nÃo hà / Lectins are a group of proteins present in various living organisms, particularly in plants and especially in Leguminosae seeds. Among this family, a species that stands out for having large amount of lectin in the seed is the Canavalia brasiliensis. One approach for the investigation of lectins on biological systems is the detection there of by techniques involving antibody. Such techniques may include from classical immunochemical assays (immunodiffusion, immunoelectrophoresis and rocketimunoeletroforese) to ELISA experiments and advanced microscopy techniques. In this sense, antibodies become biotechnological tools invaluable since they are capable of specifically recognizing epitopes of the target molecules. Immunoglobulins (Igs) are proteins present in high concentration in the blood plasma of mammals. They are vectors of humoral immunity, with the primary function to join the foreign antigens to the individual so as to neutralize them. In the case of poultry the main immunoglobulin class (IgYs) can be found not only in blood, but also in egg yolk. This fact enables fast and efficient purification of these molecules facilitating its use as biotechnological inputs. Thus, this study aimed to produce antibodies against the lectin extracted from Canavalia brasiliensis seed (ConBr) (Fabaceae) in laying hens, implementing production methodology of new biotechnological inputs for the study of lectins. For this laying hens were immunized with 200 or 400μg of lectin Canavalia brasiliensis (ConBr) for 15 weeks at 10-day intervals. Collected eggs from vaccinated birds had their isolated and precipitated yolk in 0-30% fraction with ammonium sulfate for partial purificaÃÃoo of IgYs. Confirmation of the presence of antibodies was made by immunodiffusion assays agarose gel. The fraction showed 0-30%, from the first week after the immunization, capable of recognizing ConBr IgYs. This profile appeared in all groups over the 15-week experiment. The production of specific IgYs against the lectin from Canavalia brasiliensis (ConBr) proved feasible and remained throughout the experiment, possibly in reasonable quantities. In this sense, IgYs produced in this work provide a powerful biotechnological tool to be made available for future studies. / As lectinas constituem um grupo de proteÃnas presentes em diversos organismos vivos, sobretudo em vegetais e especialmente em sementes de Leguminosae. Dentre essa famÃlia, uma espÃcie que se destaca por possuir grande quantidade de lectina na semente à a Canavalia brasiliensis. Uma das possÃveis abordagens para a investigaÃÃo de lectinas em sistemas biolÃgicos à a detecÃÃo das mesmas atravÃs de tÃcnicas que envolvam anticorpos. Tais tÃcnicas podem incluir desde ensaios clÃssicos de imunoquÃmica (imunodifusÃo, imunoeletroforese e rocketimunoeletroforese), atà experimentos de ELISA e tÃcnicas avanÃadas de microscopia. Neste sentido, os anticopos tornam-se ferramentas biotecnolÃgicas de grande valia uma vez que sÃo capazes de reconhecer especificamente epÃtopos das molÃculas alvo. As imunoglobulinas (Igs) são proteínas presentes em grande concentração no plasma sanguÃneo de mamÃferos. São os vetores da imunidade humoral, tendo como função principal unir-se aos antígenos estranhos ao indivÃduo, de modo a neutralizá-los. No caso das aves a principal classe de imunoglobulinas (IgYs) pode ser encontrada nÃo somente no sangue mas tambÃm nas gemas dos ovos. Esse fato permite purificaÃÃo rÃpida e eficiente dessas molÃculas facilitando seu uso como insumos biotecnolÃgicos. Dessa forma, este trabalho teve por objetivo produzir anticorpos contra a lectina extraÃda da semente de Canavalia brasiliensis (ConBr) (Leguminosae) em galinhas poedeiras, implementando metodologia de produÃÃo de novos insumos biotecnolÃgicos para o estudo de lectinas. Para tanto galinhas poedeiras foram imunizadas com 200 ou 400μg da lectina de Canavalia brasiliensis (ConBr) por 15 semanas em intervalos de 10 dias. Ovos coletados das aves imunizadas tiveram suas gemas isoladas e precipitadas na fraÃÃo 0-30% com sulfato de amÃnio para a purificaÃÃo parcial das IgYs. A confirmaÃÃo da presenÃa dos anticorpos foi feita por ensaios de imunodifusÃo em gel de agarose. A fraÃÃo 0-30% apresentou, a partir da primeira semana apÃs a imunizaÃÃo, as IgYs capazes de reconhecer ConBr. Esse perfil se apresentou em todos os grupos ao longo das 15 semanas de experimento. A produÃÃo das IgYs especÃficas contra a lectina de Canavalia brasiliensis (ConBr) se mostrou viÃvel e permaneceu durante todo experimento, possivelmente em quantidades razoÃveis. Neste sentido, as IgYs produzidas nesse trabalho constituem uma potente ferramenta biotecnolÃgica a ser disponibilizada para estudos futuros.

Made available in DSpace on 2014-06-12T15:52:45Z (GMT). No. of bitstreams: 2 arquivo4557_1.pdf: 827863 bytes, checksum: d2a8ca3017b898c1d5c8989791f98ed7 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2007 / Sementes de Moringa oleifera têm sido freqüentemente usadas como coagulante para o tratamento de água no Nordeste do Brasil, local com alta incidência de dengue. Lectinas constituem um grupo de proteínas heterogêneas capazes de reconhecer e ligar reversivelmente carboidratos e glicoproteínas. Inibidores de protease impedem a atividade enzimática pela formação de complexos estáveis com tripsina e/ou quimotripsina. Lectinas e inibidores de tripsina com atividade inseticida têm sido descritos. O objetivo deste trabalho foi avaliar na água tratada com sementes de M. oleifera, a partir das atividades hemaglutinante (AH), inibidora de tripsina e larvicida. Água destilada foi tratada com 1, 3, 6 ou 15 sementes de M. oleifera (MoW). Ensaio para lectina usou eritrócitos de coelho. MoW foi avaliada para atividade inibidora de tripsina usando N-&#945;-benzoil-DL-arginil-&#961;-nitroanilida (BAPNA). Bioensaio larvicida foi feito usando larvas do vetor da dengue, Aedes aegypti. Desenvolvimento, mortalidade e aspectos morfológicos das larvas foram analisados. MoW contém AH e não foi capaz de inibir tripsina. Redução significante do desenvolvimento larval foi observado após 24, 48 e 72 h de incubação com MoW. Larvas no 4º estádio (L4) foram detectadas somente no controle, MoW1 e MoW3. Significante (p<0. 0001) taxa de mortalidade foi detectada na preparação lectínica mais ativa, MoW15 (45%). Diferenças morfológicas nas larvas em L4 incubadas com MoW1 foram observadas quando comparadas ao controle por microscopia ótica invertida. Os resultados obtidos sugerem a avaliação das sementes de M. oleifera para o controle do vetor da dengue. A presença de AH na preparação ativa sobre as larvas do A. aegypti pode indicar o envolvimento de lectina no efeito larvicida de MoW

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Devido Ãs suas diferentes propriedades quÃmicas tais como capacidade de formar soluÃÃes viscosas ou gÃis em meio aquoso o isolamento e caracterizaÃÃo de galactomananas tÃm sido de grande importÃncia Nesse trabalho a galactomanana do endosperma da semente de Bauhinia pentandra foi isolada e caracterizada, apresentando-se homogÃnea por GPC Este polissacarideo foi demonstrado ser uma galactomanana clÃssica formada por uma cadeia linear de manose unidas por ligaÃÃes &#946;(1-4) com substituiÃÃes de galactose em ligaÃÃo &#945;(1-6) com uma proporÃÃo Man:Gal de 2 5.1 e viscosidade intrÃnseca em Ãgua de 10 1 dL/g A galactomanana foi avaliada quanto à capacidade de interagir com lectinas galactose ligante O polissacarÃdeo foi tratado com epicloridrina e o material obtido foi utilizado para a montagem de coluna cromatogrÃfica de afinidade Extratos ricos em lectinas de Artocarpus incisa Artocarpus integrifÃlia e Bauhinia pentandra foram aplicados e fraÃÃes lectinicas purificadas foram obtidas A capacidade da galactomanana de B. pentandra em reter a lectina (LBp) da mesma semente foi comparada com a matriz de galactomanana de Adenantera pavonina Caesalpinea pulcherrima Sophora japonica e com a matriz comercial Sepharose 4B Apesar da galactomanana de B. pentandra ter apresentado a menor capacidade de retenÃÃo frente Ãs demais, ela mostrou-se semelhante à matriz comercial sendo viÃvel a sua utilizaÃÃo / Due the chemicals properties diferences, such as the ability to make viscous solution or aqueous gels, the study of the galactomanans has been too important. In this study, the endospermic galactomannans from seeds of Bauhinia pentandra was isolated and partially characterized. This polysaccharide is a classical galalactomannan constituted by a linear chain of mannose linked by &#946;(1-4) linkages with galactose substituintions linked by &#945;(1-6) linkages, resulting in a Man:Gal ratio of 2.5:1, and water intrinsic viscosity equal to 10.1 dL/g. Galactomannan was evaluated in ability to interact with galactose-binding lectins. The polysaccharide was treated with epichlorohidrine and the material obtained was utilized to make the affinity chromatography matrix. Lectin-rich extracts from Artocarpus incisa, Artocarpus integrifolia and Bauhinia pentandra were applied and lectin fractions were obtained, thus, the affinity matrix showed to be efficient to isolate them. The retention capacity of the galactomannan from B. pentandra was compared with galactomannan matrix from Adenantera pavonina, Caesalpinea pulcherrima, Sophora japonica and commercial matrix of Sepharose 4B in regards to the isolation of the lectin from B. pentandra (LBp). Although the galactomannan matrix had been showed the smallest retention capacity in comparision with the others, it is equivalent to the commercial matrix, enabling your utilization

Zymosan is a β-glucan- and mannan-rich particle that is widely used as a cellular activator for examining the numerous responses effected by phagocytes. The macrophage mannose receptor (MR) and complement receptor 3 (CR3) have historically been considered the major macrophage lectins involved in the nonopsonic recognition of these yeast-derived particles. Using specific carbohydrate inhibitors, we show that a β-glucan receptor, but not the MR, is a predominant receptor involved in this process. Furthermore, nonopsonic zymosan binding was unaffected by genetic CD11b deficiency or a blocking monoclonal antibody (mAb) against CR3, demonstrating that CR3 was not the β-glucan receptor mediating this activity. To address the role of the recently described β-glucan receptor, Dectin-1, we generated a novel anti-Dectin-1 mAb, 2A11. Using this mAb, we show here that Dectin-1 was almost exclusively responsible for the β-glucan-dependent, nonopsonic recognition of zymosan by primary macrophages. These findings define Dectin-1 as the leukocyte β-glucan receptor, first described over 50 years ago, and resolves the long-standing controversy regarding the identity of this important molecule. Furthermore, these results identify Dectin-1 as a new target for examining the immunomodulatory properties of β-glucans for therapeutic drug design.

Myocardial infarction (MI) is an acute life-threatening disease with a high incidence worldwide. Aim of this study was to test lectin-carbohydrate binding-induced red blood cell (RBC) agglutination as an innovative tool for fast, precise and cost effective diagnosis of MI. Five lectins (Ricinus communis agglutinin (RCA), Phaseolus vulgaris erythroagglutinin (PHA), Datura stramonium agglutinin (DSA), Artocarpus agglutinin (ArA), Triticum agglutinin (TA)) were tested for ability to differentiate between agglutination characteristics in patients with MI (n = 101) or angina pectoris without MI (AP) (n = 34) and healthy volunteers (HV) as control (n =68) . RBC agglutination was analyzed by light absorbance of a stirred RBC suspension in the green to red light spectrum in an agglutimeter (amtec, Leipzig, Germany) for 15 min after lectin addition. Mean cell count in aggregates was estimated from light absorbance by a mathematical model. Each lectin induced RBC agglutination. RCA led to the strongest RBC agglutination (~500 RBCs/aggregate), while the others induced substantially slower agglutination and lead to smaller aggregate sizes (5-150 RBCs/aggregate). For all analyzed lectins the lectin-induced RBC agglutination of MI or AP patients was generally higher than for HV. However, only PHA induced agglutination that clearly distinguished MI from HV. Variance analysis showed that aggregate size after 15 min. agglutination induced by PHA was significantly higher in the MI group (143 RBCs/ aggregate) than in the HV (29 RBC-s/aggregate, p = 0.000). We hypothesize that pathological changes during MI induce modification of the carbohydrate composition on the RBC membrane and thus modify RBC agglutination. Occurrence of carbohydrate-lectin binding sites on RBC membranes provides evidence about MI. Due to significant difference in the rate of agglutination between MI > HV the differentiation between these groups is possible based on PHA-induced RBC-agglutination. This novel assay could serve as a rapid, cost effective valuable new tool for diagnosis of MI.

Thesis / Master of Science (MS)

Mannose-binding lectin (MBL) and ficolins are complement-activating proteins, and both play an important role in innate immunity by recognizing specific carbohydrate moieties on the surface of wide range of microorganisms. Previous studies have shown that porcine ficolin-α and MBL-A bind to surface polysaccharides of bacteria cultured in suspension, but their interactions with bacteria in biofilm culture have not been studied. The objectives of this thesis were to determine whether porcine plasma ficolin and MBL bind to Actinobacillus pleuropneumoniae in biofilm cultures. APP serotype 5a (APP5a) was used because it produced pronounced biofilm in plastic culture dishes, in comparison with APP5b that was previously reported to bind ficolin in suspension cultures. N-acetylglucosamine (GlcNAc) in the biofilm produced by APP5a was stained with wheat germ agglutinin conjugated with Alexa Fluor-555 and identified by confocal laser scanning microscopy (CLSM). Dispersin B prevented APP5a biofilm formation indicating the requirement of poly N-acetylglucosamine (PNAG) for bacterial cohesion. Bound purified ficolin or ficolin in plasma both were eluted with GlcNAc from APP5a biofilm cultures. To address preferential binding of ficolin-α to biofilm matrix, ficolin-α was eluted with GlcNAc from extracellular polymeric substances (EPS) in supernatant after pelleting the bacteria. Biotinylated-ficolin that retained GlcNAc-binding activity for APP5b planktonic cultures was shown to bind strongly to APP5a biofilm, as detected by fluorescent NeutrAvidin staining and CLSM, but not in the presence of GlcNAc. Further, MBL-A in ficolin-depleted porcine plasma also bound to APP5a biofilm and was eluted with a sugar solution containing GlcNAc, galactose, mannose and glucose. These studies demonstrate that both porcine ficolin-α and MBL-A bind to biofilm cultures of APP5a in a carbohydrate-dependent manner, and suggest that the production of PNAG in biofilm is a binding target for ficolin. / Natural Sciences and Engineering Research Council of Canada (NSERC)

Orientador: Valmir Fadel / Banca: Fernanda Canduri / Banca: Mario Tyago Murakami / Resumo: Este trabalho teve por objetivo cristalizar e resolver tridimensionalmente as estruturas das proteínas Canavalia gladiata lectin (CGL) e Canavalia marítima lectin (CML), ambas complexadas com o açúcar manose 1-6 manose, encontradas em sementes de leguminosas. Para o processo de cristalização foi utilizado um kit de cristalização, contendo 96 soluções previstas pelo método de cristalização da matriz esparsa, denominado Screen Index (Hampton Research). Para o processo de resolução de estrutura foi utilizado diversos métodos computacionais dentre eles, o programa CCP4 (Collaborative Computational Project n° 4). Já com a estrutura resolvida, pode-se observar o sítio de ligação da proteína e identificar quais aminoácidos fazem parte do mesmo; calcular o RMSD médio entre as estruturas nativa e complexada com o açúcar manose 1-6 manose; comparar os sítios de ligação das proteínas CGL e CML complexadas com os açúcares man 1-2 man, man 1-3 man, man 1-4 man e man 1-6 man. Enfim, o trabalho colaborou para que duas novas estruturas fossem depositadas no banco de dados PDB (Protein Data Bank), para que futuros pesquisadores possam realizar estudos utilizando essas estruturas já resolvidas. / Abstract: This study aimed to crystallize and solve the three-dimensional structures of proteins Canavalia gladiata lectin (CGL) e Canavalia maritime lectin (CML), both complexed with sugar manose 1-6 manose, found in legume seed. For the crystallization process was used a kit of crystallization, containing 96 solutions provided by the method of crystallization of the sparse matrix, called Screen Index (Hampton Research). For the process of resolution structure was used several computation methods among them, the program CCP4 (Collaborative Computational Project n° 4). Now with the structure resolved, can observe the binding siti of protein and amino acids to identify which part of the same; calculate the avarage RMSD betweem the native and complexed structures with the sugar manose 1-6 manose; compare the binding sites of CGL and CML proteins complexed with sugars man 1-2 man, man 1-3 man, man 1-4 man and man 1-6 man. Finally, the work helped the two new structures were deposited in the PDB database, for future researchers to conduct studies using these structures already solved. / Mestre

La bactérie Pseudomonas aeruginosa est un pathogène opportuniste responsable de graves infections chez les personnes affaiblies immunitairement. Présentant des souches résistantes aux antibiotiques, une nouvelle approche thérapeutique est en cours de développement avec pour objectif l’inhibition des facteurs de virulence de la bactérie. Lors de son processus d’infection, le pathogène utilise les lectines pour reconnaître et se lier de manière spécifique aux glycoconjugués des cellules-hôtes en formant une interaction lectine/glycoconjugué. Plus particulièrement, la lectine PA-IL, spécifique du galactose, a été étudiée. A l’aide de glycomimétique, il semble possible de bloquer l’action de la lectine en créant une interaction lectine/glycomimétique. Pour développer cette approche, de nombreux glycocluster sont donc été élaborés et leur affinité avec la lectine PA-IL a été évaluée par plusieurs méthodes de caractérisation (SPR, HIA, ELLA, puce à sucre,…).Dans ce projet de thèse, nous avons cherché à visualiser par microscopie à force atomique (AFM) l’arrangement des complexes lectine PA-IL/glycocluster formés pour trois glycoclusters différents. Nous avons ainsi pu montrer l’influence du cœur du glycocluster et des bras-espaceurs sur l’arrangement des complexes. Suivant le glycocluster, l’arrangement prend la forme de filaments 1D,de structures dentelées avec des bras sinueux ou encore de larges structures compactes. Dans le cas des filaments, la résolution de nos images AFM nous a permis d’identifier les lectines à l’intérieur même de la structure filaire. Nous avons aussi démontré, en observant les lectines seules, l’existence d’une interaction lectine/lectine. De plus, des expériences ont été menées pour déterminer les conditions expérimentales appropriées à leur observation à l’air et en milieu liquide. / The bacterium P. aeruginosa is an opportunistic pathogen responsible for serious infections in immunocompromised patients. It also develops some strains resistant to antibiotics. A new approach is developed to inhibit virulence factors of the bacterium. During the process of infection, the pathogen uses lectins to recognize and bind specifically to glycoconjugates of the host cells forming alectin/glycoconjugate complex. Particularly, the lectin PA-IL, specific to galactose, was studied. Using glycomimetics, it seems possible to block the action of the lectin by creating lectin/glycomimetic interaction. To develop this approach, many glycoclusters were designed and their affinity with lectin PA-IL was evaluated by various characterization techniques (SPR, HIA, ELLA, microarrays,…).In this thesis project, we have tried to visualize by Atomic Force Microscopy (AFM) the arrangement of lectin PA-IL/glycocluster complexes with three different glycoclusters. Our results show the influence of the glycocluster core and the linker on the arrangement of complexes. Depending on glycocluster, the arrangement takes the form of 1D filaments, 2D "pinked" structures with sinuous branches or large compact structures. In the case of filaments, the resolution of AFM images allows us to identify lectins along the filament. We also demonstrated the existence of lectin/lectin interactions at high concentration of lectin. In addition, some experiments were performed to determine sample preparation techniques to observe lectins in air and in liquid.

A lectina ArtinM, extraída de sementes de Artocarpus heterophyllus e caracterizada como um homotetrâmero constituído de subunidades de 16 kDa, tem alta afinidade de ligação a manotriose Man? 1-3 [Man? 1-6] Man, que constitui o core de N-glicanas. ArtinM é dotada de interessantes propriedades biológicas: (1) ativa neutrófilos a partir do reconhecimento de N-glicanas dos receptores CXCR2 e TLR2; (2) induz a desgranulação de mastócitos por interagir com N-glicanas de Fc?R ou com N-glicanas de IgE ligadas a Fc?R; (3) estimula a produção de IL-12, por reconhecer N-glicanas contidas no ectodomínio de TLR2 da superfície de células apresentadoras de antígeno (APCs); (4) exerce atividade imunomoduladora, que direciona o padrão de resposta para o perfil Th1; (5) confere resistência a infecções por patógenos intracelulares, como Paracoccidioides brasiliensis, Leishmania amazonensis e Leishmania major, Neospora caninum e Candida albicans Células T CD4+ participam de funções essenciais do sistema imune; durante o estabelecimento de uma resposta imune, podem ser desenvolvidas subpopulações de células T CD4+ adequadas para gerar respostas eficientes de combate a patógenos, manutenção da tolerância e regulação da imunidade. A ativação das células T CD4+ depende de um primeiro sinal, desencadeado pelo complexo TCR/CD3, e de um segundo sinal, oriundo de moléculas coestimulatórias como CD28. A ativação e expansão de células T CD4+ são limitadas pela ação de moléculas inibitórias, principalmente por CTLA-4. Lectinas podem ativar as células T, sendo a fitohemaglutinina (PHA) e a Concanavalin A (ConA) os exemplos mais conhecidos. Além disso, está bem caracterizado que o alvo de reconhecimento de ConA localiza-se no complexo TCR/CD3. No presente estudo buscou-se caracterizar os efeitos da lectina ArtinM sobre células T CD4+ murinas e investigar os possíveis mecanismos responsáveis pelos efeitos exercidos. Foram avaliados, inicialmente, os efeitos diretos de ArtinM sobre as células T CD4+, no que se refere à produção de citocinas, expressão de moléculas coestimulatórias e inibitórias e indução de diferenciação celular. Passou-se então à identificação de possíveis receptores de superfície reconhecidos por ArtinM e responsáveis pelo desencadeamento da ativação celular. Finalmente, buscou-se apontar moléculas sinalizadoras envolvidas nos efeitos diretos de ArtinM. A primeira evidência da interação direta de ArtinM com células T CD4+ foi proporcionada por aglutinação celular. Uma curva dose-resposta revelou que 5µg/ml foi a melhor concentração para adquirir significativa produção de citocinas Th1 (IL-2 e IFN-?) e Th17 (IL-6 e IL-17A) pelas células T CD4+. O estímulo com a concentração ótima de ArtinM mostrou que após 12 horas de incubação houve um significativo aumento nos níveis de IL-2, IFN-?, IL-6 e IL-17A no sobrenadante celular; persistindo no curso de 48 horas de observação. A secreção concomitante de IFN-? e IL-17A motivou a avaliação, por citometria de fluxo, da ocorrência de dupla marcação intracelular dessas citocinas. O estímulo, por 24 horas, com ArtinM, levou a importante aumento da frequência de células duplo-positivas para IFN-? e IL-17. Uma vez comprovado pelo padrão de citocinas secretadas que ArtinM promove a ativação das células T CD4+, investigou-se a expressão das moléculas CD25 e CTLA-4. ArtinM aumentou a expressão de ambas as moléculas, de maneira dose-dependente. Curiosamente, a detecção tanto de CD28, como de CTLA-4, foi precoce e persistente, diferindo do padrão temporal de expressão proporcionado por outros ativadores de células T CD4+. Com vistas a determinar o mecanismo através do qual ArtinM atua nas células T CD4+, alvos potenciais de reconhecimento foram ensaiados: CD3?, CD3??, CD28, CD45 e CD4. Esses receptores foram selecionados com base em predição de potenciais sítios Nglicosilados. Dessa forma, anticorpos específicos para essas moléculas foram utilizados para analisar a sua capacidade de inibir a atividade de ArtinM de induzir as células T CD4+ a produzir citocinas, como IL-2, IFN-?, IL-6 e IL-17A. Apenas o anticorpo anti-CD3?? foi capaz de impedir a secreção das citocinas induzidas por ArtinM. Além disso, esse anticorpo inibiu a marcação de células T CD4+ por ArtinM biotinilada. Esses dados indicam que ArtinM exerce sua atividade sobre células T CD4+ através do reconhecimento de glicanas na cadeia ? do receptor CD3, não excluindo-se, entretanto, a ocorrência da interação de ArtinM com outras glicoproteínas na superfície de linfócitos T CD4+. Também foi verificado que ArtinM possui alta especificidade por glicanas na superfície dessas células, pois foram necessárias elevadas concentrações de manotriose para inibir em 50% a ligação de ArtinM à superfície das células T CD4+. Através do uso de inibidores específicos para moléculas sinalizadoras, constatou-se que PI3K, PTK, p42/44MAPK, p38MAPK, JNK e PKC estão implicadas na sinalização para a produção das citocinas de perfis Th1 e Th17, induzida por ArtinM. Esse conjunto de resultados indica que ArtinM é um potente e rápido ativador de células T CD4+. A ativação celular induzida por ArtinM está relacionada com a ligação à cadeia ? do receptor CD3 e se associa à alta expressão de moléculas coestimuladoras e inibitórias. Ademais, demonstrou-se que ArtinM promove a diferenciação das células T CD4+ naive em células Th1 e Th17, utilizando moléculas sinalizadoras que são conhecidas como críticas para a indução de citocinas que caracterizam essas subpopulações celulares. / The lectin ArtinM, extracted from seeds of Artocarpus heterophyllus and characterized as a homotetramer consisted of 16 kDa subunits, has high binding affinity to the manotriose Man? 1-3 [Man? 1-6] Man, which is the core of N-glycans. ArtinM is endowed with interesting biological properties: (1) it activates neutrophils through the recognition of Nglycans attached to CXCR2 and TLR2 receptors; (2) induces degranulation of mast cells by interacting with N-glycans of Fc?R or to N-glycans of IgE bound to Fc?R; (3) stimulates the production of IL-12 through the recognition of N-glycans of the TLR2 ectodomain, expressed on the surface of antigen presenting cells (APCs); (4) exerts immunomodulatory activity, which accounts for Th1 immunity (5) confers resistance to intracellular pathogens, such as P. brasiliensis, Leishmania amazonensis and Leishmania major, Neospora caninum e Candida albicans. CD4+ T cells participate in essential functions of the immune system. During the development of an immune response, CD4+ T cells are activated and give origin to subpopulations of cells that are suitable for establishing effective responses to combat pathogens, for tolerance maintenance, and for adequate immuneregulation. The activation of CD4+ T cells depends on a first signal, triggered by the TCR/CD3 complex, and a second signal, provided by costimulatory molecules. The activation and expansion of CD4+ T cells is limited by the action of inhibitory molecules. Lectins may activate T cells, and Phytohemagglutinin (PHA) and Concanavalin A (ConA) are the best know examples. Furthermore, it is well characterized that the target for ConA recognition is localized in the TCR/CD3 complex. The present study was delineated to characterize the effects of the lectin ArtinM on murine CD4+ T cells and to investigate the possible mechanisms accounting for the observed effects. It was investigated the ArtinM direct effects on CD4+ T cells, concerning its ability to induce the production of cytokines, the expression of costimulatory and inhibitory molecules and cell differentiation. In addition, the possible surface receptors recognized by ArtinM and responsible for triggering cell activation were also assessed. Finally, signaling molecules involved in the direct effects of ArtinM were approached. The first evidence of direct interaction of ArtinM with CD4+ T cells was provided by cell agglutination. A dose-response curve has revealed that 5µg/ml was the best ArtinM concentration to achieve significant production of Th1 (IL-2 and IFN-?) and Th17 (IL-6 and IL-17A) cytokines by TCD4+ cells. Stimulus with the optimum ArtinM concentration has showed that after 12 hours incubation there was a significant augmentation of IL-2, IFN-?, IL- 6 and IL-17A levels in the cell supernatant; which has persisted in the course of 48 hours observation. The concomitant secretion of IFN-? and IL-17A led us to evaluate, by flow cytometry, the intracellular expression of these cytokines. After 24 hours stimulation with ArtinM, there was a significant increase in the frequency of cells IFN-?+IL-17+. Once the cytokines detection indicated that CD4+ T cells have been activated by ArtinM, the expression of CD25 and CTLA-4 molecules was assessed. ArtinM increased the expression of both molecules, in a dose-dependent manner. Interestingly, both cell surface molecules, CD25 and CTLA-4, were early and persistently detected a temporal pattern that is distinct from the provided by other inducers of CD4+ T cell activation. In order to determine the mechanism by which ArtinM acts on CD4+ T cells, potential targets of recognition were assessed: CD3??, CD3?, CD28, CD45 and CD4. These receptors were selected on the basis of prediction of N-glycosylation sites. Specific antibodies for these molecules were assayed regarding their ability to inhibit the ArtinM of inducing TCD4+ cells to produce cytokines, such as IL-2, IFN-?, IL-6 and IL-17A. Only anti-CD3 antibody was able to prevent the cytokines secretion induced by ArtinM. In addition, anti-CD3 antibody has inhibited the T CD4+ cell labeling by biotynil-ArtinM. These data indicate that ArtinM exerts its biological activity on T CD4+ cells through recognition of CD3 receptor ? chain glycans, without excluding the occurrence of ArtinM interactions with other glycoproteins on the surface of T CD4+ lymphocytes. The interaction of ArtinM with glycans at the surface of these cells was found to occur with great specificity, since high concentrations of the manotriose - Man? 1-3 [Man? 1-6] Man - were required to inhibit the binding. By using specific inhibitors of signaling molecules, we have found that PI3K, PTK and p42/44MAPK are relevant cytokine production profiles of Th1 and Th17 cells after stimulation with ArtinM. All toghether, these results indicate that ArtinM is a potent and rapid activator of CD4+ T cells. The activation induced by ArtinM is triggered by its binding to the CD3 receptor ? chain, which induces high expression of costimulator and inhibitory molecules. Moreover, it was demonstrated that ArtinM promotes the differentiation of naive CD4+ T cells into Th1 and Th17 cells by committing signaling molecules that are known as critical for the induction of cytokines that characterize these subpopulations of cells.