Diversos neurônios apresentam uma atividade constitutiva de NF-?B, o qual desempenha múltiplas funções fisiológicas, além da modulação de respostas patológicas. A melatonina, hormônio produzido ritmicamente pela glândula pineal na fase de escuro, é também um fator autócrino e parácrino envolvido em múltiplos processos biológicos, sendo que a citoproteção é uma ação de destaque dessa molécula. A melatonina inibe a translocação nuclear do NF-?B e a expressão do seu produto iNOS em modelos de danos celular. No presente trabalho avaliamos se o efeito citoprotetor da melatonina depende do estado de ativação do NF-?B em cultura de células granulares de cerebelo, tendo em vista que essas células apresentam uma atividade basal deste fator de transcrição fundamental para a sobrevivência das células. Além disso, questionamos se essas células em cultura produziriam melatonina e se esta teria algum papel citoprotetor. Testamos a viabilidade da cultura de células granulares de cerebelo de rato (Wistar 7-8 dias de idade) após 24 horas de incubação com melatonina na presença ou ausência de LPS. Em condição basal a melatonina diminuiu a sobrevivência das células e inibiu a morte celular induzida pelo LPS. Este efeito foi compatível com os resultados da ativação do NF-?B e da expressão da iNOS. Na presença do LPS a melatonina bloqueia a indução da translocação nuclear do NF-?B, a expressão da iNOS e a produção de NO. Quando apenas a melatonina foi incubada, observamos uma inibição transiente (15 min.) do NF-?B, seguida por um aumento do conteúdo nuclear do fator de transcrição (60 min.). A expressão da iNOS seguiu o mesmo perfil, ou seja, sofreu uma inibição transiente (30 min.) seguida de um aumento acima do nível basal após 120 minutos de incubação. Portanto, demonstramos que a melatonina afeta de forma diferente a viabilidade de células granulares de cerebelo dependo do contexto em que as células se encontram. Além disso, obtivemos evidências de que essas células expressam a enzima a AA-NAT, e produzem melatonina, que exerce função protetora para a cultura. Desta forma, nossos dados proporcionam uma base mecanicista para a compreensão da influência do contexto celular na resposta à melatonina / Several neurons constitutively express NF-?B, which plays some physiological roles, besides the well-known control of pathological responses. Melatonin, the hormone produced by the pineal gland rhythmically in the dark phase is also an autocrine and paracrine factor of immune competent cells, involved in multiple biological processes and the cytoprotective action is a highlight of this molecule. Melatonin inhibits the nuclear translocation of NF-?B and the expression of iNOS in models of cell damage. The present study evaluated whether the cytoprotective effect of melatonin depends on the state of activation of NF-?B in cultured cerebellar granule cells, given that these cells have a basal activity of this transcription factor essential for cell survival. Moreover, we questioned whether these cells in culture produce melatonin and whether it would have a cytoprotective role. We tested the viability of the rat (7-8 days old Wistar) cerebellar granule cell culture after 24 h incubation with melatonin in the presence or absence of LPS. In basal condition melatonin decreased cell survival while inhibited cell death induced by LPS. These effects were consistent with the results from the activation of NF-?B and the expression of iNOS. In the presence of LPS melatonin blocked the activation of the NF-?B , the expression of iNOS and the production of NO. When only melatonin was incubated, we observed a transient reduction (15 min) of NF-?B nuclear content, followed by an increase of its nuclear content (60 min). The iNOS expression followed the same profile, i.e. undergone a transient inhibition (30 min), followed by an increase above baseline after 120 min of incubation. Therefore, we have demonstrated that melatonin affects differently the viability of cerebellar granule cells depending on the context. Furthermore, we founded evidences that the granule cells in culture express the key enzyme in the synthesis of melatonin, AA-NAT and produce melatonin, which carries protective function for the culture. Our data provide a mechanistic basis for understanding the influence of cell context on the final output response to melatonin

O habitat natural da Neisseria meningitis é a nasofaringe humana e a transmissão da bactéria é por contato direto ou por inalação de partículas durante a fase de transmissão N. meningitis é uma bactéria Gram-negativa responsável por uma significante mortalidade em todo o mundo. Embora existam vacinas polissacárides contras os sorogrupos A, C, W135 e Y , não há uma vacina adequada para crianças menores de 4 anos para o sorogrupo B. Estudos estão sendo direcionadas para pesquisa de antígenos vacinais que são derivados da proteínas de membrana externa(NOMV). Entretanto vacinas baseadas em NOMV são consideradas pouco imunogênicas , fazendo com que o uso de adjuvantes seja necessário. Este estudo investiga a imunogenicidade da NOMV de N. meningitidis administrada pela via intranasal/intramuscular em camundongos neonatos BALC/c, usando proteína de membrana externa (NOMC) obtido de uma cepa epidêmica de N. meningitidis B:4:P1:15. As cepas usadas para imunização dos camundongos foram selecionadas por colony-blot, usando anticorpo monoclonal anti L3,7,9 para maior expressão do LPS contra o imunotipo L3,7,9 presente na cepa (B:4:P1:15 3,7,9). Como adjuvantes de mucosa foram utilizados Bordetella pertussis (células íntegras) ou sobrenadante de cultura com 48 horas ou hidróxido de alumínio [Al(OH)3]. O soro dos camundongos imunizados foram analisados pelo método de ELISA à fim de se comparar os diferentes adjuvantes utilisados. O índice de avidez também foi determinado. IgG e IgM foram detectados nos soros dos camundongos após imunização, com índices de intermediária e alta avidez. Todos os adjuvantes foram capazes de aumentar a resposta imune contra NOMV de N.meningitidis. A via intranasal foi adequada para sensibilizar as células do sistema imune que foram rapidamente estimuladas pela via intramuscular usando os adjuvantes utilizados na presente investigação. Dados sugerem que o estudo da NOMV é importante na indução da imunidade de mucosa para N. meningitidis B, e que a qualidade e magnitude da resposta imune gerada pelas vacinas de mucosa são influenciadas tanto pelo adjuvante como pelo antígeno. Concluímos que NOMV juntamente com adjuvantes de mucosa tem considerável potencial no desenvolvimento de vacinas contra o meningococo do sorogrupo B. / The natural habitat of Neisseria meningitidis is the human nasopharynx, and the bacterium is transmitted by direct mouth-to-mouth contact or by the inhalation of released mucous particles during close contact. N meningitidis is a Gram-negative bacterium responsible for significant mortality worldwide. While effective polysaccharide-based vaccines exist against serogroups A, C, W135, and Y, no similar vaccine is suitable for children under 4 years against disease caused by serogroup B strains. Current studies are searching for vaccinal antigens that are derived from the native outer membrane (NOMV). However, vaccines based on NOMV are considered weak, making the use of adjuvants necessary. This study investigated the immunogenicity of NOMV of N. meningitidis administered intranasal/intramuscular in neonate BALB/c mice, using the native outer membrane complex (NOMC) obtained from an epidemic strain of N. meningitidis B:4:P1.15. The strains used for immunization of mice were selected by colony-blot, using anti L3,7,9 monoclonal antibodies, for the highest expression of LPS among the immunotypes (B:4:P1:15 L9á). As mucosal adjuvants, we used Bordetella pertussis (whole cells) or the supernatant of 48 h culture of this bacterium, followed by an intramuscular dose of the same protein adsorbed onto , B. pertussis (whole cells) or 48-h B. pertussis culture supernatant or aluminum hydroxide [Al(OH)3]. Sera of immunized mice were evaluated by ELISA in order to compare the different adjuvants used. We also determined their avidity index. IgG and IgM were detected in the serum of mice after immunization, with avidity indices that ranged from intermediate to high. All adjuvants were capable of increasing the immune response against NOMV of N. meningitidis in the homologous prime/boost schedule used. The intranasal route was suitable for sensitizing the cells of the immune system which were quickly stimulated by the intramuscular route using the adjuvants analysed in the present invertigation. Data suggest that the NOMV studied is important in the induction of mucosal immunity to N. meningitidis B, and that the quality and magnitude of the immune responses generated by mucosal vaccines are influenced by the adjuvant as well as the antigen. In conclusion, nasal delivery of NoMV with mucosal adjuvants has considerable potential in the development of a mucosal vaccine against serogroup B meningococci.

Die meisten Lebewesen nutzen zur Abwehr von pathogenen Mikroorganismen u.a. ein weites Spektrum von antimikrobiellen, oft kationischen Peptiden. Etliche dieser natürlichen und künstlichen Peptide sind in der Lage, Bakterienprodukte wie das Lipopolysaccharid (LPS) Gram-negativer Bakterien zu binden. LPS ist als potenter Stimulator des Immunsystems ein bedeutender Faktor bei der Entstehung von Infektion und Entzündung. LPS-bindende kationische Peptide, zu denen auch von der LPS-Bindungssequenz des Limulus-anti-LPS-Faktors (LALF) abgeleiteten Ringpeptide gehören, wirken synergistisch zu den klassischen Antibiotika, ohne jedoch wie diese zu einer vermehrten LPS-Freisetzung zu führen. LALF-Peptide zeigten in bisher veröffentlichten Versuchen vielversprechende Ergebnisse bei der Bindung von LPS und in murinen Sepsismodellen. In der vorliegenden Arbeit wird in Versuchen mit humanen Monozyten und murinen Makrophagen gezeigt, dass neuartige LALF-Peptide eine durch LPS induzierte Ausschüttung des Zytokins TNF-alpha und des vasoaktiv wirksamen Stickstoffoxids wirkungsvoll unterdrücken können. Ferner wird in LPS-Bindungsassays nachgewiesen, dass dies durch eine Blockierung der LPS-Erkennung durch das LPS bindende Protein (LPB) verursacht wird. Die Verwendung von D-Aminosäuren verspricht dabei in ersten in vitro Experimenten eine Optimierung der Peptid-Eigenschaften. Die Entschlüsselung der Wirkmechanismen dieser neuartigen Peptide könnte Auswirkungen auf die Entwicklung neuer therapeutischer Interventionsstrategien bei Infektion und Sepsis haben. / In host defense against pathogenic microorganisms most organisms employ a broad spectrum of antimicrobial, often cationic peptides. Several of these natural or synthetic peptides are able to bind bacterial products like Lipopolysaccharide (LPS) of Gram-negative bacteria. As a potential stimulator of the immune system LPS is an important pathogenetic factor for infection and inflammation. Cationic LPS-binding peptides like cyclic peptides corresponding to the LPS-binding domain of the Limulus-anti-LPS-factor (LALF) have been shown to act synergistic to classic antibiotics. An advantage of these compounds, however, may be their lack of induction of LPS release. In previous studies LALF-peptides have shown promising results for binding LPS, and in murine sepsis models. Here we show in experiments with human monocytes and murine macrophages that novel LALF-derived peptides are able to effectively block the LPS-induces release of the cytokine TNF-alpha and of the vasoactive nitric oxide. In addition it is shown here by employing an LPS-binding assay that this activity is caused by inhibition of LPS-recognition brought about by the LPS binding protein (LBP). In first in vitro experiments the use of D-amino acids looks promising as they improve peptide quality. To further elucidate the mode of action of these novel peptides could lead to the development of new therapeutic strategies against infection and sepsis.

LPS wird von Gram-negativen Bakterien freigesetzt und führt mit Hilfe von LBP zur Ausschüttung proinflammatorischer Zytokine aus Monozyten und Makrophagen. Diese von LPS ausgelöste Kaskade, ist entscheidend an der Entstehung der Sepsis beteiligt. In dieser Arbeit wurde gezeigt, daß die LPS-induzierte Stimulation von Makrophagen durch murines Serum gehemmt werden kann. Außerdem konnte im Rahmen dieser Arbeit im Mausmodell verdeutlicht werden, welche Rolle Lipoproteine und LBP bei dem protektiven Serumeffekt spielen. Von den in Mausseren verschiedener Mausstämme bestimmten Parametern korrelierte der Phospholipidgehalt relativ gut mit dem inhibitorischen Serumeffekt. Eine Depletion von Lipoproteinen aus den Seren führte zu einer starken Reduktion des inhibitorischen Serumpotentials, während die Verwendung von LBP-defizienten Seren keinen Einfluß auf den Serumhemmeffekt hatte. Lipoproteine sehr geringer, geringer und hoher Dichte verursachten in Gegenwart von LBP eine deutliche Reduktion der LPS-Effekte, die gut mit ihrem Phospholipidgehalt korrelierte. In Abwesenheit von Serum und LBP konnten Lipoproteine LPS-Effekte auch bei hohen Phospholipidkonzentrationen kaum noch inhibieren. In nativen murinen Lipoproteinen hoher Dichte und lipoproteindefizientem Serum ließ sich LBP nachweisen. Die Ergebnisse dieser Arbeit zeigen, daß native Lipoproteine sehr geringer, geringer und hoher Dichten als Akzeptoren für LPS dienen können. Ihr inhibitorisches Potential korreliert am besten mit ihrem Phospholipidgehalt. Der Transport von LPS in diese Lipoproteine wird durch LBP katalysiert, was den protektiven Effekt hoher LBP-Konzentrationen erklärt. Weiterhin konnte gezeigt werden, daß LBP nicht der einzige Bestandteil von murinem Serum ist, der LPS in Lipoproteine transferiert. Vermutlich ist bei den, in dieser Arbeit verwendeten Serumkonzentrationen der PLTP-Gehalt ausreichend, diesen LPS-Transfer in Abwesenheit von LBP zu vollziehen. Bei der Interaktion zwischen Lipoproteinen und LPS handelt es sich um einen physiologischen Weg des Organismus, um auf bakterielle Endotoxine zu reagieren und so der Sepsis entgegenzuwirken. Mit genaueren Kenntnissen über diese Interaktion, bei der Lipidtransferproteine wie LBP und PLTP eine entscheidende Rolle spielen, können eventuell in Zukunft Methoden gefunden werden, diese physiologischen Vorgänge des Körpers zu unterstützen, um so eine Sepsis zu therapieren. / LPS released by gram-negative bacteria is bound by LBP and initiates the release of proinflammatory cytokines in makrophages and monocytes. These cytokines are thought to play a central role in the pathophysiology of sepsis. In these studies I was able to show an inhibitory effect of murine serum on the LPS-induced stimulation of macrophages. Furthermore the role of lipoproteins and LBP in this protective effect of serum was investigated. The inhibitory effect of serum from different mouse strains was best correlated to its phospholipid content. Depletion of serum from lipoproteins strongly reduced its LPS-inhibitory potential while depletion of serum from LBP had no effect. Murine VLDL, LDL and HDL were found to be potent inhibitors of LPS-effects in presence of LBP. In abscence of LBP the inhibitory effect was much weaker. LBP could be detected in murine HDL and murine lipoproteindeficient serum. My data shows that HDL, LDL and VLDL can act as acceptors of LPS. Their inhibitory potential is best correlated to their phospholipid content. LBP catalyses transport of LPS into lipoproteins. This could be an explanation for its protective effect in high doses. Furthermore it could be shown that LBP is not the only serum component that transfers LPS into Lipoproteins. Possibly the used serum contained enough PLTP to perform this transfer in absence of LBP. The interaction between Lipoproteins and LPS is a physiological way of the organism to react on endotoxines and inhibit the development of sepsis. PLTP and LBP play major roles in this interaction. Understanding the pathways of LPS-detoxification may help to support the organism s physiological answer and establish new methods to treat sepsis.

Linhas de Produtos de Software (LPS) abstraem a semelhança entre produtos e envolvem o conceito de reúso de software para desenvolver software em larga escala com mais rapidez e qualidade. O reúso dos artefatos em uma LPS é planejado e executado desde sua concepção, sendo assim possível armazenar esses artefatos em um repositório (núcleo de ativos reusáveis) para utilização posterior. Porém, ao se construir um repositório para uma certa LPS, este atende apenas às especificações desta arquitetura e não promove o reúso desses ativos em outras LPS, causando uma certa perda no potencial de reusabilidade dos ativos produzidos e armazenados. Além disso, a definição e os dados da execução do processo de desenvolvimento dessas LPS também não são armazenados prevendo o reúso, o que gera retrabalho para definir e instanciar um processo de software sempre que uma nova LPS é criada. Neste contexto, este trabalho tem por objetivo apresentar uma abordagem baseada em um conjunto de serviços para promover o amplo reúso de ativos produzidos e de elementos de processo utilizados, além de facilitar a criação e o desenvolvimento de linha de produtos de software, permitindo o gerenciamento do núcleo de ativos reusáveis. Por possuir uma interface baseada em serviços, esta abordagem poderá ser incorporada também a outros ambientes. Para validar a abordagem, verificar vantagens e sugerir melhorias, foi conduzida uma prova de conceito com sua aplicação. Os resultados dessa prova comprovaram a viabilidade da abordagem em cumprir os objetivos propostos. / Software Product Lines (SPL) extract the similarity among products and use the software reuse concept to produce large-scale software with increased quality and reduction in the development time. Reuse in SPL is planned and executed since the beginning for each artifact resulting from the development process, and then these artifacts are stored in a repository (reusable core assets) for later use. However, when a repository is built for a specific SPL, it supports only the specifications for that architecture and do not promote the reuse of these assets among distinct SPL, which inhibits their reuse potential. In addition, the definition and execution data of the development process applied are not stored properly for reuse, which causes a re-work to define and instantiate a software process when creating a new SPL. In this context, this work aims at present a service-based approach to promote the wide reuse of the produced assets and process elements, also to facilitate the creation and the management of SPL and to allow the management of the reusable core assets. Once this approach has a service-based interface, it can be embedded into other environments. In order to validate this approach, point out advantages and suggest improvements, a concept proof was performed applying it. The results of this proof ensured the approach viability to fulfill the proposed goals.

L'emploi des agents antibactériens est un moyen important d'une part dans la lutte contre les infections bactériennes et d'autre part pour conserver les produits alimentaires jusqu'à leur consommation. La perte d'efficacité des antibiotiques par le développement de résistance bactérienne ainsi que la toxicité des conservateurs synthétiques rend nécessaire le développement de nouveaux produits antibactériens naturels. Les protéines et les peptides antibactériens agissant sur les membranes bactériennes paraissent une alternative pour limiter l'instauration de résistances bactériennes. L'ovotransferrine est une protéine du blanc d'œuf ayant des propriétés membranotropes responsable entre autre de son activité antibactérienne. L'objectif de cette thèse est d'étudier les mécanismes membranotropes de l'ovotransferrine vis-à-vis des membranes externe et cytoplasmique d'E. coli en utilisant respectivement des monocouches de LPS (lipopolysaccharides) et de phospholipides comme modèles membranaires expérimentales. L'ovotransferrine possède une capacité d'insertion dans la monocouche de LPS qui dépend de la concentration protéique, de la compacité de la monocouche et de la conformation des molécules de LPS. L'ovotransferrine s'adsorbe faiblement à la monocouche de phospholipides. Ainsi, les monocouches sont perturbées par la désorganisation des lipides. L'analyse comparative de l'ovotransferrine chauffée à sec avec la forme native a montré la conservation des structures secondaire et tertiaire avec une augmentation de l'hydrophobie de surface et probablement de la flexibilité et une affinité plus élevée aux interfaces hydrophiles/hydrophobes (eau/air). L'activité membranaire de l'ovotransferrine est accrue après son chauffage à sec. La capacité d'insertion dans la monocouche de LPS est amplifiée avec une affinité plus importante. Une capacité d'insertion dans la monocouche de phospholipides est générée pour la forme chauffée à sec associée à une adsorption plus élevée. L'ovotransferrine chauffée à sec induit des perturbations plus importantes des monocouches à des concentrations protéiques plus faibles. / The use of antibacterial agents is very important, firstly, on the fight against bacterial infections, and secondly, to keep food products until its consumption. The loss of antibiotics effectiveness through the development of bacterial resistance and the toxicity of synthetic preservatives necessitates the development of new natural antibacterial products. Antibacterial proteins and peptides acting on the bacterial membranes appear as an alternative to limit the introduction of bacterial resistances. Ovotransferrin is an egg-white protein with membranotropic properties responsible among other things for its antibacterial activity. The aim of this thesis is to study the membranotropic mechanisms of ovotransferrin towards the outer and cytoplasmic membranes of E. coli using respectively monolayers of LPS (lipopolysaccharides) and phospholipids as experimental membrane models. Ovotransferrin has an insertion capacity in LPS monolayer that is dependent on protein concentration, monolayer compactness, and LPS molecule conformation. Ovotransferrin weakly adsorbs to the monolayer of phospholipids. Thus, the monolayers are disturbed by the disorganization of the lipids. Comparative analysis of dry-heated ovotransferrin with the native form showed conservation of secondary and tertiary structures with an increase of surface hydrophobicity and probably of flexibility and higher affinity to hydrophilic/hydrophobic interfaces (water/air). The insertion capacity in the LPS monolayer is amplified with greater affinity. Insertion capacity in the phospholipid monolayer is generated for the dry heated form associated with higher adsorption. Dry-heated ovotransferrin induces greater disruption of monolayers at lower protein concentrations.

Achyrocline satureoides (Lam) D.C., popularmente conhecida como \"marcela\", é utilizada popularmente para tratar diversas doenças, como mal estar gástrico e intestinal, inflamações, diabetes e outros. Como os mecanismos de ação do extrato de A. satureoides ainda não são conhecidos, o presente trabalho visou esclarecer os mecanismos antiinflamatórios do extrato bruto hidroalcoólico das inflorescências de A. satureoides, focando na migração e função fagocítica e microbicida de neutrófilos. Para tanto, o extrato hidroalcoólico foi administrado por gavage (50, 100 e 250mg/kg) em ratos Wistar, machos, adultos, e a inflamação foi induzida pela injeção do lipopolisacarídeo de E. coli (LPS; 2mL de solução; 500 µg/mL em PBS) no tecido subcutâneo dorsal (modelo da bolsa de ar). Animais controles receberam volumes equivalentes de veículo do extrato e indometacina (30mg/kg). Foram quantificados o número de neutrófilos (câmara de Neubauer e esfregaços corados por Panótico) e a concentração de Leucotrieno B4 e CINC-1 (ELISA) no foco de lesão; a interação leucócito-endotélio em vênulas da microcirculação mesentérica após estímulo in situ pelo lipopolisacarídeo de E.coli (LPS; 30µg/40µL; por microscopia intravital); a expressão de moléculas de adesão e do toll-like receptor (TLR-4), além da quantificação do burst oxidativo (induzido pelo miristato-acetato de forbol - PMA) e fagocitose em neutrófilos circulantes (citometria de fluxo); análise histológica e quantificação dos marcadores hepáticos (AST, ALT e Gama-GT) e renais (uréia e creatinina) no plasma por espectrofotometria. Os resultados obtidos mostraram que o tratamento com o extrato reduziu o número de neutrófilos e a concentração de LTB4 e CINC-1 na bolsa de ar; reduziu a expressão de TLR4 pelos neutrófilos circulantes e a porcentagem de neutrófilos positivos para L-selectina e β2-integrina; inibiu a adesão e o comportamento rolling de leucócitos ao endotélio microvascular; reduziu o burst induzido por PMA; aumentou o potencial de fagocitose, sem alterar o burst induzido por Staphylococcus aureus, não alterou a morfologia tecidual e a concentração sérica dos marcadores de atividade hepática e renal. Em conjunto, os dados obtidos mostram que dose, aparentemente, não tóxica do extrato de A. satureoides exerce efeito antiinflamatório in vivo frente ao LPS, quantificados pela redução da migração e pelas interferências nas atividades fagocítica e microbicida dos neutrófilos. / Achyrocline satureoides (Lam) D.C., popularly known as \"marcela\", is popularly used to treat several diseases, such as gastric and intestinal disorders, inflammation, diabetes and others. As the mechanisms of the extract of A. satureoides have not been elucidated, this study aimed to investigate the anti-inflammatory mechanisms of the crude hydroalcoholic extract of the flowers of A. satureoides, focusing on migration and phagocytic and microbicidal neutrophils function. The hydroalcoholic extract was administered by gavage (50, 100 and 250mg/kg) into male Wistar rats, adult, and inflammation was induced by injection of lipopolysaccharide E. coli (LPS; 2 mL of solution; 500µg/mL in PBS) into the dorsal subcutaneous tissue (air pouch model). Control animals received equivalent volume of extract vehicle or indomethacin solution (30mg/kg). It was quantified the numbers of neutrophils (Neubauer chamber and stained smears by Panoptic) and concentrations of leukotriene B4 (LTB4) and CINC-1 (ELISA) in the focus of the injury; the leukocyte-endothelium interactions in mesenteric venules of the microcirculation after stimulation by LPS (30µg/40µL; intravital microscopy); the adhesion molecules and toll-like receptor (TLR-4) expressions and quantification of oxidative burst and phagocytosis in circulating neutrophils (flow cytometry); histological analysis and the hepatic markers (AST, ALT and gamma-GT) and kidney (urea and creatinine) concentrations in plasma by spectrophotometry. Data obtained showed that the treatment reduced the numbers of neutrophils and concentrations of LTB4 and CINC-1 in the subcutaneous tissue; reduced the TLR4 expression by circulating neutrophils and the number of β2-integrin- and L-selectin-positive neutrophils; inhibited the leukocyte adhesion and the rolling behavior to vascular endothelium; reduced the burst evoked by PMA; increased the phagocytosis without changing the burst induced by Staphylococcus aureus; and did not alter the tissue morphology and concentration of hepatic and renal enzymes in the serum. Together, these data suggest that dose, apparently non-toxic, of extract of A. satureoides exerts anti-inflammatory effect in vivo, quantified by the reduced migration and by interference in the phagocytic and microbicidal activities of neutrophils.

Células apresentadoras de antígeno (APCs) controlam as respostas de linfócitos T por múltiplos mecanismos, que incluem a expressão de moléculas co-estimuladoras, a produção de citocinas e outros mediadores. Estes mecanismos exercem influência não só na proliferação, diferenciação e polarização dos linfócitos T, mas também interferem na sobrevivência destas células. No presente trabalho, foi demonstrado que fator(es) solúvel(eis) produzido(s) por APCs ativadas via receptores do tipo Toll (TLRs) suprimem a morte induzida por ativação (AICD) de linfócitos T. Este efeito foi observado em APCs não estimuladas, porém foi significativamente maior após estimulação das APCs com lipopolissacarídeo (LPS). Através do uso de diferentes camundongos nocautes, foi mostrado que a produção do fator protetor induzida por LPS é dependente da via de TLR4/MyD88 e independente de TLR2 e CD14. Este fator foi identificado como prostaglandina E2 (PGE2) e foi demonstrado que os sobrenadantes derivados de APC e a PGE2 sintética bloqueiam a expressão de CD95L em linfócitos T estimulados via TCR/CD3. A inibição da expressão de CD95L reduz tanto a AICD como a morte de macrófagos, alvos do ataque citotóxico dos linfócitos T ativados. Foi demonstrado também que, ao invés de bloquear a via do CD95, a PGE2 potencializa a morte induzida por anticorpos anti-CD95 agonistas. Os receptores de PGE2, EP2 e EP4, parecem ser os responsáveis por mediar os efeitos supressores da PGE2 na AICD, já que a estimulação farmacológica destes receptores mimetiza o efeito protetor da PGE2 e seus respectivos antagonistas interferem com a proteção conferida pelos sobrenadantes de APCs e pela PGE2 sintética. A ativação do EP2 e do EP4 age sinergicamente na ativação das vias dependentes da PKA e de EPAC, que contribuem para a inibição da AICD. Por fim, a ativação dos principais fatores de transcrição envolvidos com a expressão de CD95L (NFAT, AP-1 e NF-kB) não é bloqueada por PGE2. Por outro lado, PGE2 induziu a expressão de ICER, um repressor transcripcional, através da ativação de CREB. Em conjunto, estes resultados indicam que as APCs podem modular os níveis de expressão de CD95L através da secreção de PGE2 em resposta ao LPS, através de uma via dependente de TLR4 e MyD88, com conseqüências tanto para a morte de linfócitos T quanto para a sua própria sobrevivência. / Antigen-presenting cells (APCs) control T-cell responses by multiple mechanisms, including the expression of co-stimulatory molecules and the production of cytokines and other mediators that control T-cell proliferation, survival and differentiation. In this present work, it was demonstrated that soluble factor(s) produced by Toll-like receptor (TLR)-activated APCs suppress activation-induced cell death (AICD). This effect was observed in non-stimulated APCs, but it was significantly increased after lipopolysaccharide (LPS) treatment. Using different KO mice, it was found that the LPS-induced protective factor is dependent on TLR4/MyD88 and independent of TLR2 and CD14. The protective factor was identified as prostaglandin E2 (PGE2) and it was shown that both APC-derived supernatants and PGE2 prevented CD95L upregulation in T cells in response to TCR/CD3 stimulation, thereby avoiding both AICD and activated T cell killing of target macrophages. It was also demonstrated that instead of blocking CD95 pathway, PGE2 enhanced T cell death induced by agonistic anti-CD95 antibodies. The PGE2 receptors, EP2 and EP4, appear to be involved in AICD suppression since pharmacological stimulation of these receptors mimics the protective effect on T cells and their respective antagonists interfere with the protection induced by either APCs derived or synthetic PGE2. The engagement of EP2 and EP4 synergistically activates protein kinase A (PKA) and exchange protein directly activated by cAMP pathways to prevent AICD. Finally, the activation of the main transcription factors involved in CD95L expression (NFAT, AP-1 and NF-kB) is not avoided by PGE2. On the other hand, PGE2 induces the expression of ICER, a transcriptional repressor of CD95L, through CREB activation. Taken together, these results indicate that APCs can regulate T-cell levels of CD95L by releasing PGE2 in response to LPS through a TLR4/MyD88-dependent pathway, with consequences for both T cell and their own survival.

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