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.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O resveratrol à um polifenol antioxidante natural encontrado especialmente na epicarpo da uva, em nozes e na romà o qual pode inibir a ativaÃÃo de mediadores prÃ-inflamatÃrio e citocinas na fase precoce de expressÃo do gene. Jà à bem conhecido que as lectinas sÃo proteÃnas de ligaÃÃo de aÃÃcares que atuam tanto como molÃculas prÃ- e anti-inflamatÃrias. Assim, o objetivo do presente trabalho foi o de verificar a ligaÃÃo de um composto de polifenol com uma lectina de Canavalia maritima (ConM) com base na sua capacidade para inibir processos prÃ-inflamatÃrios. Para alcanÃar este objetivo, a ConM foi purificada e cristalizada, uma soluÃÃo de 5 mM de resveratrol 5 mM foi utilizada para soaking durante 2 horas de incubaÃÃo. Os cristais obtidos pertencem ao grupo espacial monoclÃnico C2, o refinamento final resultou em um Rfactor de 16,0% e uma Rfree de 25,5%. Resveratrol se liga na rÃgida atravÃs de pontes de hidrogÃnio e interaÃÃo hidrofÃbica com aminoÃcidos que compÃem a quinta e sexta fita-β da folha-β rÃgida da ConM. A ConM complexada com o resveratrol inibiu a oxidaÃÃo do DPPH, mostrando atividade sinÃrgica entre a proteÃna e o ligante com a relaÃÃo mais eficaz de 2:3. Foi verificado ainda que o sÃtio de ligaÃÃo a carboidratos nÃo està diretamente relacionado à atividade antioxidante. à a interaÃÃo entre ConM e resveratrol, que indica o sinergismo destas duas molÃculas em agir como agentes sequestrantes de radicais livres que podem estarem relacionados a capacidade de reduÃÃo do processo inflamatÃrio atravÃs da inibiÃÃo de muitos mediadores prÃ-inflamatÃrios por lecitinas. / Resveratrol is a natural antioxidant polyphenol found especially in grape epicarp, walnuts, and pomegranates, which can inhibit the activation of proinflammatory mediators and cytokines at the early gene expression stage. It is well known that lectins are sugar-binding proteins that act as both pro- and anti-inflammatory molecules. Thus, the objective of this work was to verify the binding of a polyphenol compound with a lectin of Canavalia maritima (ConM) based on their ability to inhibit pro-inflammatory processes. To accomplish this, ConM was purified and crystallized, and resveratrol was soaked at 5 mM for 2 hours of incubation. The crystal belongs to the monoclinic space group C2, the final refinement resulted in an Rfactor of 16.0% and an Rfree of 25.5%. Resveratrol binds in the rigid β-sheet through H-bonds and hydrophobic interaction with amino acids that compose the fifth and sixth β-strands of the rigid β-sheet of ConM. The ConM and resveratrol inhibited DPPH oxidation, showing synergic activity with the most effective ratio of 2:3 and carbohydrate binding site is not directly related to antioxidant activity. It is the interaction between ConM and resveratrol that indicates the synergism of these two molecules in acting as free radicals scavengers and in reducing the inflammatory process through the inhibition of many pro-inflammatory events.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2016-10-26T18:07:29Z No. of bitstreams: 1 2015 - Ileana Costa Miranda.pdf: 4377950 bytes, checksum: 0fd81889ff042fdd5ef81568986b1e7a (MD5) / Made available in DSpace on 2016-10-26T18:07:29Z (GMT). No. of bitstreams: 1 2015 - Ileana Costa Miranda.pdf: 4377950 bytes, checksum: 0fd81889ff042fdd5ef81568986b1e7a (MD5) Previous issue date: 2015-12-15 / CAPES / Animals grazing Brachiaria spp commonly present foamy macrophages isolated or grouped in the liver, and crystals within biliary ducts. The pathogenesis of formation and the nature of the material stored on these cells, however, are not completely known. Through lectin histochemistry evaluation, steroidal saponins (secondary glycosylated metabolites) have been identified in the crystals and within the cytoplasm of the foam cells, which are probably liable for damage the liver leading to accumulation of phylloerythrin. This study aims to standardize the use of lectin histochemistry to detect glycosylated metabolites in tissues of buffaloes kept in different Brachiaria spp pastures in Brazil. Fragments of liver and mesenteric lymph node from 40 animals were analyzed: 10 buffaloes that were kept in predominant pasture of B. decumbens for 12 months; 10 buffaloes that were kept in pasture with a predominance of B. brizantha for 18 months; 10 buffaloes that were kept in pasture of B. brizantha for approximately four years; and, as a negative control, 10 buffaloes that were maintained in native pasture without Brachiaria spp since birth. Fourteen lectins were tested (Con-A, SBA, WGA, DBA, UEA, RCA, PNA, GSL-I, PSA, LCA, PHA-E, PHA-L, SJA and SWGA), in a total of 1120 evaluated fragments. Previous studies demonstrated that PNA showed great binding reactivity for foamy macrophages in bovine and ovine. In the present study, SWGA presented high specificity and marked binding reactivity for foamy macrophages; WGA, GSL, PHA-E and PHA-L showed moderate to marked reactivity but low specificity for foamy macrophages; the other lectins didn't show significant reactivity or specificity. It remains unclear why there is this difference in lectins binding reactivity to foamy macrophages; it is suggested that divergences may occur depending on the species of Brachiaria ingested, the plant growth stage, the type and proportion of saponins stored in the plant due to seasonality, the differences in the metabolism of animal species, the presence of photosensitivity, the clinical course of the disease and the plant intake time. Moreover, there was no significant reactivity difference between the collected fragments of animals that grazed in B. decumbens for 12 months and B. brizantha for 18 months. However, the decreased presence of foamy macrophages and its lectin histochemical binding in animals that fed on B. brizantha for a longer time indicates that the animals can pass through an adaptation process according to the the plant intake time. Lectin histochemistry analysis can be used to characterize the material stored in foamy macrophages present in liver and mesenteric lymph node of buffaloes that graze in Brachiaria spp pastures and helps to clarify the pathogenesis of these cells. / Animais que se alimentam em pastos de Brachiaria spp comumente apresentam macr?fagos espumosos isolados ou agrupados no f?gado, al?m de cristais no interior de ductos biliares. A patog?nese da forma??o e a natureza do material armazenado nestas c?lulas, contudo, ainda n?o s?o completamente conhecidas. Atrav?s da avalia??o lectino-histoqu?mica, saponinas esteroidais (metab?litos glicosilados secund?rios) t?m sido identificadas nos cristais e no citoplasma das c?lulas espumosas, que provavelmente s?o respons?veis por danificar o f?gado e levar ao ac?mulo de filoeritrina. Por meio deste trabalho, objetivou-se padronizar e caracterizar a utiliza??o da lectino-histoqu?mica na detec??o de metab?litos glicosilados nos tecidos de b?falos mantidos em diferentes pastos de Brachiaria spp no Brasil. Fragmentos de f?gado e linfonodo mesent?rico de 40 animais foram analisados: 10 b?falos mantidos em pastagem predominante de B. decumbens por aproximadamente 12 meses; 10 b?falos mantidos em pastagem predominante de B. brizantha por aproximadamente 18 meses; 10 b?falos mantidos em pastagem de B. brizantha por aproximadamente quatro anos; e, como controle negativo, 10 b?falos mantidos em pastagem livre de Brachiaria spp desde o nascimento. Quatorze lectinas foram testadas (Con-A, SBA, WGA, DBA, UEA, RCA, PNA, GSL-I, PSA, LCA, PHA-E, PHA-L, SJA e SWGA), em um total de 1120 fragmentos avaliados. Estudos anteriores demonstraram que a lectina PNA possui marcada reatividade para macr?fagos espumosos de bovinos e ovinos. No presente estudo, a lectina SWGA apresentou acentuada reatividade e alta especificidade para macr?fagos espumosos; WGA, GSL, PHA-E e PHA-L mostraram moderada a acentuada reatividade, mas baixa especificidade aos macr?fagos espumosos; as outras lectinas n?o apresentaram reatividade ou especificidade significativas. Ainda n?o se sabe exatamente a que atribuir a diferen?a de reatividade aos macr?fagos espumosos. Sugere-se que diverg?ncias ocorram em fun??o da esp?cie de Brachiaria ingerida, da fase de crescimento da planta, do tipo e propor??o dos glicoconjugados armazenados na planta em decorr?ncia da ?poca do ano, das diferen?as no metabolismo da esp?cie do animal em quest?o, da presen?a de fotossensibiliza??o, da evolu??o cl?nica da doen?a e do tempo de ingest?o da planta. N?o houve diferen?a de marca??o significativa entre os fragmentos coletados de animais que se alimentaram de B. decumbens por 12 meses e B. brizantha por 18 meses. Por?m, a diminui??o da presen?a e marca??o lectino-histoqu?mica dos macr?fagos espumosos nos tecidos dos b?falos que ingeriram B. brizantha durante mais tempo indica que os animais podem passar por um processo de adapta??o de acordo com o tempo de ingest?o da planta. A avalia??o lectino-histoqu?mica pode ser utilizada para caracterizar o material armazenado em macr?fagos espumosos presentes no f?gado e linfonodo mesent?rico de b?falos que se alimentam em pastagens de Brachiaria spp e ajuda na compreens?o da patog?nese de forma??o destas c?lulas.

This dissertation focuses on the synthesis and characterization of glyconanomaterials, as well as their applications in studying carbohydrate-protein interactions. A new and versatile method for coupling underivatized carbohydrates to nanomaterials including gold and silica nanoparticles was developed via the photochemically induced coupling reaction of perfluorophenylazide (PFPA). A wide range of carbohydrates including mono-, oligo- and poly-saccharides were conjugated to the nanoparticles with high yields and efficiency. New analytical methods were developed to determine the binding affinities of glyconanoparticles (GNPs) with lectins; these include fluorescence-based competition assay, dynamic light scattering (DLS) and isothermal titration calorimetry (ITC). Results showed that the multivalent presentation of carbohydrate ligands significantly enhanced the binding affinity of GNPs by several orders of magnitude compared to the free ligands. Systematic studies were carried out to investigate the impact of ligand presentation, i.e., the type and length of spacer linkage, the ligand density and the nanoparticle size on the binding affinity of the resulting glyconanoparticles. We used gold GNPs to study interactions with anti-HIV lectin cyanovirin-N (CV-N), and dye-doped silica nanoparticles for labeling glyans and developing high-throughput screening technique.

Members of the C-type lectin domain (CTLD) superfamily are metazoan proteins functionally important in glycoprotein metabolism, mechanisms of multicellular integration and immunity. This thesis presents the results of several computational and experimental studies of the CTLD structure, function and evolution.¶ Core structural properties of the CTLD fold were explored in a comparative analysis of the 37 distinct CTLD structures available publicly, which demonstrate significant structural conservation despite low or undetectable sequence similarity. Pairwise structural alignments of all CTLD structures were created with three different methods (DALI, CE and LOCK) and analysed manually and using a computational algorithm developed for this purpose. The analysis revealed a set of conserved positions and interactions, which were classified based on their role in CTLD structure maintenance.¶ The CTLD family is large and diverse. To organize and annotate the several thousand of known CTLD-containing protein sequences and integrate the information on their evolution, structure and function a local database and a web-based interface to it were developed. The software is written in Perl, is based on bioperl, bioperl-db and Apache::ASP modules, and can be used for collaborative annotation of any collection of phylogenetically related sequences.¶ Several studies of CTLD genomics were performed. In one such study, carried out in collaboration with the RIKEN structural genomics centre, CTLD sequences from the Caenorhabditis elegans genome were identified and clustered into groups based on similarity. The most representative members of the groups were then selected, which if characterized structurally would tell most about the C. elegans CTLDs and provide templates for homology modelling of all C. elegans CTLD structures.¶ In the other whole-genome study, the CTLD family in the puffer fish Fugu rubripes was analysed using the draft genome sequence. This work extended and complemented three genome-level surveys on human, C. elegans and D. melanogaster reported previously. The study showed that the CTLD repertoire of Fugu rubripes is very similar to that of mammals, although several interesting differences exist, and that Fugu CTLD-encoding genes are selectively duplicated in a manner suggesting an ancient large-scale duplication event. Another important finding was the identification of several new CTLDcps, which had mammalian orthologues not recognized previously.¶ CBCP, a novel CTLD-containing protein highly conserved between fish and mammals with previously unknown domain architecture, was predicted in the Fugu study based solely on ab initio gene models from the Fugu locus and cross-species genomic DNA alignments. To test if the prediction was correct, a full-length cDNA of the mouse CBCP was cloned, its tissue distribution characterized and untranslated regions determined by RACE. The full-length mCBCP transcript is 10 kb long, encodes a protein of 2172 amino acids and confirms the original prediction. The presence of a large N-terminal NG2 domain makes CBCP a member of a small but very interesting family of Metazoan proteins.

Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. The high incidence is still found in Taiwan. There is strong evidence that host genes influence individual susceptibility to tuberculosis. Young children, like immunocompromised patients, once infected are at increased risk for TB disease and progression to extrapulmonary disease. Thus far, to identify the genes responsible for the variation in the human susceptibility/resistance to TB has remained elusive. Mannose-binding lectin (MBL) activates the complement system in an antibody-independent manner, enhances complement-mediated phagocytosis, and plays an important role in innate immunity in the regulation of inflammatory cytokine release by monocytes. It is one of the molecules that have been suggested to have a link to human susceptibility or protection against infection. According to some studies (mostly conducted in adult populations) , low levels of MBL associated with variant alleles at the promoter and exon 1 regions of MBL protect against tuberculosis. Other investigators instead claim that protection against the disease is associated with high levels of MBL. In this study we aimed to investigate the relationships between the susceptibility to TB and MBL gene polymorphisms in children with cervical mycobacterial lymphadenitis infected by M. tuberculosis.139 case patients with cervical mycobacterial lymphadenitis and 102 unrelated healthy control subjects were tested by real-time PCR for polymorphisms at the promoter and the exon 1 regions of the MBL gene. Diagnosis of mycobacterial lymphadenitis infected by M. tuberculosis, based on findings of pathological examination of the lymph nodes, was confirmed by acid-fast stain and TB PCR.The frequency of A allele was significantly higher in TB+ patients compared with TB- controls (82.7% vs 72.6%; odds ratio 1.813; p=0.007). The frequency of high-producer MBL2 genotypes (A/A) was higher in TB+ patients than in TB- subjects (70.5% vs 45.1%, odds ratio 2.91, p<0.001), while patients carried the B alleles (A/B and B/B) that have decreased levels of MBL was inversely associated with mycobacterial infectivity (29.5% vs 54.9%; odds ratio 2.910; p<0.001). The frequencies of MBL promoter -550 genotypes also revealed a significant difference between TB+ and TB- groups (p = 0.046), but in contrast, with significantly higher frequency of L/L genotype (of low MBL level) in TB+ patients (34.5% vs 21.6%; odds ratio 1.918; p=0.029). The frequencies of MBL promoter -221 genotypes (X and Y) was similar in TB+ and TB- groups.This study supports the conclusion that MBL can protect or predispose the host to tuberculosis, depending on the host¡¦s haplotype pair.

Effective methods for isolation and purification of glycoproteins are of increasing significance to the rapidly growing biopharmaceutical and diagnostic industry. Glycoproteins represent the majority of therapeutic proteins on the market and are effectively used to treat immune disorders, infections, cancers, and other diseases. Targeting these glycoproteins is also critical to an emerging field of glycoproteomics aimed to understand structure-function relationships of glycans. Architecturally, these glycoproteins are proteins with covalently linked oligosaccharide chains of varying monosaccharide composition. Affinity chromatography has proven to be an excellent method of glycoprotein purification at the bench scale. However, chromatography in large scale production has its drawbacks. Column fowling, flowrate limitations, and diffusional constraints collectively hinder the effectiveness of the method. An alternative proposed in this dissertation is the use of affinity precipitation as a purification technique. The three main objectives are 1) develop and produce dual-functional, thermo-responsive affinity ligands from a biological host, 2) characterize and optimize the accompanying affinity precipitation method, and 3) apply the ligand and process to relevant, unmodified glycoproteins. The design of the thermo-responsive affinity construct was comprised of two main functional domains. The binding capability was achieved by selection of small ligands with affinity to a specific monosaccharide moiety. Two different lectins, or sugar binding proteins, were used in the fusion design: a fucose binding lectin from Ralstonia solanacearum, and a sialic acid binding lectin from Vibrio cholera. The thermo-responsive functionality was obtained by use of an elastin-like peptide (ELP), which confers inverse solubility relationship properties to the fusion construct. A small library of varying ELP chain lengths were designed to find the optimal size fusion for both production and function. These dual functional ligands were cloned and expressed in the microbial host, E. coli. Furthermore, secretion of these constructs was achieved by employing the Tat secretion pathway in combination with an outer membrane lipoprotein deletion mutant with a leaky periplasm phenotype. This secretory mechanism allows for easy isolation, avoidance of inclusion bodies, and no additional protease inhibitors. After successful production, the ligands were tested to confirm that dual functionality was preserved in fusion form. Once binding conditions and precipitation properties were ascertained, the purification ability was tested on model glycoproteins. Experimentation was carried out monitoring the purification yield, purity, and retained activity of the target enzymes. High contaminant solutions, such as cell lysates, were spiked with the model glycoproteins to mimic crude protein solutions. The purification ability of the constructs in these models was observed. The method was then implemented on two relevant glycoprotein applications: 1) purification of soybean peroxidase from a crude protein extract and 2) targeting the therapeutic protein erythropoietin from albumin rich, used CHO cell media. By implementation of the fucose targeting fusion construct, the unmodified soybean peroxidase is isolated from a natural crude extract from the soybean hull, a by-product of the soybean industry. The affinity precipitation method parameters were optimized with respect to ratios, temperatures, recycle, and elution buffers to achieve successful isolation of the low abundance enzyme. Under the optimized conditions, >95% recovery yield and a purification of 22.7 fold of an active, pure product was attainable. The purification of erythropoietin led to additional experimentation with high-abundant glycoprotein solutions, as well as expansion of the affinity ligand platform. The concept of multi-lectin affinity precipitation, using the fucose and sialic acid binding lection sequentially, was introduced and tested for purification capability. An industrially relevant scheme involving isolation of the erythropoietin from used CHO cell media allowed for an achievable yield of about 60%, with a resulting albumin depletion of about 85%. In addition to development of a pair of novel thermo-responsive affinity ligands for glycoprotein purification, this dissertation provides insight on possible improvements and future directions with respect to the thermo-responsive affinity ligand platform. This unique concept employs novel lectin fusions to target valuable glycoproteins using a method avoiding the major drawbacks associated with chromatography.

Cells can mediate intercellular communication by the secretion and uptake of microvesicles, nano-sized membranous particles that carry signaling molecules, antigens, lipids, mRNA and miRNA between cells. The biological function of these vesicles is dependent upon their composition and cellular origin which is regulated by mechanisms that are not well understood. Based on their molecular content, microvesicles may play a role in immune regulation, cancer progression, the spread of infectious agents and numerous other important normal and pathogenic processes. The proteomic content of microvesicles from diverse sources has been intensely studied. In contrast, little is known about their glycomic content. The glycosylation pattern of a protein or lipid plays a key role in determining its functional properties in several ways. Glycans can determine the trafficking of a protein to particular regions of the cell as well as the protein’s half life. In addition, the glycan-dervied oligomerization of glycolipids and glycoproteins is a known mechanism for the activation of receptors and recognition of ligands on the surface of the cell. Glycomic analysis may thus provide valuable insights into microvesicle function. I utilized lectin microarray technology to compare the glycosylation patterns of microvesicles derived from a variety of biological sources. When compared to cellular membranes, microvesicles were enriched in high mannose, polylactosamine, α2-6 sialic acid, and complex N-linked glycans but exclude terminal blood group A and B antigens. The polylactosamine signature in microvesicles from different cell lines derives from distinct glycoprotein cohorts. After treatment of Sk-Mel-5 cells with lactose to inhibit lectin-glycan interactions, secretion of microvesicle resident proteins was severely reduced. Taken together, this work provides evidence for a role of glycosylation in microvesicle-directed protein sorting. / text

The mechanisms responsible for the enhanced inflammatory response in type 2 diabetes (T2DM) and its contribution to the severe ischemia/reperfusion (I/R) injury observed in the T2DM heart are unclear. I/R is associated with an acute inflammatory response recognized by reactive oxidant production, complement activation, and leukocyte-endothelial cell adhesion, among others. Complement activation plays an important role in the inflammatory response and is involved in the manifestation of I/R injury in the non-diabetic heart, and is a potent chemoattractant for circulating neutrophils (PMNs). The purpose of this dissertation research was to test the hypothesis that the complement system, predominantly the lectin pathway, is a significant contributor to the excessive response of the Zucker Diabetic Fatty (ZDF), a rat model of T2DM, to myocardial I/R injury. Following 30min of coronary artery occlusion and 120min of reperfusion we measured C3 deposition, PMN accumulation, PMN CD11b expression, and ICAM-1 expression. We found significantly more C3 deposition, PMN accumulation, ICAM-1 and PMN CD11b expression in diabetic samples compared to non-diabetic samples. To elucidate a role for complement system activation, we treated animals with FUT-175, a broad complement inhibitor. In vivo, FUT-175 treatment significantly decreased complement deposition (66%), PMN accumulation (59%), and infarct size (55%) compared to untreated animals in both non-diabetic Sprague-Dawley and diabetic ZDF rats. To specifically examine the role of the lectin pathway, we selectively inhibited rat MBL-A prior to myocardial I/R in ZDF rats. Anti-MBL treatment significantly decreased infarct size, C3 deposition and PMN accumulation in the ZDF post-ischemic left ventricle (LV). Genomic analysis revealed that gene expression of the pro-inflammatory cytokines IL-6 and IL-1α was enhanced in the ZDF heart following reperfusion, and quantitative RT-PCR results confirmed IL-6 upregulation. We found significantly increased complement C5a receptor (CD88) expression on diabetic neutrophils prior to ischemia, suggesting that diabetic PMNs are "primed" to respond to complement activation. Taken together, these results provide evidence that 1) the ZDF rat is a good model for chronic inflammation in the setting of T2DM, 2) lectin pathway activation plays a significant role in the inflammatory response to I/R injury in the ZDF heart, and 3) anti-complement therapy may be particularly cardio-protective in T2DM.

Walleye (Sander vitreus M.) are valuable in commercial and recreational fisheries and are affected by bacterial, fungal and viral disease. Pattern recognition receptors (PRRs) are germline-encoded and constitutively expressed and bind non-self or altered-self for immune recognition. Walleye were hypothesised to have circulating PRRs that were capable of binding diverse pathogens. These PRRs were hypothesised to increase with infection, be distributed in immunologically relevant tissues and to be strain and age specific. PRR binding was measured by affinity chromatography, plasma binding assays,SDS-PAGE, Western blots, ELISA, PCR, and immunohistochemistry. ELISA and affinity chromatography assays were developed in rainbow trout (Oncorhynchus mykiss) with known PRRs. Trout ladderlectin was confirmed as a PRR binding viral hemorrhagic septicemia virus (VHSV). These techniques were adapted to walleye using Flavobacterium columnare, chitin, VHSV and Sepharose resin. A 22 kDa protein bound to F. columnare, a 17 kDa protein bound to chitin and a 34 kDa protein bound to VHSV were identified as similar to bass apolipoprotein, carp C3 and rainbow trout intelectin, respectively. PCR and 3'-RACE-PCR were used to generate nucleotide sequence to confirm identity of walleye apolipoprotein and mannose-binding lectin (MBL)-like protein from the intelectin-like sequence. Two rabbit polyclonal antibodies were raised to 34 and 67 kDa MBL amino acid sequences and used to verify MBL-like protein as a PRR for VHSV. Healthy walleye MBL-like protein plasma concentration was 7.5 ng/ml. Significant differences were found between geographically distant strains of walleye. An ELISA demonstrated that MBL-like protein had significant differences in binding affinity between multiple strains of VHSV and different viruses found in Ontario. MBL-like protein plasma levels increased with initial infection of naïve fish with waterborne and IP VHSV (107 pfu) but did not change with IP reinfection. Previous infection with VHSV significantly decreased walleye mortality. IHC of walleye shows MBL-like protein is distributed in epithelial surfaces, primarily skin, oropharynx, gill, gastrointestinal system, renal nephrons, connective tissue of gonads and plasma. There was no qualitative difference in MBL-like protein tissue distribution in healthy and VHSV-infected walleye. This is the first evidence for fish lectins binding viruses.