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Efeitos da alga Chlorella vulgaris sobre a resposta hematopoética e capacidade funcional de neutrófilos em ratos submetidos ao estresse agudo físico e psicogênico e infectados com Listeria monocytogenes / Effects of the algae Chlorella vulgaris on hematopoietic response and functional activity of neutrophils in rats submitted to physical and psychogenic acute stress and infected with Listeria monocytogenesSouza-Queiroz, Julia de 21 June 2006 (has links)
Evidências experimentais sugerem que uma variedade de estressores ativa o controle hipotalâmico das respostas neuroendócrinas e autonômicas que estão envolvidas na produção de células do sangue e na liberação destas células da medula óssea para a circulação. A alga Chlorela vulgaris (CV) exibe várias atividades imunomoduladoras como, por exemplo, a capacidade de estimulação das células hematopoéticas e de ativação de leucócitos maduros. No presente trabalho analisamos o efeito imunoprotetor da CV em animais submetidos aos estressores físicos (estresse por choque escapável - CE e inescapável - CI) e ao estressor psicogênico (grupo de animais que testemunhou a exposição ao choque inescapável - T) e inoculados com a bactéria Listeria monocytogenes (LM). Os parâmetros imunológicos observados foram: 1) Crescimento e diferenciação de progenitores hematopoéticos para granulócitos e macrófagos (CFU-GM) da medula óssea; 2) Atividade funcional (burst e fagocitose) de neutrófilos circulantes avaliados por citometria de fluxo; 3) Sobrevida de animais inoculados com dose letal de LM e/ou submetidos aos estressores físicos e psicogênico. Nos grupos CI e T observamos uma redução no número de CFU-GM na medula óssea. Por outro lado, não houve redução deste parâmetro nos animais do grupo CE quando comparado ao medido no grupo controle. Observamos uma maior susceptibilidade do organismo a infecção por LM quando o estresse foi aplicado previamente à inoculação com dose subletal desta bactéria. No entanto, o tratamento com 200 mg/Kg/dia de CV por 5 dias consecutivos mostrou uma ação protetora, restabelecendo a mielossupressão induzida pelo choque inescapável ou pelo estressor psicogênico e/ou pela infecção. A aplicação de choques escapáveis não alterou significativamente o perfil da resposta hematopoética produzida pela infecção. O estudo da eficácia terapêutica de alga, avaliada pelo tratamento de animais infectados com dose letal de LM, demonstrou uma sobrevida de 50% no grupo infectado e de 20% nos grupos infectados e submetidos aos diferentes tipos de estresse, inclusive naquele exposto ao estresse escapável. Ao considerarmos os efeitos dos estressores sobre a atividade funcional de neutrófilos sanguíneos, observamos uma redução na capacidade de fagocitose nos grupos CI e T, e um aumento do burst induzido pela fagocitose. No grupo CE houve um aumento na capacidade de fagocitose destas células, enquanto que o burst não foi alterado. O estudo dos efeitos da CV sobre a atividade funcional de neutrófilos demonstrou uma capacidade da alga de impedir a redução da fagocitose produzida pelo estresse físico por choque inescapável e psicogênico, sem interferir com o burst oxidativo, que estava aumentado nestes grupos. Impediu também o aumento da capacidade de fagocitose verificado no grupo CE. Esses resultados sugerem que a CV possua propriedades protetoras contra os efeitos induzidos pelo diferentes tipos de estressores sobre a série granulocítica/macrofágica, a atividade funcional de neutrófilos e sobre a sobrevida de animais estressados e infectados com dose letal de LM. / Evidence suggests that a variety of stressors can activate the hypothalamic control of the neuroendocrine and autonomic responses involved in the production´s control of blood cells and their release from bone marrow to circulation. The algae Chlorella vulgaris (CV) has several imunomodulatory activities, as the stimulation of hematopoietic cells and the activation of mature leukocytes. The present study evaluated the mieloprotective effects of CV in rats exposed to physical stressors (inescapable - CI and escapable footshock - CE) and to psychogenic stressors (animals that witnessed the inescapable shock application - T), which were inoculated or not with a sublethal dose of the bacterium Lysteria monocytogenes (LM). The immunologic parameters observed were: 1) The growth and differentiation of bone marrow progenitors into granulocytes and macrophages (CFU-GM); 2) the functional activity (oxidative burst and phagocytosis) of blood neutrophils, using flow citometric methods; 3) The rate of survival of animals infected with lethal dose of LM and submitted or not to the stressors. The CI and T groups were mielossupressed. On the other hand, in the CE group, no differences in the number of CFU-GM were observed, when compared to controls. An increase in the susceptibility of the organism was observed when the animals received the inescapable shock application and the psychogenic stressor before the inoculatium of sublethal dose (7,8x108) of the 15 bacterium. However, this mielopoietic response was recovered with the pre-treatment with 200 mg/Kg/day for 5 consecutive days of CV, reestablishing the mielossupression caused by the stress and the infection. The escapable shock didn´t produce any significant difference in the hematopoetic response observed in the infection. The study of the survival rate of rats infected with the letal dose of LM showed that the pre-treatment with CV protected 50% of the animals that were only infected with LM, whereas in the group previously stressed the protection was of 20%. Here, the same response was observed in the animals submitted to the different types of stressors evaluated in this work. To assess the effect of CV treatment in the response of mature blood cells, we considered the functional activity of neutrophils of animals submitted to the stressors. We observed a reduction in phagocytosis in the CI and T groups, and an increase in the oxidative burst induced by the phagocytosis. In the CE group there was an increase in the blood neutrophil phagocytosis, while the production of oxidative burst remained equal to that of control group. The treatment with CV reestablished the changes in phagocytosis to normal values in all the groups, but it produced no changes in the respiratory burst, which was increased in the circunstances of the inescapable shock and of the psychogenic stressor, when compared to the values of control group. Considering our results, we suggest that CV has protective properties against the effects produced by the different types of stressors on the CFU-GM, the functional activity of neutrophils and on the rate of survival of animals stressed and infected with lethal dose of LM.
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Prospecção gênica e atividade antimicrobiana de b-defensina-símiles em viperídeos. / Gene survey and antimicrobial activity of beta-defensin-like in viperid.Corrêa, Poliana Garcia 30 October 2013 (has links)
As b-defensinas são pequenos peptídeos catiônicos com estrutura rica em folhas b pregueadas e seis cisteínas conservadas. São bastante estudadas em mamíferos, mas pouco em serpentes. Utilizando a PCR foram descritos 13 genes b-defensina-símiles em serpentes do gênero Bothrops e Lachesis . Eles se organizam em três éxons e dois íntrons; há alta similaridade no éxon1, íntron 1 e 2, mas os éxons 2 e 3 estão sob evolução acelerada. A análise filogenética por máxima parcimônia revelou que o gene ancestral de b-defensina-símile pode ter três éxons em vertebrados e sua evolução ocorreu de acordo com o modelo de nascimento-e-morte. Peptídeos b-defensina-símiles reduzidos, testados por ensaio de inibição de crescimento em meio líquido, apresentaram atividade inibitória contra Escherichia coli, Citrobacter freundii, Micrococcus luteus e Staphylococcus aureus. A crotamina reduzida foi mais ativa que a forma nativa. Os resultados indicam que a carga líquida era a característica bioquímica mais importante na atividade antibiótica dos peptídeos b-defensina-símiles. / b-defensins are small basic cationic peptides with b-sheet-rich fold plus six conserved cysteines. They are fully studied in mammals, but scarce in snakes. Using a PCR approach, we described 13 b-defensin-like sequences in Bothrops and Lachesis snakes. They are organized in three exons and two introns; they show high similarities in exon 1, intron 1 and intron 2, but exons 2 and 3 have undergone accelerated evolution. Phylogenetic analysis was done using maximum parsimony indicate that the ancestral b-defensin-like gene may have three exons in vertebrates and that their evolution occurred according to a birth-and-death model. Reduced b-defensin-like peptides were tested by microbroth dilution assay, showed inhibitory activity against Escherichia coli, Citrobacter freundii, Micrococcus luteus e Staphylococcus aureus. The reduced form of crotamine was more active than native. The results indicate that the positive net charge is the most important biochemical characteristic of b-defensin-like peptides to antibiotic activity.
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Efeitos da separação materna sobre o comportamento, a imunidade inata e o crescimento tumoral / Maternal separation effects on the behavior, innate immunity and tumor growthPinheiro, Milena Lobão 14 March 2008 (has links)
Um modelo de indução de estresse/ansiedade é a separação materna. Sabe-se, a este respeito, que o desenvolvimento do comportamento emocional normal em mamíferos jovens depende da interação entre a mãe e o filhote. Além disso, eventos estressantes na infância e interrupções no cuidado materno podem levar a efeitos deletérios na resposta imune e na resistência a doenças ao longo da vida. Buscamos neste trabalho estudar os efeitos da separação materna sobre o comportamento, imunidade inata e crescimento tumoral de uma prole de camundongos machos adultos. Nossos resultados mostraram que a separação materna, em camundongos A/J, (1) não produziu alterações robustas no comportamento e em parâmetros hematológicos (antes e após o crescimento tumoral), (2) aumentou a atividade de neutrófilos sanguíneos e macrófagos alveolares (antes e após o crescimento tumoral), (3) aumentou os níveis estriatais de serotonina, seu metabólito 5-HIAA e o turnover de dopamina, (4) diminuiu os níveis séricos de corticosterona e (5) não influenciou a incidência tumoral. Em camundongos C57BL/6, a separação materna produziu um comportamento ansioso e um aumento na atividade de neutrófilos sanguíneos. Tomados em seu conjunto, parece-nos possível afirmar que a separação materna tenha produzido alterações na atividade neuroimune dos animais, modificando, nos mesmos a atividade do eixo HPA e essas alterações foram influenciadas pelas características individuais das linhagens. / Maternal separation is one model of stress/anxiety induction. We know about it that the mammals normal emotional behavior development depends on the interaction between mother and younglet. Beside this, early life stressed events can induce deletery effects in immune response and to illness resistance for all life long. In this work we searched for the maternal separation effects on the behavior, innate immunity and tumor growth in an adult male mice offspring. Our results showed that the maternal separation, in A/J mice (1) had no big effects in the behavior and in the hematological parameters (before and after the tumor growth), (2) increased the neutrophil and alveolar macrophages activity (before and after the tumor growth), (3) increased the serotonin striatum levels, the 5-HIAA metabolite and the dopamine turnover (4) decreased the corticosterone serum levels and (5) had no effects on tumor incidence. In the C57BL/6 mice, the maternal separation induced an anxiety behavior and increased the neutrophil activity. These results suggest that the maternal separation could have produced alterations in neuroimmune activity, modifying the HPA axis activity and these alterations could be influenced by individual strains characteristics.
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Expressão ex vivo de fatores antivirais em mães infectadas por HIV-1 e recém-natos. / Ex vivo expression of antiviral factors in mothers infected by HIV-1 and newborns.Pereira, Natalli Zanete 16 April 2013 (has links)
A transmissão vertical mãe-recém-nato é a principal fonte de infecção pediátrica. O tratamento antirretroviral vem reduzindo a transmissão vertical, mas também tem elevado o número de infantes expostos não infectados, os quais vêm mostrando maior risco de morbidade e mortalidade. Este dado salienta a importância de avaliar as características imunológicas, relacionadas à resposta inata no binômio mãe e recém-nato. A proposta do trabalho foi avaliar a expressão de fatores antivirais em células mononucleares (CMN), tecido placentário e no colostro de mães infectadas por HIV e cordão umbilical (RN), comparadas com mães-RN controle não infectadas. Os dados mostram que há uma ativa expressão dos fatores antivirais, sejam constitutivos ou induzíveis por IFN, nas mães infectadas por HIV e nos RN expostos. No sítio de interface materno-fetal, decídua e face fetal da placenta, foi detectado um perfil alterado de expressão dos fatores antivirais, especialmente da proteína APOBEC3G. Apesar da relativa imaturidade imunológica dos RNs, a infecção materna por HIV gerou um perfil semelhante de expressão dos fatores antivirais nos RN, por uma complexa interação de fatores relacionados a gestação e a infecção. / Vertical transmission mother-newborn is the main source of pediatric infection. The antiretroviral therapy has reduced vertical transmission, but also has increased the number of exposed uninfected infants, which have shown increased risk of morbidity and mortality. This finding emphasizes the importance of evaluating the immunological characteristics, related to innate response in both the mother and newborn. The purpose of this study was to evaluate the expression of antiviral factors in mononuclear cells (MNC), placental tissue and colostrum of HIV-infected mothers and umbilical cord (RN), compared with control mothers-uninfected infants. The data show that the active expression of antiviral factors, are constitutive or inducible by IFN in HIV-infected mothers and newborns exposed. At the site of maternal-fetal interface, decidua and placental villi, a profile was detected altered expression of antiviral factors, especially the APOBEC3G protein. Despite the relative immunological immaturity of the newborn, maternal HIV infection generated a similar profile of expression of antiviral factors in RN, by a complex interaction of factors related to pregnancy and infection.
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Pattern recognition receptors and cytokine-mediated activation of human basophils: a novel link between innate immunity and allergic inflammation.January 2013 (has links)
過敏性疾病(如過敏性哮喘和過敏性皮炎)發病率在香港及世界均呈上升趨勢。過敏性哮喘是一種慢性反復發作的炎症疾病,而過敏性皮炎是一種慢性皮膚炎症。呼吸道細菌及金黃色葡萄球菌可分別加重過敏性哮喘病人氣道炎症及過敏性皮炎患者的炎症反應。人體對細菌的先天免疫反應主要通過模式識別受體(PRR)介導。NOD樣受體(NLR)和Toll樣受體(TLR)是兩種重要的PRR。 NLR 家族成員NOD2幾乎識別所有細菌中結構保守的胞壁醯二肽(MDP)。而LTR2識別範圍廣泛的病原相關分子模式,如革蘭氏陽性菌中的肽聚糖(PGN)和脂磷壁酸(LTA),以及人工合成的脂蛋白Pam3CSK4。 / 本研究包括:NOD2配體MDP,哮喘相關的腫瘤壞死因子家族成員LIGHT對共培養的人嗜鹼性粒細胞和人支氣管上皮細胞的活化作用;熱滅活的金黃色葡萄球菌(HKSA),MDP,TLR2配體PGN,LTA,以及Pam3CSK4對共培養的人嗜鹼性粒細胞和人皮膚成纖維細胞的活化作用;在體內NLR配體對卵清蛋白(OVA)致敏的哮喘小鼠的作用。 / 研究發現,在共培養體系中,MDP能顯著增強嗜鹼性粒細胞與支氣管上皮細胞表面粘附因子(細胞間粘附因子ICAM-1 及血管細胞粘附因子VCAM-1)的表達。同時,MDP能顯著促進共培養體系中炎症相關細胞因子IL-6,趨化因子CXCL8及抗菌肽β-防禦素2的釋放。在MDP刺激下,支氣管上皮細胞是共培養體系中釋放IL-6,CXCL8及β-防禦素2的主要細胞。在MDP刺激下,嗜鹼性粒細胞中包括胞核因子-kappaB(NF-κB)在內的幾個核轉錄因子的表達上升。ICAM-1,VCAM-1,IL-6,CXCL8,及β-防禦素2的表達被信號分子化學抑制劑所抑制,結果表明,嗜鹼性粒細胞與支氣管上皮細胞的相互作用受不同的信號通路(NF-κB, p38 MAPK 及 JNK)調節。OVA致敏小鼠實驗表明,NLR配體能增加分泌粘蛋白的杯狀細胞在肺氣管中的數量,使小鼠支氣管下皮結締組織纖維化並增厚。NLR配體進而提高過敏性哮喘小鼠支氣管肺泡灌洗液中CCL5與IL-13 的表達水平。 / 研究表明,在嗜鹼性粒細胞和皮膚成纖維細胞的共培養體系中,HKSA,MDP,PGN,LTA,或Pam3CSK4顯著誘導ICAM-1, IL-6, CXCL8, CCL2 和 CCL5 的表達。而嗜鹼性粒細胞與皮膚成纖維細胞的直接相互作用是釋放IL-6, CXCL8, CCL2 與 CCL5 所必需的。嗜鹼性粒細胞與皮膚成纖維細胞的相互作用並釋放細胞因子與趨化因子受p38 MAPK 及 NF-κB信號通路調控。 / 在嗜鹼性粒細胞與支氣管上皮細胞共培養體系中,LIGHT 可能通過受體HVEM 與 LTβR顯著增強支氣管上皮細胞表面粘附因子的表達,提高細胞因子IL-6, CXCL8 與 MMP-9的釋放。 / 研究結果表明,在過敏炎症中,通過與組織細胞(如支氣管上皮細胞,人皮膚成纖維細胞)相互作用,嗜鹼性粒細胞有利於組織細胞對病原相關的分子模式作出反應。因此,研究結果對細菌介導的先天性免疫應答與過敏炎症的加重之間的聯繫作出了新的解釋。以上結果也增強了我們對LIGHT在氣道重塑中的免疫病理作用及其作為氣道重塑治療靶標的認識。 / The incidences of allergic diseases such as allergic asthma and atopic dermatitis (AD) are increasing in Hong Kong and worldwide. Allergic asthma is a chronically relapsing inflammatory pulmonary disease, while AD is a chronic inflammatory skin disorder. Respiratory bacterial and Staphylococcus aureus (S. aureus) infection can provoke allergen sensitization and subsequently amplify and sustain inflammation in allergic asthma and AD, respectively. The innate immune system recognizes bacterial infection through pattern recognition receptors (PRRs), two important PRRs involving in inflammatory and immune responses are nucleotide-binding oligomerization domain-like receptors (NLRs) and Toll-like receptors (TLRs). NOD2 is one member of the NLR family, which senses the conserved structural component muramyl dipeptide (MDP) in almost all bacteria. TLR2 recognizes a wide range of pathogen-associated molecular patterns (PAMPs) including peptidoglycan (PGN) and lipoteichoic acid (LTA) from Gram-positive bacteria and synthetic triacylated lipoprotein N-palmitoyl-S-[2,3-bis (palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S] -lysine (Pam3CSK4). / In the present study, we investigated the effect of NOD2 ligand MDP, asthma-related tumor necrosis factor (TNF) family member LIGHT on human basophils co-cultured with human bronchial epithelial cells and the effect of heat-killed S. aureus, MDP, TLR2 ligands PGN, LTA and Pam3CSK4 on basophils co-cultured with human dermal fibroblasts, and the underlying intracellular mechanisms. The in vivo effect of NOD ligands on ovalbumin (OVA)-sensitized allergic asthmatic mice was also studied. / It was found that MDP could significantly enhance the cell surface expression of adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on basophils and primary human bronchial epithelial cells (HBE) in the co-culture system (all p < 0.05). MDP could further enhance the release of inflammatory cytokine interleukin (IL)-6, chemokine CXCL8, and epithelium derived anti-microbial peptide β-defensin 2 in the co-culture. HBE cells were the major source while basophils were the minor source to release IL-6, CXCL8 and β-defensin 2 in the co-culture upon MDP stimulation. The activities of several nuclear transcription factors, including NF-κB, were up-regulated in human basophils upon MDP stimulation. The cell surface expression of ICAM-1 and VCAM-1 and the release of IL-6, CXCL8 and β-defensin 2 were suppressed by the signaling molecule inhibitors, implying that the interaction between basophils and primary human bronchial epithelial cells could be differentially regulated by the NF-κB, p38 MAPK and JNK pathways. The animal study showed that iE-DAP and MDP could increase the number of mucin-secreting goblet cells, the thickness and fibrosis of the bronchial subepithelial tissue of airways from the OVA-sensitized mice. The iE-DAP and MDP could further promote the levels of CCL5 and IL-13 (all p < 0.05) in bronchoalveolar lavage fluid (BALF) of allergic asthmatic mice. / It was found that the induction of ICAM-1, IL-6, CXCL8, CCL2 and CCL5 was significantly promoted upon the interaction between human basophils and dermal fibroblasts activated by heat-killed S. aureus, MDP, PGN, LTA or Pam3CSK4. The release of IL-6, CXCL8, CCL2 and CCL5 might depend on the direct interaction of basophils and dermal fibroblasts. The p38 MAPK and NF-κB pathways should be involved in the release of the cytokines and chemokines upon the interaction of basophils and human dermal fibroblasts. / LIGHT could significantly promote the cell surface expression of adhesion molecule, the release of IL-6, CXCL8 and MMP-9 from human bronchial epithelial cells upon the interaction with basophils, probably through the receptors HVEM and LTβR. / The results suggest that, through the interaction with tissue-resident cells such as bronchial epithelial cells and dermal fibroblasts, basophils may facilitate the activation of tissue-resident cells in response to the PAMPs in allergic inflammation. The results therefore provide a new insight of the crucial link between the bacterial-mediated innate immune response and the exacerbation of allergic inflammation. The above results also enhance our understanding on the immunopathological roles of LIGHT in airway remodeling, and the potential therapeutic target for airway remodeling. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Qiu, Huaina. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 165-196). / Abstract also in Chinese. / Acknowledgements --- p.i / Abbreviations --- p.iii / Abstract --- p.vi / 摘要 --- p.ix / Publications --- p.xi / Table of Contents --- p.xiii / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- Asthma and atopic dermatitis (AD) --- p.1 / Chapter 1.2 --- Human basophils in allergic inflammation --- p.3 / Chapter 1.2.1 --- Development and morphology of basophils --- p.3 / Chapter 1.2.2 --- Receptors and products of basophils --- p.4 / Chapter 1.2.3 --- Cell surface markers on basophils --- p.7 / Chapter 1.2.4 --- Basophils in allergic inflammation --- p.7 / Chapter 1.3 --- Human bronchial epithelial cells in airway inflammation --- p.10 / Chapter 1.4 --- Human fibroblasts in AD --- p.11 / Chapter 1.5 --- Staphylococcus aureus (S. aureus) in AD --- p.12 / Chapter 1.6 --- NOD2 and TLR2 in allergic inflammation --- p.14 / Chapter 1.7 --- IL-33 in allergic inflammation --- p.18 / Chapter 1.8 --- IL-6 in allergic inflammation --- p.18 / Chapter 1.9 --- CXCL8 in allergic inflammation --- p.20 / Chapter 1.10 --- CCL2 in allergic inflammation --- p.21 / Chapter 1.11 --- CCL5 in allergic inflammation --- p.22 / Chapter 1.12 --- β-defensin 2 (HBD-2) in allergic inflammation --- p.23 / Chapter 1.13 --- ICAM-1 and VCAM-1 in allergic inflammation --- p.25 / Chapter 1.14 --- LIGHT and airway remodeling in allergic asthma --- p.25 / Chapter 1.15 --- Signal transduction pathways in allergic inflammation and pharmacological inhibitors --- p.26 / Chapter 1.15.1 --- Signal transduction pathways in allergic inflammation --- p.26 / Chapter 1.15.2 --- Signaling molecule inhibitors as new drugs for inflammatory diseases --- p.31 / Chapter 1.16 --- Aims and scope of the study --- p.32 / Chapter Chapter 2: --- Materials and Methods --- p.35 / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Reagents and buffers for the purification of human basophils --- p.35 / Chapter 2.1.2 --- Primary cells and cell lines --- p.36 / Chapter 2.1.3 --- Heat-killed Staphyloccocus aureus (HKSA) --- p.38 / Chapter 2.1.4 --- Ligands for NLR and TLR2 --- p.39 / Chapter 2.1.5 --- Recombinant human cytokines --- p.39 / Chapter 2.1.6 --- Reagents and buffer solutions for flow cytometry --- p.40 / Chapter 2.1.7 --- RNA extraction, reverse transcription-polymerase chain reaction (RT-PCR), and real-time quantitative PCR (qPCR) --- p.45 / Chapter 2.1.8 --- Cytometric Bead Array (CBA) Kits --- p.48 / Chapter 2.1.9 --- MILLIPLEX® MAP Human Cytokine/Chemokine Magnetic Bead Panel Kit --- p.49 / Chapter 2.1.10 --- Enzyme-linked immunosorbent assay (ELISA) kits --- p.49 / Chapter 2.1.11 --- Procarta Transcription Factor Assay kit --- p.50 / Chapter 2.1.12 --- Signal transduction inhibitors --- p.50 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Purification of primary human basophils and basophil culture --- p.50 / Chapter 2.2.2 --- Culture of KU812 cells --- p.51 / Chapter 2.2.3 --- Culture of primary human bronchial epithelial cells --- p.51 / Chapter 2.2.4 --- Culture of BEAS-2B cells --- p.52 / Chapter 2.2.5 --- Culture of human dermal fibroblasts --- p.52 / Chapter 2.2.6 --- Co-culture of primary human bronchial epithelial cells/human bronchial epithelial cell line (BEAS-2B) cells and basophils/KU812 cells --- p.52 / Chapter 2.2.7 --- Co-culture of human dermal fibroblasts and basophils/KU812 cells --- p.52 / Chapter 2.2.8 --- Co-culture of fixed primary human bronchial epithelial cells and basophils --- p.53 / Chapter 2.2.9 --- Co-culture of human dermal fibroblasts and basophils in the presence of transwell inserts --- p.53 / Chapter 2.2.10 --- CBA assay --- p.53 / Chapter 2.2.11 --- ELISA --- p.54 / Chapter 2.2.12 --- Human Transcription Factor Plex Assay --- p.54 / Chapter 2.2.13 --- Milliplex Human Cytokine / Chemokine Magnetic Panel assay --- p.54 / Chapter 2.2.14 --- Bio-Plex mouse cytokine assay --- p.55 / Chapter 2.2.15 --- Flow cytometric analysis of cell surface expression of target molecules --- p.55 / Chapter 2.2.16 --- Flow cytometric analysis of intracellular expression of target molecules --- p.55 / Chapter 2.2.17 --- Allergic asthmatic mice model --- p.57 / Chapter 2.2.18 --- Statistical analysis --- p.57 / Chapter Chapter 3: --- Muramyl Dipeptide Mediated Activation of Human Bronchial Epithelial Cells Interacting with Basophils: A Novel Mechanism of Airway Inflammation --- p.59 / Chapter 3.1 --- Introduction --- p.59 / Chapter 3.2 --- Results --- p.61 / Chapter 3.2.1 --- Cell surface expression of CD203c on basophils --- p.61 / Chapter 3.2.2 --- Intracellular expression of NOD2 protein --- p.63 / Chapter 3.2.3 --- Cell surface expression of adhesion molecules on basophils and primary human bronchial epithelial cells activated by MDP --- p.67 / Chapter 3.2.4 --- Induction of cytokine, chemokine and β-defensin 2 upon the interaction of basophils and bronchial epithelial cells stimulated by MDP --- p.71 / Chapter 3.2.5 --- Bronchial epithelial cells were the main source for the release of IL-6, CXCL8 and β-defensin 2 in co-culture --- p.74 / Chapter 3.2.6 --- Effects of signaling inhibitors on MDP-induced cytokines and adhesion molecules --- p.77 / Chapter 3.2.7 --- Differential activation of intracellular signaling pathways involved in the interaction of KU812 and BEAS-2B upon MDP stimulation --- p.84 / Chapter 3.2.8 --- In vivo effect of NOD1,2 ligands on IgE and chemokine production in serum and BALF in allergic asthmatic mice --- p.89 / Chapter 3.3 --- Discussion --- p.93 / Chapter Chapter 4: --- NOD2 and TLR2 Ligands Mediated Activation of Basophils Interacting with Human Dermal Fibroblasts in Atopic Dermatitis --- p.100 / Chapter 4.1 --- Introduction --- p.100 / Chapter 4.2 --- Results --- p.102 / Chapter 4.2.1 --- Cell surface expression of adhesion molecules ICAM-1 on human dermal fibroblasts activated by heat-killed Staphyloccocus aureus (HKSA) --- p.102 / Chapter 4.2.2 --- Induction of chemokines upon the interaction of basophils and human dermal fibroblasts stimulated by HKSA --- p.104 / Chapter 4.2.3 --- Expression of NOD2 and TLR2 protein --- p.107 / Chapter 4.2.4 --- Cell surface expression of adhesion molecule ICAM-1 on human dermal fibroblasts activated by MDP, PGN, LTA or Pam3CSK4 --- p.110 / Chapter 4.2.5 --- Induction of cytokine and chemokines upon the interaction of basophils (with or without IL-33 priming) and human dermal fibroblasts stimulated by MDP, PGN, LTA or Pam3CSK4 --- p.112 / Chapter 4.2.6 --- Direct interaction between human dermal fibroblasts and basophils was required for the release of IL-6, CXCL8, CCL2 and CCL5 upon the stimulation of MDP, PGN, LTA and Pam3CSK4 --- p.118 / Chapter 4.2.7 --- Effect of signaling molecular inhibitors on the expression of adhesion molecule ICAM-1 --- p.121 / Chapter 4.2.8 --- Effect of signaling molecule inhibitors on the release of cytokine and chemokines upon the stimulation by NOD2 and TLR2 ligands --- p.123 / Chapter 4.2.9 --- Differential activation of intracellular signaling pathways involved in the interaction of human dermal fibroblasts and basophilic KU812 upon stimulation of NOD2 and TLR2 ligands --- p.127 / Chapter 4.3 --- Discussion --- p.131 / Chapter Chapter 5: --- Effect of Tumor Necrosis Factor Family Member LIGHT on the Activation of Basophils Interacting with Bronchial Epithelial Cells: Potential Therapeutic Target for Airway Remodeling --- p.138 / Chapter 5.1 --- Introduction --- p.138 / Chapter 5.2 --- Results --- p.139 / Chapter 5.2.1 --- Cell surface expression of HVEM and LTβR --- p.139 / Chapter 5.2.2 --- Effect of LIGHT on the expression of ICAM-1 on basophil or BEAS-2B alone or co-culture --- p.141 / Chapter 5.2.3 --- Induction of cytokine and chemokine upon the interaction of basophils and BEAS-2B cells stimulated by LIGHT --- p.144 / Chapter 5.2.4 --- Induction of MMP-9 upon the interaction of basophils and BEAS-2B cells stimulated by LIGHT --- p.147 / Chapter 5.2.5 --- Effect of LIGHT on the release of TGFβ-1, histamine and periostin upon the interaction of basophils and BEAS-2B cells --- p.149 / Chapter 5.3 --- Discussion --- p.152 / Chapter Chapter 6: --- Conclusion and Future Perspectives --- p.156 / Chapter 6.1 --- General conclusions --- p.156 / Chapter 6.2 --- Future perspectives --- p.160 / Appendix --- p.163 / References --- p.165
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Efeitos do diazepam sobre o crescimento tumoral e imunidade de animais portadores do tumor ascítico de Ehrlich / Diazepam effects on tumor growth and on immunity of Ehrlich tumor bearing miceMônica Sakai 07 December 2004 (has links)
Benzodiazepínicos (BDZ) são fármacos amplamente utilizados devido às suas propriedades ansiolíticas e sedativas, mediadas pelo complexo GABAA no Sistema Nervoso Central (SNC). Além destes receptores centrais, os BDZ possuem afinidade por receptores do tipo periféricos (PBR) os quais estão presentes em células do sistema imune, como linfócitos e macrófagos, em células tumorais e em glândulas adrenais. O presente trabalho avaliou os efeitos do diazepam, um BDZ freqüentemente utilizado, sobre o crescimento tumoral e a imunidade de animais portadores do Tumor Ascítico de Ehrlich (TAE). Mais especificamente, este trabalho avaliou os seguintes parâmetros da resposta imune: atividade de macrófagos, populações de linfócitos B, T helper e citotóxicos esplênicos, citotoxicidade de células Natural Killer (NK). Além disso, a marcação para PBR em células do TAE e avaliação de possíveis efeitos do diazepam ou Ro5-4864 in vitro sobre o ciclo celular do TAE foram avaliadas. Os resultados mostraram os seguintes efeitos do tratamento com diazepam in vivo (3,0 mg/kg): (1) aumento do crescimento do TAE; sem modificação das fases do ciclo celular do tumor, após 7 dias, (2) diminuição do número de leucócitos da cavidade peritoneal, da produção de NO e do índice de espraiamento; mas sem interferência com a produção de peróxido de hidrogênio e o índice de fagocitose, após 2 dias (3) não modificou o peso relativo do baço e a porcentagem de linfócitos esplênicos, após 2 dias ou 7 dias (4) aumento da citotoxicidade de células NK, após 3 dias (5) diminuição da porcentagem de células do TAE marcadas para PBR, após 7 dias. O tratamento in vitro com diazepam ou Ro5-4864 mostrou um aumento da proliferação de células do TAE. Já o tratamento in vivo com diazepam em doses menores (0,3 mg/kg e 1mg/kg) não modificou o crescimento do TAE, após 7 dias. Desta forma, sugere-se que o diazepam na dose de 3,0 mg/kg tenha aumentado o crescimento do TAE e diminuído a resposta imune inata, observada por meio da diminuição da atividade dos macrófagos peritoneais. Parece-nos plausível excluir de nossos resultados a participação de linfócitos B, T helper e citotóxicos. Por outro lado, não foi possível precisar a relevância das células NK para o desenvolvimento do tumor. Além disso, pode-se afirmar que há expressão de PBRs em células do TAE e que o tratamento in vitro com diazepam ou Ro5-4684 aumentou a proliferação destas células. Desta forma, os resultados dos dois últimos experimentos sugerem que a existência de efeitos do diazepam sobre crescimento tumoral in vivo pode também ser atribuída, ao menos em parte, a uma ação direta deste fármaco sobre células do TAE / Benzodiazepines (BDZ) are drugs widely used due to their anxiolytic and sedative properties, acting on specific sites coupled to GABAA complex in the Central Nervous System (CNS). Besides these central receptors, BDZ have affinity for peripheral-type receptors (PBR), which have been found in immune cells, such as lymphocytes and macrophages, in tumor cells and in the adrenal glands. The present study evaluated the effects of diazepam, a commonly used BDZ, on tumor growth and immunity of mice bearing Ehrlich Ascitic Tumor (EAT). Specifically, this study evaluated the following parameters of the immune system: macrophage activity, populations of B, helper and cytotoxic T lymphocytes, and Natural Killer (NK) cells cytotoxicity. Furthermore, the evaluation of PBR expression in EAT cells and possible in vitro effects of diazepam or Ro5-4864 on EAT cell cycle were performed. Results showed the following diazepam effects in vivo (3.0 mg/kg per day): (1) increased tumor growth without changes in cell cycle, after 7 days; (2) decreased the number of leucocytes in the peritoneal cavity, the production of NO and the spreading index, but did not modify the production of hydrogen peroxide and the phagocytosis index, after 2 days; (3) did not modify the relative spleen weight and the population of lymphocytes after 2 or 7 days, (4) increased NK cytotoxicity after 3 days; (5) reduced the percentage of EAT cells expressing PBR after 7 days. Experiments performed in vitro showed that diazepam or Ro5-4864 increased the proliferation of EAT cells. Diazepam treatment in vivo using lower doses (0.3 mg/kg and 1mg/kg) did not modify tumor growth. Therefore, diazepam in the dose of 3.0 mg/kg increased the growth of EAT and reduced innate immunity, probably through the decrease in the activity of peritoneal macrophages. A role of B and helper or cytotoxic T lymphocytes in our experiments seems unlikely since the population of these cells types remained unchanged. On the other hand, it was not possible to determine the relevance of NK cells cytotoxicity on tumor development. The expression of PBR in EAT cells and the increase of their proliferation induced by in vitro treatment with diazepam or Ro5-4684 were observed. The results of these two last experiments suggest that the increase on tumor growth following diazepam treatment in vivo can be attributed, at least in part, to a direct action of this drug on EAT cells
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Função fagocítica em leucócitos humanos silenciados ou mutados para AIRE. / Phagocytic function of human leukocytes silenced or mutated AIRE.Carvalho, Marina Uchôa Wall Barbosa de 19 June 2013 (has links)
A APECED é uma doença que apresenta autoimunidade e susceptibilidade a Candida albicans. Nosso grupo observou que a proteína AIRE participa da via da Dectina-1, importante contra a C. albicans. Neste projeto, investigamos como a ausência de AIRE influencia em eventos para a eliminação do patógeno via Dectina-1. Assim, avaliamos o burst oxidativo, expressão de moléculas do sistema NADPH oxidase e fagocitose por células de paciente com APECED e células THP-1 silenciadas para AIRE. Não houve diferença no burst oxidativo e na expressão dos componentes do sistema NADPH oxidase por estas células e as células silenciadas fagocitam menos que as células selvagens. Observamos que não há diferença na expressão flavocitocromo b558 e p40phox do paciente comparado ao controle. Em paralelo, mostramos que as células do paciente apresentaram um burst oxidativo e fagocitose diminuídos comparado ao controle. Estes resultados sugerem que há um defeito no reconhecimento via Dectina-1, gerando uma diminuição da fagocitose que pode dificultar sua eliminação. / The APECED is a syndrome with autoantibodies and Candida albicans susceptibility. Our group has noted that AIRE protein is required for dectin-1 signaling, important against C. albicans. In this project, we investigate how the absence of AIRE influences in events for elimination of pathogen via dectin-1. We evaluated reactive oxygen species production, expression of NADPH oxidase molecules and phagocytosis by APECED pacient cells or AIRE silent THP-1 cells. We didnt observe differences in oxidative burst and expression of NADPH oxidase components by these cells and silent cells phagocytize less than wild-type cells. We observed no difference in flavocytochrome b558 and p40phox expression in paciente cells and control. In parallel, we showed that pacient cells has a decrease in burst oxidative and phagocytosis compared to control. Our results suggest that there is a defect in pathogen recognition via dectin-1, resulting in decrease on phagocytosis that can hamper their elimination.
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Efeitos do diazepam sobre o crescimento tumoral e imunidade de animais portadores do tumor ascítico de Ehrlich / Diazepam effects on tumor growth and on immunity of Ehrlich tumor bearing miceSakai, Mônica 07 December 2004 (has links)
Benzodiazepínicos (BDZ) são fármacos amplamente utilizados devido às suas propriedades ansiolíticas e sedativas, mediadas pelo complexo GABAA no Sistema Nervoso Central (SNC). Além destes receptores centrais, os BDZ possuem afinidade por receptores do tipo periféricos (PBR) os quais estão presentes em células do sistema imune, como linfócitos e macrófagos, em células tumorais e em glândulas adrenais. O presente trabalho avaliou os efeitos do diazepam, um BDZ freqüentemente utilizado, sobre o crescimento tumoral e a imunidade de animais portadores do Tumor Ascítico de Ehrlich (TAE). Mais especificamente, este trabalho avaliou os seguintes parâmetros da resposta imune: atividade de macrófagos, populações de linfócitos B, T helper e citotóxicos esplênicos, citotoxicidade de células Natural Killer (NK). Além disso, a marcação para PBR em células do TAE e avaliação de possíveis efeitos do diazepam ou Ro5-4864 in vitro sobre o ciclo celular do TAE foram avaliadas. Os resultados mostraram os seguintes efeitos do tratamento com diazepam in vivo (3,0 mg/kg): (1) aumento do crescimento do TAE; sem modificação das fases do ciclo celular do tumor, após 7 dias, (2) diminuição do número de leucócitos da cavidade peritoneal, da produção de NO e do índice de espraiamento; mas sem interferência com a produção de peróxido de hidrogênio e o índice de fagocitose, após 2 dias (3) não modificou o peso relativo do baço e a porcentagem de linfócitos esplênicos, após 2 dias ou 7 dias (4) aumento da citotoxicidade de células NK, após 3 dias (5) diminuição da porcentagem de células do TAE marcadas para PBR, após 7 dias. O tratamento in vitro com diazepam ou Ro5-4864 mostrou um aumento da proliferação de células do TAE. Já o tratamento in vivo com diazepam em doses menores (0,3 mg/kg e 1mg/kg) não modificou o crescimento do TAE, após 7 dias. Desta forma, sugere-se que o diazepam na dose de 3,0 mg/kg tenha aumentado o crescimento do TAE e diminuído a resposta imune inata, observada por meio da diminuição da atividade dos macrófagos peritoneais. Parece-nos plausível excluir de nossos resultados a participação de linfócitos B, T helper e citotóxicos. Por outro lado, não foi possível precisar a relevância das células NK para o desenvolvimento do tumor. Além disso, pode-se afirmar que há expressão de PBRs em células do TAE e que o tratamento in vitro com diazepam ou Ro5-4684 aumentou a proliferação destas células. Desta forma, os resultados dos dois últimos experimentos sugerem que a existência de efeitos do diazepam sobre crescimento tumoral in vivo pode também ser atribuída, ao menos em parte, a uma ação direta deste fármaco sobre células do TAE / Benzodiazepines (BDZ) are drugs widely used due to their anxiolytic and sedative properties, acting on specific sites coupled to GABAA complex in the Central Nervous System (CNS). Besides these central receptors, BDZ have affinity for peripheral-type receptors (PBR), which have been found in immune cells, such as lymphocytes and macrophages, in tumor cells and in the adrenal glands. The present study evaluated the effects of diazepam, a commonly used BDZ, on tumor growth and immunity of mice bearing Ehrlich Ascitic Tumor (EAT). Specifically, this study evaluated the following parameters of the immune system: macrophage activity, populations of B, helper and cytotoxic T lymphocytes, and Natural Killer (NK) cells cytotoxicity. Furthermore, the evaluation of PBR expression in EAT cells and possible in vitro effects of diazepam or Ro5-4864 on EAT cell cycle were performed. Results showed the following diazepam effects in vivo (3.0 mg/kg per day): (1) increased tumor growth without changes in cell cycle, after 7 days; (2) decreased the number of leucocytes in the peritoneal cavity, the production of NO and the spreading index, but did not modify the production of hydrogen peroxide and the phagocytosis index, after 2 days; (3) did not modify the relative spleen weight and the population of lymphocytes after 2 or 7 days, (4) increased NK cytotoxicity after 3 days; (5) reduced the percentage of EAT cells expressing PBR after 7 days. Experiments performed in vitro showed that diazepam or Ro5-4864 increased the proliferation of EAT cells. Diazepam treatment in vivo using lower doses (0.3 mg/kg and 1mg/kg) did not modify tumor growth. Therefore, diazepam in the dose of 3.0 mg/kg increased the growth of EAT and reduced innate immunity, probably through the decrease in the activity of peritoneal macrophages. A role of B and helper or cytotoxic T lymphocytes in our experiments seems unlikely since the population of these cells types remained unchanged. On the other hand, it was not possible to determine the relevance of NK cells cytotoxicity on tumor development. The expression of PBR in EAT cells and the increase of their proliferation induced by in vitro treatment with diazepam or Ro5-4684 were observed. The results of these two last experiments suggest that the increase on tumor growth following diazepam treatment in vivo can be attributed, at least in part, to a direct action of this drug on EAT cells
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Inflammasome regulation and activation in the intestinal epitheliumLei, Andrea January 2017 (has links)
Microbiota colonisation of the intestinal tract makes it difficult for pattern recognition receptors (PRR) to discriminate between beneficial microbes and harmful pathogens. We aim to define the roles of cytosolic Nod-like receptors (NLR) in intestinal immunity and homeostasis. Upon activation, some NLR form inflammasomes that mediate the release of inflammatory cytokines and pyroptosis, an inflammatory form of cell death. NLR activation in the non-hematopoietic compartment was shown to be protective during acute intestinal infection. To identify the cell type responsible for this protection, we generated transgenic mice in which the key inflammasome adaptor molecule Asc is selectively ablated in intestinal epithelial cells (IEC) (Asc<sup>ΔVC</sup>) and observed that inflammasomes are important for controlling Citrobacter rodentium clearance in these mice. To further dissect the importance of pathogen clearance by IEC inflammasome, ex vivo cultures of primary IEC organoids were established. Thus far this system has revealed profound differences in inflammasome regulation between IEC organoids and bone marrow-derived macrophages (BMDM). This research will inform our understanding of cell type-specific regulation of inflammasomes.
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Função fagocítica em leucócitos humanos silenciados ou mutados para AIRE. / Phagocytic function of human leukocytes silenced or mutated AIRE.Marina Uchôa Wall Barbosa de Carvalho 19 June 2013 (has links)
A APECED é uma doença que apresenta autoimunidade e susceptibilidade a Candida albicans. Nosso grupo observou que a proteína AIRE participa da via da Dectina-1, importante contra a C. albicans. Neste projeto, investigamos como a ausência de AIRE influencia em eventos para a eliminação do patógeno via Dectina-1. Assim, avaliamos o burst oxidativo, expressão de moléculas do sistema NADPH oxidase e fagocitose por células de paciente com APECED e células THP-1 silenciadas para AIRE. Não houve diferença no burst oxidativo e na expressão dos componentes do sistema NADPH oxidase por estas células e as células silenciadas fagocitam menos que as células selvagens. Observamos que não há diferença na expressão flavocitocromo b558 e p40phox do paciente comparado ao controle. Em paralelo, mostramos que as células do paciente apresentaram um burst oxidativo e fagocitose diminuídos comparado ao controle. Estes resultados sugerem que há um defeito no reconhecimento via Dectina-1, gerando uma diminuição da fagocitose que pode dificultar sua eliminação. / The APECED is a syndrome with autoantibodies and Candida albicans susceptibility. Our group has noted that AIRE protein is required for dectin-1 signaling, important against C. albicans. In this project, we investigate how the absence of AIRE influences in events for elimination of pathogen via dectin-1. We evaluated reactive oxygen species production, expression of NADPH oxidase molecules and phagocytosis by APECED pacient cells or AIRE silent THP-1 cells. We didnt observe differences in oxidative burst and expression of NADPH oxidase components by these cells and silent cells phagocytize less than wild-type cells. We observed no difference in flavocytochrome b558 and p40phox expression in paciente cells and control. In parallel, we showed that pacient cells has a decrease in burst oxidative and phagocytosis compared to control. Our results suggest that there is a defect in pathogen recognition via dectin-1, resulting in decrease on phagocytosis that can hamper their elimination.
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