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- ItemSomente MetadadadosDistribution of Trypanosoma cruzi stage-specific epitopes in cardiac muscle of Calomys callosus, BALB/c mice, and cultured cells infected with different infective forms(Elsevier B.V., 2007-07-01) Taniwaki, Noerni N.; Silva, Claudio Vieira da; Silva, Solange da; Mortara, Renato A.; Universidade Federal de São Paulo (UNIFESP); Secao Microscopia Eletron Inst Adolfo LutzTo examine whether distinct parasite infective forms or the mammalian host could affect the distribution of Trypanosoma cruzi stage-specific epitopes defined by monoclonal antibodies (Mabs) raised against mammalian-stage parasite forms, immunofluorescence studies followed the intracellular life cycle of the parasite in the cardiac muscle of Calomys callosus and BALB/c mice in the acute phase of the disease and in LLC-MK(2) Cultured cells. Animals and cells were infected either with tissue-culture derived trypomastigotes (TCT) or bloodstream trypornastigotes (BT) from the Y strain of T cruzi. Samples were examined under confocal fluorescence microscopy after labeling with Mabs 2C2, 1D9, 2B7, 3G8, 3B9, and 4B9 that react with carbohydrate epitopes on Ssp-4, a major amastigote surface glycoprotein; Mab 4B5 that identifies a noncarbohydrate epitope on all intracellular parasites stages, and Mab 3B2 that also recognizes a noncarbohydrate epitope expressed only in flagellated forms. Samples were double labeled with DAPI to visualize parasites' kinetoplasts and nuclei. Most of the Mabs used in this work displayed a surface labeling pattern on amastigotes present in Calomys and mice hearts, and in LLC-MK2 cultured cells infected with BT or TCT. Mab 2B7, however, displayed a marked polymorphic distribution in antigen expression between both mammalian hosts, independent on the infective form. Beyond the polymorphic distribution of amastigote surface epitopes, Calomys, and mice heart sections presented several inflammatory cells around amastigotes and trypornastigotes nests. (c) 2007 Elsevier B.V. All rights reserved.
- ItemAcesso aberto (Open Access)Features of host cell invasion by different infective forms of Trypanosoma cruzi(Instituto Oswaldo Cruz, Ministério da Saúde, 1999-09-01) Mortara, Renato Arruda [UNIFESP]; Procópio, Daniela O [UNIFESP]; Barros, Helena C [UNIFESP]; Verbisck, Newton V [UNIFESP]; Andreoli, Walter K [UNIFESP]; Silva, Ricardo Bs [UNIFESP]; Silva, Solange da [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Through its life cycle from the insect vector to mammalian hosts Trypanosoma cruzi has developed clever strategies to reach the intracellular milieu where it grows sheltered from the hosts' immune system. We have been interested in several aspects of in vitro interactions of different infective forms of the parasite with cultured mammalian cells. We have observed that not only the classically infective trypomastigotes but also amastigotes, originated from the extracellular differentiation of trypomastigotes, can infect cultured cells. Interestingly, the process of invasion of different parasite infective forms is remarkably distinct and also highly dependent on the host cell type.
- ItemSomente MetadadadosInvolvement of Ssp-4-related carbohydrate epitopes in mammalian cell invasion by Trypanosoma cruzi amastigotes(Elsevier B.V., 2006-07-01) Silva, Claudio Vieira da; Luquetti, Alejandro O.; Rassi, Anis; Mortara, Renato Arruda; Universidade Federal de São Paulo (UNIFESP); Universidade Federal de Goiás (UFG)We examined whether the expression of Ssp-4-related carbohydrate epitopes defined by monoclonal antibodies ID9 and 2B7 was related to cell invasion by Trypanosonia cruzi amastigotes from different isolates and whether the highest expression of the epitope defined by MAb ID9 would confer greater infectivity. Confocal microscopy showed that both epitopes localize to the membrane of amastigotes from 569, 588, 573, 587 and SC2005 isolates, similar to the G isolate, whereas the CL isolate showed a punctate and diffuse staining. Flow cytometry revealed inter and intra-isolate variable expression of these epitopes. Apart from the lower expression of MAb 2B7 epitope by intracellular amastigotes of the SC2005 isolate, amastigotes from chagasic patient isolates expressed both epitopes similar to the G isolate, in contrast to CL isolate, that showed lower expression of both epitopes. MAb ID9 did not react with CL isolate on immunoblots and reacted poorly with 588 and 587 parasites. MAb 2B7 preferentially reacted with an epitope on an 84 kDa component in G and 573 isolates. Invasion assays revealed that despite the fact that arnastigotes from chagasic patient isolates displayed high levels of the epitope defined by MAb 1D9, only isolate 588 invaded host cells in levels comparable to that of isolate G. Both MAbs specifically inhibited cell invasion by G and 588, but not CL. These results suggested that the highest expression of MAb 1D9 epitope was not sufficient to confer higher infectivity on the isolate, and besides the two epitopes, other factors may modulate the invasiveness of extracellular amastigotes from the different isolates. (c) 2006 Elsevier SAS. All rights reserved.
- ItemSomente MetadadadosLeishmania (L.) amazonensis: Fusion between parasitophorous vacuoles in infected bone-marrow derived mouse macrophages(Elsevier B.V., 2008-05-01) Real, F. [UNIFESP]; Pouchelet, M.; Rabinovitch, M. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); INSERM[Leishmania (L.)] amazonensis amastigotes reside in macrophages within spacious parasitophorous vacuoles (PVs) which may contain numerous parasites. After sporadic fusion events were detected by time-lapse cinemicrography, PV fusion was examined in two different models. in single infections, it was inferred from the reduction in PV numbers per cell. in a reinfection model, macrophages infected with unlabeled amastigotes were reinfected with GFP-transfected- or carboxyfluorescein diacetate succinimidyl ester-labeled parasites, and fusion was detected by the colocalization of labeled and unlabeled amastigotes in the same PVs. the main findings were: (1) as expected, fusion frequency increased with the multiplicity of infection; (2) most fusion events took place in the first 24 h of infection or reinfection, prior to the multiplication of incoming parasites; (3) resident and incoming parasites multiplied at similar rates in fused PVs. the model should be useful in studies of parasite and host cell factors and mechanisms involved in PV fusogenicity. (C) 2007 Elsevier Inc. All rights reserved.
- ItemSomente MetadadadosNovel strategy in Trypanosoma cruzi cell invasion: Implication of cholesterol and host cell microdomains(Elsevier B.V., 2007-11-01) Fernandes, Maria Cecilia; Cortez, Mauro; Geraldo Yoneyama, Kelly Aparecida; Straus, Anita Hilda; Yoshida, Nobuko; Mortara, Renato Arruda; Universidade Federal de São Paulo (UNIFESP)Trypanosoma cruzi, the etiological agent of Chagas' disease, is an obligatory intracellular parasite in the mammalian host. in order to invade a wide variety of mammalian cells, T cruzi engages parasite components that are differentially expressed among strains and infective forms. Because the identification of putative protein receptors has been particularly challenging, we investigated whether cholesterol and membrane rafts, sterol- and sphingolipid-enriched membrane domains, could be general host surface components involved in invasion of metacyclic trypomastigotes and extracellular amastigotes of two parasite strains with distinct infectivities. HeLa or Vero cells treated with methyl-beta-cyclodextrin (M beta CD) are less susceptible to invasion by both infective forms, and the effect was dose-dependent for trypomastigote but not amastigote invasion. Moreover, treatment of parasites with MPCD only inhibited trypomastigote invasion. Filipin labeling confirmed that host cell cholesterol concentrated at the invasion sites. Binding of a cholera toxin B subunit (CTX-B) to ganglioside GM1, a marker of membrane rafts, inhibited parasite infection. Cell labeling with CTX-B conjugated to fluorescein isothiocyanate revealed that not only cholesterol but also GM1 is implicated in parasite entry. These findings thus indicate that microdomains present in mammalian cell membranes, that are enriched in cholesterol and GM1, are involved in invasion by T cruzi infective forms. (c) 2007 Australian Society for Parasitology Inc. Published by Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosTrypanosoma cruzi cell invasion and traffic: Influence of Coxiella burnetii and pH in a comparative study between distinct infective forms(Elsevier B.V., 2007-07-01) Fernandes, Maria Cecilia; L'Abbate, Carolina [UNIFESP]; Andreoli, Walter Kindro; Mortara, Renato Arruda; Universidade Federal de São Paulo (UNIFESP)Previous studies have shown that Coxiella burnetii, an intracellular bacterium that resides within acidified vacuoles with secondary lysosomal characteristics, is an effective modulator of the intracellular traffic of trypomastigote forms of Trypanosoma cruzi.. in addition, vacuolar and cellular pH are related to fusion events that result in doubly infected phagosomes. T cruzi, the etiological agent of Chagas' disease, occurs as different strains grouped in two major phylogenetic lineages: T cruzi I, associated with the sylvatic cycle, and T cruzi II, linked to the human disease. in this work we compared extracellular amastigotes (EA), metacyclic trypomastigotes (NIT) and tissue Culture derived trypomastigotes (TCT) belonging to T. cruzi I or T. cruzi II for their ability to invade and escape from their parasitophorous vacuole (PV), in Vero cells or Vero cells harboring the bacterium, C burnetti. Distinct invasion patterns were observed between different infective stages and between infective forms of different strains. Studies on the transference kinetics revealed that pH modulates the intracellular traffic of each infective stage, but this influence is not exclusive for each phylogenetic group. Endosomal to lysosomal sequential labeling with EEA-1 and LAMP-1 of the PV formed during the entry of each infective form revealed that the phagosome maturation processes are distinct but not strain-dependent. Due to their low hemolysin and trans-sialidase activities, MTs are retained for longer periods in LAMP-1 positive vacuoles. Our results thus suggest that despite the contrasting invasion capabilities, parasites of distinct phylogenetic group behave in similar fashion once inside the host cell. (c) 2007 Elsevier B.V. All rights reserved.
- ItemAcesso aberto (Open Access)Trypanosoma cruzi: amastigote polymorphism defined by monoclonal antibodies(Associação Brasileira de Divulgação Científica, 1998-12-01) Verbisck, Newton Valério [UNIFESP]; Da-Silva, S. [UNIFESP]; Mortara, Renato Arruda [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)We have raised monoclonal antibodies (mAbs) directed towards amastigote forms of Trypanosoma cruzi, and shown that mAbs 1D9 and 4B9 are carbohydrate while mAb 4B5 activity is resistant to periodate oxidation of the antigen. Here we used an ELISA to quantitate and compare the expression of surface epitopes on fixed parasites among different parasite isolates. The expression of markers varied among T. cruzi amastigotes isolated from infected cells or after extracellular differentiation of trypomastigotes. Moreover, we also observed an extensive polymorphic expression of these epitopes among amastigotes derived from different strains and clones. For instance, mAb 2C2 strongly and evenly reacted with 9 strains and clones (G, Y, CL, Tulahuen, MD, and F, and clones Sylvio X-10/4, D11, and CL.B), with absorbance at 492 nm (A492 nm) from 0.6 to 0.8. By contrast, mAb 4B5 had a higher expression in Tulahuen amastigotes (around 0.9 at 492 nm) whereas its reactivity with amastigotes from clones CL.B, Sylvio X-10/4 and D11 was much lower (around 0.4). mAb 1D9 displayed an interesting pattern of reactivity with amastigotes of the different strains and clones (A492 nm of G>D11³Sylvio X-10/4 = MD>Tulahuen = F = Y>CL>CL.B). Finally, we observed that mAb 4B9 had the lowest reaction with the parasites studied, with higher values of A492 nm with Y strain (around 0.6) and lower values with Tulahuen, F and CL.B strains (around 0.2). Immunoblotting analysis also showed extensive variations among amastigotes of the various parasite isolates and mAbs 4B9, 1D9 and 4B5 revealed significant differences in expression between clones and parental strains. These data describe a previously uncharacterized polymorphism of T. cruzi amastigote surface components.