Browsing by Author "Coelho, Paulo Marcos Zech"
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Item Ageing down-modulates liver inflammatory immune responses to schistosome infection in mice.(2010) Faria, Elaine Speziali de; Aranha, Claudio Henrique Magalhães; Carvalho, Andréa Teixeira de; Santiago, Andrezza Fernanda; Oliveira, Rafael Pires de; Rezende, Michelle Carvalho de; Carneiro, Claudia Martins; Corrêa, Deborah Aparecida Negrão; Coelho, Paulo Marcos Zech; Faria, Ana Maria Caetano deAgeing is associated with several alterations in the immune system. Our aim in this study was to compare the development of immunity to Schistosoma mansoni infection in young versus aged C57Bl ⁄ 6 mice using the liver as the main organ to evaluate pathological alterations and immune responses. In the acute phase, young mice had large liver granulomas with fibrosis and inflammatory cells. Chronic phase in young animals was associated with immunomodulation of granulomas that became reduced in size and cellular infiltrate. On the other hand, aged animals presented granulomas of smaller sizes already in the acute phase. Chronic infection in these mice was followed by no alteration in any of the inflammatory parameters in the liver. In concert with this finding, there was an increase in activated CD4+ T, CD19+ B and NK liver cells in young mice after infection whereas old mice had already higher frequencies of activated B, NK and CD4+ T liver cells and infection does not change these frequencies. After infection, liver production of inflammatory and regulatory cytokines such as IFN-c, IL-4 and IL-10 increased in young but not in old mice that had high levels of IL-4 and IL-10 regardless of their infection status. Our data suggest that the unspecific activation status of the immune system in aged mice impairs inflammatory as well as regulatory immune responses to S. mansoni infection in the liver, where major pathological alterations and immunity are at stage. This poor immune reactivity may have a beneficial impact on disease development.Item Characterisation of major vault protein during the life cycle of the human parasite Schistosoma mansoni.(2014) Reis, Eneida Virgínia; Pereira, Roberta Verciano; Gomes, Matheus de Souza; Passos, Liana Konovaloff Jannotti; Babá, Élio Hideo; Coelho, Paulo Marcos Zech; Mattos, Ana Carolina Alves de; Couto, Flávia Fernanda Búbula; Borges, William de Castro; Cota, Renata Guerra de SáVaults are ribonucleoproteins (13MDa) highly conserved among lower and higher eukaryotes. Their association produces a complex composed of three proteins named Major Vault Protein (MVP), vault (PolyADP-ribose) polymerase (VPARP) and Telomerase-associated protein (TEP1), plus a small untranslated RNA. The exact function of this complex is unknown, although the biological role of vaults has been associated with multidrug resistance phenotypes and signal transduction pathways. Genomic analysis showed that model organisms, such as Caenorhabditis elegans and Drosophila melanogaster, do not possess genes encoding vaults. However, we have found that vault-related genes are present in the Schistosoma mansoni genome. These observations raised questions on the involvement of vaults in mechanisms of adaptation of the parasite in its mammalian host. Therefore, molecular characterisation of the putative Major Vault Protein performed using bioinformatics tools showed that this vault component is highly conserved in S. mansoni. The MVP expression level was quantified by qRT-PCR using total RNA from susceptible (LE) and resistant (LE-PZQ) adult worm lineages, cercariae and mechanically transformed schistosomula(MTS) cultured for 3.5, 24, 48 and 72h in vitro. Our results suggest a stage-specific expression in all developmental stages analysed. Western blotting has shown upregulation of SmMVP in the MTS-3.5, 72 h and resistant adult worms, and similar levels in all other stages. Furthermore, SmMVPwas found differentially expressed in adult males and females fromthe susceptible lineage. Further studies should clarify whether SmMVP is somehow linked to drug resistance in S. mansoni.Item Deep sequencing of small RNAs reveals the repertoire of miRNAs and piRNAs in Biomphalaria glabrata.(2020) Queiroz, Fábio Ribeiro; Portilho, Laysa Gomes; Jeremias, Wander de Jesus; Babá, Élio Hideo; Amaral, Laurence Rodrigues do; Silva, Luciana Maria; Coelho, Paulo Marcos Zech; Caldeira, Roberta Lima; Gomes, Matheus de SouzaBACKGROUND Biomphalaria glabrata snails are widely distributed in schistosomiasis endemic areas like America and Caribe, displaying high susceptibility to infection by Schistosoma mansoni. After the availability of B. glabrata genome and transcriptome data, studies focusing on genetic markers and small non-coding RNAs have become more relevant. The small RNAs have been considered important through their ability to finely regulate the gene expression in several organisms, thus controlling the functions like cell growth, metabolism, and susceptibility/resistance to infection. OBJECTIVE The present study aims on identification and characterisation of the repertoire of small non-coding RNAs in B. glabrata (Bgl-small RNAs). METHODS By using small RNA sequencing, bioinformatics tools and quantitative reverse transcription polymerase chain reaction (RT-qPCR), we identified, characterised, and validated the presence of small RNAs in B. glabrata. FINDINGS 89 mature miRNAs were identified and five of them were classified as Mollusk-specific. When compared to model organisms, sequences of B. glabrata miRNAs showed a high degree of conservation. In addition, several target genes were predicted for all the mature miRNAs identified. Furthermore, piRNAs were identified in the genome of B. glabrata for the first time. The B. glabrata piRNAs showed strong conservation of uridine as first nucleotide at 5’ end, besides adenine at 10th position. Our results showed that B. glabrata has diverse repertoire of circulating ncRNAs, several which might be involved in mollusk susceptibility to infection, due to their potential roles in the regulation of S. mansoni development. MAIN CONCLUSIONS Further studies are necessary in order to confirm the role of the Bgl-small RNAs in the parasite/host relationship thus opening new perspectives on interference of small RNAs in the organism development and susceptibility to infection.Item Diagnosis of Schistosoma mansoni infections : what are the choices in Brazilian low-endemic areas?(2019) Moraes, Vanessa Silva; Shollenberger, Lisa Marie; Siqueira, Liliane Maria Vidal; Borges, William de Castro; Harn, Donald A.; Grenfell, Rafaella Fortini Queiroz; Rabello, Ana Lúcia Teles; Coelho, Paulo Marcos ZechThe population of Brazil is currently characterised by many individuals harbouring low-intensity Schistosoma mansoni infections. The Kato-Katz technique is the diagnostic method recommended by the World Health Organization (WHO) to assess these infections, but this method is not sensitive enough in the context of low egg excretion. In this regard, potential alternatives are being employed to overcome the limits of the Kato-Katz technique. In the present review, we evaluated the performance of parasitological and immunological approaches adopted in Brazilian areas. Currently, the diagnostic choices involve a combination of strategies, including the utilisation of antibody methods to screen individuals and then subsequent confirmation of positive cases by intensive parasitological investigations.Item Genome-wide identification, characterisation and expression profiling of the ubiquitin-proteasome genes in Biomphalaria glabrata.(2019) Portilho, Laysa Gomes; Duarte, Bruna Custódio Dias; Queiroz, Fábio Ribeiro; Ribeiro, Thales Henrique Cherubino; Jeremias, Wander de Jesus; Babá, Elio Hideo; Coelho, Paulo Marcos Zech; Morais, Enyara Rezende; Cabral, Fernanda Janku; Caldeira, Roberta Lima; Gomes, Matheus de SouzaBACKGROUND Biomphalaria glabrata is the major species used for the study of schistosomiasis-related parasite-host relationships, and understanding its gene regulation may aid in this endeavor. The ubiquitin-proteasome system (UPS) performs post-translational regulation in order to maintain cellular protein homeostasis and is related to several mechanisms, including immune responses. OBJECTIVE The aims of this work were to identify and characterise the putative genes and proteins involved in UPS using bioinformatic tools and also their expression on different tissues of B. glabrata. METHODS The putative genes and proteins of UPS in B. glabrata were predicted using BLASTp and as queries reference proteins from model organism. We characterised these putative proteins using PFAM and CDD software describing the conserved domains and active sites. The phylogenetic analysis was performed using ClustalX2 and MEGA5.2. Expression evaluation was performed from 12 snail tissues using RPKM. FINDINGS 119 sequences involved in the UPS in B. glabrata were identified, which 86 have been related to the ubiquitination pathway and 33 to proteasome. In addition, the conserved domains found were associated with the ubiquitin family, UQ_con, HECT, U-box and proteasome. The main active sites were lysine and cysteine residues. Lysines are responsible and the starting point for the formation of polyubiquitin chains, while the cysteine residues of the enzymes are responsible for binding to ubiquitin. The phylogenetic analysis showed an organised distribution between the organisms and the clades of the sequences, corresponding to the tree of life of the animals, for all groups of sequences analysed. The ubiquitin sequence was the only one with a high expression profile found in all libraries, inferring its wide range of performance. MAIN CONCLUSIONS Our results show the presence, conservation and expression profile of the UPS in this mollusk, providing a basis and new knowledge for other studies involving this system. Due to the importance of the UPS and B. glabrata, this work may influence the search for new methodologies for the control of schistosomiasis.Item Identification of 170 new long noncoding RNAs in Schistosoma mansoni.(2018) Oliveira, Victor Fernandes de; Moraes, Lauro Ângelo Gonçalves de; Mota, Ester Alves; Passos, Liana Konovaloff Jannotti; Coelho, Paulo Marcos Zech; Mattos, Ana Carolina Alves de; Couto, Flávia Fernanda Búbula; Caffrey, Brian E.; Marsico, Annalisa; Cota, Renata Guerra de SáLong noncoding RNAs (lncRNAs) are transcripts generally longer than 200 nucleotides with no or poor protein coding potential, and most of their functions are also poorly characterized. Recently, an increasing number of studies have shown that lncRNAs can be involved in various critical biological processes such as organism development or cancer progression. Little, however, is known about their effects in helminths parasites, such as Schistosoma mansoni. Here, we present a computational pipeline to identify and characterize lncRNAs from RNA-seq data with high confidence from S. mansoni adult worms. Through the utilization of different criteria such as genome localization, exon number, gene length, and stability, we identified 170 new putative lncRNAs. All novel S. mansoni lncRNAs have no conserved synteny including human and mouse. These closest protein coding genes were enriched in 10 significant Gene Ontology terms related to metabolism, transport, and biosynthesis. Fifteen putative lncRNAs showed differential expression, and three displayed sex-specific differential expressions in praziquantel sensitive and resistant adult worm couples. Together, our method can predict a set of novel lncRNAs from the RNA-seq data. Some lncRNAs are shown to be differentially expressed suggesting that those novel lncRNAs can be given high priority in further functional studies focused on praziquantel resistance.Item Prevalência das infecções parasitárias intestinais em aldeias da etnia indígena Maxakali, Minas Gerais, Brasil.(2019) Nacife, Maria Beatriz Pena e Silva Leite; Coelho, George Luiz Lins Machado; Siqueira, Liliane Maria Vidal; Katz, Naftale; Coelho, Paulo Marcos Zech; Coelho, George Luiz Lins MachadoA prevalência de parasitas intestinais é reconhecidamente elevada entre populações ameríndias e há sérias desigualdades no que se refere à saúde e à assistência de saúde das populações indígenas no Brasil. A etnia Maxakali, localizada na região nordeste de Minas Gerais em fronteira com o sul da Bahia, é um grupo indígena que sobreviveu ao extermínio e até hoje preservam muitos de seus aspectos culturais. Possuem hábitos como andar descalços, viver de forma aglomerada, deixar expostos no ambiente os alimentos, defecar no chão e nas coleções hídricas, dentre outros, que propiciam a transmissão de parasitos intestinais. Além disso, são seminômades o que dificulta a implementação de medidas de saneamento pelos órgãos competentes e a determinação da real prevalência desses parasitos intestinais. Desta forma estudos epidemiológicos nesta área se fazem necessários para que medidas preventivas e de controle sejam implementadas, podendo desta forma melhorar a qualidade de vida destes povos. As parasitoses intestinais são umas das principais causas de morbimortalidade nestes índios. Objetivo: O objetivo deste estudo foi descrever o perfil epidemiológico da esquistossomose e de outras infecções parasitárias intestinais em indivíduos que vivem nos polos base Água Boa e Pradinho, pertencentes à etnia Maxakali. Materiais e métodos: Os exames parasitológicos foram realizados pelas técnicas de Kato-Katz (uma lâmina de uma amostra) e TF-Test® (três lâminas de uma amostra). Um total de 545 amostras foram analisadas, correspondendo a quantidade de indivíduos que completaram as duas técnicas diagnósticas. Resultados: Foi encontrada uma prevalência de 84,2% para helmintos, sendo a maior positividade obtida para infecção por ancilostomídeos (59,3%), seguida por uma taxa de 51,9% para Schistosoma mansoni, de 13,9% para Hymenolepis nana, 3,9% para Trichuris trichiura, 0,7% para Taenia sp., detectados pela combinação das duas técnicas diagnósticas. Ascaris lumbricoides foi detectado apenas pela técnica de Kato-Katz com uma taxa de 0,6%, Strongyloides stercoralis e Enterobius vermicularis foram detectados apenas pela técnica de TF-Test®, apresentando positividade de 13,0% e 0,4%, respectivamente. Considerando os protozoários intestinais, foi encontrada uma prevalência de 84,8%, dos quais 28,3% eram patogênicos, 16,1% for Entamoeba histolytica/dispar e 15,2% por Giardia duodenalis. Dentre os protozoários não patogênicos, as taxas de positividade foram de 74,5% para Entamoeba coli, 56,7% para Endolimax nana e 29,9% para Iodamoeba butschii. Em relação ao diagnóstico da esquistossomose mansoni, a avaliação do desempenho da técnica de TF-Test® comparado a técnica de Kato-Katz demonstrou uma taxa de positividade pelo Kato-Katz de 45,7%, e pelo TF-Test® de 33,2%, e combinando as duas técnicas a positividade foi de 51,9%. A amplitude da carga parasitária foi de 24 a 4.056 ovos por grama de fezes (opg), com uma média geométrica de 139 opg. A sensibilidade, especificidade e acurácia pelo TF-Test® em relação ao Kato-Katz foram de 59,0%, 88,5%, e 75,0%, respectivamente. A concordância entre as duas técnicas foi moderada (k = 0,486), conforme determinado pelo índice kappa. Conclusão: A combinação de um método quantitativo e qualitativo realizado com apenas uma amostra fecal foi eficaz para a detecção de uma maior prevalência de S. mansoni e outros helmintos. A alta prevalência de parasitos intestinais pode ser atribuída à falta de infraestrutura sanitária, aos hábitos de higiene propícios à transmissão, às condições ambientais e também à alta densidade de hospedeiros intermediários, este último especificamente para a infecção da esquistossomose. Isso indica a necessidade da implementação de medidas de controle como saneamento básico, educação em saúde, além do tratamento em massa.Item Serological proteomic screening and evaluation of a recombinant egg antigen for the diagnosis of low-intensity Schistosoma mansoni infections in endemic area in Brazil.(2019) Moraes, Vanessa Silva; Shollenberger, Lisa Marie; Borges, William Castro; Rabello, Ana Lucia Teles; Harn, Donald A.; Medeiros, Lia Carolina Soares; Jeremias, Wander de Jesus; Siqueira, Liliane Maria Vidal; Pereira, Caroline Stephane Salviano; Pedrosa, Maria Luysa Camargos; Almeida, Nathalie Bonatti Franco; Almeida, Aureo; Lambertucci, Jose Roberto; Carneiro, Nídia Francisca de Figueiredo; Coelho, Paulo Marcos Zech; Grenfell, Rafaella Fortini QueirozBackground Despite decades of use of control programs, schistosomiasis remains a global public health problem. To further reduce prevalence and intensity of infection, or to achieve the goal of elimination in low-endemic areas, there needs to be better diagnostic tools to detect low- intensity infections in low-endemic areas in Brazil. The rationale for development of new diagnostic tools is that the current standard test Kato-Katz (KK) is not sensitive enough to detect low-intensity infections in low-endemic areas. In order to develop new diagnostic tools, we employed a proteomics approach to identify biomarkers associated with schisto- some-specific immune responses in hopes of developing sensitive and specific new meth- ods for immunodiagnosis. Methods and findings Immunoproteomic analyses were performed on egg extracts of Schistosoma mansoni using pooled sera from infected or non-infected individuals from a low-endemic area of Brazil. Cross reactivity with other soil-transmitted helminths (STH) was determined using pooled sera from individuals uniquely infected with different helminths. Using this approach, we identified 23 targets recognized by schistosome acute and chronic sera samples. To identify immunoreactive targets that were likely glycan epitopes, we compared these targets to the immunoreactivity of spots treated with sodium metaperiodate oxidation of egg extract. This treatment yielded 12/23 spots maintaining immunoreactivity, suggesting that they were protein epitopes. From these 12 spots, 11 spots cross-reacted with sera from individuals infected with other STH and 10 spots cross-reacted with the negative control group. Spot number 5 was exclusively immunoreactive with sera from S. mansoni-infected groups in native and deglycosylated conditions and corresponds to Major Egg Antigen (MEA). We expressed MEA as a recombinant protein and showed a similar recognition pattern to that of the native protein via western blot. IgG-ELISA gave a sensitivity of 87.10% and specificity of 89.09% represented by area under the ROC curve of 0.95. IgG-ELISA performed better than the conventional KK (2 slides), identifying 56/64 cases harboring 1–10 eggs per gram of feces that were undiagnosed by KK parasitological technique. Conclusions The serological proteome approach was able to identify a new diagnostic candidate. The recombinant egg antigen provided good performance in IgG-ELISA to detect individuals with extreme low-intensity infections (1 egg per gram of feces). Therefore, the IgG-ELISA using this newly identified recombinant MEA can be a useful tool combined with other techniques in low-endemic areas to determine the true prevalence of schistosome infection that is underestimated by the KK method. Further, to overcome the complexity of ELISA in the field, a second generation of antibody-based rapid diagnostic tests (RDT) can be developed.Item Serological proteomic screening and evaluation of a recombinant egg antigen for the diagnosis of low-intensity Schistosoma mansoni infections in endemic area in Brazil.(2019) Moraes, Vanessa Silva; Shollenberger, Lisa Marie; Borges, William de Castro; Rabello, Ana Lúcia Teles; Harn, Donald A.; Medeiros, Lia Carolina Almeida Soares; Jeremias, Wander de Jesus; Siqueira, Liliane Maria Vidal; Pereira, Caroline Stephane Salviano; Pedrosa, Maria Luysa de Camargos; Almeida, Nathalie Bonatti Franco; Oliveira, Áureo Almeida de; Lambertucci, José Roberto; Carneiro, Nídia Francisca de Figueiredo; Coelho, Paulo Marcos Zech; Grenfell, Rafaella Fortini QueirozBackground: Despite decades of use of control programs, schistosomiasis remains a global public health problem. To further reduce prevalence and intensity of infection, or to achieve the goal of elimination in low-endemic areas, there needs to be better diagnostic tools to detect low-intensity infections in low-endemic areas in Brazil. The rationale for development of new diagnostic tools is that the current standard test Kato-Katz (KK) is not sensitive enough to detect low-intensity infections in low-endemic areas. In order to develop new diagnostic tools, we employed a proteomics approach to identify biomarkers associated with schistosome-specific immune responses in hopes of developing sensitive and specific new methods for immunodiagnosis. Methods and findings: Immunoproteomic analyses were performed on egg extracts of Schistosoma mansoni using pooled sera from infected or non-infected individuals from a low-endemic area of Brazil. Cross reactivity with other soil-transmitted helminths (STH) was determined using pooled sera from individuals uniquely infected with different helminths. Using this approach, we identified 23 targets recognized by schistosome acute and chronic sera samples. To identify immunoreactive targets that were likely glycan epitopes, we compared these targets to the immunoreactivity of spots treated with sodium metaperiodate oxidation of egg extract. This treatment yielded 12/23 spots maintaining immunoreactivity, suggesting that they were protein epitopes. From these 12 spots, 11 spots cross-reacted with sera from individuals infected with other STH and 10 spots cross-reacted with the negative control group. Spot number 5 was exclusively immunoreactive with sera from S. mansoni-infected groups in native and deglycosylated conditions and corresponds to Major Egg Antigen (MEA). We expressed MEA as a recombinant protein and showed a similar recognition pattern to that of the native protein via western blot. IgG-ELISA gave a sensitivity of 87.10% and specificity of 89.09% represented by area under the ROC curve of 0.95. IgG-ELISA performed better than the conventional KK (2 slides), identifying 56/64 cases harboring 1–10 eggs per gram of feces that were undiagnosed by KK parasitological technique. Conclusions: The serological proteome approach was able to identify a new diagnostic candidate. The recombinant egg antigen provided good performance in IgG-ELISA to detect individuals with extreme low-intensity infections (1 egg per gram of feces). Therefore, the IgG-ELISA using this newly identified recombinant MEA can be a useful tool combined with other techniques in low-endemic areas to determine the true prevalence of schistosome infection that is underestimated by the KK method. Further, to overcome the complexity of ELISA in the field, a second generation of antibody-based rapid diagnostic tests (RDT) can be developed.Item The effect of chronic ingestion of ethanol on modulation of granulomatous inflammation in experimental schistosomiasis in mice.(1993) Castro, Lúcia Porto Fonseca de; Bambirra, Eduardo Alves; Coelho, Paulo Marcos Zech; Silva, Marcelo EustáquioWe studied the role of ethanol on the modulation of liver granulomata around Schistosoma mansoni eggs in mice. Albino mice, receiving 7% ethanol as the sole drinking liquid, at 60 and 90 days post-infection, presented smaller granulomata than controls did, when sacrificed at 120 days post-infection. No differences in diameters could observed, when ethanol was given 4 months before up to 120 days after infection. The results suggested that modulation of schistosomose granulomata by ethanol ingestion varies with time and duration of drug consumption.