Microscopia confocal de fluorescência: uma ferramenta poderosa no estudo da doença de Chagas

Confocal scanning fluorescence microscopy has become widely used in cell biology and pathology. In conjunction with monoclonal antibodies it may turn out to be a powerful diagnostic tool that also enables detailed studies of tissue forms of Trypanosoma cruzi.A microscopia confocal por varredura a laser vem se tornando extremamente útil na biologia celular e patologia. Com o uso de anticorpos monoclonais, pode ser uma poderosa ferramenta de diagnóstico assim como para estudos detalhados das diferentes formas do Trypanosoma cruzi em vários tecidos infectados.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de Microbiologia, Imunologia e ParasitologiaInstituto Adolfo Lutz Seção de Microscopia EletrônicaUNIFESP, EPM, Depto. de Microbiologia, Imunologia e ParasitologiaSciEL

1,4-disubstituted-1,2,3-triazole compounds induce ultrastructural alterations in leishmania amazonensis promastigote: An in vitro antileishmanial and in silico pharmacokinetic study

Funding Information: This research was funded by the Coordination for the Improvement of Higher Education Personnel (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior do Brazil; CAPES) grant number Finance Code 001; and the Carlos Chagas Filho Foundation for Research Support of the State of Rio de Janeiro (Fundacao Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro; FAPERJ) grant number E-26/010.001759/2019. The APC was funded by the Oswaldo Cruz Institute (Instituto Oswaldo Cruz; IOC). Dr. Fernando Almeida-Souza is a postdoctoral researcher fellow of CAPES grant number 88887.363006/2019-00. Dra. Ana Lucia Abreu-Silva is a research productivity fellow of National Scientific and Technological Development Council (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico; CNPq) grant number 309885/2017-5.The current standard treatment for leishmaniasis has remained the same for over 100 years, despite inducing several adverse effects and increasing cases of resistance. In this study we evaluated the in vitro antileishmanial activity of 1,4-disubstituted-1,2,3 triazole compounds and carried out in silico predictive study of their pharmacokinetic and toxicity properties. Ten compounds were analyzed, with compound 6 notably presenting IC50: 14.64 ± 4.392 µM against promastigotes, IC50: 17.78 ± 3.257 µM against intracellular amastigotes, CC50: 547.88 ± 3.256 µM against BALB/c peritoneal macrophages, and 30.81-fold selectivity for the parasite over the cells. It also resulted in a remarkable decrease in all the parameters of in vitro infection. Ultrastructural analysis revealed lipid corpuscles, a nucleus with discontinuity of the nuclear membrane, a change in nuclear chromatin, and kinetoplast swelling with breakdown of the mitochondrial cristae and electron-density loss induced by 1,4-disubstituted-1,2,3-triazole treatment. In addition, compound 6 enhanced 2.3-fold the nitrite levels in the Leishmania-stimulated macrophages. In silico pharmacokinetic prediction of compound 6 revealed that it is not recommended for topical formulation cutaneous leishmaniasis treatment, however the other properties exhibited results that were similar or even better than miltefosine, making it a good candidate for further in vivo studies against Leishmania parasites.publishersversionpublishe

Ultrastructural Changes and Death of Leishmania infantum

The search for new treatments against leishmaniasis has increased due to high frequency of drug resistance registered in endemics areas, side effects, and complications caused by coinfection with HIV. Morinda citrifolia Linn., commonly known as Noni, has a rich chemical composition and various therapeutic effects have been described in the literature. Studies have shown the leishmanicidal activity of M. citrifolia; however, its action on the parasite has not yet been elucidated. In this work, we analyzed leishmanicidal activity and ultrastructural changes in Leishmania infantum promastigotes caused by M. citrifolia fruit juice treatment. M. citrifolia fruit extract showed a yield of 6.31% and high performance liquid chromatography identified phenolic and aromatic compounds as the major constituents. IC50 values were 260.5 µg/mL for promastigotes and 201.3 µg/mL for intracellular amastigotes of L. infantum treated with M. citrifolia. Cytotoxicity assay with J774.G8 macrophages showed that M. citrifolia fruit juice was not toxic up to 2 mg/mL. Transmission electron microscopy showed cytoplasmic vacuolization, lipid inclusion, increased exocytosis activity, and autophagosome-like vesicles in L. infantum promastigotes treated with M. citrifolia fruit juice. M. citrifolia fruit juice was active against L. infantum in the in vitro model used here causing ultrastructural changes and has a future potential for treatment against leishmaniasis

Bacteria arise at the border of mycoplasma-infected HeLa cells, containing cytoplasm with either malformed cytosol, mitochondria and endoplasmic reticulum or tightly adjoined smooth vacuoles

A study with transmission electron microscopy of mycoplasma-contaminated HeLa cells using five cell donors referred to as donors A, B, C, D and E, observations are herein presented. Experiments performed with cells from donors B, C and D, revealed the presence of Mycoplasma hyorhinis after PCR and sequencing experiments. Bacteria probably originated from a cytoplasm with compacted tiny granular particles replacing the normal cytosol territories, or from the contact with the cytoplasm through a clear semi-solid material. The compact granularity (CG) of the cytoplasm was crossed by stripes of smooth and rough endoplasmic reticulum cisternae. Among apparently normal mitochondria, it was noted, in variable proportions, mitochondria with crista-delimited lucent central regions that expand to and occupied the interior of a crista-less organelle, which can undergo fission. Other components of the scenarios of mycoplasma-induced cell demolition are villus-like structures with associated 80-200 nm vesicles and a clear, flexible semi-solid, process-sensitive substance that we named jam-like material. This material coated the cytoplasmic surface, its recesses, irregular protrusions and detached cytoplasmic fragments. It also cushioned forming bacteria. Cyst-like structures were often present in the cytoplasm. Cells, mainly apoptotic, exhibiting ample cytoplasmic sectors with characteristic net-like profile due to adjoined vacuoles, as well as ovoid or elongated profiles, consistently appeared in all cells from the last four cell donors. These cells were named “modified host cells” because bacteria arose in the vacuoles. The possibility that, in some samples, there was infection and/or coinfection of the host cell by another organism(s) cannot be ruled out

Distribuition of Trypanosoma cruzi and myofibrillar components in cardiac muscle of Colomy callosus chronically infected and immunosuppressed

Um modelo experimental de reativação da doença de Chagas crônica com um agente imunossupressor foi desenvolvido a fim de se obter informações sobre do ciclo de vida intracelular do Trypanosoma cruzi e o arranjo das miofíbrilas cardíacas, com o propósito de entender melhor o processo de reativação que ocorre no homem. Calomys caffosus cronicamente infectados com as cepas Y e CL de T. cruzi sofreram reagudização quando tratados com ciclofosfamida, um agente imunossupressor. Cortes de 5 - 8 mm de parafina foram processados para microscopia confocal de imunofluorescência. Anticorpos monoclonais (AcM) produzidos contra as formas do T. cruzi foram usados com base em reatividades previamente descritas: AcM 2C2, reage com um epitopo carboidrato em Ssp-4, uma glicoproteína majoritária da superfície dos amastigotas, enquanto AcM 1 D9, 2137, 3139, 4139 identificam epitopos sobre Ssp-4 diferentes do AcM 2C2. Amostras foram coradas com DAPI para visualizar o cinetoplasto e o núcleo dos parasitas. AcM 2C2 apresentou um padrão de fluorescência homogeneamente distribuído sobre a superfície dos amastigotas; AcM 4139 e 3139 apresentaram a mesma distribuição observada em 2C2. Marcação muito fraca e negativa foi observada com AcM 1 D9 e 2137, respectivamente. AcM 3132, que reage com um epitopo nãocarboidrato de formas flageladas e AcM 4135 que também detecta um epitopo nãocarboidrato, revelou a bolsa flagelar em amastigotas alongados e a membrana plasmática dos parasitas que estavam sofrendo divisão. Para observação das proteínas miofibrilares, a técnica de recuperação antigênica foi utilizada nos cortes de parafina. Os cortes foram submetidos a tripla marcação; 1. AcM para visualizar as diferentes proteínas miofibrilares (miosina, actina, desmina, tropomiosina, troponina T, titina e a-actinina) do músculo cardíaco; 2. Soro chagásico humano para marcar a suprefície dos parasitas; 3. DAPI (para marcar DNA) que coram o núcleo das células e cinetoplasto e núcleo dos parasitas. Em paralelo, culturas primárias de cardiomiócitos de Calomys caflosus recémnascidos foram desenvolvidas para obtenção de informações adicionais sobre o arranjo ou desarranjo das miofibrilas durante a proliferação do T cruzi nestas células. Tanto amostras in vivo como in vitro apresentaram regiões com perda de estriação transversal, acúmulo de. marcação e desarranjo das miofibrilas. Ao E microscópio eletrônico de transmissão também foram observados desarranjos das ...(au).BV UNIFESP: Teses e dissertaçõe