No description available.

No description available.

The neutrophil is the first haemopoetic cell to arrive at the site of infection. In acute respiratory distress syndrome (ARDS), dense neutrophilic infiltrates are found in the lung in response to bacterial infection as well as generalised inflammatory stimuli, such as pancreatitis. At sites of infection, phagocytosis of bacteria by neutrophils enhances their subsequent apoptosis and clearance by macrophages however at inflammatory sites, the lifespan of the neutrophil is influenced by both pro- and antiapoptotic factors in the inflammatory milieu. Furthermore subsequent macrophage phagocytosis of apoptotic neutrophils induces the macrophage to switch to an antiinflammatory phenotype thereby hastening resolution of inflammation. The Fas death receptor pathway is important in T lymphocyte apoptosis but its role in neutrophil apoptosis is controversial. We have shown that neutrophils express the Fas receptor (CD95) on their surface but there is no evidence of expression of its natural ligand (FasL). An agonistic anti-Fas monoclonal antibody (CH-11) accelerated neutrophil apoptosis under certain culture conditions. Lipopolysaccharide (LPS) originating from Gram-negative bacteria is often found at sites of inflammation. We have shown that LPS attenuated CH-11 - induced neutrophil apoptosis unless the Fas/FasL death receptor pathway was activated prior to the LPS signalling pathway. This LPS-mediated attenuation did not involve the p42/44 ERK, protein kinase C or phosphatidylinositol 3-kinase signalling pathway however the p38 MAPK and NF-κB pathway appeared to be partially involved. We have shown that neutrophils express the protein cFLIPs and that CH-11 and inflammatory mediators altered its expression. Although macrophages are principally phagocytes, they are also important in determining the composition of the milieu at an inflammatory site. Macrophages have been shown to express FasL which can be shed and may contribute to the pools of sFasL found in the bronchoalveolar lavage fluid (BALF) in ARDS patients. We have shown that the conditioned supernatants from LPS-activated macrophages induced neutrophil apoptosis at early time points. The pro-apoptotic activity was mediated by TNF-α and was found in the fraction containing proteins with molecular weights greater than 50kD. Macrophage phagocytosis of apoptotic neutrophils suppressed TNF-α production by LPS-activated macrophages and this was associated with loss of the pro-apoptotic activity. In summary, our data suggest that Fas/FasL fratricide does not appear to be involved in spontaneous neutrophil apoptosis. However LPS attenuates Fas-induced apoptosis unless the Fas/FasL death receptor pathway is activated prior to LPS signalling pathways. The signalling pathways involved in this attenuation are not clear but may involve cellular FLIP. Furthermore, activated macrophages secrete inflammatory mediators and at early time points, TNF-α appears to be the most important in inducing neutrophil apoptosis.

No description available.

No description available.

<p>Type 1 diabetes (T1D) is due to a selective loss of the insulin producing β-cells. However, the process responsible for this loss is still unknown. There is accumulating evidence that enteroviruses (EVs) are involved in T1D. In addition to direct virus-induced cytolysis, EVs could facilitate β-cell destruction by inducing inflammatory cytokines. Induction of such genes has previously been shown in EV-infected islets <i>in vitro</i>. Modulation of inflammatory mediators expressed in the islets could be a possible strategy to reduce β-cell destruction.</p><p>In the first paper we screened uninfected isolated human islets for genes with the potential to induce or modulate an immune response. We found that several of the genes expressed in the islets encode proteins with a powerful biological activity, such as IL-1β, IL-8, MIP-2α, MCP-1 and MIF. This indicates that the islets themselves can express several triggers of inflammation, and if expressed <i>in vivo</i> these mediators would probably contribute to β-cell destruction.</p><p>The vitamin B3 derivate, nicotinamide (NA), has been shown to modulate expression of factors important for coagulation and inflammatory responses. Addition of NA into isolated islet cultures resulted in a reduced expression of the pro-inflammatory chemokine MCP-1 and the coagulation activator tissue factor, suggesting that NA may have implications for both inflammatory responses and the pro-coagulant activity of islets.</p><p>We successfully isolated EVs from three newly diagnosed T1D patients. All isolates showed tropism for human islets and β-cells <i>in vitro</i> and clearly affected islet function. We also found that EV infection induced islet secretion of the chemokines IP-10 and MCP-1and that this induction could be blocked or reduced by addition of NA to the culture medium. Interestingly, NA also reduced viral replication and virus-induced islet destruction.</p><p>To conclude, this thesis provides new information about expression and modulation of inflammatory mediators in infected and uninfected human islets that could trigger inflammatory reactions leading to β-cell destruction. Moreover, it further strengthens the causal relationship between EV and T1D.</p>

Type 1 diabetes (T1D) is due to a selective loss of the insulin producing β-cells. However, the process responsible for this loss is still unknown. There is accumulating evidence that enteroviruses (EVs) are involved in T1D. In addition to direct virus-induced cytolysis, EVs could facilitate β-cell destruction by inducing inflammatory cytokines. Induction of such genes has previously been shown in EV-infected islets in vitro. Modulation of inflammatory mediators expressed in the islets could be a possible strategy to reduce β-cell destruction. In the first paper we screened uninfected isolated human islets for genes with the potential to induce or modulate an immune response. We found that several of the genes expressed in the islets encode proteins with a powerful biological activity, such as IL-1β, IL-8, MIP-2α, MCP-1 and MIF. This indicates that the islets themselves can express several triggers of inflammation, and if expressed in vivo these mediators would probably contribute to β-cell destruction. The vitamin B3 derivate, nicotinamide (NA), has been shown to modulate expression of factors important for coagulation and inflammatory responses. Addition of NA into isolated islet cultures resulted in a reduced expression of the pro-inflammatory chemokine MCP-1 and the coagulation activator tissue factor, suggesting that NA may have implications for both inflammatory responses and the pro-coagulant activity of islets. We successfully isolated EVs from three newly diagnosed T1D patients. All isolates showed tropism for human islets and β-cells in vitro and clearly affected islet function. We also found that EV infection induced islet secretion of the chemokines IP-10 and MCP-1and that this induction could be blocked or reduced by addition of NA to the culture medium. Interestingly, NA also reduced viral replication and virus-induced islet destruction. To conclude, this thesis provides new information about expression and modulation of inflammatory mediators in infected and uninfected human islets that could trigger inflammatory reactions leading to β-cell destruction. Moreover, it further strengthens the causal relationship between EV and T1D.

No description available.

Ácido clorogênico (ACG) é o termo utilizado para designar um grupo de compostos fenólicos oriundos da reação de esterificação entre ácidos hidroxicinâmicos (p-cumárico, caféico e ferúlico) e ácido quínico. São amplamente encontrados em produtos naturais e exercem ações antioxidantes, citotóxicas, antitumorais, antibacterianas, antifúngicas e antiinflamatórias. Apesar da descrição dos seus efeitos antiinflamatórios em diferentes modelos experimentais, a literatura é carente quanto suas ações específicas em funções inflamatórias de neutrófilos. Assim, o objetivo do presente trabalho foi investigar os efeitos do ACG sobre funções de neutrófilos in vitro. Neutrófilos foram obtidos do lavado peritoneal de ratos Wistar, machos, 4 horas após a injeção local de glicogênio de ostra a 1% e foram incubados, na presença ou ausência de LPS, com ACG nas concentrações de 25, 50, 100 ou 1000 &#181;M. A viabilidade celular (exclusão por trypan-blue), a secreção de citocinas e prostaglandina E2 (ensaio imunoenzimático); a produção de óxido nítrico (reação de Griess); a expressão de moléculas de adesão (citometria de fluxo); a aderência e a quimiotaxia foram avaliadas. Os resultados obtidos mostraram que o ACG não afetou as secreções do fator de necrose tumoral-&#945;,de interleucina 1&#946;, do óxido nítrico e de prostaglandina E2 em condições basais ou após estimulação pelo LPS. Diferentemente, a incubação com ACG inibiu a aderência de neutrófilos à cultura primária de célula endotelial de microcirculação de ratos e a quimiotaxia in vitro frente ao peptídeo formilado (N-formilmetionil- leucil-fenilalanina). Estes últimos efeitos podem estar associados à ação do ACG sobre a expressão de moléculas de adesão, já que o ACG elevou a expressão de L-selectina e reduziu as expressões de &#946;2 integrina e da molécula de adesão plaqueta e endotélio. Os dados obtidos não foram dependentes de alterações da viabilidade celular. Em conjunto, os resultados mostram que o ACG possui efeito direto sobre funções de neutrófilos responsáveis pela interação ao endotélio microvascular e pela migração orientada em resposta a estímulo inflamatório. Estes efeitos podem contribuir, pelo menos em parte, para redução da migração de neutrófilos para focos de inflamação na vigência de tratamento com ACG amplamente descrita na literatura. / Chlorogenic acid (CGA) is the term utilized to design a group of phenolic compounds from the sterification reaction between the hydroxycinnamic acids (p-coumaric, caffeic and ferulic acid) and the quinic acid. They are largely found in natural products and exert anti-oxidant, citoxic, anti-tumoral, anti-bactericidal, anti-fungicidal and anti-inflammatory activity. Besides the description of its anti-inflammatory effect in different experimental models, the literature is scarse regarding its specific actions in neutrophil inflammatory functions. Therefore, the aim of this present work was to investigate the CGA effects on neutrophils functions in vitro. Neutrophils were obtained from the peritoneal lavage of male Wistar rats four hours after a local injection of oyster glycogen 1% and were incubated, in the presence or absence of LPS, with CGA in the concentrations of 25, 50, 100 or 1000 &#181;M. The cellular viability (trypan-blue exclusion), the cytokines secretions (enzyme-linked immunosorbend assay), production of nitric oxide (Greiss reaction); adhesion molecules expression (flow citometry), adherence and chemotaxis in vitro were assessed. The results shows that the CGA did not affect the secretion of the tumor necrosis factor-&#945; , of nitric oxide and prostaglandin E2 and only the incubation with 50&#181;M of CGA inhibited the secretion of Interleukin 1&#946; after stimulation with LPS. Differently, the incubation with CGA inhibited the adherence of neutrophils on the primary culture of endothelial cell from the micro-circulation of rats and the chemotaxis in vitro against formylated peptide (fenyl-metyl-leucyl-alanin). This lasts effects might be associated with the action of CGA on the expression of adhesion molecules, hence this compound was capable of elevate the expression of L-selectin and reduce the expression of &#946;2 integrin and PECAM-1. The data here obtained are not dependent of cellular viability alterations. In conjunct, the data here obtained shows that the CGA has direct effect on neutrophils functions responsible of the interaction with the micro vascular endothelium and the oriented migration in response to an inflammatory stimuli. These effects can contribute, at least in part, to the decreased neutrophil migration in the inflammatory focus in the presence of CGA treatment.

Doenças da córnea estão entre as principais causas de cegueira e os mecanismos de lesão e reparação estão em grande parte concentradas no epitélio, porém os mediadores e alvos para possíveis intervenções terapêuticas são pouco conhecidos.Nas agressões ao epitélio da córnea, a ativação dos receptores vanilóides de potencial transitório 1 (TRPV1) leva a resposta inflamatória e cicatricial. O objetivo desse trabalho é identificar os mecanismos de sinalização e resposta do TRPV1 na córnea de animais experimentais. O cultivo de células epiteliais de córnea de ratos, a identificação de mediadores por western blot, medida do influxo de cálcio, resposta de citocinas medidas por ensaio quantitativo de PCR em tempo real (RNAm) e ELISA (proteína) após estímulos nocioceptivos ou inflamatórios; e a investigação histológica e imunohistoquimica em modelos animais, sejam ratos com diabetes mellitus (DM) por 8 semanas ou expostos a cloreto de benzalcônio a 0,2% (BAC) por 7 dias. Camundongos TRPV1-/- comparados aos controles C57 foram comparados sem estimulo, ou sob estimulo único com CAP a 1µM, BAC 0,2 % por 7 dias ou após queimadura alcalina com NaOH a 1M. Os resultados mostraram a presença de TRPV1 em epitélio de córnea em cultura primária, mostraram que essas células respondem com influxo de cálcio ao estímulo com o agonista Capsaicina a 1µM e aumento de TNF? e IL-1? (sendo máximo nas combinações CAP+CPZ e Win+CAP, respectivamente). Em ratos com DM e BAC ocorre a redução da expressão do RNAm de IL-1? (p=0,0269) na córnea, entre outras alterações. Os camundongos TRPV1-/- tem fenótipo físico e ocular normal, porém reduzida sensibilidade a CAP 1µM. O tratamento com BAC leva a diminuição da secreção lacrimal (p=0,0011) e de IL-1? e TNF? na glândula lacrimal (GL) de TRPV1-/- machos (p=0,0177, p=0,0245, respectivamente). A queimadura alcalina com NaOH 1M resultou em melhor cicatrização e maior espessura epitelial no grupo TRPV1-/-. Em ix conclusão, o TRPV1 está presente na córnea e atua em resposta a agressões externas. Situações adquiridas como DM e toxicidade por BAC, reconhecidas como neuropáticas diminuem a expressão de mediadores inflamatórios. A ausência genética e TRPV1 não altera o fenótipo, mas apresenta menor sensibilidade e melhor restauração da superfície ocular após queimadura alcalina / Corneal diseases are among the leading causes of blindness.Damage and repair mechanisms are largely concentrated in the epithelium, but the mediators and targets for possible therapeutic interventions are poorly understood. The aggressions to the corneal epithelium, the activation of vanilloid receptors transient potential 1 (TRPV1) leads to inflammatory and wound healing. The aim of this study is to identify the signaling mechanisms and TRPV1 response in the cornea of experimental animals.The culture of epithelial cells from rat cornea, to identify mediators by western blot analysis, measurement of calcium influx, cytokine response measured by PCR real time (mRNA) and ELISA (protein) nocioceptivos or after inflammatory stimuli; and the histological and immunohistochemical investigation in animal models are mice with diabetes mellitus (DM) for 8 weeks, and exposed to benzalkonium chloride at 0.2% (BAC) for 7 days. Mice TRPV1-/- compared to control C57 were compared with no stimulus, or under single stimulation with 1 µM Capsaicine (CAP), BAC 0.2% for 7 days or after the alkali burn with NaOH 1 M.The results showed the presence of TRPV1 in corneal epithelium in primary culture, showed that these cells respond to calcium influx the stimulus with capsaicin agonist 1?M and increase of TNF? and IL-1? (being maximum at the CAP + CPZ combinations and Win + CAP, respectively). In rats with DM and BAC occurs reducing the expression of IL-1? mRNA (p = 0.0269) in the cornea, among other changes. The TRPV1-/- mice have physical phenotype and normal ocular but low sensitivity to CAP 1?M. Treatment with BAC leads to decreased tear secretion (p = 0.0011) and IL-1? and TNF? in the lacrimal gland (GL) of TRPV1-/- mice (p = 0.0177, p = 0.0245, respectively) . The alkaline burn with NaOH 1M resulted in better healing and greater epithelial thickness in TRPV1-/- group .In conclusion, TRPV1 is present in the cornea and operates in response to external aggressions. Situations existing as DM and xi toxicity BAC recognized as neuropathic decrease the expression of inflammatory mediators. Genetic absence and TRPV1 does not alter the phenotype, but has lower sensitivity and better restoration of the ocular surface after alkaline burn