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- ItemSomente MetadadadosActin-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.
- ItemSomente MetadadadosB and T cell responses elicited by monoclonal anti-idiotypic antibody (Ab2 beta) mimicking gp43 from Paracoccidioides brasiliensis(Blackwell Publishing Ltd, 2004-07-01) Souza, E. B.; Lopes, Jose Daniel [UNIFESP]; Almeida, SR; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)Paracoccidioidomycosis is a systemic mycosis endemic in Latin America, with a high prevalence in Brazil, Argentina, Colombia and Venezuela. the aetiological agent of disease is the thermal dimorphic fungus, Paracoccidioides brasiliensis. A glycoprotein of 43 kD (gp43) is the major antigen of P. brasiliensis. Antibodies directed to this antigen are detected in the sera of all patients with paracoccidioidomycosis (PCM). Recently, it has been shown that mice immunized with anti-gp43 monoclonal antibodies (MAbs) (Ab1), induce the idiotypic cascade in the gp43 system, which produced both, anti-Id antibodies (Ab2) and anti-anti-Id antibodies (Ab3). To further characterize the idiotypic cascade modulation in mice immunized with anti-gp43 MAb 17c, hybridomas were produced. Ab2 MAbs named 7.B12 inhibited (>95%) the binding of gp43 to MAb 17c (Ab1), suggesting that this anti-Id MAb bind to the idiotope, thus fulfilling the internal image criteria. To elucidate whether Ab2 MAb could act as antigen in serological assays, instead of gp43, sera from PCM patients were tested. Using an ELISA test, it was observed that antibodies from patients and not normal serum bound to Ab2. However, the ELISA test using Ab2 bound to the solid phase made possible to serologically monitor the patients after antifungal therapy, showing an equivalent curve when compared with ELISA test employing purified gp43. Our results also showed that, when mice were immunized with Ab2beta and their cells were exposed to gp43 in vitro, a T cell proliferation response was observed.
- ItemSomente MetadadadosDistribution 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.
- ItemSomente MetadadadosEvidence of idiotypic modulation in the immune response to gp43, the major antigenic component of Paracoccidioides brasiliensis in both mice and humans(Blackwell Science Ltd, 1998-10-01) Souza, A. R.; Gesztesi, Jean-Luc [UNIFESP]; Moraes, Jane Zveiter de [UNIFESP]; Cruz, CRB; Sato, J.; Mariano, Mario [UNIFESP]; Lopes, Jose Daniel [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Latin America, with a high prevalence in Brazil, Argentina, Colombia and Venezuela. The aetiologic agent of disease is a thermal dimorphic fungus, Paracoccidioides brasiliensis. A glycoprotein of 43 000D (gp43) is the major antigen of P. brasiliensis. Antibodies directed to this antigen are detected in the sera of all patients with PCM. Gp43 binds to laminin, thus participating in adhesion, invasion and pathogenesis of the fungus. As the role of antibodies in PCM is not fully understood, we decided to investigate the outcome of mice immunization with three distinct anti-gp43 MoAbs (17c, 8a and 24a) coupled with keyhole limpet haemocyanin (KLH). Results show not only the expected presence of anti Id (AB2) antibodies in the sera of these animals but also a spontaneous and increasing amount of anti-anti-Id (AB3) antibodies after the third course of immunization. Hybridomas producing both AB2 and AB3 MoAbs were obtained using spleen cells from mice immunized with MoAb 17c. AB3 MoAbs were also obtained with spleen cells of mice immunized with MoAbs 8a and 24a. It was also shown that human PCM patients' sera with high titres of anti-gp43 antibodies generate anti-Id antibodies. These data suggest that the immune response to P. brasiliensis can be spontaneously modulated by the idiotypic network.
- ItemSomente MetadadadosExpression of GP82 and GP90 surface glycoprotein genes of Trypanosoma cruzi during in vivo metacyclogenesis in the insect vector Rhodnius prolixus(Elsevier B.V., 2008-01-01) Cordero, Esteban M. [UNIFESP]; Gentil, Luciana G. [UNIFESP]; Crisante, Gladys; Ramirez, Jose Luis; Yoshida, Nobuko [UNIFESP]; Anez, Nestor; Silveira, Jose Franco da [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Los Andes; Fdn Inst Estudios Avanzados IDEA MCTTrypanosoma cruzi, the parasite causing Chagas' disease, relies on triatomines for its transmission. T cruzi metacyclic trypomastigotes express GP82 and GP90, which are developmentally regulated surface proteins that have been implicated in host cell invasion. We used quantitative RT-PCR to quantify GP90 and GP82 mRNA levels expressed by T cruzi in the digestive tract of experimentally infected Rhodnius prolixus at different times post infection. Translation of these transcripts was assessed by immunofluorescence using specific monoclonal antibodies against GP90 and GP82. We found that although GP82 and GP90 proteins were not detected in epimastigote cells by immunofluorescence, transcripts were present at lower levels. Increased levels of GP90 and GP82 transcripts and the appearance of these proteins on the parasite surface were accompanied by morphological differentiation from epimastigotes into metacyclic forms. Our data suggest that during in vivo metacyclogenesis there is a coordinated mechanism that links stabilization of GP90 and GP82 mRNAs with their translation. (C) 2007 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosGlycosphingolipid antigens from Leishmania (L) amazonensis amastigotes. Binding of anti-glycosphingolipid monoclonal antibodies in vitro and in vivo(Assoc Bras Divulg Cientifica, 1997-03-01) Straus, Anita Hilda [UNIFESP]; Valero, Valderez Bastos [UNIFESP]; Takizawa, C. M.; Levery, S. B.; Toledo, Marcos Sergio de [UNIFESP]; Suzuki, E.; Salyan, MEK; Hakomori, S.; Barbieri, Clara Lucia [UNIFESP]; Takahashi, Helio Kiyoshi [UNIFESP]; UNIV GEORGIA; PACIFIC NW RES FDN; UNIV WASHINGTON; Universidade Federal de São Paulo (UNIFESP)Specific glycosphingolipid antigens of Leishmania (L.) amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs) ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC). Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L.) amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of I-125-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues.
- ItemSomente MetadadadosImmunochemical and subcellular localization of the 43 kDa glycoprotein antigen of Paracoccidioides brasiliensis with monoclonal antibodies(Blackwell Science Ltd, 1996-06-01) Straus, A. H.; Freymuller, E.; Travassos, Luiz Rodolpho [UNIFESP]; Takahashi, H. K.; Universidade Federal de São Paulo (UNIFESP)Two monoclonal antibodies ST-7 (IgG1) and ST-8 (IgG2b), directed against a 43 kDa glycoprotein (gp43) of Paracoccidioides brasiliensis were produced. It was possible to detect the gp43 by ELISA in amounts as low as 100 ng per well, and by Western blot about 300 ng were detected. Mild treatment of the gp43 with sodium metaperiodate did not alter its reactivity with ST-7 and ST-8, which suggests that these MAbs recognize peptide epitopes. Confirming the periodate oxidation data, the 38 kDa protein resulting from deglycosylation of the gp43 with trifluoromethanesulphonic acid (TMFS), was reactive with ST-7 and ST-8. Immunoelectron microscopy showed that the gp43 is stored inside large dense core vesicles, which flowed into the plasma membrane and extruded from cell membrane into the cell wall. Finally the antigen was secreted into the extracellular space as dense membrane-free material. Secretion of the gp43 occurred at scattered sites interspersed along the cell surface.
- ItemAcesso aberto (Open Access)Interaction between Shiga Toxin and Monoclonal Antibodies: Binding Characteristics and in Vitro Neutralizing Abilities(Mdpi Ag, 2012-09-01) Rocha, Leticia B.; Luz, Daniela E.; Moraes, Claudia T. P.; Caravelli, Andressa; Fernandes, Irene; Guth, Beatriz Ernestina Cabilio [UNIFESP]; Horton, Denise S. P. Q.; Piazza, Roxane M. F.; Butantan Inst; Universidade Federal de São Paulo (UNIFESP)Monoclonal antibodies (MAbs) have been employed either for diagnosis or treatment of infections caused by different pathogens. Specifically for Shiga toxin-producing Escherichia coli (STEC), numerous immunoassays have been developed for STEC diagnosis, showing variability in sensitivity and specificity when evaluated by reference laboratories, and no therapy or vaccines are currently approved. Thus, the aim of this work was the characterization of the interaction between MAbs against Stx1 and Stx2 toxins and their neutralizing abilities to enable their use as tools for diagnosis and therapy. the selected clones designated 3E2 (anti-Stx1) and 2E11 (anti-Stx2) were classified as IgG1. 3E2 recognized the B subunit of Stx1 with an affinity constant of 2.5 x 10(-10) M, detected as little as 6.2 ng of Stx1 and was stable up to 50 degrees C. in contrast, 2E11 recognized the A subunit of Stx2, was stable up to 70 degrees C, had a high dissociation constant of 6.1 x 10(-10) M, and detected as little as 12.5 ng of Stx2. Neutralization tests showed that 160 ng of 3E2 MAb inhibited 80% of Stx1 activity and 500 mu g 2E11 MAb were required for 60% inhibition of Stx2 activity. These MAb amounts reversed 25 to 80% of the cytotoxicity triggered by different STEC isolates. in conclusion, these MAbs show suitable characteristics for their use in STEC diagnosis and encourage future studies to investigate their protective efficacy.
- ItemAcesso aberto (Open Access)Monoclonal antibodies to identify Tomato mosaic tobamovirus (ToMV)(Sociedade Brasileira de Microbiologia, 2001-10-01) Duarte, Keila M.R.; Gomes, Luiz Humberto; Gesztesi, Jean-Luc [UNIFESP]; Lopes, Jose Daniel [UNIFESP]; Tavares, Flávio C.A.; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)Monoclonal antibodies were obtained against Tomato mosaic tobamovirus (ToMV) isolated in Brazil. One antibody (8G7G2) isotyped as IgG2b (kappa light chain) showed strong specificity and very low cross reaction with the Tobacco mosaic virus (TMV). It can be used in identification of tomato mosaic virus (ToMV).
- ItemSomente MetadadadosA surface 75-kDa protein with acid phosphatase activity recognized by monoclonal antibodies that inhibit Paracoccidioides brasiliensis growth(Elsevier B.V., 2007-10-01) Xander, Patricia [UNIFESP]; Vigna, Ana Flavia; Feitosa, Luciano dos Santos; Pugliese, Livia; Bailao, Alexandre Melo; Soares, Celia Maria de Almeida; Mortara, Renato Arruda; Mariano, Mario; Lopes, Jose Daniel; Universidade Federal de São Paulo (UNIFESP); Universidade Federal de Goiás (UFG)Paracoccidioides brasiliensis is a thermo-dimorphic fungus responsible for paracoccidioidomycosis (PCM), a systemic granulomatous mycosis prevalent in Latin America. the fungus releases many antigens which may be transiently bound to its cell surface. Some of them may show enzymatic functions essential for maintaining many cell processes and survival of the microorganism at different conditions. in this study, we report the characterization of a secreted 75 kDa protein from P. brasiliensis with phosphatase activity. Biologic function of the molecule was demonstrated using two specific mAbs produced and characterized as IgM and IgG isotypes. Confocal microscopy and flow cytometry analysis demonstrated that both mAbs recognized the protein on the fungus surface, mainly in its budding sites. in vitro experiments showed that fungal growth was inhibited by blocking the protein with mAbs. in addition, opsonized yeast cells with both mAbs facilitated phagocytosis by murine peritoneal macrophages. Passive immunization using mAbs before P. brasiliensis mice infection reduced colony-forming units (CFU) in the lungs as compared with controls. Histopathology showed smaller inflammation, absence of yeast cells and no granuloma formation. (c) 2007 Elsevier Masson SAS. All rights reserved.
- ItemSomente MetadadadosToxicological considerations for intravitreal drugs(Informa Healthcare, 2011-08-01) Penha, Fernando M. [UNIFESP]; Rodrigues, Eduardo B. [UNIFESP]; Furlani, Bruno A. [UNIFESP]; Dib, Eduardo [UNIFESP]; Melo, Gustavo B. [UNIFESP]; Farah, Michel E. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Introduction: Intravitreal injections are a very common procedure and are the most effective route of drug delivery to the retina. There are currently several drugs available and even more are in development; therefore, safety is a very important concern.Areas covered: the toxicological considerations of the most common drugs used for intravitreal pharmacotherapy such as anti-VEGFs, corticosteroids and antibiotics. Emerging agents such as anti-TNFs, VEGF-trap and kinase inhibitors are also discussed. An assessment of the efficacy and safety issues of the most relevant drugs including bevacizumab, ranibizumab and triamcinolone is presented.Expert opinion: the toxicology and safety profiles are available for several drugs that are either in use or will be available for intravitreal injections. Retinal pharmacotherapy is very effective for different retinal diseases; however safety is a very important issue when intravitreal injections are applied and the possibility of retinal toxicity should always be kept in mind. Bevacizumab and ranibizumab are effective for the therapy of wet-age-related macular degeneration and macular edema, while triamcinolone remains an alternative agent to treat secondary macular edema. It is important, as some of these drugs will be used for extended periods of time, that their long-term toxicological effects are better understood.
- ItemSomente MetadadadosTrypanosoma 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.
- 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.
- ItemSomente MetadadadosViés de publicação em ensaios clínicos sobre anticorpos monoclonais e repercussão jurídica do direito fundamental à saúde baseada em evidências(Universidade Federal de São Paulo (UNIFESP), 2014-12-31) Santos, Douglas Henrique Marin dos [UNIFESP]; Atallah, Alvaro Nagib Atallah [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Aims: We aimed to assess the proportion of publication and reporting of results of clinical trials on monoclonal antibodies adalimumab, bevacizumab, rituximab, trastuzumab and infliximab registered at ClinicalTrials.gov. We also aimed to evaluate the circumstances associated to publications bias and the effectiveness of FDAAA legislation in controlling it. Methods: In this cross-sectional, we searched ClinicalTrials.gov for protocols of interventional studies, phases III and IV, on monoclonal antibodies adalimumab, bevacizumab, rituximab, trastuzumab and infliximab, interventional (n = 243). After, we searched Pubmed, Embase, Lilacs, Cochrane Central and Google Scholar in order to evaluate and find published papers. Results: Among the 243 trials that comprised our initial sample, 178 (?73,2%) were published and 73 (?30%) had results reported at ClinicalTrials.gov. Industry sponsored trials were 169 (? 69.5%). Regarding the methodological quality of protocols, 159 (?65,4%) trials were designed with two or more comparison groups (intervention versus control), 149 (?61,3%) were randomized and 84 (?34,5%) were masked. Only 82 trials (?33,7%) were cumulatively designed with control groups, randomized allocation of participants, and masking. Among published studies (n=178), 118 (?66,3%) reported positive results, 18 (? 10%) reported negative results, 11 (?6,2%) found neutral or inconclusive results, and 24 (?13,5%) were partially positive. In the subsample of trials under FDAAA mandatory reporting (n=57), 48 (?84.2%) were published and 40 (?70,2%) had their results disclosed at ClinicalTrials.gov. Placebo-controlled trials were significantly more common among industry-funded trials when compared with independent trials (n=44/169 [26%] versus 9/74 [?12,2%]; p=0,025). Treatment as usual controlled trials were significantly more common among independent studies when compared to industry-funded trials (n=36/74 [?48.6%] versus 44/169 [?26%]; p<0,001). Conclusions: Publication bias in clinical trials is intense, despite the nature of intervention investigated (in this case, monoclonal antibodies). The source of funding (independent or industry) did not change the patterns of publication, suggesting that publication bias is evenly spread among different sponsors. However, studies involving placebo or single arm studies were more common in industry-funded trials. Our findings also suggest a higher prevalence of positive results among published trials. Lack of transparency, therefore, goes beyond selective publications and encompasses poor designed and biased protocols. Clinical trials subject to the FDAAA legislation had a greater proportion of published studies and disclosed results at ClinicalTrials.gov, suggesting the effectiveness of U.S. law in controlling publication bias and expanding in clinical research.