Orientadores: Renata de Oliveira Mattos Graner, Flávia Sammartino Mariano Rodrigues / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-26T11:30:42Z (GMT). No. of bitstreams: 1 Oliveira_ThaisRossinide_M.pdf: 1438459 bytes, checksum: a127a667898930e41649ac3faebb8d1a (MD5) Previous issue date: 2014 / Resumo: Streptococcus sanguinis é uma espécie pioneira comensal das superfícies dos dentes, que também está envolvida na endocardite infecciosa. Sua alta prevalência na cavidade oral indica capacidade de adaptar-se e sobreviver a diversos fatores de defesa presentes nesse nicho. Para se adaptar ao ambiente e a fatores do hospedeiro, as bactérias utilizam-se de sistemas reguladores de transcrição de dois componentes (SDC), que modulam a regulação de genes em respostas a diferentes estímulos. Neste estudo avaliou-se o papel do SDC VicRK e CovR, na susceptibilidade de S. sanguinis a opsonização pelo sistema complemento. Para isso, foram analisados os níveis de deposição de C3b / iC3b na presença de soro em um total de sete cepas clínicas de S. sanguinis, e as frequências de fagocitose por polimorfonucleares (PMNs) do sangue humano foram comparados entre as cepas S. sanguinis SK36 e mutantes knockout de vicK (SKvic) e covR (SKcov), genes estes que codificam componentes VicK e CovR, respectivamente. A cepa de S. mutans U159 foi utilizada como referência. Resumidamente, as cepas foram incubadas com soro humano a 2 ou 20% por 30 min (37 ° C, 10% de CO2), lavadas, e a presença de C3b associada à superfície foi detectado utilizando anticorpos anti-C3b humano (conjugado com FITC), sendo quantificadas por citometria de fluxo. Para avaliar as frequências de fagocitose por PMNs, as cepas foram incubadas com sangue humano durante tempos de 5, 15, 30 e 60 min (37 ° C, 10% de CO2), fixadas e coradas com Giemsa. PMNs com bactérias intracelulares, foram contados utilizando um microscópio de luz (1000 x) e os resultados expressos em relação a análise de um total de 200 PMNs. Resultados: As percentagens de deposição de C3b em SKvic, SKcov e SK36 foram de 11,3 (± 2,61), 40,2 (± 1,46) e 37,9% (± 3,97), respectivamente. Percentagem de superfície C3b foi significativamente menor em cepas de S. sanguinis em comparação a cepa de S.mutans (Kruskal Wallis, p <0,05), e em SKvic em comparação a cepa SK36 (Kruskal Wallis, p <0,05). As frequências médias de fagocitose por PMNs não foram afetadas, e foram 88, 99,3 e 99% em SKvic, SKcov e SK36, respectivamente. Conclusão: a inativação do VicK, mas não de CovR, reduz a deposição de C3b em S. sanguinis SK36. Cepas de S. sanguinis também são menos suscetíveis a deposição de C3b em comparação a S. mutans / Abstract: Streptococcus sanguinis is a commensal pioneer species of the tooth surfaces, which is also involved in infectious endocarditis. Its high prevalence in the oral cavity indicates ability to survive to several host defense factors present in the oral niches. To sense and respond to environmental and host factors, bacteria apply regulatory two-component systems (TCSs), which modulate gene transcription in response to different stimuli. This study evaluated the role of TCS VicRK and CovR in S. sanguinis susceptibility to opsonization by the complement system. To this aim, a total of seven S. sanguinis strains were analyzed, and levels of deposition of C3b/iC3b on the present of serum, and the frequencies of phagocytosis by polymorphonuclear (PMNs) in human blood were compared between parent S. sanguinis strain SK36 and knockout mutants of vicK (SKvic) and covR (SKcov) (genes encoding VicK and CovR components, respectively). S. mutans strain U159 was used as reference. Briefly, strains were incubated with 2 or 20% of human serum during 30 min (37°C, 10%CO2), washed, and the presence of surface-associated C3b was detected using anti-human C3b antibodies (FITC conjugated), which were quantified by flow cytometry. To assess the frequencies of phagocytosis by PMN, strains were incubated with human blood during 5, 15, 30 and 60 min (37°C, 10% CO2), fixed and stained with Giemsa. PMN with intracellular bacteria were counted using a light microscope (1000 x) and expressed in relation to a total of 200 PMN analyzed. Results: The percentages of C3b deposition on SKvic, SKcov and SK36 were 11.3 (± 2.61), 40.2 (± 1.46) and 37.9% (± 3.97), respectively. Percentage of surface C3b was significantly lower in S. sanguinis strains compared to S. mutans (Kruskal Wallis, p < 0.05), and in SKvic compared to SK36 (Kruskal Wallis, p < 0.05). The mean frequencies of phagocytosis by PMN was not affected, and were 88, 99.3 and 99% in SKvic, SKcov and SK36, respectively. Conclusion: the inactivation of the vicK, but not of covR, reduces the deposition of C3b in S. sanguinis SK36. S. sanguinis strains are also less susceptible to C3b deposition compared to S. mutans / Mestrado / Microbiologia e Imunologia / Mestra em Biologia Buco-Dental

Adult female guinea pigs received subcutaneous implants of diethylstilbestrol-cholestrol pellets which produced splenomegaly and increased numbers of splenic Kurloff cells. Latex spheres subsequently injected intravenously were not phagocytized by Kurloff cells within the lungs and spleen as examined with the electron microscope. This is considered as evidence that Kurloff cells are probably not phagocytic. The origin of these cells is discussed.

Dectin-1 is a pattern-recognition receptor recognizing β-(1,3)-glucans found on fungal cell walls. Dectin-1 plays an important role in immunity to fungi by mediating phagocytic clearance of fungal particles and inducing transcription of innate response genes. We show here that the two processes are linked and that Dectin-1 signalling for inflammation is attenuated by phagocytosis. Blocking Dectin-1 ligand-dependent internalization using either actin polymerization or dynamin inhibitors, large non-phagocytosable β-glucan particles or poorly phagocytic cells leads in all cases to enhanced and sustained activation of downstream signalling pathways and culminates in production of high levels of proinflammatory cytokines. These findings establish the importance of phagocytosis not only in the clearance of pathogens, but also in the modulation of pattern-recognition receptor signalling and strongly suggest that internalization is the first step to attenuation of Dectin-1-mediated pro-inflammatory responses.

Recognition of the fungal cell wall carbohydrate β-glucan by the host receptor Dectin-1 elicits broad immunomodulatory responses, such as phagocytosis and activation of oxidative burst. These responses are essential for engulfing and killing fungal pathogens. Phagocytic monocytes are key mediators of these early host inflammatory responses to infection. Remarkably, whether phagocytosis of fungal β-glucan leads to an inflammatory response in human monocytes remains to be established. Here, we show that phagocytosis of heat-killed Candida albicans is essential to trigger inflammation and cytokine release. By contrast, inhibition of actin-dependent phagocytosis of particulate (1-3,1-6)-β-glucan induces a strong inflammatory signature. Sustained monocyte activation, induced by fungal β-glucan particles upon actin cytoskeleton disruption, relies on Dectin-1 and results in the classical caspase-1 inflammasome formation through NLRP3, generation of an oxidative burst, NF-κB activation, and increased inflammatory cytokine release. PI3K and NADPH oxidase were crucial for both cytokine secretion and ROS generation, whereas Syk signaling mediated only cytokine production. Our results highlight the mechanism by which phagocytosis tightly controls the activation of phagocytes by fungal pathogens and strongly suggest that actin cytoskeleton dynamics are an essential determinant of the host's susceptibility or resistance to invasive fungal infections.

No description available.

Caffeine is one of the most widely used neurostimulants in the world and there is considerable debate on its effect in immune cells. One of its main targets is proposed to be adenosine receptors which mediate an anti-inflammatory switch in activated immune cells while another target is phosphodiesterase where it acts as an inhibitor. In macrophages, caffeine has been shown to cause both pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. If the primary effect of caffeine on macrophages were to antagonize adenosine receptors we would expect cells exposed to caffeine to have a prolonged M1 response. However, we show that caffeine suppresses phagocytosis at physiological concentrations (an indicator of M2 phenotype). This suppression was reversed when macrophages were pretreated with protein kinase A inhibitor, suggesting that at physiological concentrations caffeine's phagocytic suppression may be due to its function as a phosphodiesterase inhibitor, pushing cells towards an M2 fate. However, mRNA expression profiling suggests that caffeine can modulate A2A receptor expression and suppress MKP-1 expression, a hallmark of M1 macrophages. Caffeine is, therefore an immunomodulator that can suppress or prolong inflammatory responses in macrophages, which may account for the abundance of contradicting evidence in the literature. Additionally, these effects are complicated by regular caffeine intake and fitness level, emphasizing that tolerance and immune robustness are important factors in macrophage activation.

No description available.

Thesis (D.P.H.)--University of Michigan.

Thesis (D.P.H.)--University of Michigan.

The successful resolution of inflammation requires removal of neutrophils from the inflammatory site to prevent release of histotoxic contents that may potentiate inflammatory processes and promote progression to a chronic state associated with impaired repair mechanisms and/or autoimmune responses. Macrophages are “professional” phagocytes required for rapid and efficient clearance of apoptotic neutrophils. Macrophage phagocytic capacity can be critically regulated by a number of environmental factors, including cytokines, bacterial products, and glucocorticoids. We have hypothesised that modulation of macrophage phagocytic capacity may represent an effective strategy for promoting resolution of inflammation in diseases where clearance of neutrophils may be impaired or inefficient. The aim of this thesis was to investigate the molecular mechanisms underlying glucocorticoid-augmentation of macrophage phagocytosis. We have demonstrated that long-term exposure of human peripheral blood monocytes to the synthetic glucocorticoid dexamethasone dramatically increases phagocytic capacity for “early” membrane-intact apoptotic neutrophils. Increased phagocytic potential was associated with a “switch” from a serum-independent to a serum-dependent apoptotic cell recognition mechanism. We initially employed an “add back” approach to rule out several well-defined opsonins in apoptotic neutrophil clearance, including immune complexes, IgG, complement proteins, pentraxin-3, fibronectin, annexin I, and platelet-derived factors. Using a multi-step purification scheme involving anion exchange and gel filtration chromatography, we purified a high molecular weight fraction that contained the prophagocytic activity of serum and analysis by mass spectrometry identified C4-binding protein as a candidate protein. C4-binding protein circulates in human plasma bound predominately in a >570kDa complex with protein S and the presence of protein S in high molecular weight fractions was confirmed by immunoblotting. We found that protein S was equivalent to unfractionated serum in its ability to enhance phagocytosis of apoptotic neutrophils by dexamethasone-treated monocyte-derived macrophages (Dex-MDMo) and that immunodepletion of protein S resulted in loss of prophagocytic activity. Protein S was found to opsonise apoptotic neutrophils in a calcium-dependent manner and enhanced phagocytic potential by Dex-MDMo through stimulation of Mer tyrosine kinase (Mertk), a receptor that is upregulated on the surface of Dex-MDMo compared to untreated MDMo. The studies presented in this thesis have provided novel insight into the underlying molecular mechanisms required for high capacity clearance of apoptotic neutrophils by macrophages following treatment with glucocorticoids and may form the foundations for further studies investigating glucocorticoid action for development of safer and more selective therapies.