter uma linha pura de parasitas. Algumas das linhas obtidas foram ainda analisadas por Southern blot, Western blot e Northern blot que revelaram uma correcta integração, expressão e transcrição, respectivamente, dos genes introduzidos. Apesar do sucesso da transfecção com os vectores de fluorescência, pelo método SKIM, não foi detectada por PCR e FIGE, uma correcta integração da cassette de genes transmission-blocking de interesse nas duas linhas criadas, possivelmente devido à incorrecta administração da 5-fluorocitosina. Em conclusão, os resultados preliminares indicam que é uma técnica com elevado potencial de aplicação revelando um menor tempo necessário à obtenção de linhas puras de parasitas transfectados, livres de marcador de selecção bem como uma elevada eficiência de transfecção. No entanto, como sugerido pelas linhas onde a integração não foi bem sucedida, é necessário optimizar as condições de selecção negativa de modo a garantir a eficiência do método. Como objectivos futuros, pretendemos analisar a expressão e localização dos antigénios introduzidos bem como realizar ensaios de imunogenicidade
Revue systématique des tests sérologiques à base de peptides pour le diagnostic de la leishmaniose
International audienceSerological methods should meet the needs of leishmaniasis diagnosis due to their high sensitivity and specificity, economical and adaptable rapid diagnostic test format, and ease of use. Currently, the performances of serological diagnostic tests, despite improvements with recombinant proteins, vary greatly depending on the clinical form of leishmaniasis and the endemic area. Peptide-based serological tests are promising as they could compensate for antigenic variability and improve performance, independently of Leishmania species and subspecies circulating in the endemic areas. The objective of this systematic review was to inventory all studies published from 2002 to 2022 that evaluate synthetic peptides for serological diagnosis of human leishmaniases and also to highlight the performance (e.g., sensitivity and specificity) of each peptide reported in these studies. All clinical forms of leishmaniasis, visceral and tegumentary, and all Leishmania species responsible for these diseases were considered. Following PRISMA statement recommendations, 1,405 studies were identified but only 22 articles met the selection criteria and were included in this systematic review. These original research articles described 77 different peptides, of which several have promising performance for visceral or tegumentary leishmaniasis diagnosis. This review highlights the importance of and growing interest in synthetic peptides used for serological diagnosis of leishmaniases, and their performances compared to some widely used tests with recombinant proteins.Serological methods should meet the needs of leishmaniasis diagnosis due to their high sensitivity and specificity, economical and adaptable rapid diagnostic test format, and ease of use. Currently, the performances of serological diagnostic tests, despite improvements with recombinant proteins, vary greatly depending on the clinical form of leishmaniasis and the endemic area. Peptide-based serological tests are promising as they could compensate for antigenic variability and improve performance, independently of Leishmania species and subspecies circulating in the endemic areas. The objective of this systematic review was to inventory all studies published from 2002 to 2022 that evaluate synthetic peptides for serological diagnosis of human leishmaniases and also to highlight the performance (e.g., sensitivity and specificity) of each peptide reported in these studies. All clinical forms of leishmaniasis, visceral and tegumentary, and all Leishmania species responsible for these diseases were considered. Following PRISMA statement recommendations, 1,405 studies were identified but only 22 articles met the selection criteria and were included in this systematic review. These original research articles described 77 different peptides, of which several have promising performance for visceral or tegumentary leishmaniasis diagnosis. This review highlights the importance of and growing interest in synthetic peptides used for serological diagnosis of leishmaniases, and their performances compared to some widely used tests with recombinant proteins. Re ´sume ´-Revue systématique des tests sérologiques à base de peptides pour le diagnostic de la leishmaniose. D'une sensibilité et d'une spécificité élevées, faciles à réaliser, économiques et adaptables à un format de test de diagnostic rapide, les méthodes sérologiques devraient répondre aux besoins du diagnostic de la leishmaniose. Actuellement, les performances des tests de diagnostic sérologique, malgré des améliorations avec les protéines recombinantes, varient fortement selon la forme clinique de la leishmaniose et les zones d'endémie. Les tests sérologiques à base de peptides sont prometteurs car ils pourraient compenser la variabilité antigénique et améliorer les performances, indépendamment des espèces et sous-espèces de Leishmania circulant dans les zones endémiques. L'objectif de cette revue systématique était d'inventorier toutes les études publiées de 2002 à 2022 qui évaluent les peptides synthétiques pour le diagnostic sérologique des leishmanioses humaines et également de mettre en évidence les performances (dont la sensibilité et la spécificité) de chaque peptide rapporté dans ces études. Toutes les formes cliniques de leishmanioses, viscérales et tégumentaires, et toutes les espèces de Leishmania responsables de ces maladies ont été considérées. Suite aux recommandations de la déclaration PRISMA, 1405 études ont été identifiées mais seuls 22 articles répondaient aux critères de sélection et ont été inclus dans cette revue systématique. Ces articles de recherche originaux décrivent 77 peptides différents, dont plusieurs sont prometteurs pour le diagnostic de la leishmaniose viscérale ou tégumentaire. Cette revue met en évidence l'importance et l'intérêt croissant accordés aux peptides synthétiques utilisés pour le diagnostic sérologique des leishmanioses, et leurs performances par rapport à certains tests largement utilisés avec des protéines recombinantes
Hospitalizations for Chronic Obstructive Pulmonary Disease Exacerbation During COVID-19
International audienceThis cross-sectional study investigates changes in the number of chronic obstructive pulmonary disease (COPD)–related admissions before, during, and after the COVID-19 pandemic in France