Navegando por Palavras-chave "confocal microscopy"

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    Acidification modulates the traffic of Trypanosoma cruzi trypomastigotes in Vero cells harbouring Coxiella burnetii vacuoles
    (Elsevier B.V., 2003-02-01) Andreoli, W. K.; Mortara, R. A.; Universidade Federal de São Paulo (UNIFESP)
    We studied the fate of different Trypanosoma cruzi trypomastigote forms after they invade Vero cells persistently colonised with Coxiella burnetii. When the invasion step was examined we found that persistent C. burnetii infection per se reduced only tissue-culture trypomastigote invasion, whereas raising vacuolar pH with Bafilomycin Al and related drugs, increased invasion of both metacyclic and tissue-culture trypomastigotes when compared with control Vero cells. Kinetic studies of trypomastigote transfer indicated that metacyclic trypomastigotes parasitophorous vacuoles are more efficiently fused to C. burnetii vacuoles. the higher tissue-culture trypomastigote hemolysin and transialidase activities appear to facilitate their faster escape from the parasitophorous vacuole. Sialic acid deficient Lec-2 cells facilitate the escape of both forms. Endosomal-lysosomal sequential labelling with EEA1, LAMP-1, and Rab7 of the parasitophorous vacuoles formed during the entry of each infective form revealed that the phagosome maturation processes are also distinct. Measurements of C. burnetii vacaolar pH disclosed a marked preference for trypomastigote fusion with more acidic rickettsia vacuoles. Our results thus suggest that intravacuolar pH modulates the traffic of trypomastigote parasitophorous vacuoles in these doubly infected cells. (C) 2003 Australian Society for Parasitology Inc. Published by Elsevier B.V. All rights reserved.
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    Actin-rich structures formed during the invasion of cultured cells by infective forms of Trypanosoma cruzi
    (Urban & Fischer Verlag, 1999-12-01) Procopio, Daniela de Oliveira [UNIFESP]; Barros, Helena Cristina [UNIFESP]; Mortara, Renato Arruda [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Previous work has shown that Trypanosoma cruzi extracellular amastigotes as well as metacyclic trypomastigotes infect cultured cells in a highly specific parasite form-cell type interaction. In this work we have investigated the mode of interaction of both forms with HeLa and Vero cells using scanning electron and confocal fluorescence microscopy. We examined the distribution of several host cell components as well as extracellular matrix elements during cell invasion by both T. cruzi infective forms. Scanning electron microscopy showed that membrane expansions formed during the invasion of cells by extracellular amastigotes. These expansions correspond to small cup-like structures in HeLa cells and are comparatively larger crater-like in Vero cells. We detected by confocal microscopy actin-rich structures associated with the internalisation of both infective forms of the parasite that correspond to the membrane expansions. Confocal fluorescence microscopy combining DIC images of cells labelled with monoclonal antibodies to phosphotyrosine, cytoskeletal elements, integrins, and extracellular matrix components revealed that some of the components like gelsolin and a-actinin accumulate in actin-rich structures formed in the invasion of amastigotes of both cell types. Others, like vinculin and alpha 2 integrin may be present in these structures without evident accumulation. And finally some actin-rich processes may be devoid of components like fibronectin or alpha V integrin. These studies provide evidence that the repertoire of host cell/extracellular matrix components that engage in the invasion process of T, cruzi forms is cell type- and parasite form-dependent.
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    Diagnosis of epithelial ingrowth after penetrating keratoplasty with confocal microscopy
    (Lippincott Williams & Wilkins, 2006-10-01) Forseto, Adriana dos Santos; Santos, Myrna Serapiao dos; Sampaio, Angelica; Mascaro, Vera; Nose, Walton; Eye Clin Day Hosp; Universidade Federal de São Paulo (UNIFESP); Hosp Servidor Publ Estadual
    Purpose: To report confocal microscopy use in the clinical diagnosis of epithelial ingrowth after penetrating keratoplasty (PKP).Methods: A 36-year-old female patient with keratoconus developed a well-delimited posterior hazy membrane covering the inferior two thirds of the cornea 3 months after an uneventful PKP. A posterior corneal line was present resembling an endothelial graft rejection line, but with no keratic precipitates or corneal edema. Ocular hypertension was not observed, Confocal microscopy was performed to elucidate the diagnosis.Results: Confocal microscopy showed epithelium and stroma with normal findings. Two distinct cellular types were presented at the endothelium layer. Enlarged endothelial cells were observed in the superior part of the cornea up to the leading edge of the hazy membrane. in the middle and inferior part of the graft, the cells were larger, with polygonal shape and easily recognizable hyperreflective nuclei, suggestive of epithelial cells. With these confocal microscopy findings, the patient was promptly submitted to another PKP. Histologic analysis confirmed the diagnosis of epithelial ingrowth.Conclusion: Confocal microscopy imaging technique seems to be a useful tool in the early diagnosis of epithelial ingrowth after PKP.
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    Disruption of myofibrillar proteins in cardiac muscle of Calomys callosus chronically infected with Trypanosoma cruzi and treated with immunosuppressive agent
    (Springer, 2005-10-01) Taniwaki, N. N.; Andreoli, W. K.; Calabrese, K. S.; Silva, S. da; Mortara, R. A.; Universidade Federal de São Paulo (UNIFESP); Inst Adolfo Lutz Registro; Dept Protozool
    Calomys callosus (Rodentia: Cricetidae) chronically infected with CL strain of Trypanosoma cruzi undergo recrudescence of the acute phase when treated with the immunosuppressor cyclophosphamide. the distribution of cytoskeletal proteins in cardiac tissue of immunosuppressed animals was mapped by immunofluorescence and electron microscopy to evaluate myofibrillar distribution during the intracellular life cycle of T. cruzi. Cardiac muscle sections showed enhancement of myocarditis and parasite proliferation after immunosuppression. Immunofluorescence using monoclonal antibodies against myosin, actin, desmin, titin, tropomyosin, and troponin T demonstrated disruption and loss of contractile proteins, such as myosin and actin. Desmin and titin were irregularly distributed in close proximity to parasite nests. Ultrastructural observations confirmed alterations of cardiac cells with Z-line fragmentation, indistinguishable I-bands and A-bands, and loss of myofibrillar elements. the disruption of the muscle cell architecture was greater as infection progressed, probably as a result of increased myocarditis and physical displacement due to the activity of flagellated parasites.
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    Distribution of epitopes of Trypanosoma cruzi amastigotes during the intracellular life cycle within mammalian cells
    (Soc Protozoologists, 1997-07-01) Barros, H. C.; Verbisck, N. V.; DaSilva, S.; Araguth, M. F.; Mortara, R. A.; Universidade Federal de São Paulo (UNIFESP)
    In this study we have examined the distribution of epitopes defined by monoclonal antibodies raised against Trypanosoma cruzi amastigotes during the intracellular life cycle of the parasite. We have raised monoclonal antibodies towards amastigote forms and performed preliminary immunochemical characterization of their reactivities. MAB 1D9, 3G8, 2B7, 3B9, and 4B9 react with carbohydrate epitopes of the parasite major surface glycoprotein-Ssp-4 defined by MAB 2C2 [5]; MAB 4B5 reacts with a noncarbohydrate epitope in all developmental stages of the parasite, and MAB 3B2 also detects a noncarbohydrate epitope preferentially in T. cruzi flagellated forms. Vero cells infected with tissue culture-derived trypomastigotes of clone D11 (G strain) were fixed at different times during the intracellular proliferation of parasites, and processed for immune-electron microscopy and confocal immunofluorescence with the different monoclonal antibodies. We observed that while the surface distribution of MAB 2C2 and 4B9 epitopes was uniform throughout the cycle, MAB 1D9, 3G8, and 2B7 reacted with cytoplasmic membrane-bound compartments of the amastigotes. MAB 3B9 displayed a unique surface dentate pattern in some amastigotes. MAB 4B5 recognized a curved-shaped structure at the flagellar pocket region in some intracellular amastigotes and localized to the membrane in dividing forms. in intracellular trypomastigotes, MAB 4B5 also displayed a punctate pattern near the flagellar pocket.
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    Diterpenoids from Azorella compacta (Umbelliferae) active on Trypanosoma cruzi
    (Instituto Oswaldo Cruz, Ministério da Saúde, 2003-04-01) Araya, Jorge E; Neira, Iván; Silva, Solange da [UNIFESP]; Mortara, Renato Arruda [UNIFESP]; Manque, Patricio; Cordero, Esteban Mauricio [UNIFESP]; Sagua, Hernán; Loyola, Alberto; Bórquez, Jorge; Morales, Glauco; González, Jorge; Universidad de Antofagasta Departamento de Tecnología Médica Unidad de Parasitología; Universidad de Antofagasta Departamento de Química Laboratorio de Productos Naturales; Universidade Federal de São Paulo (UNIFESP)
    The anti-Trypanosoma cruzi activity of natural products isolated from Azorella compacta was evaluated, with particular emphasis on their effect against intracellular amastigotes. Five diterpenoids from A. compacta derived from mulinane and azorellane were isolated and identified. Only two products, named azorellanol (Y-2) and mulin-11,3-dien-20-oic acid (Y-5), showed trypanocidal activity against all stages of T. cruzi including intracellular amastigotes. At 10 µM, these compounds displayed a strong lytic activity. It ranged from 88.4 ± 0.6 to 99.0 ± 1 % for all strains and stages evaluate, with an IC50 /18 h values of 20-84 µM and 41-87 µM, respectively. The development of intracellular amastigotes was also inhibited by nearly 60% at 25 µM. The trypanocidal molecules Y-2 and Y-5 did show different degrees of cytotoxicity depending on the cell line tested, with an IC50 /24 h ranging from 33.2 to 161.2 µM. We evaluated the effect of diterpenoids against intracellular T. cruzi forms by immunofluorescent identification of a specific membrane molecular marker (Ssp-4 antigen) of the T. cruzi amastigote forms. The accuracy and reproducibility of the measurements were found to be outstanding when examined by confocal microscopy.
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    Heterologous expression of a Trypanosoma cruzi surface glycoprotein (gp82) in mammalian cells indicates the existence of different signal sequence requirements and processing
    (Soc Protozoologists, 1999-11-01) Ramirez, M. I.; Boscardin, S. B.; Han, S. W.; Paranhos-Baccala, G.; Yoshida, N.; Kelly, J. M.; Mortara, R. A.; Silveira, J. F. de; Universidade Federal de São Paulo (UNIFESP); Ecole Normale Super Lyon; London Sch Hyg & Trop Med
    Metacyclic trypomastigotes of Trypanosoma cruzi express a developmentally regulated 82 kDa surface glycoprotein (gp82) that has been implicated in the mammalian cell invasion. When the non-infective epimastigote stage of the parasite was transfected with a vector containing the gp82 gene, an 82 kDa surface glycoprotein, which was indistinguishable from the metacyclic stage protein, was expressed. in contrast, when the same gene was expressed in transfected mammalian cells, although a large amount of protein was produced, it was not imported into the endoplasmic reticulum and glycosylated. This blockage in targeting and processing could be partially compensated for by the addition of a virus haemagglutinin signal peptide to the amino terminus of gp82. Thus, the requirements for membrane protein processing are distinct in mammals and T cruzi, and an intrinsic feature of the gp82 prevents subsequent sorting to the mammalian cell surface. These results could be useful in the development of new DNA vaccines against T. cruzi employing parasite genes encoding immunodominant surface glycoproteins.
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    Motility, morphology and phylogeny of the plasmodial worm, Ceratomyxa vermiformis n. sp (Cnidaria: Myxozoa: Myxosporea)
    (Cambridge Univ Press, 2017) Adriano, E. A. [UNIFESP]; Okamura, B.
    The Myxozoa demonstrate extensive morphological simplification and miniaturization relative to their free-living cnidarian ancestors. This is particularly pronounced in the highly derived myxosporeans, which develop as plasmodia and pseudoplasmodia. To date, motility in these stages has been linked with membrane deformation (e.g. as pseudopodia and mobile folds). Here we illustrate a motile, elongate plasmodium that undergoes coordinated undulatory locomotion, revealing remarkable convergence to a functional worm at the cellular level. Ultrastructural and confocal analyses of these plasmodia identify a highly differentiated external layer containing an actin-rich network, long tubular mitochondria, abundant microtubules, a secreted glycocalyx layer, and an internal region where sporogony occurs and which contains homogeneously distributed granular/fibrillar material. We consider how some of these features may support motility. We also describe the species based on spore morphology and SSU rDNA sequence data, undertake molecular phylogenetic analysis to place it within an early-diverging clade of the ceratomyxids, and evaluate the resultant implications for classification (validity of the genus Meglitschia) and for inferring early host environments (freshwater) of ceratomyxids.
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    Phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of GPI-anchored surface molecules of Trypanosoma cruzi triggers in vitro morphological reorganization of trypomastigotes
    (Soc Protozoologists, 2001-01-01) Mortara, R. A.; Minelli, LMS; Vandekerchove, F.; Nussenzweig, V; Ramalho-Pinto, F. J.; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); NYU Med Ctr
    Trypanosoma cruzi trypomastigotes treated with phosphatidylinositol-specific phospholipase C (PI-PLC) in vitro are rapidly induced to differentiate into round forms. Using confocal microscopy, we were able to show that trypomastigotes treated with PI-PLC initiate the process of flagellum remodeling by 30 sec after contact with the enzyme and amastigote-like forms are detected as early as 10 min after PI-PLC treatment. Scanning and transmission electron microscopy indicate that trypomastigotes undergo a previously undescribed process of flagellum circularization and internalization. Analysis of the flagellar complex with monoclonal antibody 4D9 shows heterogeneous labeling among the parasites, suggesting a remodeling of these molecules. After PI-PLC treatment, parasites rapidly lose the surface marker Ssp-3 and 24 h post-treatment they begin to exhibit a circular nucleus and a rod-shaped kinetoplast. By flow cytometry analysis and confocal microscopy, the Ssp-4 amastigote-specific epitope can be detected on the parasite surface. This indicates that thr release of trypomastigote GPI-anchored molecules by exogenous PI-PLC in vitro can trigger morphological changes.
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    Praziquantel and albendazole damaging action on in vitro developing Mesocestoides corti (Platyhelminthes : Cestoda)
    (Elsevier B.V., 2006-03-01) Markoski, M. M.; Trindade, E. S.; Cabrera, G.; Laschuk, A.; Galanti, N.; Zaha, A.; Nader, H. B.; Ferreira, H. B.; Univ Fed Rio Grande Sul; Universidade Federal de São Paulo (UNIFESP); Univ Chile
    Parasitic flatworms present several steps of body architecture rearrangement during their fast transition from one developmental stage to another, which are, at least in part, responsible for their evasion from host immune response. Besides, different developmental stages present different degrees of susceptibility to drug action, and the identification of more susceptible stages is of importance for the definition of therapeutical approaches. Mesocastoides corti (syn. Mesocestoides vogae) is considered a good model to Study cestode biology because it can be easily manipulated both in vivo and in vitro and due to its relatively close relationship to cestodes of medical relevance, such as those from genera Echinococcus or Taenia. We have analyzed the damaging action of two broad spectrum anthelmintic drugs (praziquantel and albendazole) throughout the in vitro strobilization process of M. corti in order to identify developmental stages or body structures more Susceptible to these drugs. Tetrathyridia (larval stage) and segmented-induced worms were cultivated and treated with praziquantel and albendazole. Whole mounted samples, taken from different developmental stages, were fixed and stained with fluorophore-labeled WGA lectin and phalloidin for the analysis of tegument and Muscles, respectively. Conflocal laser scanning microscopy was used to identify anatomical changes and lesions caused by each anthelmintic drug in a 3D view. We demonstrated that both praziquantel and albendazole cause extensive tissue damage, especially on tegument, and that adult forms were the most Susceptible to drug exposure. (c) 2005 Elsevier Ireland Ltd. All rights reserved.
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    Reflection imaging of China ink-perfused brain vasculature using confocal laser-scanning microscopy after clarification of brain tissue by the Spalteholz method
    (Wiley, 2017) Gutierre, R. C. [UNIFESP]; Vannucci Campos, D. [UNIFESP; Mortara, R. A. [UNIFESP; Coppi, A. A.; Arida, R. M. [UNIFESP
    Confocal laser- scanning microscopy is a useful tool for visualizing neurons and glia in transparent preparations of brain tissue from laboratory animals. Currently, imaging capillaries and venules in transparent brain tissues requires the use of fluorescent proteins. Here, we show that vessels can be imaged by confocal laser- scanning microscopy in transparent cortical, hippocampal and cerebellar preparations after clarification of China inkinjected specimens by the Spalteholz method. This method may be suitable for global, three- dimensional, quantitative analyses of vessels, including stereological estimations of total volume and length and of surface area of vessels, which constitute indirect approaches to investigate angiogenesis.
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    Study of acute chagasic mice under immunosuppressive therapy by cyclosporin A: modulation and confocal analysis of inflammatory reaction
    (Elsevier B.V., 2000-04-01) Calabrese, K. S.; Paradela, ASRC; Valle, T. Z. do; Tedesco, R. C.; Silva, S.; Mortara, R. A.; Costa, SCG da; Ist Oswaldo Cruz; Universidade Federal de São Paulo (UNIFESP)
    The in vivo effects of cyclosporin A (CsA) on Trypanosoma cruzi infection were examined using different schedules of the drug in mice infected with the Y strain. Parasitaemia at day 8 after infection among CsA-treated animals was usually higher than control infected non-treated mice. On the other hand, mortality analysis showed that animals CsA-treated either with 200 mg/kg 2 days before infection or with therapeutic doses (10 mg/kg every other day) showed almost the same mean time of death (35.8 and 38.2 days, respectively). in these groups mice died 50% less than control infected non-treated ones. the mean time of death in the animals treated with 200 mg/kg 5 days after infection and in infected non-treated control mice were respectively 29.0 and 22.6 days. the kinetics analysis of the leukocyte population of animals treated with a single dose of 200 mg/kg of CsA before or after infection did not show the alternate pattern of leukopenia/leukocytosis observed in control groups of infected mice but differential cell counts indicated a modulatory action upon circulating leukocytes of therapeutic doses of CsA. the animals treated with any of the CsA schedules showed a moderate to intense diffuse inflammatory reaction exhibiting mainly mononuclear cells in the heart. Immunofluorescence analysis by confocal microscopy revealed that macrophages are a major component of the inflammatory infiltrate in all groups of CsA-treated mice and also in the control group. (C) 2000 Elsevier Science B.V. All rights reserved.
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    T cell subpopulations in myocardial inflammatory infiltrates detected by confocal microscopy: dose dependence in mice treated with cyclophosphamide during acute Trypanosoma cruzi infection
    (Elsevier B.V., 2003-04-01) Calabrese, K. S.; Paradela, ASRC; Valle, T. Z. do; Tedesco, R. C.; Leonardo, R.; Mortara, R. A.; Costa, SCG da; Inst Oswaldo Cruz; Universidade Federal de São Paulo (UNIFESP)
    In this article, we have characterized cell subpopulations found in the hearts of mice presenting acute Chagas' disease by immunocytochemistry and subjected to different schedules of an immunosuppressive therapy with cyclophosphamide (CY). in this comparative study, CY treatment with different doses was carried out before or after infection with Trypanosoma cruzi Y strain trypomastigotes, enabling us to discriminate the parasitemic kinetics and inflammatory processes in the heart, 12 d after infection. Animals treated with 200 mg/kg of CY 2 d before infection presented high parasitaemia as well as heavy inflammation and low parasite loads in the heart. Mice treated 5 d after infection with the same dose, developed the same parasitaemic peak but were not able to control it. Their heart did not present inflammation, but a high number of parasites could be seen. Animals treated with five 3 mg/kg doses of CY every other day presented heavy inflammatory reaction and low parasitaemia. in this group, as well as the one treated before infection, immunocytochemistry studies have shown predominance of CD8(+) T cells in the myocardium. On the other hand, mice treated with 200 mg/kg of CY 5 d after infection, presented small amounts of CD4(+) T cells while no CD8(+) could be found. These results have confirmed the dose dependence influence of this drug on the T cell populations in the inflammatory infiltrates as well as the importance of the schedule employed. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.
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    Trypanosoma cruzi disrupts myofibrillar organization and intracellular calcium levels in mouse neonatal cardiomyocytes
    (Springer, 2006-06-01) Taniwaki, N. N.; Machado, F. S.; Massensini, A. R.; Mortara, R. A.; Universidade Federal de São Paulo (UNIFESP); Inst Adolofo Lutz; Universidade de São Paulo (USP); Universidade Federal de Minas Gerais (UFMG)
    Immunofluorescence studies of normal and Trypanosoma cruzi-infected primary cultures of heart muscle cells were performed to gather information about the arrangement of myofibrillar components during the intracellular life cycle of this parasite. By using a panel of monoclonal antibodies against various myofibrillar proteins, a progressive disruption and loss of contractile proteins (such myosin and actin) of the host cell was detected during infection. the host cell formed a loose network of myofibrillar proteins around the parasites. Breakdown of the myofibrils occurred in regions where the parasites were present, and heavily infected cells showed myofibrillar proteins at their periphery. in parallel, we investigated the effect of T. cruzi infection on intracellular calcium levels by using a Ca(2+) fluorescent indicator (confocal microscopy). Infected cardiomyocytes displayed a marked impairment in contractility, and calcium influxes became irregular and less intense when compared with those of non-infected cells. Our results demonstrate that T. cruzi infection dramatically affects calcium fluxes and causes myofibrillar breakdown disturbing cardiomyocyte contractility.