Navegando por Autor "Bartholomeu, Daniella Castanheira"
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Item Avaliação do desempenho da TcI/TcVI/TcII Chagas-Flow ATE-IgG2a no sorodiagnóstico universal e genótipo-específico de infecções experimentais simples e mistas pelo Trypanosoma cruzi.(2017) Alessio, Glaucia Diniz; Lana, Marta de; Martins Filho, Olindo Assis; Vieira, Paula Melo de Abreu; Menezes, Evandro Marques de; Bartholomeu, Daniella Castanheira; Pereira, Rosiane Aparecida da Silva; Lana, Marta deO Segundo Consenso Taxonômico do Trypanosoma cruzi subdividiu a espécie em seis grupos genéticos distintos (DTUs- “Discrete Typing Unity”), TcI a TcVI. Nesse contexto, diversos estudos vêm associando a variabilidade genética do T. cruzi com as características biológicas, epidemiológicas e clínicas da doença de Chagas (DCh). Assim, torna-se cada vez mais importante correlacionar a diversidade genética do parasito com o diagnóstico da DCh. Recentemente, foi padronizada a técnica de pesquisa de anticorpos IgG anti-amastigotas (AMA), tripomastigotas (TRIPO) e epimastigotas (EPI) do T. cruzi por citometria de fluxo (Chagas-Flow ATE), com excelente desempenho no diagnóstico e na monitoração pós-terapêutica da DCh. Assim, esse trabalho busca otimizar a Chagas-Flow ATE no diagnóstico universal e genótipoespecífico da infecção experimental simples e mista pelo T. cruzi, utilizando distintas DTUs do parasito como antígeno. Foram avaliados pela TcI/TcVI/TcII Chagas-Flow ATE-IgG2a amostras de soros de camundongos não infectados e com infecções pelo T. cruzi simples: Colombiana (COL)/TcI, CL/TcVI e Y/TcII e mistas: Colombiana/TcI + CL/TcVI, CL/TcVI + Y/TcII e Colombiana/TcI + Y/TcII nas infecções recentes (IR) e tardias (IT). Os dados demonstraram o excelente desempenho da TcI/TcVI/TcII Chagas-Flow ATE-IgG2a no diagnóstico universal da infecção experimental pelo T. cruzi, com 100% de sensibilidade e especifidade. Essa metodologia apresentou ainda um bom desempenho no diagnóstico genótipo-específico de infecções IR simples, com uma acurácia de 69% para discriminar infecções por TcI (COL)/TcVI (CL)/TcII (Y) e de 94% para diferenciar infecções por TcI (COL)/TcII (Y). Além do mais, essa técnica demonstrou uma boa performance para distinguir infecções pelo T. cruzi nas IR e IT, com uma acurácia de 81%. Os resultados evidenciaram ainda o bom desempenho dessa metodologia para discriminar infecções simples e mistas na IR e na IT, com uma acurácia de 85% e 84%, respectivamente. Além do mais, a TcI/TcVI/TcII Chagas-Flow ATE-IgG2a apresentou uma boa performance no diagnóstico genótipo-específico de infecções experimentais simples e mistas pelo T. cruzi na IR, com uma acurácia de 72% e 80%, respectivamente, e de infecções simples e mistas na IT, com uma acurácia de 69% e 76%, respectivamente. Em relação à análise dos algoritmos sequenciais, a metodologia proposta demonstrou uma acurácia de 81% como um teste diagnóstico complementar para a infecção experimental pelo T. cruzi. Os resultados indicam o potencial da TcI/TcVI/TcII Chagas-Flow ATE-IgG2a para o diagnóstico universal e genótipo-específico da infecção experimental simples e mista pelo T. cruzi, na IR e na IT. A padronização da metodologia Chagas-Flow ATE para o diagnóstico genótipoespecífico da DCh será de extrema importância para a proposta clínica, estudos epidemiológicos, e de monitoração pós-terapêutica.Item Comparative genomics of canine-isolated Leishmania (Leishmania) amazonensis from an endemic focus of visceral leishmaniasis in Governador Valadares, southeastern Brazil.(2017) Valdivia Rodríguez, Hugo Oswaldo; Almeida, Laila Viana de; Roatt, Bruno Mendes; Cunha, João Luís Reis; Pereira, Agnes Antônia Sampaio; Gontijo, Célia Maria Ferreira; Fujiwara, Ricardo Toshio; Reis, Alexandre Barbosa; Sanders, Mandy J.; Cotton, James A.; Bartholomeu, Daniella CastanheiraLeishmaniasis is a highly diverse group of diseases caused by kinetoplastid of the genus Leishmania. These parasites are taxonomically diverse, with human pathogenic species separated into two subgenera according to their development site inside the alimentary tract of the sand fly insect vector. The disease encompasses a variable spectrum of clinical manifestations with tegumentary or visceral symptoms. Among the causative species in Brazil, Leishmania (Leishmania) amazonensis is an important etiological agent of human cutaneous leishmaniasis that accounts for more than 8% of all cases in endemic regions. L. (L.) amazonensis is generally found in the north and northeast regions of Brazil. Here, we report the first isolation of L. (L.) amazonensis from dogs with clinical manifestations of visceral leishmaniasis in Governador Valadares, an endemic focus in the southeastern Brazilian State of Minas Gerais where L. (L.) infantum is also endemic. These isolates were characterized in terms of SNPs, chromosome and gene copy number variations, confirming that they are closely related to a previously sequenced isolate obtained in 1973 from the typical Northern range of this species. The results presented in this article will increase our knowledge of L. (L.) amazonensis-specific adaptations to infection, parasite survival and the transmission of this Amazonian species in a new endemic area of Brazil.Item Epitope mapping of the HSP83.1 protein of Leishmania braziliensis discloses novel targets for immunodiagnosis of tegumentary and visceral clinical forms of leishmaniasis.(2014) Souza, Daniel Menezes; Mendes, Tiago Antônio de Oliveira; Gomes, Matheus de Souza; Cunha, João Luís Reis; Nagem, Ronaldo Alves Pinto; Carneiro, Cláudia Martins; Coelho, Eduardo Antônio Ferraz; Galvão, Lúcia Maria da Cunha; Fujiwara, Ricardo Toshio; Bartholomeu, Daniella CastanheiraGold standard serological diagnostic methods focus on antigens that elicit a strong humoral immune response that is specific to a certain pathogen. In this study, we used bioinformatics approaches to identify linear B-cell epitopes that are conserved among Leishmania species but are divergent from the host species Homo sapiens and Canis familiaris and from Trypanosoma cruzi, the parasite that causes Chagas disease, to select potential targets for the immunodiagnosis of leishmaniasis. Using these criteria, we selected heat shock protein 83.1 of Leishmania braziliensis for this study. We predicted three linear B-cell epitopes in its sequence. These peptides and the recombinant heat shock protein 83.1 (rHSP83.1) were tested in enzyme-linked immunosorbent assays (ELISAs) against serum samples from patients with tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL) and from dogs infected with Leishmania infantum (canine VL [CVL]). Our data show that rHSP83.1 is a promising target in the diagnosis of TL. We also identified specific epitopes derived from HSP83.1 that can be used in the diagnosis of human TL (peptide 3), both human and canine VL (peptides 1 and 3), and all TL, VL, and CVL clinical manifestations (peptide 3). Receiver operating characteristic (ROC) curves confirmed the superior performance of rHSP83.1 and peptides 1 and 3 compared to that of the soluble L. braziliensis antigen and the reference test kit for the diagnosis of CVL in Brazil (EIE-LVC kit; Bio-Manguinhos, Fiocruz). Our study thus provides proof-of-principle evidence of the feasibility of using bioinformatics to identify novel targets for the immunodiagnosis of parasitic diseases using proteins that are highly conserved throughout evolution.Item Genetic profiling of Trypanosoma cruzi directly in infected tissues using nested PCR of polymorphic microsatellites.(2008) Pimenta, Juliana Ramos; Freitas, Jorge Marcelo de; Duffy, Tomás; Bartholomeu, Daniella Castanheira; Oliveira, Riva de Paula; Chiari, Egler; Moreira, Maria da Consolação Vieira; Brasileiro Filho, Geraldo; Schijman, Alejandro Gabriel; Franco, Glória Regina; Machado, Carlos Renato; Pena, Sérgio Danilo Junho; Macedo, Andréa MaraThe investigation of the importance of the genetics of Trypanosoma cruzi in determining the clinical course of Chagas disease will depend on precise characterisation of the parasites present in the tissue lesions. This can be adequately accomplished by the use of hypervariable nuclear markers such as microsatellites. However the unilocal nature of these loci and the scarcity of parasites in chronic lesions make it necessary to use high sensitivity PCR with nested primers, whose design depends on the availability of long flanking regions, a feature not hitherto available for any known T. cruzi microsatellites. Herein, making use of the extensive T. cruzi genome sequence now available and using the Tandem Repeats Finder software, it was possible to identify and characterise seven new microsatellite loci – six composed of trinucleotide (TcTAC15, TcTAT20, TcAAT8, TcATT14, TcGAG10 and TcCAA10) and one composed of tetranucleotide (TcAAAT6) motifs. All except the TcCAA10 locus were physically mapped onto distinct intergenic regions of chromosome III of the CL Brener clone contigs. The TcCAA10 locus was localised within a hypothetical protein gene in the T. cruzi genome. All microsatellites were polymorphic and useful for T. cruzi genetic variability studies. Using the TcTAC15 locus it was possible to separate the strains belonging to the T. cruzi I lineage (DTU I) from those belonging to T. cruzi II (DTU IIb), T. cruzi III (DTU IIc) and a hybrid group (DTU IId, IIe). The long flanking regions of these novel microsatellites allowed construction of nested primers and the use of full nested PCR protocols. This strategy enabled us to detect and differentiate T. cruzi strains directly in clinical specimens including heart, blood, CSF and skin tissues from patients in the acute and chronic phases of Chagas disease.Item Hookworm products ameliorate dextran sodium sulfate-induced colitis in BALB/c mice.(2011) Cançado, Guilherme Grossi Lopes; Fiuza, Jacqueline Araújo; Paiva, Nívia Carolina Nogueira de; Lemos, Lucas de Carvalho Dhom; Ricci, Natasha Delaqua; Guimarães, Pedro Henrique Gazzinelli; Martins, Virgílio Gandra; Bartholomeu, Daniella Castanheira; Corrêa, Deborah Aparecida Negrão; Carneiro, Cláudia Martins; Fujiwara, Ricardo ToshioBackground: Several lines of evidence have shown that helminthiasis can significantly reduce disease severity in animal models of intestinal inflammation, airway inflammation/hyperreactivity, diabetes, and multiple sclerosis. Identification and characterization of helminth-derived immunomodulatory molecules that contribute to anticolitis effects could lead to new therapeutic approaches in inflammatory bowel diseases (IBDs) without the need for helminth infection. We evaluated the therapeutic potential of adult human hookworm, Ancylostoma ceylanicum, crude (Aw) and excreted/secreted (ES) products on dextran sulfate sodium (DSS)-induced colitis in BALB/c mice. Methods: Colitis was induced by 5% DSS oral administration for 7 days. Clinical disease severity was monitored daily during concomitant intraperitoneal treatment with helminth-derived products. Additionally, several pathways of immunological modulation induced by A. ceylanicum products (MPO, EPO, Th1, Th2, and Th17 cytokine responses) in the inflamed intestinal microenvironment were assessed. Finally, the histopathological profile of the colon was characterized. Results: Hookworm products are able to modulate the potent proinflammatory response induced by DSS, mainly through the downregulation of Th1 and Th17 cytokines. These proteins also reduce clinical and colonic microscopic inflammation scores as well as EPO and MPO activity. Conclusions: Ancylostoma ceylanicum Aw and ES mediators have an important therapeutic potential in experimental colitis in mice, which may provide a more socially acceptable form of therapy for patients with IBDs as opposed to using living worms. Our results support the urgency of further isolation and recombinant expression of active hookworm products responsible for the beneficial effects on colitis.Item Leishmania amazonensis from distinct clinical forms/hosts has polymorphisms in Lipophosphoglycans, displays variations in immunomodulatory properties and, susceptibility to antileishmanial drugs.(2022) Rêgo, Felipe Dutra; Cardoso, Camila Almeida; Moreira, Paulo Otávio Lourenço; Nogueira, Paula Monalisa; Araújo, Márcio Sobreira Silva; Borges, Valéria Matos; Laurenti, Marcia Dalastra; Bartholomeu, Daniella Castanheira; Reis, Alexandre Barbosa; Monte Neto, Rubens Lima do; Soares, Rodrigo Pedro PintoLipophosphoglycan (LPG), the major Leishmania glycoconjugate, induces pro‐ inflammatory/immunosuppressive innate immune responses. Here, we evaluated functional/biochemical LPG properties from six Leishmania amazonensis strains from different hosts/clinical forms. LPGs from three strains (GV02, BA276, and LV79) had higher pro‐inflammatory profiles for most of the mediators, including tumor necrosis factor alpha and interleukin 6. For this reason, glycoconjugates from all strains were biochemically characterized and had polymorphisms in their repeat units. They consisted of three types: type I, repeat units devoid of side chains; type II, containing galactosylated side chains; and type III, containing glucosylated side chains. No relationship was observed between LPG type and the pro‐inflammatory properties. Finally, to evaluate the susceptibility against antileishmanial agents, two strains with high (GV02, BA276) and one with low (BA336) pro‐inflammatory activity were selected for chemotherapeutic tests in THP‐1 cells. All analyzed strains were susceptible to amphotericin B (AmB) but displayed various responses against miltefosine (MIL) and glucantime (GLU). The GV02 strain (canine visceral leishmaniasis) had the highest IC50 for MIL (3.34 μM), whereas diffuse leishmaniasis strains (BA276 and BA336) had a higher IC50 for GLU (6.87–12.19 mM). The highest IC50 against MIL shown by the GV02 strain has an impact on clinical management. Miltefosine is the only drug approved for dog treatment in Brazil. Further studies into drug susceptibility of L. amazonensis strains are warranted, especially in areas where dog infection by this species overlaps with those caused by Leishmania infantum.Item Replacement of Leishmania (Leishmania) infantum populations in an endemic focus of visceral leishmaniasis in Brazil.(2022) Valdivia Rodríguez, Hugo Oswaldo; Roatt, Bruno Mendes; Baptista, Rodrigo de Paula; Ottino, Jennifer; Santos, Anderson Coqueiro dos; Sanders, Mandy J.; Reis, Alexandre Barbosa; Cotton, James A.; Bartholomeu, Daniella CastanheiraVisceral leishmaniasis is an important global health problem with an estimated of 50,000 to 90,000 new cases per year. VL is the most serious form of leishmaniasis as it can be fatal in 95% of the cases if it remains untreated. VL is a particularly acute problem in Brazil which contributed with 97% of all cases reported in 2020 in the Americas. In this country, VL affects mainly the poorest people in both urban and rural areas and continues to have a high mortality rate estimated around 8.15%. Here, we performed a temporal parasite population study using whole genome sequence data from a set of 34 canine isolates sampled in 2008, 2012 and 2015 from a re-emergent focus in Southeastern Brazil. Our study found the presence of two distinct sexual subpopulations that corresponded to two isolation periods. These subpopulations diverged hundreds of years ago with no apparent gene flow between them suggesting a process of rapid replacement during a two-year period. Sequence comparisons and analysis of nucleotide diversity also showed evidence of balancing selection acting on transport-related genes and antigenic families. To our knowledge this is the first population genomic study showing a turn-over of parasite populations in an endemic region for leishmaniasis. The complexity and rapid adaptability of these parasites pose new challenges to control activities and demand more integrated approaches to understand this disease in New World foci.