Células dendríticas são importantes na interação dos sistemas imune inato e adaptativo, apresentando antígenos para linfócitos T CD4+ e CD8+ e induzindo respostas adaptativas contra estes. Elas são alvo de interesse no desenvolvimento de novas estratégias vacinais que visam o direcionamento de antígenos para as diferentes populações de DCs in vivo. Consiste na administração de anticorpos contra receptores da superfície da DC em fusão com o antígeno. Nós comparamos o direcionamento da proteína NS1 de DENV2 para as duas principais populações de DCs no baço, na presença de diferentes agonistas. Poly I:C é capaz de induzir as respostas de Linfócitos T CD8+ mais robustas, mas somente quando o antígeno é direcionado para a população de DCs CD8+DEC205+. Com CpG observamos resposta de células T CD8+ equivalente quando a proteína foi direcionada para qualquer uma das populações estudadas. Direcionamento com Poly I:C gerou proteção contra desafio por DENV2 quando NS1 é direcionada para a população de DCs DEC205+, sendo esta proteção dependente de Linfócitos T CD8+ e CD4+. / Dendritic Cells are critical in the interaction between innate and the adaptive immune system, presenting antigens to T lymphocytes and inducing adaptive immune responses against these. DCs are target for development of new vaccine strategies based on targeting antigens to different DCs populations in vivo. This consists on the administration of antibodies specific to DC surface endocytic receptor fused to an antigen. In this study, we evaluated the differences in adaptive responses when the NS1 protein from the DENV2 was directed to the two main populations of DCs in the spleen along with different agonistic molecules. Highest CD8+ T cell responses were seen when Poly I:C was used, but only when directed to the CD8+DEC205+ DC population. In the presence of agonist CpG we observed similar responses when the protein was targeted to either CD8+DEC205+ or CD8-DCIR2+ DCs. In the presence of Poly I:C, only targeting to the DEC205+ DCs was able to protect mice against a challenge with DENV2. This protection was dependent on both CD8+ and CD4+ T cell responses.

The consumption of fungal- and plant-derived non-starch polysaccharides (NSP) have been associated with reduced risk of cardiovascular diseases and cancer. In addition to promote physiochemical effects on the gastrointestinal tract and serve as substrate for the intestinal microbiota to produce short-chain fatty acids, NSP can interact with immune system cells including macrophages, which are crucial for tissue repair, lipid metabolism and host defense against foreign substances and pathogens. However, the effects of NSP in macrophages depends on their structure. Recently, it was showed that the chayote (Sechium edule) and the fungus Pleurotus albidus are promising sources of NSP with potential immunomodulatory effects in macrophages. In this study, it was explored the effects of cooking on the composition of NSP from chayote and evaluated their biological effects in macrophages. Furthermore, it was optimized a method for the extraction of mushroom NSP and characterized the structure and biological effects of NSP from P. albidus in macrophages. Results showed that the NSP from chayote pulp regulate cytokine secretion and phagocytosis by macrophages, and minor changes in composition during cooking influences their effects in macrophages. Furthermore, NSP from chayote induces cholesterol efflux and inhibits the expression of genes required for NLRP3 inflammasome activation in macrophages previously exposed to cholesterol crystals. Then, it was showed that the optimized method for the extraction of NSP from mushroom reduces by up to half the extraction time commonly required. Furthermore, results showed that P. albidus is source of easily extractable glucans with biological effects in macrophages. Results also suggest that glucans from P. albidus inhibit lipid-induced inflammation and foam-cell formation at distinct levels, with significant effects on NLRP3 inflammasome activation. Taken together, the results suggest that the benefits of chayote NSP is beyond their physical properties on the gastrointestinal tract, and that the P. albidus NSP offers potential health benefits that might be of relevance as a functional food ingredient. / O consumo de polissacarídeos não-amido (PNA) de fungos e plantas tem sido associado a redução do risco de doenças cardiovasculares. Além de promoverem efeitos físicos no trato gastrointestinal e serem utilizados como substratos pela microbiota intestinal, os PNA podem interagir com células do sistema imune, como macrófagos, cruciais no reparo tecidual, metabolismo lipídico, e na defesa do organismo contra patógenos. Entretanto, os efeitos em macrófagos dependem da estrutura do PNA. Recentemente, foi observado que o chuchu (Sechium edule) e o fungo Pleurotus albidus são fontes de PNA com potencial efeito sobre macrófagos. Assim, foram avaliados os efeitos dos PNA do chuchu fresco e cozido em macrófagos. Além disso, foi otimizado um método para extração de polissacarídeos de cogumelo, e avaliada a estrutura e os efeitos biológicos dos PNA do P. albidus em macrófagos. Foi observado que os PNA do chuchu regulam a secreção de citocinas e o processo de fagocitose por macrófagos, e alterações na composição de PNA durante o cozimento tem um impacto em seus efeitos biológicos. Além disso, os PNA do chuchu induzem o efluxo de colesterol e regulam a expressão de genes necessários para a ativação do inflamassoma NLRP3 em macrófagos previamente tratados com cristais de colesterol. Também foi demonstrado que o método otimizado de extração de PNA de cogumelos reduz em até pela metade o tempo de extração normalmente empregado. Além disso, foi verificado que o P. albidus é fonte para extração de glicanos com efeitos em macrófagos. Os resultados também sugerem que os glicanos obtidos do P. albidus inibem em diferentes níveis a inflamação induzida por lipídeos e a formação de células espumosas, com efeitos significativos sobre a ativação do inflamassoma NLRP3. Tais diferenças parecem estar associadas à estrutura dos glicanos. Por fim, os resultados sugerem que os benefícios dos PNA do chuchu estão além dos seus efeitos físicos sobre o trato gastrointestinal, e que os PNA do P. albidus promovem benefícios que podem ser relevantes para explorar sua utilização como um alimento ou fonte para extração de ingredientes funcionais.

Os peptídeos bioactivas são fundamentais no sistema imune inato (SII), devido a que eles proveem informação importante sobre o SII. O objetivo deste estudo é caracterizar os componentes e reações do SII em quilópodes. Para isto, analisamos o transcriptoma dos hemócitos de Scolopendra subspinipes subspinipes; assim como também, extraímos, purificamos e caracterizamos os peptídeos antimicrobianos presentes na exúvia, o extrato total e a hemolinfa de S. s. subspinipes e S. viridicornis. Várias frações com atividade antimicrobiana foram isoladas da hemolinfa. Dois novos peptídeos antimicrobianos foram caracterizados e sintetizados: a Pinipesina, de S. s. subspinipes e a Lacraina, de S. viridicornis. A análise preliminar do transcriptoma apresentou que os contigs relacionados ao SII foram transcritos com mais frequência moléculas com atividade regulatória (33,7%) e de reconhecimento (11,6%), e as relacionadas a cascata de coagulação (21,4%). Uma análise mais completa do transcriptoma e das moléculas bioactivas é apresentado. / Bioactive peptides are vital in the innate immune system (IIS), because they provide substantial information about the IIS. This study aims to characterize the components and reactions that constitute the ISS in Chilopoda. For this, we analyzed the hemocytes transcriptome from Scolopendra subspinipes subspinipes; as also extracted, purified and characterized the bioactive peptides presents in the exuviae, the body extract and the hemolymph from S. s. subspinipes and S. viridicornis. Several fractions with antimicrobial activity from the hemolymph were isolated. Two new antimicrobial peptides were characterized and synthetized as well: The Pinipesin, from S. s. subspinipes, and the Lacrain, from S. viridicornis. The preliminary analysis from the transcriptome show that the contigs related to the IIS were transcribed more frequently molecules with regulatory (33,7 %) and recognition (11,6%) activities, and those involved in the coagulation cascade (21,4%). A more complete analysis of the transcriptome and the bioactive molecules is presented.

A doença periodontal é uma doença infecciosa na qual em resposta à microbiota subgengival, o hospedeiro libera uma série de citocinas inflamatórias que levam à degradação dos tecidos periodontais. Esses mediadores não se restringem ao nível gengival e disseminam-se pelo organismo, causando impacto na saúde sistêmica. Atualmente, tem sido sugerido que a inflamação associada ao câncer é similar àquela vista em inflamações crônicas. Citocinas dentro de processos neoplásicos podem atuar na resposta imune antitumoral ou estimular o desenvolvimento do câncer. Desta forma, este trabalho objetivou caracterizar o perfil inflamatório apresentado por pacientes portadoras de câncer de mama com e sem periodontite crônica, avaliando os parâmetros clínicos periodontais (índice de placa IP, sangramento à sondagem SS, profundidade de sondagem PS, nível clínico de inserção NCI) e ao dosar as citocinas IL-2, IL-4, IL-6, IL-10, IL-17, TNF-&alpha;, IFN-y, TGF-&beta; e IL-1&beta; em fluido crevicular gengival (FCG). Para isso, foram selecionadas 90 mulheres voluntárias, alocadas em 4 grupos: saudáveis (SAU) (n=20), doença periodontal (DP) (n=20), câncer de mama (CAN) (n=25) e câncer de mama com DP (CAN-DP) (n=25). As concentrações das citocinas foram determinadas pela metodologia LUMINEX. Além disso, foram realizados exames laboratoriais como proteína C-reativa, glicemia em jejum, triglicérides, colesterol total, colesterol LDL e HDL e leucograma em amostras de sangue periférico. Todos dados foram estatisticamente analisados. Os grupos DP e CAN-DP apresentaram maiores valores de IP, SS, PS e NCI (p<0,05). Entre os quatro grupos, não houve diferença significativa nos exames hematológicos (p>0,05), exceto o grupo DP que apresentou níveis de neutrófilos mais elevados que os grupos SAU, CAN e CAN-DP (p<0,05). Não houve diferença estatística nos níveis de IL-2, IL-4, TNF-&alpha;, IFN-y e IL-10 entre os quatro grupos (p>0,05). O grupo DP apresentou significantes níveis de IL-17 e TGF-&beta;, IL-6 e IL-1&beta; em relação ao grupo SAU (p<0,05). No grupo CAN-DP, níveis IL-17 e TGF-&beta; foram estatisticamente maiores do que no grupo CAN (p<0,05). As citocinas IL-6 e IL-1&beta; também estavam mais elevado no CAN-DP, porém sem diferença em relação ao grupo CAN (p>0,05). Mulheres com câncer de mama e doença periodontal apresentaram um perfil de citocinas com características pró-inflamatórias, evidenciado pela significante expressão do perfil Th17/IL-17. Além disso, o perfil Treg (TGF-&beta;) também estava aumentado. A combinação Th17/IL-17 e Treg (TGF-&beta;) pode ter impacto nas atividades pró-tumorigênicas no microambiente tumoral. Diante disso, a doença periodontal comportou-se como um fonte de mediadores inflamatórios relacionados com imunopatogênese do câncer de mama. / Periodontal disease is an infectious disease which in response to subgingival microbiota, the host releases a number of inflammatory cytokines that lead to degradation of periodontal tissues. These mediators are not restricted to the gingival level, and spread on blood, causing systemic health impact. Currently, it has been suggested that inflammation associated with cancer is similar to those seen in chronic inflammation. Cytokines in neoplastic processes may act in anti-tumor immune response or stimulate cancer development. Thus, this study aimed to characterize the inflammatory profile presented by patients with breast cancer with and without chronic periodontitis, evaluating the clinical periodontal parameters (plaque index - PI, bleeding on probing - BOP, probing depth - PD, clinical attachment level - CAL) and dose cytokines IL-2, IL-4, IL-6, IL-10, IL-17, TNF-&alpha;, IFN-y, TGF-&beta; and IL-1&beta; in gingival crevicular fluid (GCF). Ninety volunteers women were selected, divided into 4 groups: healthy (H) (n=20), chronic periodontitis (CP) (n=20), breast cancer (BC) (n=25) and breast cancer with CP (BC-CP) (n=25). Concentrations of cytokines were determined by Luminex method. In addition, laboratory tests were performed as C-reactive protein, fasting blood glucose, triglycerides, total cholesterol, LDL and HDL cholesterol and white blood cell count in peripheral blood samples. All data were statistically analyzed. CP and BC-CP groups had higher values PI, BOP, PD, and CAL (p<0.05). Among four groups, there was no significant difference in blood tests (p>0.05), except the PC group that showed higher levels of neutrophils than H, BC, BC-CP groups (p<0.05). There was no statistical difference in IL-2 levels, IL-4, TNF-&alpha;, IFN-y- and IL-10 among the four groups (p>0.05). CP group showed significant levels of IL-17 and TGF&beta;, IL-6 and IL-1&beta; compared to H group (p<0.05). In CAN-PD group, IL-17 and TGF-&beta; levels were significantly higher than the CAN group (p<0.05). IL-6 and IL-1&beta; cytokines were also higher in CAN-DP, but without difference compared to the CAN group (p>0.05). Women with breast cancer and periodontal disease showed a pro-inflammatory profile of cytokines, as evidenced by significant expression profile Th17/IL-17. Furthermore, Treg profile (TGF-&beta;) was also increased. Th17/Treg and IL-17 (TGF-&beta;) in combination may have an impact on pro-tumorigenic activities in the tumor microenvironment. Therefore, periodontal disease behaved as a source of inflammatory mediators related to breast cancer immunopathogenesis.

by Poon Yam Kau. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 174-183). / Abstract --- p.1 / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Discovery of melatonin --- p.4 / Chapter 1.2 --- Biosynthesis of melatonin --- p.4 / Chapter 1.3 --- Physiology of melatonin and other pineal indoles --- p.5 / Chapter 1.4 --- Relationship between pineal indoles and cancers --- p.6 / Chapter 1.5 --- Macrophages --- p.9 / Chapter 1.6 --- Lymphocytes --- p.11 / Chapter Chapter 2 --- Effects of different light/dark cycles on serum melatonin level in mice and effect of melatonin-feeding on serum glutamate-oxaloacetate transaminase (GOT) activity in mice / Chapter 2.1 --- Introduction --- p.14 / Chapter 2.2 --- Materials and methods --- p.15 / Chapter 2.3 --- Results --- p.22 / Chapter 2.4 --- Discussion --- p.23 / Chapter Chapter 3 --- Actions of endogenous and exogenous melatonin on murine peritoneal macrophages / Chapter 3.1 --- Introduction --- p.27 / Chapter 3.2 --- Materials and methods --- p.28 / Chapter 3.3 --- Results --- p.33 / Chapter 3.4 --- Discussion --- p.36 / Chapter Chapter 4 --- Actions of endogenous and exogenous melatonin on murine splenic lymphocytes / Chapter 4.1 --- Introduction --- p.55 / Chapter 4.2 --- Materials and methods --- p.56 / Chapter 4.3 --- Results --- p.62 / Chapter 4.4 --- Discussion --- p.69 / Chapter Chapter 5 --- In vitro effects of melatonin on murine peritoneal macrophages and splenic lymphocytes / Chapter 5.1 --- Introduction --- p.105 / Chapter 5.2 --- Materials and methods --- p.106 / Chapter 5.3 --- Results --- p.109 / Chapter 5.4 --- Discussion --- p.113 / Chapter Chapter 6 --- Effects of methoxytryptamine on murine peritoneal macrophages and splenic lymphocytes / Chapter 6.1 --- Introduction --- p.125 / Chapter 6.2 --- Materials and methods --- p.126 / Chapter 6.3 --- Results --- p.129 / Chapter 6.4 --- Discussion --- p.132 / Chapter Chapter 7 --- In vitro effects of pineal indoles on cultured tumor cell lines / Chapter 7.1 --- Introduction --- p.145 / Chapter 7.2 --- Materials and methods --- p.146 / Chapter 7.3 --- Results --- p.148 / Chapter 7.4 --- Discussion --- p.152 / Chapter Chapter 8 --- General Discussion --- p.170 / References --- p.174

A doença periodontal é uma doença infecciosa na qual em resposta à microbiota subgengival, o hospedeiro libera uma série de citocinas inflamatórias que levam à degradação dos tecidos periodontais. Esses mediadores não se restringem ao nível gengival e disseminam-se pelo organismo, causando impacto na saúde sistêmica. Atualmente, tem sido sugerido que a inflamação associada ao câncer é similar àquela vista em inflamações crônicas. Citocinas dentro de processos neoplásicos podem atuar na resposta imune antitumoral ou estimular o desenvolvimento do câncer. Desta forma, este trabalho objetivou caracterizar o perfil inflamatório apresentado por pacientes portadoras de câncer de mama com e sem periodontite crônica, avaliando os parâmetros clínicos periodontais (índice de placa IP, sangramento à sondagem SS, profundidade de sondagem PS, nível clínico de inserção NCI) e ao dosar as citocinas IL-2, IL-4, IL-6, IL-10, IL-17, TNF-&alpha;, IFN-y, TGF-&beta; e IL-1&beta; em fluido crevicular gengival (FCG). Para isso, foram selecionadas 90 mulheres voluntárias, alocadas em 4 grupos: saudáveis (SAU) (n=20), doença periodontal (DP) (n=20), câncer de mama (CAN) (n=25) e câncer de mama com DP (CAN-DP) (n=25). As concentrações das citocinas foram determinadas pela metodologia LUMINEX. Além disso, foram realizados exames laboratoriais como proteína C-reativa, glicemia em jejum, triglicérides, colesterol total, colesterol LDL e HDL e leucograma em amostras de sangue periférico. Todos dados foram estatisticamente analisados. Os grupos DP e CAN-DP apresentaram maiores valores de IP, SS, PS e NCI (p<0,05). Entre os quatro grupos, não houve diferença significativa nos exames hematológicos (p>0,05), exceto o grupo DP que apresentou níveis de neutrófilos mais elevados que os grupos SAU, CAN e CAN-DP (p<0,05). Não houve diferença estatística nos níveis de IL-2, IL-4, TNF-&alpha;, IFN-y e IL-10 entre os quatro grupos (p>0,05). O grupo DP apresentou significantes níveis de IL-17 e TGF-&beta;, IL-6 e IL-1&beta; em relação ao grupo SAU (p<0,05). No grupo CAN-DP, níveis IL-17 e TGF-&beta; foram estatisticamente maiores do que no grupo CAN (p<0,05). As citocinas IL-6 e IL-1&beta; também estavam mais elevado no CAN-DP, porém sem diferença em relação ao grupo CAN (p>0,05). Mulheres com câncer de mama e doença periodontal apresentaram um perfil de citocinas com características pró-inflamatórias, evidenciado pela significante expressão do perfil Th17/IL-17. Além disso, o perfil Treg (TGF-&beta;) também estava aumentado. A combinação Th17/IL-17 e Treg (TGF-&beta;) pode ter impacto nas atividades pró-tumorigênicas no microambiente tumoral. Diante disso, a doença periodontal comportou-se como um fonte de mediadores inflamatórios relacionados com imunopatogênese do câncer de mama. / Periodontal disease is an infectious disease which in response to subgingival microbiota, the host releases a number of inflammatory cytokines that lead to degradation of periodontal tissues. These mediators are not restricted to the gingival level, and spread on blood, causing systemic health impact. Currently, it has been suggested that inflammation associated with cancer is similar to those seen in chronic inflammation. Cytokines in neoplastic processes may act in anti-tumor immune response or stimulate cancer development. Thus, this study aimed to characterize the inflammatory profile presented by patients with breast cancer with and without chronic periodontitis, evaluating the clinical periodontal parameters (plaque index - PI, bleeding on probing - BOP, probing depth - PD, clinical attachment level - CAL) and dose cytokines IL-2, IL-4, IL-6, IL-10, IL-17, TNF-&alpha;, IFN-y, TGF-&beta; and IL-1&beta; in gingival crevicular fluid (GCF). Ninety volunteers women were selected, divided into 4 groups: healthy (H) (n=20), chronic periodontitis (CP) (n=20), breast cancer (BC) (n=25) and breast cancer with CP (BC-CP) (n=25). Concentrations of cytokines were determined by Luminex method. In addition, laboratory tests were performed as C-reactive protein, fasting blood glucose, triglycerides, total cholesterol, LDL and HDL cholesterol and white blood cell count in peripheral blood samples. All data were statistically analyzed. CP and BC-CP groups had higher values PI, BOP, PD, and CAL (p<0.05). Among four groups, there was no significant difference in blood tests (p>0.05), except the PC group that showed higher levels of neutrophils than H, BC, BC-CP groups (p<0.05). There was no statistical difference in IL-2 levels, IL-4, TNF-&alpha;, IFN-y- and IL-10 among the four groups (p>0.05). CP group showed significant levels of IL-17 and TGF&beta;, IL-6 and IL-1&beta; compared to H group (p<0.05). In CAN-PD group, IL-17 and TGF-&beta; levels were significantly higher than the CAN group (p<0.05). IL-6 and IL-1&beta; cytokines were also higher in CAN-DP, but without difference compared to the CAN group (p>0.05). Women with breast cancer and periodontal disease showed a pro-inflammatory profile of cytokines, as evidenced by significant expression profile Th17/IL-17. Furthermore, Treg profile (TGF-&beta;) was also increased. Th17/Treg and IL-17 (TGF-&beta;) in combination may have an impact on pro-tumorigenic activities in the tumor microenvironment. Therefore, periodontal disease behaved as a source of inflammatory mediators related to breast cancer immunopathogenesis.

Concentrações séricas basais da proteína amiloide sérica A (SAA) estão significativamente aumentadas em pacientes com câncer e alguns autores sugerem uma relação causal. Trabalho anterior do grupo mostrou que a SAA induz a proliferação de duas linhagens de glioblastoma humano e afeta os processos de invasividade in vitro, sustentando um papel pró-tumoral para esta proteína. Com base nesse trabalho, investigamos a abrangência dos efeitos de SAA para outro tipo de célula tumoral e para isso escolhemos um painel de linhagens de melanoma humano e uma linhagem primária obtida a partir de aspirado de linfonodo de paciente com melanoma, por nós isolada. Observamos que apesar da célula precursora de melanomas, isto é, melanócito, não produzir SAA, todas as linhagens de melanoma produziram a proteína e expressaram alguns dos seus receptores. Além disso, quando estas células foram estimuladas com SAA houve uma inibição da proliferação em tempos curtos de exposição (48 horas) e efeitos citotóxicos após um tempo maior (7 dias). A SAA também afetou processos de invasividade e a produção das citocinas IL-6, IL-8 e TNF-&#945;. Aos avaliarmos o efeito da SAA na interação das células de melanoma com células do sistema imune, vimos que a SAA ativou uma resposta imune anti-tumoral aumentando a expressão de moléculas co-estumolatórias, como CD69 e HLA-DR, e sua função citotóxica. Ainda, vimos que a produção de TNF-&#945;, IFN-&#947, IL-10, IL-1&#946; e IL-8 estimuladas por SAA podem contribuir com os efeitos desta. De forma geral estes resultados nos levam a crer que a SAA tem atividade anti-tumoral em melanomas. Finalizando, com base na importância do desenvolvimento da resistência às terapias atuais para o melanoma, observamos que em células resistentes ao PLX4032, um inibidor de BRAF, os efeitos imunomodulatórios induzidos pela SAA estão abolidos, possivelmente identificando um novo componente da resistência. / Basal serum concentrations of the protein serum amyloid A are significantly increased in cancer patients and some authors suggest a causal relationship. Previous work of our research group showed that SAA induces proliferation of two cell lines of human glioblastoma and affects invasiveness processes in vitro, supporting a pro-tumor role for this protein. Based on this work, we investigated the extent of SAA effects to another type of tumor cell and we chose a panel of human melanoma cell lines and primary line obtained from a patient with melanoma by lymph node aspirate. Melanoma cells were isolated by us. We observed that while the precursor cells of melanoma, melanocytes, do not produce SAA, all melanoma cell lines expressed the protein and produced some of their receptors. Moreover, when these cells were stimulated with SAA there was an inhibition of proliferation in short exposure times (48 hours) and cytotoxic effects after a longer period (7 days). SAA also affected invasive procedures and the production of the cytokines IL-6, IL-8 and TNF-&#945;. To evaluate the SAA effect in the interaction of melanoma cells with immune system cells, we found that SAA activated an anti-tumor immune response by increasing the expression of co-estimulatory molecules such as CD69 and HLA-DR, and their cytotoxic function. Furthermore, we found that the production of TNF-&#945;, IFN-&#947;, IL-10, IL-1&#946; and IL-8 stimulated by SAA can contribute to this effect. In general these results lead us to believe that the SAA has anti-tumor activity in melanomas. Finally, based on the importance of the resistance development to current therapies for melanoma we observed that in cells resistant to PLX4032, a BRAF inhibitor, the immunomodulatory effects induced by SAA are abolished, possibly identifying a new resistance component.

O mercúrio (Hg), na forma química de metilmercúrio (MeHg) é considerado um dos contaminantes ambientais mais nocivos à saúde humana e ao ambiente, podendo provocar efeitos adversos diversos. A exposição ocorre principalmente pelo consumo de peixes contaminados e é freqüente em populações ribeirinhas da Amazônia Brasileira, incluindo o médio Tapajós. Um dos principais efeitos tóxicos associados à exposição ao MeHg é o estresse oxidativo. Também tem sido relatada a indução do processo inflamatório. Entretanto, a dieta pode modificar consideravelmente os efeitos tóxicos decorrentes da exposição ao MeHg. Além disso, na Região Amazônica a sazonalidade é importante na oferta de alimentos, podendo assim, influenciar nos efeitos da exposição ao Hg, pela diferente oferta de micronutrientes que minimizariam os efeitos após a exposição. Neste sentido, o presente trabalho teve como objetivo o de avaliar, em diferentes estações do ano e comunidades ribeirinhas amazônicas, características sociais e demográficas, a exposição ao MeHg, o status de metais essenciais (Cu, Mn, Se e Zn), carotenóides (?-caroteno e licopeno) e vitaminas (A e E) no organismo; bem como os biomarcadores do estresse oxidativo GSH, atividade das enzimas antioxidantes (CAT, GSH-Px e SOD), concentração de MDA, atividade e reativação da ALA-D e marcadores de inflamação (PCRus e as interleucinas 6 e 10). Foi observado que a sazonalidade e a localização da comunidade influenciavam na exposição ao MeHg, no teor de micronutrientes e nos marcadores do estresse oxidativo. A exposição ao MeHg também esteve associada ao aumento da GSH-Px, do MDA e reativação da ALA-D, bem como a diminuição da atividade da ALA-D. Cabe também ressaltar que um aumento da PCRus e expressão das ILs 6 e 10 também se correlacionaram com a exposição ao MeHg. No entanto, para alguns micronutrientes os teores em sangue foram dependentes de fatores sócio-demográficos. Finalmente, observou-se que a dieta é um importante preditor para os biomarcadores do estresse oxidativo GSH, GSH-Px, MDA e ALA-D nos indivíduos expostos ao MeHg. No entanto, dentre os marcadores inflamatórios avaliados somente a IL-10 foi influenciada pelo status nutricional. / Methylmercury (MeHg) is a well-known environmental contaminant and it may cause severe adverse effects to human health. The exposure to this organic form of mercury occurs mainly through consumption of contaminated fish and this source of exposure is common in riparian communities of the Brazilian Amazon, including the Middle Tapajós. One of the main toxic effects related to MeHg exposure is the oxidative stress. MeHg exposure has also been reported to induce inflammatory response. However, components of diet can modify the toxic effects after MeHg exposure. Mopreover, seasonality in the Amazon region is an important factor in food availability and it may also influence the effects of the Hg exposure depending on different populations. Therefore, this study aimed to evaluate, in different seasons and Amazonian riverside communities, social and demographic characteristics, exposure to MeHg, the status of essential metals (Cu, Mn, Se and Zn), carotenoids (?- carotene and lycopene) and vitamins (A and E) in the body as well as biomarkers of oxidative stress GSH, antioxidant enzymes (CAT, GSH-Px and SOD), MDA concentration, activity and reactivation of ALA-D and markers of inflammation (CRPhs and interleukins 6 and 10). The seasonality and community location influenced on the MeHg exposure, micronutrients content and oxidative stress biomarkers. MeHg exposure was also associated with increased GSH-Px, MDA and ALA-D reactivation as well as the decrease on ALA-D activity. The elevations of the CRPhs and interleukins expression were also associated with MeHg exposure. For some micronutrients their levels in blood were dependent on socio-demographic factors. Finally, we found that diet is an important predictor for oxidative stress biomarkers GSH, GSH-Px, MDA and ALA-D in the individuals exposed to MeHg. However, among the inflammatory markers here evaluated only IL-10 was influenced by nutritional status.

Apesar da forte ativação do sistema imune que ocorre durante a infecção pelo Plasmodium, a memória imunológica à infecção é restrita a pacientes residentes em áreas endêmicas. Dessa forma é importante a geração de métodos capazes de induzir uma resposta imune eficaz e duradoura contra o parasito. Nesse contexto o direcionamento de antígenos para células centrais do sistema imune tem se apresentado como uma alternativa promissora. Produzimos e caracterizamos um anticorpo híbrido específico para a molécula DEC205, um receptor endocítico presente nas células dendríticas, acoplado à proteína MSP-1(19) de P. chabaudi, para fins de imunização e análise da resposta imune celular e humoral. Ensaios de imunização mostraram a indução de resposta humoral em camundongos imunizados com anticorpo híbrido e seu controle isotípico, caracterizada pela produção de IgM. Nossos resultados prévios indicam que o direcionamento de antígenos aliado a outras estratégias de imunizações podem resultar na ativação da resposta imune específica ao parasita. / Despite the strong activation of the immune system that occurs during infection by Plasmodium, the immunological memory to infection is restricted to patients residing in endemic areas. Thus it is important to the generation of methods to induce an effective immune response against the parasite. In this context, the targeting of antigens to the central cells of the immune system has emerged as a promising alternative. We produce and characterize a hybrid antibody molecule specific for DEC205, an endocytic receptor present on dendritic cells, coupled to protein MSP-1(19) of P. chabaudi, for immunization and analysis of cellular and humoral immune response. Immunization tests showed the induction of humoral response in mice immunized with hybrid antibody and isotype control, characterized by production of IgM. Our previous results indicate that targeting antigens combined with other strategies for immunization may result in the activation of specific immune response to the parasite.

Tse Pui Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves [191]-216). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABBREVIATIONS --- p.III / ABSTRACT --- p.VIII / 摘要 --- p.X / PUBLICATIONS --- p.XII / TABLE OF CONTENTS --- p.XIII / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Human Immune System and Cancer --- p.1 / Chapter 1.1.1 --- Brief Introduction of the Human Immune System --- p.1 / Chapter 1.1.2 --- Prevalence of Cancer in Hong Kong --- p.4 / Chapter 1.1.3 --- The Role of the Immune System in Tumorigenesis --- p.4 / Chapter 1.1.4 --- Cancer Treatment --- p.5 / Chapter 1.1.5 --- Cancer Prevention --- p.5 / Chapter 1.2 --- Mushroom Polysaccharides --- p.6 / Chapter 1.2.1 --- General Aspects of Mushroom Polysaccharides --- p.6 / Chapter 1.2.2 --- Structure of Mushroom Polysaccharides --- p.9 / Chapter 1.2.2.1 --- Beta (P)-D-glucans --- p.9 / Chapter 1.2.2.2 --- Heteroglucans and Protein-bound Polysaccharides --- p.10 / Chapter 1.2.2.3 --- Structure-Function Interactions of Polysaccharides --- p.12 / Chapter 1.2.3 --- Molecular Interactions of Polysaccharides --- p.14 / Chapter 1.2.4 --- Biological Activities of Polysaccharides --- p.15 / Chapter 1.2.4.1 --- Anti-tumor Activities of Polysaccharides --- p.15 / Chapter 1.2.4.2 --- Immunomodulatory Activities of Polysaccharides --- p.16 / Chapter 1.3 --- Yun Zhi (Coriolus versicolor) --- p.17 / Chapter 1.3.1 --- General Features of Yun Zhi --- p.17 / Chapter 1.3.2 --- Traditional Uses of Yun Zhi --- p.20 / Chapter 1.3.3 --- Active Ingredients of Yun Zhi --- p.20 / Chapter 1.3.3.1 --- "Origin, Properties and Composition of PSK" --- p.21 / Chapter 1.3.3.2 --- "Origin, Properties and Composition of PSP" --- p.22 / Chapter 1.3.4 --- Pharmacological Actions of PSP and PSK --- p.25 / Chapter 1.3.4.1 --- Immunomodulatory Activities --- p.25 / Chapter 1.3.4.2 --- Anti-tumor Activities --- p.32 / Chapter 1.3.4.2 --- Antiviral and Antimicrobial Activities --- p.35 / Chapter 1.3.4.3 --- Antioxidant Activities --- p.36 / Chapter 1.3.5 --- Human Clinical Studies on Yun Zhi --- p.36 / Chapter 1.3.6 --- Toxicology of Yun Zhi --- p.42 / Chapter 1.4 --- Danshen (Salvia miltiorrhiza) --- p.43 / Chapter 1.4.1 --- General Features of Danshen --- p.43 / Chapter 1.4.2 --- Traditional Uses of Danshen --- p.46 / Chapter 1.4.3 --- Active Ingredients of Danshen --- p.47 / Chapter 1.4.4 --- Pharmacological Actions of Danshen --- p.50 / Chapter 1.4.4.1 --- Cardiovascular Effects --- p.50 / Chapter 1.4.4.2 --- Scavenging Effects on Free Radicals --- p.52 / Chapter 1.4.4.3 --- Hepatoprotective Effects --- p.54 / Chapter 1.4.4.4 --- Anti-tumor Effects --- p.56 / Chapter 1.4.4.5 --- Renal Protective Effects --- p.56 / Chapter 1.4.5 --- Human Clinical Studies --- p.57 / Chapter 1.4.6 --- Toxicity of Danshen --- p.59 / Chapter 1.5 --- Aims and Scopes of This Investigation --- p.60 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Normal Subjects --- p.62 / Chapter 2.1.1 --- Inclusion and Exclusion Criteria of Recruitment --- p.62 / Chapter 2.1.2 --- Study Design and Procedure --- p.63 / Chapter 2.1.3 --- Treatment and Blinding --- p.65 / Chapter 2.1.4 --- Blood Sampling --- p.66 / Chapter 2.1.5 --- Blood Processing for Assessment of Immunological Functions --- p.67 / Chapter 2.2 --- Materials --- p.69 / Chapter 2.2.1 --- Endotoxin Assay --- p.69 / Chapter 2.2.2 --- Reagents for Whole Blood Assay --- p.69 / Chapter 2.2.2.1 --- Plain RPMI 1640 Medium --- p.69 / Chapter 2.2.2.2 --- Phosphate-Buffered Saline (PBS) --- p.69 / Chapter 2.2.2.3 --- Mitogens --- p.70 / Chapter 2.2.3 --- Reagents for Total RNA Extraction --- p.70 / Chapter 2.2.3.1 --- Ficoll-Paque Density Gradient Solution --- p.70 / Chapter 2.2.3.2 --- RNA Extraction Kit --- p.70 / Chapter 2.2.3.3 --- RNase-Free DNase Set --- p.71 / Chapter 2.2.3.4 --- β-Mercaptoethanol (β-ME) Solution --- p.71 / Chapter 2.2.4 --- Reagents for Flow Cytometric Analysis of T/B/NK Cell Ratios --- p.71 / Chapter 2.2.4.1 --- MultiTEST IMK Kit with TruCOUNT Tubes --- p.71 / Chapter 2.2.4.2 --- FACSFlo´wёØ Sheath Fluid --- p.74 / Chapter 2.2.4.3 --- CaliBRITE 3 and APC Beads --- p.74 / Chapter 2.2.5 --- Immunoassay Kits for Measuring Cytokines Level --- p.75 / Chapter 2.2.5.1 --- Enzyme-linked Immunosorbent Assay (ELISA) Kits of Cytokines --- p.75 / Chapter 2.2.5.2 --- Human Thl/Th2 Cytokine Cytometric Bead Array (CBA) Kit-II --- p.75 / Chapter 2.2.6 --- Reagents and Buffers for Gel Electrophoresis --- p.78 / Chapter 2.2.6.1 --- Ethidium Bromide (EtBr) --- p.78 / Chapter 2.2.6.2 --- Gel Loading Solution (5X) --- p.78 / Chapter 2.2.6.3 --- Tris-Acetate-EDTA (TAE) Buffer --- p.78 / Chapter 2.2.6.4 --- Agarose Gel --- p.78 / Chapter 2.2.6.5 --- 100 base pair DNA Ladder --- p.79 / Chapter 2.2.7 --- Kits and Reagents for Messenger RNA (mRNA) Expression Array --- p.79 / Chapter 2.2.7.1 --- Human Inflammatory Cytokine/Receptor GEArraýёØ Q Series Kit --- p.79 / Chapter 2.2.7.2 --- Deoxynucleoside Triphosphates (dNTPs) --- p.84 / Chapter 2.2.7.3 --- Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLVRT) --- p.84 / Chapter 2.2.7.4 --- Rnasin Ribonuclease Inhibitor --- p.84 / Chapter 2.2.7.5 --- Biotin-16-2'-deoxy-uridine-5'-triphosphate (Biotin-16-dUTP) --- p.85 / Chapter 2.2.7.6 --- Salmon Sperm DNA Solution --- p.85 / Chapter 2.2.7.7 --- 100 % Sodium Dodecyl Sulfate (SDS) Solution --- p.86 / Chapter 2.2.7.8 --- 20X SSC --- p.86 / Chapter 2.2.7.9 --- ECL Films (Hyperfilm 226}0ёØ ECL 226}0ёØ) --- p.86 / Chapter 2.3 --- Methods / Chapter 2.3.1 --- Endotoxin Assay --- p.87 / Chapter 2.3.2 --- Whole Blood Assay (WBA) --- p.88 / Chapter 2.3.3 --- Isolation and Preparation of Plasma and Peripheral Blood Mononuclear Cells (PBMC) from EDTA Blood --- p.88 / Chapter 2.3.4 --- Total RNA extraction --- p.89 / Chapter 2.3.5 --- Flow Cytometric Analysis of T/B/NK Cell Ratios --- p.90 / Chapter 2.3.6 --- Immunoassays of Plasma Samples or Culture Supernatant in WBA --- p.92 / Chapter 2.3.6.1 --- Enzyme-linked Immunosorbent Assay (ELISA) --- p.92 / Chapter 2.3.6.2 --- Human Thl/Th2 Cytokine Cytometric Bead Assay (CBA) --- p.93 / Chapter 2.3.7 --- mRNA Expression Study --- p.94 / Chapter 2.3.7.1 --- Agarose Gel Electrophoresis --- p.94 / Chapter 2.3.7.2 --- cDNA Expression Array Analysis --- p.95 / Chapter 2.3.8 --- Statistical Analysis --- p.96 / Chapter CHAPTER 3: --- ENDOTOXIN LEVEL OF YUN ZHI-DANSHEN CAPSULES & SAFETY MEASURES ON STUDY POPULATION IN THE CLINICAL TRIAL / Chapter 3.1 --- Introduction --- p.98 / Chapter 3.2 --- Results --- p.101 / Chapter 3.2.1 --- Endotoxin Level of the Yun Zhi and Danshen Active Capsule --- p.101 / Chapter 3.2.2 --- Study Population --- p.103 / Chapter 3.2.3 --- Dropout Cases --- p.103 / Chapter 3.2.4 --- Safety Parameters --- p.104 / Chapter 3.2.5 --- Compliance Rates --- p.104 / Chapter 3.3 --- Discussion --- p.109 / Chapter CHAPTER 4: --- FLOW CYTOMETRIC ANALYSIS OF T/B/NK CELL RATIOS OF HEALTHY SUBJECTS TAKING YUN ZHI-DANSHEN CAPSULES / Chapter 4.1 --- Introduction --- p.112 / Chapter 4.2 --- Results --- p.118 / Chapter 4.2.1 --- The Percentage and Absolute Count of T Lymphocytes (CD3+) --- p.118 / Chapter 4.2.2 --- The Percentage and Absolute Count of T Helper (TH) Lymphocytes (CD3+ CD4+) --- p.121 / Chapter 4.2.3 --- The Percentage and Absolute Count of Cytotoxic T (CTL) and T Suppressor (Ts) Lymphocytes (CD3+ CD8+) --- p.124 / Chapter 4.2.4 --- The Ratio of T Helper Lymphocytes (CD3+ CD4+) and Cytotoxic T (CTL) and T Suppressor (Ts) Lymphocyes (CD3+ CD8+) --- p.127 / Chapter 4.2.5 --- The Percentage and Absolute Count of B Lymphocytes (CD19+) --- p.129 / Chapter 4.2.6 --- The Percentage and Absolute Count of NK Lymphocytes (CD3- CD 16+ and/or CD56+) --- p.132 / Chapter 4.2.7 --- The Absolute Count of Lymphocytes (CD45+) --- p.135 / Chapter 4.3 --- Discussion --- p.138 / Chapter CHAPTER 5: --- PLASMA CONCENTRATION OF SOLUBLE CYTOKINE RECEPTOR AND EX VIVO CYTOKINE PRODUCTION OF HEALTHY SUBJECTS TAKING YUN ZHI-DANSHEN CAPSULES / Chapter 5.1 --- Introduction --- p.142 / Chapter 5.2 --- Results --- p.147 / Chapter 5.2.1 --- Plasma Concentration of Soluble IL-2 Receptor --- p.147 / Chapter 5.2.2 --- Ex vivo Cytokine Production --- p.147 / Chapter 5.2.3 --- Mitogen Induced IL-6 Production --- p.150 / Chapter 5.2.4 --- Mitogen Induced IFN- γ Production --- p.150 / Chapter 5.2.5 --- Mitogen Induced TNF- a Production --- p.153 / Chapter 5.2.6 --- Mitogen Induced IL-10 Production --- p.153 / Chapter 5.3 --- Discussion --- p.156 / Chapter CHAPTER 6: --- "GENE EXPRESSION OF CYTOKINES, CHEMOKINES AND RECEPTORS OF PBMC OF HEALTHY SUBJECTS TAKING YUN ZHI- DANSHEN CAPSULES" / Chapter 6.1 --- Introduction --- p.162 / Chapter 6.2 --- Results --- p.165 / Chapter 6.2.1 --- Gene Expression of IL-2 Receptor β chain --- p.165 / Chapter 6.2.2 --- Gene Expression of IL-2 Receptor γ chain --- p.165 / Chapter 6.2.3 --- Gene Expression of IL-6 Receptor --- p.166 / Chapter 6.2.4 --- "Gene Expression of Other Cytokines, Chemokines and Receptors" --- p.169 / Chapter 6.3 --- Discussion --- p.172 / Chapter CHAPTER 7: --- CONCLUDING REMARKS AND FUTURE / PERSPECTIVES --- p.176 / APPENDICES --- p.184 / REFERENCES --- p.192