β-Glucans have beneficial health effects due to their immune modulatory properties. Oral administration of β-glucans affects tumour growth, microbial infection, sepsis, and wound healing. We hypothesized that pre-treatment with orally delivered soluble and particulate β-glucans could ameliorate the development of aggravate dextran sulfate sodium (DSS) induced intestinal inflammation. To study this, mice were orally pre-treated with β-glucans for 14 days. We tested curdlan (a particulate β-(1,3)-glucan), glucan phosphate (a soluble β-(1,3)-glucan), and zymosan (a particle made from Saccharomyces cerevisiae, which contains around 55% β-glucans). Weight loss, colon weight, and feces score did not differ between β-glucan and vehicle treated groups. However, histology scores indicated that β-glucan-treated mice had increased inflammation at a microscopic level suggesting that β-glucan treatment worsened intestinal inflammation. Furthermore, curdlan and zymosan treatment led to increased colonic levels of inflammatory cytokines and chemokines, compared to vehicle. Glucan phosphate treatment did not significantly affect cytokine and chemokine levels. These data suggest that particulate and soluble β-glucans differentially affect the intestinal immune responses. However, no significant differences in other clinical colitis scores between soluble and particulate β-glucans were found in this study. In summary, β-glucans aggravate the course of dextran sulfate sodium (DSS)-induced intestinal inflammation at the level of the mucosa.

The CD5 lymphocyte surface receptor is a group B member of the ancient and highly conserved scavenger receptor cysteine-rich superfamily. CD5 is expressed on mature T and B1a cells, where it is known to modulate lymphocyte activation and/or differentiation processes. Recently, the interaction of a few group B SRCR members (CD6, Spα, and DMBT1) with conserved microbial structures has been reported. Protein binding assays presented herein indicate that the CD5 ectodomain binds to and aggregates fungal cells (Schizosaccharomyces pombe, Candida albicans, and Cryptococcus neoformans) but not to Gram-negative (Escherichia coli) or Gram-positive (Staphylococcus aureus) bacteria. Accordingly, the CD5 ectodomain binds to zymosan but not to purified bacterial cell wall constituents (LPS, lipotheicoic acid, or peptidoglycan), and such binding is specifically competed by β-glucan but not by mannan. The K d of the rshCD5/(1→3)-β-D-glucan phosphate interaction is 3.7 ± 0.2 nM as calculated from tryptophan fluorescence data analysis of free and bound rshCD5. Moreover, zymosan binds to membrane-bound CD5, and this induces both MAPK activation and cytokine release. In vivo validation of the fungal binding properties of the CD5 ectodomain is deduced from its protective effect in a mouse model of zymosan-induced septic shock-like syndrome. In conclusion, the present results indicate that the CD5 lymphocyte receptor may sense the presence of conserved fungal components [namely, (1→3)-β-D- glucans] and support the therapeutic potential of soluble CD5 forms in fungal sepsis.

β-(1,3)-Glucan is present in mould cell walls and frequently detected in house dust mite (HDM) faeces. β-Glucan exposure is thought to be associated with pulmonary allergic inflammation in mouse and man, although the published data are inconsistent. Here, we show that highly purified β-glucan exacerbates HDM-induced eosinophilic, T helper 2 type airway responses by acting as an adjuvant, promoting activation, proliferation and polarisation of HDM-specific T cells (1-Derβ T cells). We therefore provide definitive evidence that β-glucan can influence allergic pulmonary inflammation.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aflatoxin B1 (AFB1) is one of the main mycotoxins that can be identified in foods, and has relevance for agricultural economics and public health because of its immunotoxic properties. A functional immune system is a basic requirement for a healthy life in modern animal production. The interaction involving nutrition and immunity is a strategic factor to obtain a high quality performance in the poultry industry. Immunomodulators such as β-glucans have an immunostimulating activity, which enables the host ability to resist opportunistic infections. To contribute to the elucidation of the mechanism of action of β-glucans in broiler chicken lymphocytes, the effects of the concentrations of 0.1, 1 and 10% of β-glucans derived from Saccharomyces cerevisiae were investigated in lymphocytes exposed to increasing concentrations of AFB1 (0, 0.1, 1, 10, 20 μg/ml). Lymphocytes were separated by Ficoll-Histopaque density and cultured in 96 well-plates containing AFB1 and/or β-glucans in a 5% CO2 atmosphere at 39°C. MTT, PicoGreen® and 2'-7'-dichlorofluorescein diacetate cytotoxicity tests were evaluated at 24, 48 and 72 h of incubation. The comet assay to elucidate the DNA damage was also performed. The percentage of viable cells decreased in the presence of 10 μg/ml AFB1 at 48 h (p < 0.05) and 10 and 20 μg/ml AFB1 at 72 h (p < 0.001 and p < 0.01, respectively), when compared to the control group (0 μg/ml). Furthermore, an increase in cell-free DNA in AFB1 concentrations > 1 μg/ml (p < 0.001) and the generation of ROS at 24 h were also observed. DNA damage increased approximately 2.3 fold in lymphocytes exposed to 20 μg/ml of AFB1 when compared to the control group. Conversely, β-glucans showed cytoprotective effects (p < 0.001), and the concentration of 1% reverted the AFB1-induced lymphocyte damage. β-glucans at 10% significantly increased (p < 0.001) the formation of reactive oxygen species (ROS), potentiating the AFB1-induced ROS formation. In conclusion, this study showed that AFB1 and β-glucans exert influence on lymphocyte oxidative metabolism and have dose-dependent potentiating effects. The results also showed genoprotective in vitro effect of β-glucans in poultry lymphocytes exposed to AFB1, being the concentration of 1% β-glucans able to maintain DNA integrity. / A aflatoxina B1 (AFB1) é uma das principais micotoxinas que podem ser identificadas em alimentos, possuindo relevância agroeconômica e para a saúde pública, por ser considerada imunotóxica. Um sistema imune funcional é um requisito básico para uma vida saudável na produção moderna de animais. A interação envolvendo nutrição e imunidade é um fator estratégico para obter um bom desempenho em frangos de corte. Devido as suas atividades imunomodulatórias, substâncias como as β-glucanas proporcionam ao hospedeiro uma maior capacidade de resistir a infecções oportunistas. Para melhor compreender o mecanismo de ação das β-glucanas, sobre os linfócitos de frangos de corte, investigou-se os efeitos das concentrações de 0.1, 1 e 10% de β-glucanas derivadas do fungo Saccharomyces cerevisiae em linfócitos expostos a crescentes concentrações de AFB1 (0, 0.1, 1, 10, 20 μg/ml). Os linfócitos foram separados através do reagente de densidade Ficoll-Histopaque e cultivados em placas de 96 poços, contendo as concentrações de AFB1 e/ou β-glucanas em atmosfera de 5% de CO2 a 39°C durante 24, 48 e 72 h. A citotoxicidade celular foi avaliada através dos testes MTT e PicoGreen®, e a formação de espécies reativas de oxigênio (EROS) através do ensaio 2′-7′- diacetato diclorofluoresceína. Também foi utilizado o teste cometa para elucidar os danos ao DNA. A viabilidade celular reduziu na presença de 10 μg/ml de AFB1 em 48 h (p < 0.05) e em 10 and 20 μg/ml de AFB1 (p < 0.01 e p < 0.001, respectivamente) em 72 h quando comparada ao grupo controle. Além disso, as concentrações de AFB1 > 1 μg/ml aumentaram significativamente (p < 0.001) a liberação de fita dupla de DNA (dsDNA) e a produção de EROS em 24 h. Os danos causados ao DNA foram confirmados através do momento cauda do cometa e aumentaram cerca de 2,3 vezes em linfócitos expostos a 20 μg/ml de AFB1 quando comparados ao grupo controle. Por outro lado, as β-glucanas exerceram efeitos citoprotetores (p < 0.001), sendo que a concentração de 1% foi capaz de reverter os danos genotóxicos causados pela ação da AFB1. Já a concentração de 10% de β-glucanas aumentou significativamente (p < 0.001) a formação de EROS, potencializando a ação da AFB1. Em conclusão, este estudou evidenciou que a AFB1 e as β-glucanas exercem influência sobre o metabolismo oxidativo dos linfócitos e possui efeito potencializador dose-dependente. Os resultados também evidenciaram o efeito genoprotetor in vitro de β-glucanas em linfócitos de frangos expostos a AFB1, sendo a concentração de β-glucanas a 1% capaz de manter a integridade do DNA.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-10-20T17:56:33Z No. of bitstreams: 2 Dissertação - Estefânia Viano da Silva - 2014.pdf: 4645592 bytes, checksum: f3321dacb1ccfd2f2b017d08f8e3b578 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-10-20T17:58:09Z (GMT) No. of bitstreams: 2 Dissertação - Estefânia Viano da Silva - 2014.pdf: 4645592 bytes, checksum: f3321dacb1ccfd2f2b017d08f8e3b578 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-10-20T17:58:09Z (GMT). No. of bitstreams: 2 Dissertação - Estefânia Viano da Silva - 2014.pdf: 4645592 bytes, checksum: f3321dacb1ccfd2f2b017d08f8e3b578 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-06-26 / The mushrooms have been used as food and medicine for thousands of years. Among them, Grifola frondosa (“Maitake”) may be on the most versatile and promising medicinal mushrooms for use as a dietary supplement. Besides the high nutritional value, they are a good source of compounds such as carbohydrates that can act as biological response modifiers. In this work, β-D-glucans (PSF2-GfM and MRSF2K-GfM fractions) and heteropolysaccharides unusual in the literature (MEPF3CW-GfM and PF2K-GfM fractions) were isolated from the mycelium, grown in solid culture, of the G. frondosa, by successive aqueous and alkaline extractions, followed by fractionation through freeze-thawing, precipitation of soluble material with a Fehling solution, and dialysis using a membrane with an exclusion limit of 1000 kDa. The chemical structures were established based on monossacharide composition, periodate oxidation, methylation analysis, HPSEC-MALLS and NMR studies. The branched β-D-glucans (GLC-GfM) found were similar to those previously described for the fungus, which contains a (13)- linked β-Glcp main-chain, substituted at O-6 by single-unit -Glcp side-chains, on the average of one to every third residues of the backbone, but with different molar masses and water solubility. In adittion, novel heteropolymers were obtained. One was a fucomannogalactan (FMG-GfM), with a molar mass of 175 x 103 g mol-1, was shown to have a main chain of (1→6)-linked α-D-Galp and 3-O-Me-α-DGalp, both of which are partially substituted at O-2, principally, by 3-O-α-D-Manp-α-LFucp groups. The other was a fucoxylomannan (FXM-GfM) (Mw 202 x 103 g.mol-1), which consisted of backbone of (13)-linked α-D-Manp units, totally substituted at O-4 mainly by 4-O-α-L-Fucp-β-D-Xylp disaccharide. Thus, the polymers obtained from Grifola frondosa may could be good candidates for evaluation of its biological effect, considering the results obtained for others similar mushrooms polysaccharides. / Os cogumelos têm sido usados como alimentos e remédios por milhares de anos. Entre eles, o “Maitake” (Grifola frondosa) pode ser um dos cogumelos medicinais mais versáteis e promissores para o uso como um suplemento dietético. Além do valor nutricional elevado, eles são uma boa fonte de compostos, como os carboidratos, que podem agir como modificadores de resposta biológica. Neste trabalho, foram isolados do micélio (cultivado em meio sólido) de G. frondosa as β-D-glucanas (frações PSF2KGfM e MRSF2K-GfM) e heteropolissacarídeos (frações MePF3CW-GfM e PF2K-GfM) não comumente encontrados na literatura, por sucessivas extrações aquosas e alcalinas, seguido por fracionamento através do processo de congelamento/degelo, precipitação das frações solúveis em água com solução de Fehling e diálise em membranas com limite de exclusão de 1000 kDa. A estrutura química foi determinada através das análises de composição mossacarídica, oxidação com periodato de sódio, metilação, HPSEC-MALLS e RMN. As β-D-glucanas (GLC) isoladas foram similares àquelas previamente descritas para este fungo, as quais contém uma cadeia principal formada por unidades de 3-O-β-D-Glcp, podendo ser substituídas em O-6 por terminais não redutores de β-Glcp (1 ramificação a cada três unidades da cadeia principal), distinguindo-as quanto à solubilidade em água e tamanho (massa molar). Além destas, foram obtidos novos heteropolímeros. Um deles foi uma fucomanogalactana (FMGGfM), com uma massa molar de 175 x 103 g mol-1, a qual possui um core constituído por unidades de 6-O-α-D-Galp e 6-O-(3-O-Me-α-D-Galp), sendo ambas parcialmente substituídas em O-2 por 3-O-α-D-Manp-α-L-Fucp. O outro heteropolissacarídeo isolado consiste em uma fucoxilomanana (FXM-GfM) (Mw 202 x 103 g.mol-1) formada por unidades de α-D-Manp unidas por ligações glicosídicas do tipo (13) as quais se encontram ramificadas em O-4 principalmente pelo dissacarídeo 4-O-α-L-Fucp-β-DXylp. Considerando os efeitos terapêuticos apresentados por moléculas similares descritas para macrofungos, estes podem ser bons candidatos para avaliação do efeito biológico.

Les ß-glucanes (BG) sont les polysaccharides les plus abondants de la paroi de Saccharomyces cerevisiae. Depuis des millénaires, ils sont utilisés pour leurs propriétés immunostimulantes et leurs potentiels thérapeutiques. L'objectif de ce travail était de caractériser la réponse immunitaire induite par les BG et de comprendre leurs modes d'action sur les macrophages murins en contexte infectieux. Nous avons montré que (i) les extraits de paroi enrichis en BG n'induisent qu'une faible production de cytokines par les macrophages contrairement aux extraits bruts, (ii) la réponse inflammatoire médiée par les extraits bruts résulte de la signalisation des TLRs et non de Dectin-1 et (iii) les BG stimulent la synthèse tardive de GM-CSF via Dectin-1. En conditions infectieuses, les BG enrichis confèrent une forte signature inflammatoire aux macrophages prétraités conduisant à l'amplification de la production cytokinique, à la synthèse de ROS et l'optimisation de la clairance bactérienne. En conclusion, cette étude souligne les enjeux de l'utilisation des BG enrichis comme adjuvants dans l'amélioration de la résistance des individus aux infections. / ß-glucans (BG) are the most abundant polysaccharides of the Saccharomyces cerevisiae cell wall. For decades, they have been extensively used because of their immuno-modulatory properties and their potential therapeutic effects. The aim of this study was to characterize the immune response induced by BG and to understand their mechanisms of action on murine macrophages occurring upon bacterial infections. We demonstrated that (i) BG-enriched extracts trigger low amounts of cytokine production in contrast with crude products, (ii) the immune response mediated by crude extracts results from TLRs and not from Dectin-1 signaling and (iii) BG-enriched compounds stimulate the late and strong induction of GM-CSF in a Dectin-1-dependent manner. Upon bacteria exposure, BG-enriched extracts confer a strong inflammatory to pretreated macrophages leading to synergistic increase of cytokine release, ROS production and better clearance of pathogens. Altogether, our findings emphasize the relevancy of using BG-enriched extracts for the design of novel adjuvant formulations contributing to individuals' resistance to infections.