Interactions of cells with the extracellular matrix (ECM) have important roles in various physiological and pathological processes, including tissue morphogenesis during embryonic development, wound healing and tumor invasion. Although most of the proteins involved in cell-ECM interactions have been identified, the underlying mechanisms and involved signaling pathways are incompletely understood. Here, atomic force microscope-based imaging and single-cell force measurements were used to characterize the interactions of different cell types with ECM proteins. The interplay between cells and ECM is complex. However, two interaction types, protein-protein and protein-carbohydrate, predominate. Integrins, adhesion receptors for ECM, mediate the former, galectins, a family of animal lectins, the latter. In the second chapter of this thesis, the contributions of both receptor families to the interactions of epithelial MDCK cells with ECM proteins are presented. It was found that galectins-3 and 9 are highly expressed in MDCK cells and required for optimal long-term adhesion (90 minutes) to ECM proteins collagen-I and laminin-111. Interestingly, early adhesion (< 2 minutes) to laminin-111, was integrin-independent and instead mediated by carbohydrate interactions and galectins. In contrast, early adhesion to collagen-I was exclusively mediated by integrins. Moreover, cells frequently entered an enhanced adhesion state, marked by a significant increase in the force required for cell detachment. Although adhesion was mediated by integrins, adhesion enhancement was especially observed in cells depleted for galectin-3. It was proposed that galectin-3 influences integrin-mediated adhesion complex formation by altering receptor clustering. To control their attachment to ECM proteins, cells regulate integrin receptors. One regulatory process is integrin crosstalk, where the binding of one type of integrin influences the activity of another type. In the third chapter, the implementation of a single-cell force spectroscopy assay to identify such crosstalks and gain insight into their mechanisms is described. In this assay the interactions of integrin receptors being specifically attached to one ligand are characterized in dependence of another ligand-bond receptor pair. With this assay a crosstalk between collagen-binding integrin α1β1 and fibronectin-binding integrin α5β1 was identified in HeLa cells. This crosstalk was directional from integrin α1β1 to integrin α5β1 and appeared to regulate integrin α5β1 by inducing its endocytosis. In the fourth and final chapter, mechanisms of matrix-induced cell alignment were studied by imaging cells on two-dimensional matrices assembled of highly aligned collagen fibrils. Integrin α2β1 was identified as the predominant receptor mediating cell polarization. Time-lapse AFM demonstrated that during alignment cells deform the matrix by reorienting individual collagen fibrils. Cells deformed the collagen matrix asymmetrically, revealing an anisotropy in matrix rigidity. When matrix rigidity was rendered uniform by chemical cross-linking or when the matrix was formed from collagen fibrils of reduced tensile strength, cell polarization did not occur. This suggested that both the high tensile strength and pliability of collagen fibrils contribute to the anisotropic rigidity of the matrix and lead to directional cellular traction and cell polarization. During alignment, cellular protrusions contacted the collagen matrix from below and above. This complex entanglement of cellular protrusions and collagen fibrils may further promote cell alignment by maximizing cellular traction. The work presented here adds to the understanding of cell-ECM interactions. Atomic force microscopy imaging allowed characterizing the behavior of cells on nanopatterned collagen matrices whereas single-cell force spectroscopy revealed insights into the regulation of cell adhesion by galectins. Furthermore, methodological advances in the single-cell force spectroscopy assay allowed the intracellular regulation of receptor molecules to be studied. The work demonstrates that atomic force microscopy is a versatile tool to study cell-ECM interactions.

No description available.

A galectina-1 (LGALS1) participa em diferentes etapas da neoplasia, mas sua função na leucemia mielóide crônica (LMC) é desconhecida. Neste trabalho, a expressão etópica de BCR-ABL selvagem, mas não de BCR-ABL quinase deficiente, em linhagens celulares hematopoéticas, resultou em aumento de LGALS1. O efeito foi revertido com a inibição da tirosina quinase pelo mesilato de imatinibe. Este resultado indicou que a galectina-1 é modulada pela atividade tirosina-quinase de BCR-ABL. Em pacientes com LMC, a maior expressão de LGALS1 foi correlacionada a altos níveis de BCR-ABL, progressão da doença e a um tempo de sobrevida menor. Adicionalmente, as células K562 com LGALS1 inibida por RNA de interferência exibiram crescimento mais lento do que as células K562 com LGALS1 intacta, em camundongos nude. Portanto, o pior prognóstico de pacientes com altos níveis de galectina-1 sugere um efeito cooperativo de galectina-1 na tumorigênese de BCR-ABL reforçando o conceito de que a galectina-1 é um forte candidato para intervenção terapêutica na LMC. / Galectin-1 (LGALS1) participates in different steps of neoplasia but its role in chronic myeloid leukemia (CML) is unknown. In this work, ectopic expression of wild-type but not kinase-deficient BCR-ABL in different hematopoietic cells resulted in LGALS1 upregulation. Tyrosine-kinase inhibition by imatinib mesylate reversed this effect. This result indicate that galectin-1 is modulated by tyrosine kinase activity. In CML patients, the elevated expression of LGALS1 was correlated with high BCR-ABL levels, disease progression and shorter survival time. Additionally, in nude mice, LGALS1-deficient K562 cells obtained by RNA interference were less efficient in tumor formation than control K562 cells. Therefore, the worst prognosis in patients bearing high LGALS1 levels suggests a cooperative role for galectin-1 in BCR-ABL-positive leukemia and support the concept that galectin-1 is a strong candidate for CML therapeutic intervention.

Interactions of cells with the extracellular matrix (ECM) have important roles in various physiological and pathological processes, including tissue morphogenesis during embryonic development, wound healing and tumor invasion. Although most of the proteins involved in cell-ECM interactions have been identified, the underlying mechanisms and involved signaling pathways are incompletely understood. Here, atomic force microscope-based imaging and single-cell force measurements were used to characterize the interactions of different cell types with ECM proteins. The interplay between cells and ECM is complex. However, two interaction types, protein-protein and protein-carbohydrate, predominate. Integrins, adhesion receptors for ECM, mediate the former, galectins, a family of animal lectins, the latter. In the second chapter of this thesis, the contributions of both receptor families to the interactions of epithelial MDCK cells with ECM proteins are presented. It was found that galectins-3 and 9 are highly expressed in MDCK cells and required for optimal long-term adhesion (90 minutes) to ECM proteins collagen-I and laminin-111. Interestingly, early adhesion (< 2 minutes) to laminin-111, was integrin-independent and instead mediated by carbohydrate interactions and galectins. In contrast, early adhesion to collagen-I was exclusively mediated by integrins. Moreover, cells frequently entered an enhanced adhesion state, marked by a significant increase in the force required for cell detachment. Although adhesion was mediated by integrins, adhesion enhancement was especially observed in cells depleted for galectin-3. It was proposed that galectin-3 influences integrin-mediated adhesion complex formation by altering receptor clustering. To control their attachment to ECM proteins, cells regulate integrin receptors. One regulatory process is integrin crosstalk, where the binding of one type of integrin influences the activity of another type. In the third chapter, the implementation of a single-cell force spectroscopy assay to identify such crosstalks and gain insight into their mechanisms is described. In this assay the interactions of integrin receptors being specifically attached to one ligand are characterized in dependence of another ligand-bond receptor pair. With this assay a crosstalk between collagen-binding integrin α1β1 and fibronectin-binding integrin α5β1 was identified in HeLa cells. This crosstalk was directional from integrin α1β1 to integrin α5β1 and appeared to regulate integrin α5β1 by inducing its endocytosis. In the fourth and final chapter, mechanisms of matrix-induced cell alignment were studied by imaging cells on two-dimensional matrices assembled of highly aligned collagen fibrils. Integrin α2β1 was identified as the predominant receptor mediating cell polarization. Time-lapse AFM demonstrated that during alignment cells deform the matrix by reorienting individual collagen fibrils. Cells deformed the collagen matrix asymmetrically, revealing an anisotropy in matrix rigidity. When matrix rigidity was rendered uniform by chemical cross-linking or when the matrix was formed from collagen fibrils of reduced tensile strength, cell polarization did not occur. This suggested that both the high tensile strength and pliability of collagen fibrils contribute to the anisotropic rigidity of the matrix and lead to directional cellular traction and cell polarization. During alignment, cellular protrusions contacted the collagen matrix from below and above. This complex entanglement of cellular protrusions and collagen fibrils may further promote cell alignment by maximizing cellular traction. The work presented here adds to the understanding of cell-ECM interactions. Atomic force microscopy imaging allowed characterizing the behavior of cells on nanopatterned collagen matrices whereas single-cell force spectroscopy revealed insights into the regulation of cell adhesion by galectins. Furthermore, methodological advances in the single-cell force spectroscopy assay allowed the intracellular regulation of receptor molecules to be studied. The work demonstrates that atomic force microscopy is a versatile tool to study cell-ECM interactions.

No description available.

Comme nous l’indiquions dans le But du Travail, le mélanome figure parmi les cancers associés aux pronostics les plus sombres, et ce en raison de son taux de réponse très faible à la radiothérapie et à la chimiothérapie. Cette résistance à la radiothérapie et à la chimiothérapie provient essentiellement du fait que les cellules de mélanomes sont résistantes à l’apoptose, et que la radiothérapie ainsi que bon nombre d’agents chimiothérapiques induisent la mort des cellules cancéreuses en y induisant l’apoptose. Nous avons voulu investiguer les rôles de la gastrine et de la galectine 1 sur le comportement biologique des cellules de mélanomes afin de voir s’il était possible de proposer la gastrine et/ou la galectine 1 comme nouvelles cibles thérapeutiques potentielles dans le cas du mélanome. Notre stratégie de recherche est basée sur le principe (démontré sur le plan expérimental par de nombreuses études) selon lequel les cellules cancéreuses migrantes résistent à l’apoptose, et sont dès lors protégées contre les effets pro-apoptotiques de la chimiothérapie et de la radiothérapie qui représentent la quasi totalité de l’arsenal thérapeutique dont disposent les oncologues pour combattre les cancers. Diverses études expérimentales ont démontré que le fait de réduire le taux de migration de cellules cancéreuses résistantes à l’apoptose conférait à celles-ci une sensibilité accrue aux agents pro-apoptotiques. Nos résultats démontrent que la gastrine modifie de manière très significative les propriétés migratoires des cellules de mélanomes, sans toutefois modifier leur sensibilité à des agents pro-apoptotiques. Au contraire, la gastrine protègerait les cellules de mélanomes contre l’apoptose. Nous démontrons également dans notre travail, in vivo, un rôle pro-angiogénique pour la gastrine au sein de xénogreffes de mélanomes humains. Signalons que notre travail est le premier à démontrer un rôle potentiel de la gastrine au niveau de la biologie des mélanomes, tout au moins sur le plan expérimental. Tout comme nous l’avons observé pour la gastrine, la galectine 1 semble également conférer aux cellules de mélanomes un certain degré de résistance aux agressions chimiothérapiques. Cette fois, le fait de diminuer le taux d’expression de la galectine 1 au sein de cellules du mélanome murin expérimental B16F10 (qui exprime des quantités importantes de galectine 1) renforce l’effet thérapeutique du témozolomide qui est une molécule cytotoxique. Cet effet semble survenir, tout au moins partiellement, suite à une diminution du taux d’expression de la protéine Hsp70 (suite à la diminution du taux d’expression de la galectine 1), avec pour conséquence une augmentation de la mort cellulaire par perméabilisation de la membrane des lysosomes. Nous proposons une nouvelle approche thérapeutique pour combattre les mélanomes en faisant appel à la technique des petits ARN interférants (siRNA), dirigés dans le cas présent contre la galectine 1.

Doenças Mieloproliferativas Crônicas são desordens hematológicas malignas caracterizadas pela alteração na célula-tronco hematopoética e independência ou hipersensibilidade dos progenitores hematopoéticos a citocinas. Em 2008 a OMS renomeou esse grupo como Neoplasias Mieloproliferativas (NMPs), no qual estão inclusas as entidades nosológicas Policitemia Vera (PV), Trombocitemia Essencial (TE) e Mielofibrose Primária (MFP), doenças alvo desse estudo. Apesar dos avanços no diagnóstico das NMPs e nos mecanismos envolvidos com a fisiopatologia dessas doenças, sua patogênese permanece desconhecida. Alterações na maquinaria apoptótica parecem estar envolvidas em na fisiopatologia das NMP e por isso a compreensão dos mecanismos de regulação da apoptose e a interferência das galectinas-1 e 3 nesse processo, em pacientes com NMPs, é relevante para a busca de novos alvos terapêuticos. Neste contexto, os objetivos deste trabalho foram: avaliar em leucócitos de sangue periférico e células tronco hematopoéticas CD34+ de medula óssea dos pacientes com PV, TE e MFP os níveis de expressão das LGALS1 e LGALS3 e a concentração de galectina-3 plasmática. Foram determinadas as correlações dos níveis de expressão de LGALS1 e LGALS3 e da concentração da galectina-3 plasmática com os níveis de expressão do RNAm das moléculas reguladoras da apoptose e com os dados clínico-laboratoriais dos pacientes como a concentração de hemoglobina, percentagem de hematócrito, porcentagem de alelos mutados JAK2V617F, contagem de leucócitos e esplenomegalia. A expressão de LGALS1 estava diminuída em células CD34+ em PV e MFP e em leucócitos de sangue periférico de pacientes com MFP. Os pacientes de TE apresentaram aumento na expressão de LGALS3 em leucócitos de sangue periférico e alta concentração de galectina-3 no plasma. Houve correlação entre os níveis de expressão de LGALS1 e a porcentagem de alelos mutados e a contagem de leucócitos, em pacientes com PV. Foi detectada a correlação entre os níveis de expressão de LGALS3 a porcentagem de alelos mutados e o tamanho do baço, em pacientes com MFP. Com relação aos genes reguladores da apoptose, foram observadas correlações entre os níveis de expressão de LGALS1 e BCL-2 em células CD34+ de pacientes PV e entre LGALS3 e A1, MCL-1, BAX e C-FLIP em leucócitos de de pacientes com TE. Os resultados obtidos indicam que as NPM apresentam expressão diferencial de LGALS1 e LGALS3 e sugerem a associação entre a expressão de galectinas e o status da mutação JAK2V617F, principalmente em pacientes com MFP / Chronic myeloproliferative diseases are haematological malignant disorders characterized by the presence of an altered haematopoietic stem cell and independence or hypersensibility of their hematopoietic progenitors to cytokines. In 2008, WHO renamed this group of diseases as Myeloproliferative Neoplasms (MPN) in which is included Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). There have been advances concerning the knowledge about the mechanisms involved in MPN pathophysiology, however their pathogenesis remains unknow. Deregulation in apoptotic machinery seems to be involved in MPN pathophysiology. Fully understanding of apoptotic machinery and the influence of galectin-1 and 3 in this process in NMP patients might unveil novel targets for manipulation. The aims of the present study were to evaluate in leukocytes and CD34+ hematopoietic stem cells from PV, ET and PMF patients the LGALS1 and LGALS3 expression levels, the Galectin-3 plasma levels and to correlate LGALS1 and LGALS3 expression levels with galectin-3 plasma levels, apoptosis-related genes expression, JAK2 mutation status and clinic-laboratorial parameters. PV and PMF patients showed decreased expression levels of LGALS1 in CD34+ cells and also decreased LGALS1 expression levels in PMF leukocytes. ET patients presented an increased expression level of galectin-3 in leukocytes and plasma. We detected the correlations between LGALS3 gene expression with JAK2 allele burden and with leukocytes number in PV patients. We also observed in PMF patients the correlation between LGALS3 expression levels with JAK2 allele burden and spleen size. We also detected the correlation between LGALS1 expression levels BCL-2 gene expression in PV CD34+ HSC cells and between LGALS3 expression and A1, MCL-1, BAX and C-FLIP gene expression in TE leukocytes. Taken together, the results suggest the LGALS1and LGALS3 differential expression in NMP and the relation between JAK2V617F status with galectins expression, especially in PMF patients.

Assim como tecidos normais, tumores possuem uma demanda de nutrientes e oxigênio, suprida através da vasculatura a eles associada que resulta do processo de angiogênese. Fatores pró-angiogênicos são capazes de atrair monócitos, os quais se diferenciam em macrófagos associados a tumores (TAMs). TAMs comumente apresentam fenótipo M2, cujas características são consideradas pró-tumorais, como a promoção da angiogênese e a degradação de matriz extracelular. Estudos indicam que galectina-3 (gal-3), uma proteína pleiotrópica que se liga a ?-galactosídeos, participa do controle da angiogênese e da infiltração de macrófagos M2 na massa tumoral, mas pouco se sabe sobre os mecanismos envolvidos. No presente estudo, utilizamos um modelo de sarcoma induzido por carcinógeno em camundongos selvagens (WT) e knockout para gal-3 (Gal- 3 KO). Comparando os tumores de animais WT e Gal-3 KO, não observamos diferenças no padrão de crescimento tumoral, na área necrótica relativa, na proliferação celular e na quantificação de fibras de colágeno. Demonstramos que, embora ambos os grupos desenvolvam tumores, a angiogênese foi inibida em um microambiente desprovido de gal-3. Entretanto, não houve diferença na produção do fator de crescimento endotelial vascular (VEGF). As imagens obtidas in vivo indicaram que gal- 3 também influencia na formação estrutural de vasos adjacentes ao tumor. Além de mediar aspectos morfológicos relacionados à angiogênese, demonstramos que gal-3 também contribuiu para a funcionalidade vascular, pois houve uma redução na velocidade de fluxo sanguíneo nos vasos intratumorais de animais Gal-3 KO. Nossos dados sugeriram que há menos macrófagos no tumor que não expressa gal-3 e, dentre os TAMs, há mais M2 em comparação ao tumor gal-3-positivo. A análise do tecido onde o tumor se desenvolve, na fase inicial da tumorigênese, indicou que a ausência de gal-3 está relacionada a uma maior densidade de macrófagos M2. Considerando que a presença maior de macrófagos M2 nos sarcomas gal-3-negativos não resultou em maior produção de VEGF, mas sim na inibição da angiogênese, nossos resultados apontam para uma participação significativa de gal-3 na mediação da angiogênese pelos macrófagos / As well as normal tissues, tumors require nutrients and oxygen, which are supplied by the associated vasculature that results from the process of angiogenesis. Pro-angiogenic factors are able to attract monocytes and they differentiate into tumor-associated macrophages (TAMs). TAMs commonly exhibit M2 phenotype, which has characteristics considered pro-tumoral, such as angiogenesis promotion and degradation of extracellular matrix. Studies show that galectin-3 (gal-3), a pleiotropic ?-galactosidebinding protein, participates in angiogenesis control and M2 macrophage infiltration into the tumor mass, but little is known about the mechanisms involved. In this work, we established a model of carcinogen-induced sarcoma in wild-type (WT) and gal-3 knockout (Gal-3 KO) mice. Comparing tumors from WT and Gal-3 KO animals, there were no differences in the pattern of tumor growth, relative necrotic area, cell proliferation and collagenous fibers. We demonstrated that, although both groups develop tumors, angiogenesis was inhibited in a microenvironment devoid of gal-3. However, there was no difference in the production of vascular endothelial growth factor (VEGF). The images obtained in vivo indicated that gal-3 also influenced the structural formation of vessels adjacent to the tumor. In addition to mediating morphological aspects related to angiogenesis, we demonstrated that gal-3 also contributes to vascular functionality, since there was a reduction in blood flow velocity in intratumoral vessels from Gal-3 KO animals. Our data suggested that there are fewer macrophages in tumors without gal-3 and, among TAMs, there are more M2 compared to gal-3-positive tumors. Analysis of the tissue where the tumor develops, in early stages of tumorigenesis, indicated that the lack of gal-3 is related to an increased density of M2 macrophages. Since the greater number of M2 macrophages in gal-3-negative fibrosarcomas did not result in increased VEGF production, but inhibited angiogenesis, our results suggest a significant role of gal-3 in regulation of angiogenesis by macrophages

A galectina-1 (Gal-1) é uma proteína que reconhece -galactosídeos e participa de vários processos biológicos, incluindo a modulação da resposta imunológica. Vários relatos da literatura reportam o potencial uso terapêutico da Gal-1 para doenças auto-imunes, inflamatórias, degenerativas e infecciosas. Entretanto, são escassos os relatos sobre o envolvimento da Gal-1 na doença causada por Tripanosoma cruzi. O presente trabalho tem como objetivos o estudo da participação da Gal-1 endógena e exógena na evolução da infecção aguda experimental por T. cruzi. Galectina-1 recombinante, camundongos C57BL/6 deficientes (nocaute - Gal-1-/-) ou não (selvagem - Gal-1+/+) do gene da galectina-1 e macrófagos desses animais foram utilizados em experimentos de infecção in vivo e/ou in vitro. Os animais foram infectados com tripomastigotas de T. cruzi da cepa Y, por via intraperitoneal. Os parâmetros analisados na caracterização do processo de infecção foram: parasitemia e sobrevivência; histopatologia do tecido cardíaco; dosagem de óxido nítrico, pelo método de Griess; imunofenotipagem de leucócitos, por citometria de fluxo; dosagem de citocinas por ELISA e taxa de liberação de parasitas em cultura de macrófagos. Os camundongos Gal-1-/- ou Gal-1+/+ tratados com Gal-1 exógena apresentaram as menores taxas de parasitemia. De modo interessante, todos os camundongos Gal-1-/- sobreviveram à infecção, enquanto que os selvagens apresentaram uma drástica redução de sobrevivência após o desafio com T. cruzi. A ausência da Gal-1 endógena ou o tratamento com a Gal-1 exógena provocou no músculo cardíaco de camundongos infectados uma significativa redução infiltrado inflamatório. As dosagens de citocinas séricas indicaram que animais selvagens infectados e tratados com Gal-1 apresentaram uma diminuição de IFN- em relação aos não tratados. Além disso, soros de animais nocautes infectados apresentaram níveis inferiores de diferentes citocinas (TNF-, IFN- , IL-4, IL-10 e IL-12) em comparação com amostras séricas de animais selvagens infectados. As porcentagens de diferentes tipos de células esplênicas (T, B, macrófagos, NKT e NK) foram, geralmente, maiores em camundongos selvagens do que em nocautes após a infecção por T. cruzi. Curiosamente, na cavidade peritoneal de camundongos Gal-1-/- ocorreu um aumento de neutrófilos e macrófagos após 12h ou 24h da infecção, respectivamente. O pico de produção de NO induzido por T. cruzi em macrófagos Gal-1-/- foi mais precoce e intenso do que o obtido por macrófagos Gal-1+/+. Além disso, os macrófagos Gal-1-/- liberaram menos parasitas in vitro em comparação como os macrófagos Gal-1+/+. Com base nesse conjunto de resultados sugerimos que a ausência de Gal-1 endógena ou o tratamento de animais com Gal-1 exógena promoveram perfis imunológicos (resposta inata e adaptativa) favorecedores da resolução da infecção experimental aguda por T. cruzi. / Galectin-1 (Gal-1) is a -galactoside-binding protein and participates in several biological processes, including modulation of immune response. In the literature, there are several reports about the potential therapeutic use of Gal-1 for autoimmune diseases, inflammatory, degenerative and infectious diseases. However, there are few reports on the involvement of Gal-1 in disease caused by Trypanosoma cruzi. Thus, this work was conducted to study the participation of endogenous and exogenous Gal-1 in acute experimental infection by T. cruzi. Recombinant Gal-1, galectin-1-deficient mice (KO - Gal-1-/-) or wild type (WT - Gal-1+/+) mice and macrophages from these animals were used to perform the in vivo and in vitro assays. The animals were infected with trypomastigotes of T. cruzi (strain Y), intraperitoneally. The biological parameters analyzed were parasitemia and survival; histopathology of heart tissue, measurement of nitric oxide by Griess reaction; leukocyte immunophenotyping by flow cytometry; cytokine detection by ELISA and the release rate of parasites in cultured macrophages. Infected-Gal-1-/- mice or infected-Gal-1+/+ mice treated with Gal-1 showed the lowest levels of parasitemia. Interestingly, all infected-KO mice survived after the infection, whereas the infected-WT mice showed a drastic reduction in survival. The absence of endogenous Gal-1 or the exogenous Gal-1 treatment promoted a drastic reduction on inflammatory cells infiltrate in the cardiac muscle of infected mice. The sera of infected-WT mice treated with Gal-1, but not untreated animals, showed high levels of IFN-. Additionally, sera from infected-KO mice showed lower levels of different cytokines (TNF-, IFN-, IL-4, IL-10 and IL-12) compared with serum samples from infected-WT animals. The number of spleen cells (T, B cells, macrophages, NK and NKT) were generally higher in WT mice than in KO mice after infection with T. cruzi. Interestingly, the peritoneal cavity of infected-KO mice presented an increased numbers of neutrophils and macrophages after 12h or 24h of infection, respectively. The peak of NO production induced by T. cruzi in Gal-1-/- macrophages was earlier and more intense than that obtained by Gal-1+/+ macrophage. Furthermore, Gal-1-/- macrophage released fewer parasites in vitro in comparison to Gal-1+/+ macrophages. Taken together, these results suggest that the absence of endogenous Gal-1 or treatment with exogenous Gal-1 promoted immunological profiles (innate and adaptative responses) that cooperate to the resolution of acute experimental infection by T. cruzi

A galectina-1 (Gal-1) pertence à uma família de proteínas ligantes de ?-galactosídeos e participa de vários processos biológicos, tais como a modulação da resposta inflamatória. Os mastócitos desempenham um importante papel em eventos inflamatórios e alérgicos. Entretanto, o impacto da Gal-1 na biologia dos mastócitos é pouco conhecido. Neste trabalho, foram analisados os aspectos morfológicos e funcionais de células RBL-2H3 tratadas com Gal-1. Além disso, foi investigado o efeito da ausência de Gal-1 endógena sobre a desgranulação in vivo. A avaliação da interação de Gal-1Texas-Red com células RBL-2H3, por citometria de fluxo, indicou que esta lectina ligou-se às superfícies dessas células de modo dose-dependente. A Gal-1 foi capaz de promover a exposição de fosfatidilserina (FS, um marcador de apoptose) nas superfícies dessas células por meio de reconhecimento de carboidratos. Entretanto, as células RBL-2H3 tratadas com Gal-1 não apresentaram apoptose e/ou necrose, como indicado pelos resultados obtidos dos ensaios de TUNEL, DNA laddering, hipodiploidia, citotoxicidade e microscopia eletrônica de transmissão. As análises de microscopia eletrônica de varredura e Differential Interference Contrast (DIC) de células tratadas com Gal-1 (10 µM-1 hora) indicaram que essa lectina induziu ondulações apenas nas porções apicais das membranas plasmáticas de células RBL-2H3. Além disso, a Gal-1 modulou negativamente a formação de ondulações nas superfícies de células RBL-2H3 estimuladas via Fc?RI. A distribuição de componentes de Lipid Rafts relacionados ao processo de ativação celular via Fc?RI (Lyn, LAT e GD1b) foi modificada pelo tratamento dessas células com Gal-1 (10 µM), por 1 hora. As células RBL-2H3 tratadas com Gal-1(10µM-45 minutos) apresentaram níveis de liberação da enzima ?-hexosaminidase (?-HEX) semelhantes ao do controle negativo, sugerindo que essa lectina não promove a desgranulação de células RBL-2H3. Por outro lado, a Gal-1 inibiu a desgranulação de células RBL-2H3 ativadas via Fc?RI. O valor máximo de inibição (80%) da liberação de ?-HEX foi atingido quando as células foram tratadas com 10 µM de Gal-1 por 24 horas. Este efeito inibitório não foi detectado quando as células foram tratadas com Gal-1 na presença de ?-D-Tiogalactopiranosídeo (TDG), quando estimuladas com ionóforo de cálcio ou tratadas com a forma monomérica da Gal-1. Os dados de microscopia confocal mostraram que os grânulos secretórios de células submetidas ao procedimento de estimulação via Fc?RI, foram fracamente marcados com o anticorpo AD1 e apresentaram uma distribuição citoplasmática difusa. Entretanto, células tratadas com Gal-1 (10 µM - 24 horas) e estimuladas via Fc?RI foram intensamente marcadas com anticorpo AD1 e mostraram um padrão perinuclear, como detectado em células não estimuladas. De modo interessante, camundongos deficientes para o gene de Gal-1 quando submetidos ao ensaio de anafilaxia passiva cutânea (PCA) apresentaram uma reação significativamente maior que os animais selvagens. Com base no conjunto de resultados obtidos sugere-se que a Gal-1 pode participar da homeostase de mastóctios sem provocar apoptose e/ou necrose dessas células. Além disso, a Gal-1 pode modular o processo de exocitose de mastócitos por meio das propriedades lectínica e de dimerização dessa proteína e esse efeito modulatório parece estar associado a eventos de sinalização celular anteriores ao influxo de cálcio. / Galectin-1 (Gal-1) belongs to a family of ?-galactoside-binding proteins and is involved in several biological processes, including modulation of the inflammatory response. Mast cells play a critical role in allergic and inflammatory events, however, little is known about the impact of Gal-1 on mast cell biology. In this study, we examined the role of Gal-1 in mast cell function using RBL-2H3 (Rat Basophilic Leukemia) and galectin-1 deficient mice. We report that Gal-1 recognized glycoconjugates on mast cells and this interaction promotes phosphatidylserine (PS) exposure in the absence of cell death or apoptosis. Morphological analysis of Gal-1-treated RBL-2H3 cells, by scanning electron microscopy and Differential Interference Contrast (DIC), indicated that Gal-1 induces modifications on cell membranes and modulation of ruffles formation on cell surface of stimulated RBL-2H3 cells. Interesting, Gal-1 treatment of RBL-2H3 cells, with or without stimulation, promotes alterations in the distribution of the components (Lyn, LAT and GD1b) of Lipid Rafts. Gal-1 did not promote degranulation on RBL-2H3 with or without prior sensitization with IgE. However, Gal-1 treatment inhibits the cell degranulation mediated by via Fc?RI and this effect was time and dose-dependent. Also, this inhibition was related to carbohydrate recognition domain and required Gal-1 dimerization. Importantly, confocal microscopy analysis showed that Gal-1 was distributed in cytoplasm close to secretory granules stained AD-1 antibody on RBL-2H3 with or without prior stimulation with Fc?RI. In addition, we found significantly increased passive cutaneous anaphylaxis reaction in Gal-1 deficient mice. The results demonstrated that Gal-1 may participate of homeostasis and exocytose in mast cells suggesting that Gal-1 can have important role in allergic and inflammatory process.