Fucus vesiculosus et Ascophyllum nodosum sont des algues brunes comestibles et abondantes au Québec. Cependant, elles ont été négligées en raison de leur valeur potentielle inconnue et de la période de récolte limitée. Afin de valider leur utilité, la composition chimique de ces algues et l’activité inhibitrice des enzymes digestives de l'amidon par les fucoïdanes extraits de ces deux espèces d'algues ont été étudiés en fonction de la saison. Les composants principaux des algues sont dans l’ordre: les polysaccharides> minéraux> protéines> fucoïdane> lipides> > composés phénoliques, et leur quantité est très variable selon la période de récolte. F. vesiculosus contenait une plus grande quantité de protéines et de minéraux, alors que A. nodosum avait relativement plus de polysaccharides. Par conséquent, F. vesiculosus serait plus avantageux comme source d’éléments nutritifs. L’algue A. nodosum récoltée en Juillet a permis d’obtenir le fucoïdane ayant la pureté la plus élevée et le meilleur rendement. Les fucoïdanes extraits des deux espèces d’algues ont inhibé l’activité de l’α-glucosidase alors que seul celui extrait d’A. nodosum a pu, de plus, inhiber l’α-amylase. Le fucoïdane d’A. nodosum était un inhibiteur plus puissant que le fucoïdane de F. vesiculosus pour l’α-glucosidase avec des IC50 variant de 0,013 ~ 0,047 mg/ml, tout comme pour l’α-amylase avec des IC50 de 0,12 ~ 4,62 mg/mL selon le mois. Pour comprendre les facteurs clés expliquant les différences d’inhibition d’α-amylase entre les fucoïdane d’A. nodosum et F. vesiculosus, certaines caractéristiques structurales ont été analysées et comparées à du galactofucoidane qui a servi de contrôle. A partir des résultats obtenus, il est confirmé que la masse moléculaire plus faible (637 kDa) du fucoïdane d’A. nodosum et la présence de sulfates sont liées à son activité inhibitrice. Nous avons émis l’hypothèse que les faibles masses moléculaires permettent d’exposer facilement les groupements sulfate qui peuvent agir sur l’α-amylase par interaction électrostatique, et donc d'inhiber son activité. En conclusion, les algues brunes du Québec présentent un potentiel d’utilisation important pour leur valeur nutritionnelle et leurs composés bioactifs. Le fucoïdane a montré une activité d'inhibition des enzymes digestives de l'amidon (α-amylase et α-glucosidase) et cette activité est différente selon les espèces d'algues et la période de récolte. Une meilleure compréhension du mécanisme inhibiteur par le fucoïdane peut être utile afin de développer un ingrédient fonctionnel permettant de prévenir le diabète de Type-2. / Fucus vesiculosus and Ascophyllum nodosum are edible brown seaweed and abundantly available in Quebec. However, they have been neglected because of their unknown value and technical limitation in harvest. In order to validate their usefulness, chemical composition in seaweeds and starch digestive enzyme inhibition activity by fucoidan extracted from two seaweed species were investigated with different seasons. The major components in both seaweeds were in order: polysaccharide > minerals > protein > fucoidan > lipid > phenol, and their quantity was quite variable depending on harvesting timing. F. vesiculosus contained larger amount of proteins and minerals, while A. nodosum had relatively more polysaccharides. Therefore, F. vesiculosus are advantageous as a nutritional source. Especially, from A. nodosum harvested in July, a fucoidan having higher purity and better yield was obtained. Fucoidans from two seaweeds species inhibited α-glucosidase activity while, only fucoidan from A. nodosum could inhibit α-amylase activity. A. nodosum fucoidan was a more potent inhibitor than F. vesiculosus fucoidan for α-glucosidase with IC50 of 0.013 ~ 0.047 mg/mL, and for α-amylase with IC50 of 0.12 ~ 4.62 mg/mL depending on harvest month. To understand the key factors explaining the difference in α-amylase inhibition between A. nodosum fucoidan and F. vesiculosus fucoidan, structural characteristic was analyzed and compared with galactofucoidan as a control. From the obtained results, it is confirmed that smaller molecular weight (637 kDa) of A. nodosum fucoidan and the presence of sulfates are related to its inhibitory activity. It is proposed that small molecular weight permits to expose easily sulfate groups for interaction with α-amylase throughout electrostatic interactions, and therefore inhibiting its activity. In conclusion, brown seaweeds in Quebec have a considerable importance for nutrition and bioactive products. Fucoidan shows the inhibition activity for starch digestive enzymes (α-amylase and α-glucosidase) and its activity is different depending on seaweed species and harvesting period. Further understanding of the inhibitory mechanism by fucoidan can be useful to develop a functional ingredient to help preventing for Type-2 diabetes.

In order to develop an environmentally friendly seaweed extract treatment which will benefit both pre- and post-harvest qualities of vegetables, the effects of pre-harvest application of the brown algae Ascophyllum nodosum extracts on the nutritional quality and post-harvest storability of spinach (Spinacia oleracea L.) was investigated. Plants treated with A. nodosum extracts accumulated higher concentrations of iron, potassium, total soluble protein, and total phenolics as compared to untreated controls. 1H NMR and LC-MS analysis revealed a roughly 50% enhanced accumulation of the 9 flavonoids identified, which is partially confirmed by the elevated chalcone isomerase activity. A. nodosum extract treatment caused an increase in transcription of the genes related to plant growth, osmolyte accumulation, and antioxidative activities. Post-harvest analysis revealed that A. nodosum extract treatment caused an enhanced storability of spinach leaves in terms of visual quality, weight loss, and senescence. Lipid peroxidation and ascorbate content were correlated with visual quality during storage. Animal experiments using the Caenorhabditis elegans nematode model revealed that spinach extracts prolonged the life span of C. elegans, and A. nodosum extract-enhanced polyphenols exerted improved beneficial effects in C. elegans against oxidative and heat stresses. Taken together, the results suggest that A. nodosum extracts enhance both pre- and post-harvest quality of spinach through stimulation of flavonoid pathways, thus leading to accumulation of flavonoids and promotion of anti-radical capacity in spinach leaves, which may protect the plant tissue against reactive oxygen species and subsequent decay. Furthermore, the increased flavonoid content in spinach exerted beneficial effects in C. elegans against oxidative and heat stresses via different mechanisms.

The effect of Ascophyllum nodosum extracts on the nodulation and growth of alfalfa was investigated. Plant growth assay revealed that alfalfa treated with 2 g L-1 ANE exhibited a significant increase in leaf area. Under salt stress, alfalfa treated with 0.5 g L-1 ANE exhibited a significant increase in total length compared to controls. A root hair deformation assay indicated that ANE 0.5 g L-1 stimulated the synthesis of Nod factors secreted by rhizobia thus accelerate root hair deformation of alfalfa. Similarly, ANE 0.5 g L-1 caused an increase in nodC gene expression suggesting that ANE may act similarly to flavonoids in the rhizobium-legume symbiosis. Under field conditions, ANE increased the total number of functional nodules, total root length and total leaf area. Taken together, the results suggest that ANE may contain compound(s) that promote specific metabolic pathway both in alfalfa and bacterium thus enhance the symbiotic relationship.

Made available in DSpace on 2014-06-11T19:25:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-01-15Bitstream added on 2014-06-13T18:26:25Z : No. of bitstreams: 1 almeida_mgj_me_arafcf_parcial.pdf: 82683 bytes, checksum: 49424d5182616aaa8e926f756efd65e1 (MD5) Bitstreams deleted on 2015-02-09T14:35:42Z: almeida_mgj_me_arafcf_parcial.pdf,Bitstream added on 2015-02-09T14:36:22Z : No. of bitstreams: 1 000710077.pdf: 903439 bytes, checksum: 7ad6ad625787ddd4aff6f1d6cad4169e (MD5) Bitstreams deleted on 2015-02-09T17:15:05Z: 000710077.pdf,Bitstream added on 2015-02-09T17:15:41Z : No. of bitstreams: 1 000710077.pdf: 903439 bytes, checksum: 7ad6ad625787ddd4aff6f1d6cad4169e (MD5) / A tendência atual na indústria cosmética é o desenvolvimento de produtos multifuncionais, ou seja, aqueles com capacidade de apresentar diferentes funções, possibilitando diferentes resultados para o consumidor. Seguindo esta tendência, ativos naturais são alternativas interessantes como substitutos a um único composto ativo, uma vez que são matrizes complexas e apresentam diversos componentes que podem possuir diferentes mecanismos de ação e, assim, conferir ao produto mais de uma função. Por este motivo, nos últimos anos o uso de extratos naturais tem crescido muito nesta área. Com o aumento da aplicação de extratos naturais em produtos cosméticos, há também a necessidade de estudos que comprovem sua eficácia e segurança. Uma das principais funções buscadas em produtos cosméticos é a ação antienvelhecimento, ou seja, a capacidade dos produtos em atuar neutralizando radicais livres. Neste contexto, o objetivo deste trabalho foi avaliar a eficácia e segurança do extrato de Ascophyllum nodosum, além de desenvolver um sistema emulsionado para sua incorporação e verificar a eficácia deste sistema. Os resultados de citotoxicidade mostraram que este extrato pode ser utilizado com segurança na concentração proposta (1,2% m/m). Além disso, foi avaliado, por metodologias in vitro, o potencial antioxidante e o potencial de inibir a atividade da tirosinase, sendo verificado que o extrato de A. nodosum apresenta a capacidade de atuar neutralizando radicais livres e inibindo a atividade enzimática da tirosinase no processo de melanogênese. Apesar de serem necessários estudos complementares para garantir a eficácia, o fitocosmético desenvolvido com o extrato aquoso de A. nodosum apresenta enorme potencial de aplicação podendo resultar em preparações cosméticas multifuncionais / The current trend in the cosmetic industry is the development of multifunctional products, or, those with the ability to have different functions, facilitating the results to the consumer. Following this trend, natural actives are interesting alternatives as substitutes for a single active compound, since they are complex matrices and present various components that may have different mechanisms of action and thereby give the product more than one function. For this reason, in recent years the use of natural extracts has grown a lot in this area. With the increased use of natural extracts in cosmetic products, there is also a need for studies to prove its effectiveness and safety. A major function is sought in cosmetic anti-aging action, for instance, the ability of the products act by neutralizing free radicals. In this context, the aim of this study was to evaluate the effectiveness and safety of the extract of Ascophyllum nodosum, and develop a system for incorporation emulsified and verify the effectiveness of this system. The results of cytotoxicity have shown that this extract can be safely used in the proposed concentration (1.2% w/w). Furthermore, it was evaluated by in vitro methods, the antioxidant potential and the potential to inhibit the activity of tyrosinase, and found that the extract of A. nodosum has the ability to act by neutralizing free radicals and inhibiting the enzymatic activity of tyrosinase in the process of melanogenesis. Although additional studies are needed to ensure effectiveness, phytocosmetic developed with aqueous extract of A. nodosum has enormous potential application may result in multifunctional cosmetic preparations

Submitted by Renata Bruna dos Santos Coscolin null (rbscoscolin@fca.unesp.br) on 2017-01-16T22:27:20Z No. of bitstreams: 1 definitivo tese 8_VF_Completa.pdf: 3379003 bytes, checksum: 659bf515460d03427a658914feaa471a (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-01-19T18:33:58Z (GMT) No. of bitstreams: 1 coscolim_rbs_dr_bot.pdf: 3379003 bytes, checksum: 659bf515460d03427a658914feaa471a (MD5) / Made available in DSpace on 2017-01-19T18:33:58Z (GMT). No. of bitstreams: 1 coscolim_rbs_dr_bot.pdf: 3379003 bytes, checksum: 659bf515460d03427a658914feaa471a (MD5) Previous issue date: 2016-11-17 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O amendoim é uma cultura de interesse econômico para o estado de São Paulo, principalmente para renovação de canaviais e pastagens na região oeste do estado. Nesta região, os crescentes custos de produção do amendoim acompanhados por baixo rendimento por área devido à suscetibilidade às variações climáticas são influências negativas e contribuem para desestimular a produção dessa cultura. Nesse âmbito, a associação com fungos micorrízicos arbusculares (FMAs) e suplementação com bioestimulante a base de extrato solúvel de algas (Ascophyllum nodosum) (ESA) podem promover melhorias no crescimento e desenvolvimento da planta, além de atenuarem os efeitos negativos provocados pela deficiência hídrica. As plantas foram cultivadas em estufa agrícola, com monitoramento das relações hídricas além do estudo de trocas gasosas, análise de crescimento, análises microbiológicas e de produção, bem como das respostas metabólicas das plantas em função dos tratamentos. A presente pesquisa valida os benefícios para a cultura do amendoim quando em associação com os FMAs e / ou com a suplementação com o ESA, pois além de incrementarem o crescimento e taxa de assimilação líquida de carbono nas plantas em condições hidratadas, mantiveram o status hídrico das plantas e proveram também o acionamento de enzimas do complexo antioxidativo nas plantas em condições de deficiência hídrica moderada e severa. / Water is essential element for plant development and its absence or deficiency induces physiological changes with severe consequences for productivity. Peanut is a culture of economic interest to the State of São Paulo especially in the western region. In this region, the increasing of production costs in peanuts is associate by low yield due to susceptibility to climate variations, such as dry seasons. They are negative influences and do not contribute to production in that crop. In this context, the association with arbuscular mycorrhizal fungi (AMF) and supplementation with biostimulant based on soluble algal extract (Ascophyllum nodosum) (ESA) can promote improvements in plant growth and development, as well as attenuate the negative effects caused by water deficiency. The plants grown in a greenhouse with monitoring of water relations in addition to the study of gas exchange parameters, growth analysis, microbiological analysis, production, and metabolic responses of plants in the treatments. The present study validates the benefits for peanut cultivation with fungus and/or seaweed extract supplementation. Plants under hydrated conditions had better performance in growth and carbon assimilation rate and under conditions of moderate and severe water deficiency.of, they maintained water status of the plants and also provided the activation of enzymes of the antioxidative complex. / CNPq: 141167/2014-9

Orientador: Chung Man Chin / Coorientador: Vera Lucia Borges Isaac / Banca: Marcos Antônio Correa / Banca: Helena Margarida Ribeiro / Resumo: A tendência atual na indústria cosmética é o desenvolvimento de produtos multifuncionais, ou seja, aqueles com capacidade de apresentar diferentes funções, possibilitando diferentes resultados para o consumidor. Seguindo esta tendência, ativos naturais são alternativas interessantes como substitutos a um único composto ativo, uma vez que são matrizes complexas e apresentam diversos componentes que podem possuir diferentes mecanismos de ação e, assim, conferir ao produto mais de uma função. Por este motivo, nos últimos anos o uso de extratos naturais tem crescido muito nesta área. Com o aumento da aplicação de extratos naturais em produtos cosméticos, há também a necessidade de estudos que comprovem sua eficácia e segurança. Uma das principais funções buscadas em produtos cosméticos é a ação antienvelhecimento, ou seja, a capacidade dos produtos em atuar neutralizando radicais livres. Neste contexto, o objetivo deste trabalho foi avaliar a eficácia e segurança do extrato de Ascophyllum nodosum, além de desenvolver um sistema emulsionado para sua incorporação e verificar a eficácia deste sistema. Os resultados de citotoxicidade mostraram que este extrato pode ser utilizado com segurança na concentração proposta (1,2% m/m). Além disso, foi avaliado, por metodologias in vitro, o potencial antioxidante e o potencial de inibir a atividade da tirosinase, sendo verificado que o extrato de A. nodosum apresenta a capacidade de atuar neutralizando radicais livres e inibindo a atividade enzimática da tirosinase no processo de melanogênese. Apesar de serem necessários estudos complementares para garantir a eficácia, o fitocosmético desenvolvido com o extrato aquoso de A. nodosum apresenta enorme potencial de aplicação podendo resultar em preparações cosméticas multifuncionais / Abstract: The current trend in the cosmetic industry is the development of multifunctional products, or, those with the ability to have different functions, facilitating the results to the consumer. Following this trend, natural actives are interesting alternatives as substitutes for a single active compound, since they are complex matrices and present various components that may have different mechanisms of action and thereby give the product more than one function. For this reason, in recent years the use of natural extracts has grown a lot in this area. With the increased use of natural extracts in cosmetic products, there is also a need for studies to prove its effectiveness and safety. A major function is sought in cosmetic anti-aging action, for instance, the ability of the products act by neutralizing free radicals. In this context, the aim of this study was to evaluate the effectiveness and safety of the extract of Ascophyllum nodosum, and develop a system for incorporation emulsified and verify the effectiveness of this system. The results of cytotoxicity have shown that this extract can be safely used in the proposed concentration (1.2% w/w). Furthermore, it was evaluated by in vitro methods, the antioxidant potential and the potential to inhibit the activity of tyrosinase, and found that the extract of A. nodosum has the ability to act by neutralizing free radicals and inhibiting the enzymatic activity of tyrosinase in the process of melanogenesis. Although additional studies are needed to ensure effectiveness, phytocosmetic developed with aqueous extract of A. nodosum has enormous potential application may result in multifunctional cosmetic preparations / Mestre

Syfte: Syftet med denna studie var att studera huruvida algen Ascophyllum Nodosum (AN) utövar någon effekt på proteasaktivitet i oralt plack, samt om effekten finns i algen från början eller om man måste inta den oralt för att få en systemisk effekt.En förhöjd proteasaktivitet har förknippats med gingivit och parodontit. Vi förväntar oss en minskad proteasaktivitet hos försökspersonerna, och därmed en minskad risk för gingivit och parodontit, efter intag av AN under en månads tid. Material och metod: Ett in vitro med en pilotstudie, och ett in vivo försök utfördes. I in vitro försöket användes pulveriserat och upplöst Ascophyllum Nodosum. I in vitro studien deltog 5 personer i åldersgruppen 20 till 30 år. Plackprover på försökspersonerna togs före och efter intag av algen i 4 veckor. Under båda försökstillfällena fick försökspersonerna inte ha borstat tänderna på 12h innan försöket. Resultat: Vid kombinering av pilotstudien och in vitro studien ses ingen signifikant skillnad gällande Ascophyllum Nodosums proteasaktivitet i pulveriserad form. Våra resultat erhållna från in vivo studien visar att det har skett en ökad proteasaktivitet i placket hos försökspersonerna efter en månads intag av Ascophyllum Nodosum. Konklusion: Denna studie visar på en tendens till en ökad proteasaktivitet orsakad av Ascophyllum nodosum. Studien har inte undersökt vilka proteaser som påverkats. På grund av komplexiteten i den orala miljön och de många olika typerna av proteaser, behöver fler studier utföras för att studera de exakta effekterna på den orala miljön. / Aims: The purpose of this study was to investigate if the alga Ascophyllum Nodosum (AN) exerts any effect on protease activity in plaque, if such an effect is present in the algae from the beginning or if it has to be taken orally to exert a systemic effect.Increased protease activity has been associated with gingivitis and periodontitis. We expected a reduced protease activity, and thus a potentially reduced risk for gingivitis and periodontitis, after ingestion of AN for a month.Materials and methods: One in vitro trial with a pilot study, and one in vivo trial was carried out. In the in vitro trial pulverized and dissolved AN was used to make a solution, tested for protease activity.In the in vitro study 5 subjects, aged 20 to 30 years, participated. Plaque samples were taken before and after ingestion of the algae for 4 weeks. Subjects were instructed not to brush their teeth 12 h before sampling.Results: When combining the results from the pilot and in vitro studies, no AN protease activity could be detected. Our in vivo results showed an increased protease activity in the plaque after a month of AN intake.Conclusion: This study indicates a tendency to an increased protease activity caused by Ascophyllum Nodosum. However, the study did not examine which protease was affected. Because of the complexity in the oral environment and the many different types of protease, more studies need to be executed to study the exact effects of AN on the oral environment.

A hanseníase é geralmente agravada pelo aparecimento de reações, que são quadros inflamatórios de difícil tratamento e a principal causa de seqüelas. Nossa hipótese é de que deficiência e/ou perda da função das células T reguladoras (Tregs) podem estar envolvidas no desenvolvimento das reações. Além da avaliação da frequência das Tregs circulantes em pacientes com reação tipo 1 (R1) e reação tipo 2 (R2), também foi avaliada a frequência in situ de FoxP3, IL-17, IL-6 e TGFbeta. Pacientes com R2 apresentaram expressiva diminuição na frequência das Tregs circulantes e in situ em comparação com pacientes com R1 e com os controles. Paralelamente a diminuição das Tregs nas R2 foi observado aumento da expressão de IL-17 in situ e diminuição da expressão de TGFbeta. Biópsias obtidas de pacientes com R1 e R2 antes do episódio reacional mostraram números de células FoxP3+ e IL-17+ similares entre os dois grupos. Entretanto, nas biópsias obtidas durante a reação foi observado diminuição de Tregs e aumento de células IL-17+ em pacientes com R2, enquanto que pacientes com R1 apresentaram o oposto: aumento de Tregs e diminuição de células IL-17+. Além disso, foi observada diminuição da expansão das Tregs frente ao estímulo in vitro com Mycobacterium leprae e uma tendência a baixa expressão de FoxP3 e da molécula imunossupressora CTLA-4 em Tregs de pacientes com R2. Nossos resultados sugerem que nas R2, a diminuição na frequência de Tregs possa estar favorecendo o desenvolvimento de uma resposta Th17, a qual é característica deste tipo de reação. Adicionalmente, com a finalidade de obter um número suficiente de Tregs para realização de ensaios funcionais com estas células, uma vez que se trata de uma subpopulação com baixa freqüência no sangue periférico ( < 10%), foram estabelecidos e avaliados três protocolos distintos para expansão in vitro de Tregs: protocolo Rapamicina, protocolo TGFbeta e protocolo Vitamina D3. Todos os protocolos foram capazes de induzir expansão de Tregs viáveis nos grupos estudados (paucibacilares e multibacilares sem reação, R1 e R2). Em todos os grupos estudados as Tregs expandidas apresentaram capacidade de suprimir a proliferação de linfócitos TCD4+ e TCD8+. Apesar dos três protocolos testados apresentarem capacidade de expandir Tregs in vitro, selecionamos para ensaios futuros os protocolos Rapamicina e TGFbeta por apresentarem melhor custo-benefício. A expansão in vitro será utilizada para estudos funcionais das Tregs buscando melhor entendimento do envolvimento desta subpopulação na patogenia das reações hansênicas / Leprosy is frequently complicated by the appearance of reactions that are difficult to treat and are the main cause of sequelae. We speculated that disturbances in regulatory T-cells (Tregs) could play a role in leprosy reactions. We determined the frequency of circulating Tregs in patients with type 1 reaction (T1R) and type 2 reaction (T2R). The in situ frequency of FoxP3 and interleukin (IL)-17, IL-6, and transforming growth factor beta (TGF)-beta expressing cells was also determined. T2R patients showed markedly lower number of circulating and in situ Tregs than T1R patients and controls. This decrease was paralleled by increased in situ IL-17 expression but decreased TGF-beta expression. Biopsies from T1R and T2R patients before the reaction episodes showed similar number of forkhead box protein P3 + (FoxP3+) and IL-17+ cells. However, in biopsies taken during the reaction, T2R patients showed a decrease in Tregs and increase in IL-17+ cells, whereas T1R patients showed the opposite: Tregs increased but IL17+ cells decreased. We also found decreased expansion of Tregs upon in vitro stimulation with Mycobacterium leprae and a trend for lower expression of FoxP3 and the immunosuppressive molecule CTLA-4 in T2R Tregs. Our results provide some evidence to the hypothesis that, in T2R, downmodulation of Tregs may favor the development of T-helper-17 responses that characterize this reaction. In addition, aiming to provide sufficient number of Tregs to perform functional assays with these cells, as they correspond to a subtle subpopulation among the peripheral blood mononuclear cells ( < 10%), we established and analyzed three different protocols for in vitro Tregs: a Rapamycin protocol, a TGFbeta protocol and a Vitamina D3 protocol. All three protocols were able to induce the expansion of viable in the four types of patients of the study (paucibacillary and multibacillary patients without reaction, and R1 and R2 patients). In these four groups the tregs were able to suppress the proliferative response of TCD4+ e TCD8+ lymphocytes. Although the three protocols resulted in expansion of Tregs, we selected two of them, Rapamycin and TGFbeta for further assays since they showed better cost-benefit. The in vitro expansion will be used to perform functional assays of the Tregs aiming at a better understanding of the involvement of this subpopulation in the pathogenesis of the leprosy reaction episodes

A hanseníase é geralmente agravada pelo aparecimento de reações, que são quadros inflamatórios de difícil tratamento e a principal causa de seqüelas. Nossa hipótese é de que deficiência e/ou perda da função das células T reguladoras (Tregs) podem estar envolvidas no desenvolvimento das reações. Além da avaliação da frequência das Tregs circulantes em pacientes com reação tipo 1 (R1) e reação tipo 2 (R2), também foi avaliada a frequência in situ de FoxP3, IL-17, IL-6 e TGFbeta. Pacientes com R2 apresentaram expressiva diminuição na frequência das Tregs circulantes e in situ em comparação com pacientes com R1 e com os controles. Paralelamente a diminuição das Tregs nas R2 foi observado aumento da expressão de IL-17 in situ e diminuição da expressão de TGFbeta. Biópsias obtidas de pacientes com R1 e R2 antes do episódio reacional mostraram números de células FoxP3+ e IL-17+ similares entre os dois grupos. Entretanto, nas biópsias obtidas durante a reação foi observado diminuição de Tregs e aumento de células IL-17+ em pacientes com R2, enquanto que pacientes com R1 apresentaram o oposto: aumento de Tregs e diminuição de células IL-17+. Além disso, foi observada diminuição da expansão das Tregs frente ao estímulo in vitro com Mycobacterium leprae e uma tendência a baixa expressão de FoxP3 e da molécula imunossupressora CTLA-4 em Tregs de pacientes com R2. Nossos resultados sugerem que nas R2, a diminuição na frequência de Tregs possa estar favorecendo o desenvolvimento de uma resposta Th17, a qual é característica deste tipo de reação. Adicionalmente, com a finalidade de obter um número suficiente de Tregs para realização de ensaios funcionais com estas células, uma vez que se trata de uma subpopulação com baixa freqüência no sangue periférico ( < 10%), foram estabelecidos e avaliados três protocolos distintos para expansão in vitro de Tregs: protocolo Rapamicina, protocolo TGFbeta e protocolo Vitamina D3. Todos os protocolos foram capazes de induzir expansão de Tregs viáveis nos grupos estudados (paucibacilares e multibacilares sem reação, R1 e R2). Em todos os grupos estudados as Tregs expandidas apresentaram capacidade de suprimir a proliferação de linfócitos TCD4+ e TCD8+. Apesar dos três protocolos testados apresentarem capacidade de expandir Tregs in vitro, selecionamos para ensaios futuros os protocolos Rapamicina e TGFbeta por apresentarem melhor custo-benefício. A expansão in vitro será utilizada para estudos funcionais das Tregs buscando melhor entendimento do envolvimento desta subpopulação na patogenia das reações hansênicas / Leprosy is frequently complicated by the appearance of reactions that are difficult to treat and are the main cause of sequelae. We speculated that disturbances in regulatory T-cells (Tregs) could play a role in leprosy reactions. We determined the frequency of circulating Tregs in patients with type 1 reaction (T1R) and type 2 reaction (T2R). The in situ frequency of FoxP3 and interleukin (IL)-17, IL-6, and transforming growth factor beta (TGF)-beta expressing cells was also determined. T2R patients showed markedly lower number of circulating and in situ Tregs than T1R patients and controls. This decrease was paralleled by increased in situ IL-17 expression but decreased TGF-beta expression. Biopsies from T1R and T2R patients before the reaction episodes showed similar number of forkhead box protein P3 + (FoxP3+) and IL-17+ cells. However, in biopsies taken during the reaction, T2R patients showed a decrease in Tregs and increase in IL-17+ cells, whereas T1R patients showed the opposite: Tregs increased but IL17+ cells decreased. We also found decreased expansion of Tregs upon in vitro stimulation with Mycobacterium leprae and a trend for lower expression of FoxP3 and the immunosuppressive molecule CTLA-4 in T2R Tregs. Our results provide some evidence to the hypothesis that, in T2R, downmodulation of Tregs may favor the development of T-helper-17 responses that characterize this reaction. In addition, aiming to provide sufficient number of Tregs to perform functional assays with these cells, as they correspond to a subtle subpopulation among the peripheral blood mononuclear cells ( < 10%), we established and analyzed three different protocols for in vitro Tregs: a Rapamycin protocol, a TGFbeta protocol and a Vitamina D3 protocol. All three protocols were able to induce the expansion of viable in the four types of patients of the study (paucibacillary and multibacillary patients without reaction, and R1 and R2 patients). In these four groups the tregs were able to suppress the proliferative response of TCD4+ e TCD8+ lymphocytes. Although the three protocols resulted in expansion of Tregs, we selected two of them, Rapamycin and TGFbeta for further assays since they showed better cost-benefit. The in vitro expansion will be used to perform functional assays of the Tregs aiming at a better understanding of the involvement of this subpopulation in the pathogenesis of the leprosy reaction episodes

Prior immunity to a related or unrelated pathogen greatly influences the host’s immune response to a subsequent infection and can cause a dramatic difference in disease course, a phenomenon known as heterologous immunity. Heterologous immunity can influence protective immunity, immunopathology and/or immune deviation of cytokine-producing T cell subsets. Examples of heterologous immunity have been well documented in mouse models, as well as during human infections. For example, prior immunity to lymphocytic choriomeningitis virus (LCMV) provides partial protection against vaccinia virus (VV), as LCMV-immune mice show reduced VV titers and increased survival upon lethal dose VV infection. Heterologous protection against VV challenge, as a result of LCMV immunity, is mediated by LCMV-specific CD4 and CD8 T cells, as transfer of LCMV-specific memory T cells can mediate this protective effect in naïve mice. The recognition of a single TCR with more than one MHC-peptide complex is referred to as T cell cross-reactivity. A VV Kb-restricted epitope a11r198 was identified to be able to induce cross-reactive responses from LCMV-specific CD8 T cells. During VV infection, LCMV-specific memory T cells that are cross-reactive to VV epitopes produce IFN-γ early in VV infection. IFN-γ is essential for mediating the protection against VV in LCMV-immune mice, as this heterologous protection is absent in IFN-γR-/-and IFN-γ blocking antibody-treated LCMV-immune mice. In addition to protective immunity, cross-reactive LCMV-specific memory T cells and IFN-γ also induce an altered immunopathology during heterologous VV challenge. LCMV-immune mice show moderate to severe levels of inflammation of the fat tissue, known as panniculitis, in the visceral fat pads upon VV challenge. In humans, panniculitis is a painful condition, most commonly presenting as erythema nodosum. Erythema nodosum is a disease of unknown etiology with no known treatment. It may occur following intracellular bacterial and viral infections, and occasionally happens after vaccination with VV for smallpox. During infections there can be a delicate balance between the ability of immune responses to provide protective immunity, and the tendency to induce immunopathology. By using the mouse model of heterologous immunity between LCMV and VV, we tried to understand how the immunity to LCMV biased the balance between the protective immunity and immunopathology, and what effector molecules were responsible for the pathogenesis of panniculitis in this system. TNF is a pleiotropic cytokine, which is required for normal innate and adaptive immune responses. Its functions range from inducing proliferative responses including cell survival, to destructive responses such as promoting apoptosis and programmed necrosis. In response to inflammatory stimuli, activated macrophages/ monocytes produce large amounts of TNF, and upon activation, T cells, B cells and NK cells also produce TNF. In vitro and in vivo studies have shown that TNF in synergy with IFN-γ plays an important role in mediating host defense against pathogens, such as Listeria monocytogenesand poxviruses in mice and hepatitis B virus and human immunodeficiency virus in humans. However, inappropriate expression of TNF often results in tissue damage. Considering the important role TNF plays in both host defense and mediating autoimmune diseases, we hypothesized that TNF was required for mediating both protective and pathogenic effects in the heterologous immunity between LCMV and VV. We first examined whether TNF was involved in mediating protective heterologous immunity. LCMV-immune mice, that were TNF-deficient as a consequence of genetic deletion (TNF-/-) or receptor blockade by treatment with etanercept (TNFR2: Fc fusion protein), were challenged with VV. These TNF-deficient mice showed normal recruitment and selective expansion of cross-reactive LCMV-specific memory CD8 T cells. They also exhibited efficient clearance of VV similar to LCMV-immune mice with normal TNF function. Thus, we concluded that neither TNF nor lymphotoxin (LT), which uses the same receptors as TNF, was required in mediating protective heterologous immunity against VV. Indeed, prior immunity to LCMV could completely compensate for the role of TNF in protection of naïve mice against VV infection, even under conditions of lethal dose inoculum. Thus, heterologous immunity may help explain why treatment of humans with etanercept is reasonably well tolerated with relatively few infectious complications. One of the histological characteristics of panniculitis is necrosis of adipose tissue. It is known that three members in the TNF superfamily, i.e. TNF/LT, FasL and TRAIL are able to induce necrosis of a target cell. It is also known that TNF is able to induce VV-infected cells to go through necrosis, when apoptosis is blocked in these cells by VV protein. Furthermore, TNF and FasL have already been shown to be associated with some skin and fat pathology. Thus, we hypothesized that TNF, FasL and TRAIL were involved in the pathogenesis of panniculitis in VV infected LCMV-immune mice. By using blocking antibodies or genetically deficient mice, we demonstrated that both TNF/LT and FasL were crucial for inducing panniculitis. Although TNFR1 has been reported to induce programmed necrosis, our data indicated that TNFR2, not TNFR1, was involved in mediating tissue damage in the fat pads of LCMV-immune mice infected with VV. We also found that TNF signaled through TNFR2 to up-regulate the expression of Fas on adipocytes. Thus, the engagement of Fas on the adipocytes with FasL expressed on activated VV-specific and cross-reactive LCMV-specific CD8 T cells in the fat pads could lead to panniculitis. Thus, our data may identify a potential mechanism in the pathogenesis of human panniculitis, and may suggest a possible treatment for this painful disease. Recent reports suggest that heterologous immunity may contribute to the tremendous variation in symptoms between individuals, from subclinical to death, upon viral infection. Even in genetically identical mice, variations in immunopathology from none to life-threatening levels of pathology are observed in LCMV-immune mice during VV infection. By adoptive transfer of splenocytes from a single LCMV-immune donor into two recipients, we showed that similar levels of pathology were generated in mice receiving the same splenocytes. However, the level of pathology varied among recipients receiving splenocytes from different LCMV-immune donors. The difference in levels of VV-induced pathology observed in individual LCMV-immune mice was a reflection of the private specificity of the T cell repertoire, which is a unique characteristic of each individual immune host. The goal of this doctoral thesis is to understand how heterologous immunity contributes to the pathogenesis of panniculitis. Our data demonstrate that TNF/LT and FasL directly contribute to development of panniculitis in LCMV-immune mice during VV infection, and suggest that anti-TNF treatment might be a useful treatment for diseases, such as erythema nodosum and lupus-induced acute fatty necrosis in humans.