Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 115-123).

Galectina-3, uma proteína que se liga a -galactosídeos, é expressa por neutrófilos e inúmeras evidências indicam que esta molécula atua como uma possível reguladora da resposta imune. Sabe-se que galectina-3 ao ligar com LPS pode levar a formação de oligômeros, que podem alterar o limiar de ativação de células da resposta imune inata. Apesar de existirem diversos estudos que mostram a influência de galectina-3 na resposta de neutrófilos frente a componentes bacterianos, os resultados são em sua maioria contraditórios e inconclusivos. Para elucidar a influência da galectina-3 na reposta imune inata a patógenos periodontais, o presente trabalho avaliou a atividade antimicrobiana in vitro de neutrófilos, isolados de camundongos selvagens (WT) ou geneticamente deficientes de galectina-3 (Gal-3KO), previamente estimulados com LPS de Aa e Pg. Os resultados não evidenciaram diferenças significativas no número de unidades formadoras de colônia (UFC) recuperadas das culturas de neutrófilos provenientes de animais deficientes de galectina-3 e do grupo controle (WT). Contudo, a estimulação de neutrófilos com LPS por 18 horas levou a redução no número de UFC recuperadas das culturas, quando comparado com as culturas estimuladas com LPS por apenas 3 horas. / Galectin-3, a protein that binds -galactosides, is expressed by neutrophils and numerous evidences indicate that this molecule acts as a possible regulator of the immune response. It is known that galectin-3 binding to LPS can lead to the formation of oligomers and thus changing the activation threshold of cells of the innate immune response. Although there are several studies that show the influence of galectin-3 in neutrophil response against bacterial components, the results are conflicting and inconclusive in their majority. To elucidate the influence of galectin-3 in the innate immune response to periodontal pathogens, the present study evaluated the in vitro antimicrobial activity of neutrophils, isolated from wild-type or galectin-3 deficient mice, previously stimulated with LPS of Aa and Pg. The results showed no significant differences in the number of colony forming units (CFU) recovered from cultured galectin-3 deficient neutrophils or control group. However, in a 18 hours time course of LPS stimulation, we observed reduction in the number of CFU, when compared to 3 hours of LPS stimulation.

O lipopolissacarídeo (LPS) é uma endotoxina de bactérias gram-negativas utilizada experimentalmente para estimular processos inflamatórios e ativar o sistema imune através da liberação de citocinas pró-inflamatórias no sistema nervoso central (SNC). A administração experimental de LPS a ratas prenhes resulta em alterações que podem ser verificadas tanto nos animais submetidos ao tratamento quanto na prole destas fêmeas. O objetivo deste estudo foi o de verificar a existência alterações relacionadas à aprendizagem e à memória na prole masculina de ratas submetidas ao LPS durante a gestação. Para tanto, foi administrado LPS (100mg/kg) no 9,5º dia da prenhes de ratas Wistar. O presente trabalho avaliou os efeitos desta administração no reconhecimento dos animais utilizando o olfato tanto na infância quanto na idade adulta, assim como os níveis de neurotransmissores no bulbo olfatório. A atividade geral dos animais foi analisada no campo aberto e também o comportamento no labirinto em cruz elevado. A aprendizagem dos animais foi observada na caixa de Skinner e também foi analisado reconhecimento de objetos dos animais. Os níveis de neurotransmissores do hipocampo na idade adulta também foram dosados. Os resultados mostraram que a administração de LPS no 9,5º de gestação causou: 1) diminuição do reconhecimento do odor materno nos filhotes na infância e que essa alteração não permanece na idade adulta; 2) diminuição nos níveis de dopamina no bulbo olfatório dos animais tratados pré-natalmente; 3) ausência de interferência na atividade geral e de locomoção dos animais quando comparados com o grupo controle; 4) ausência de alterações na aquisição do aprendizado resultando porém em diminuição no tempo de reversão do aprendizado; 5) nenhuma diferença no reconhecimento de novos objetos assim como ausência de alterações nos níveis de neurotransmissores no hipocampo comparado ao grupo controle. Esses dados sugerem que infecções maternas podem interferir no ambiente intra-uterino e desta forma interferir no desenvolvimento e na atividade do SNC da ninhada em longo prazo / The lipopolysaccharide (LPS) is an endotoxin of gram-negative bacteria used experimentally to stimulate inflammatory processes and activate the immune system through the release of pro-inflammatory cytokines in the central nervous system (CNS). The experimental administration of the LPS in pregnant rats results in changes that can be viewed both in animals subjected to the treatment and the offspring of these females. The aim of this study was to verify the changes related to learning and memory in male offspring of rats submitted to LPS during gestation. It was administered LPS (100mg/kg) in 9,5th day of pregnant of Wistar rats. This work has investigates the effects of this administration in recognition of animals using the olfaction in childhood and adulthood as well as levels of neurotransmitters in the olfactory bulb. The general activity of the animals was analyzed in the open field task and also the behavior in the elevated pluz maze. The learning process of the animals was observed in the Skinner box and also studied animal objects recognition. The levels of neurotransmitters of hippocampus were also analyzed. The results showed that the administration of LPS on 9,5 gestation day caused: (1) decrease of the smell recognition in childhood and do not remains in adulthood; (2) decrease in levels of dopamine in the olfactory bulb of the animals treated; (3) not interfered in general activity of animals when compared with the control group; (4) there was no difference in the acquisition of learning but presented decrease in time reversal of learning; (5) did not make differences in recognition of the new objects as well as not submitted changes in levels of neurotransmitters in hippocampus compared to the group control. These data suggest that maternal infection can interfere in the intrauterine environment and thus interfere with development and activity of the SNC

Orientador: Mario Jose Abdalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T21:44:54Z (GMT). No. of bitstreams: 1 Zambon_RenataAlvaresBagarolli_D.pdf: 7972972 bytes, checksum: 0c4feccde8df219c21aef1c3e8740fc0 (MD5) Previous issue date: 2013 / Resumo: A obesidade é caracterizada por processo inflamatório crônico e resistência à insulina (RI), os quais são responsáveis por grande parte de suas doenças associadas. Sabe-se que diversas moléculas do sistema imune inato estão associadas à RI e obesidade, destacando-se o receptor toll-like-receptor 4 (TLR4). Sua via de sinalização está ativada na obesidade, devido à presença aumentada na circulação de seu principal ligante, lipopolissacarídeo (LPS). Acredita-se que esta endotoxemia metabólica seja causada por alterações na microbiota e na permeabilidade intestinais, o que torna o intestino e as bactérias que o habitam, grandes alvos para o tratamento da obesidade. O objetivo deste presente trabalho foi avaliar os efeitos dos probióticos (PB) na sensibilidade à insulina e na sinalização de TLR4 em tecidos insulino-sensíveis, além de verificar suas possíveis ações na microbiota intestinal. Dessa forma, camundongos Swiss foram divididos em 4 grupos: controles (C), controles tratados com PB (C+PB), obesos (DIO) e obesos tratados com PB (DIO+PB). O tratamento teve a duração de 5 semanas. O uso de PB em animais obesos proporcionou grandes melhoras nos parâmetros fisiológicos e moleculares de RI, além de atenuar a ativação da via de sinalização do TLR4, provavelmente pela redução dos níveis circulantes de LPS. O grupo DIO+PB ainda mostrou mudanças positivas na distribuição dos filos de bactérias intestinais e menor permeabilidade intestinal, quando comparado com o grupo DIO. Analisando o hipotálamo, observou-se que o uso de PB nesse modelo de obesidade regulou de forma favorável os mecanismos centrais de controle da fome, bem como alguns parâmetros de inflamação. Assim, conclui-se que a regulação da microbiota intestinal promovida pela administração de probióticos pode trazer benefícios no controle da obesidade, por reduzir ou atenuar mecanismos moleculares de resistência à insulina / Abstract: Obesity is the main risk factor to the development of insulin resistance and type 2 diabetes. The common basis among these events is an inflammatory process characterized by the activation of toll-like receptor 4 (TLR4) by its main ligand lipopolysaccharide, LPS. Its concentration is higher in obese people and it is believed that changes in composition of the gut microbiota and epithelial functions may play a role in the inflammation associated with obesity. The aim of the study was to evaluate the effects of probiotic on the insulin sensitivity, TLR4 signaling, intestinal permeability and microbiota composition in diet-induced obese mice. Male adult Swiss mice composed randomly 2 groups: chow diet (CTL) and high-fat diet by 5 consecutive weeks (DIO). During these 5 weeks, some mice of the DIO and CTL groups received daily a pool of probiotics. The DIO animals that received probiotic presented an expressive improvement in their glucose tolerance test, fasting glucose and in parallel a significant increase in the phosphorylation levels of insulin induced IR, IRS1 and Akt in muscle, liver and adipose tissue. There was a relevant reduction in the TLR4-Myd88 interaction, IKK? and JNK phosphorylation and iNOS expression in DIO mice treated with probiotic. This treatment also improved the expression of ileal tight-junctions proteins (ZO-1, Occludin), decreased LPS portal levels and the concentrations of bacteria of the phylum Firmicutes (associated with obesity) in feces. Analyzing the hypothalamus, it was observed that the use of probiotics in this model of obesity favorably regulated central mechanisms of food intake, as well as some inflammation parameters. In conclusion, our results show that probiotics, through their effects on intestinal permeability and microbiota composition, can improve insulin sensitivity and signaling of DIO mice, reducing their inflammation and suggesting potential beneficial effects in the treatment of insulin resistance and type 2 diabetes / Doutorado / Medicina Experimental / Doutora em Ciências

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 1982 / Vita. / Includes bibliographical references. / by Charles Joseph Reynold Verret. / Ph. D. / Ph. D. Massachusetts Institute of Technology, Department of Chemistry

Mounting evidence has pointed to associations between Alzheimer's disease (AD) and dysbiotic microbiota in the oral cavity and GI tract. However, no studies have compared the role of specific oral and GI-tract pathogens in the development of AD or the roles inflammation severity and sex may play. We compared the effect of chronic exposure to two doses of lipopolysaccharides (LPS) from a periodontal pathogen, Porphyromonas gingivalis, and a GI-tract pathogen, Escherichia coli, on neuroinflammation, cognition, and AD-like pathology. P. gingivalis-LPS, E. coli-LPS, or endotoxin-free PBS was continuously subcutaneously administered to adult male and female C57BL/6 mice for 28 days via osmotic pumps at 250 µg/kg/day (low-dose) or 500 µg/kg/day (high-dose). After 21 days, spatial learning and reference memory were assessed using the Morris Water Maze (MWM) and Y-Maze. After 28 days of LPS treatment, levels of proinflammatory cytokines, TNF-α and IL-β, in serum and brain tissue were measured by ELISA. Markers of AD pathology and inflammation were detected in situ using immunohistochemistry (IHC) in paraffin-embedded brain sections. Western blot (WB) analysis was used to compare the expression of Toll-Like Receptors (TLR) 2 and 4 in brain, bone, liver, and kidney tissues. Sustained exposure to high-dose, but not low-dose, P. gingivalis-LPS or E. coli-LPS significantly impaired cognition in male mice only. P. gingivalis-LPS triggered worse cognitive impairment and neuroinflammation, whereas E. coli-LPS caused more pronounced serum inflammation. IHC analysis revealed that P. gingivalis-LPS-treated male mice had increased amyloid precursor protein and hyperphosphorylated tau protein in both the hippocampus and neocortex, while E. coli-LPS treatment had a less prominent effect. P. gingivalis-LPS also induced a greater degree of astrogliosis. The mechanisms underlying these observations may be related to differential TLR2 and TLR4 expression, as WB analysis showed TLR2 was predominantly expressed in the brain while TLR4 was constitutively expressed in both the brain and peripheral tissues. Overall, P. gingivalis-LPS treatment triggered more prominent neuroinflammation, cognitive impairment, and AD-like pathological brain changes than E. coli-LPS treatment. Moreover, these phenotypes were dose-dependent and sex-dependent. In conclusion, chronic periodontitis may be a significant risk factor for AD, and this relationship warrants further investigation.

Innate immune memory has become an increasingly popular area of research in the last decade. However, much of the work done on innate immune memory using inflammatory agents such as BCG, C. albicans, and β-glucan has been pursued through systemic administration, which has been shown to induce training in circulating monocytes. In addition, little is known about whether microbial ligands can induce training. Here, we show that local mucosal exposure to an acute dose of LPS induces long-lasting phenotypic changes in airway macrophage populations. LPS-exposed macrophages display increased glycolytic metabolism and differential cytokine expression upon restimulation, whereas circulating monocytes are not affected. Finally, we show that LPS exposure provides long-lasting protection against Streptococcus pneumoniae in the lung, likely due to the higher acquisition of CD11b, which is indicative of macrophage activation and phagocytosis. As much of the work on innate immune memory has been done through systemic administration of training agents, this project aims to fill existing knowledge gaps in the induction of innate immune memory upon local mucosal exposure to inflammatory agents. / Thesis / Master of Science in Medical Sciences (MSMS) / The innate immune system is one of the first defenders in our bodies that fight against a variety of pathogens. In the last decade, the innate immune system was found to be capable of having memory, meaning it reacts faster or at a heightened magnitude in response to a wide range of subsequent pathogens after it is trained by an agent. This project explores the effect a bacteria wall component, LPS, has on the lung environment and examines if it will induce memory in the lung. Our findings show that intranasal exposure to LPS changes the cellular landscape in the lung. LPS-exposed airway innate immune cells become more activated and provide subsequent protection against bacterial infections. This work has implications for using LPS as a vaccine adjuvant in order to provide protection against a variety of pathogens in addition to specific protection brought by the vaccine.

No description available.

Lipopolysaccharides (LPS) représentent le composant principal de la membrane externe des bactéries Gram-négatives. Étant libérés dans le flux sanguin, les LPS induisent une forte réponse immunitaire accompagnée d'une coagulation intensifiée du sang activée à la fois par l'endommagement de la paroi vasculaire et par l'activation de la voie de contact. Dans cette thèse, nous développons des modèles théoriques pour élucider les détails de la coagulation sanguine induite par les molécules LPS. Dans les deux premiers chapitres, nous décrivons l'état de l'art du problème et les méthodes utilisées. Le troisième chapitre est consacré à l'analyse des modèles mathématiques de la coagulation sanguine. Nous déterminons les conditions de l'existence de solutions en ondes progressives dans le modèle de la croissance du caillot, estimons la vitesse de leur propagation et démontrons l'existence de la solution en forme de pulse déterminante la valeur critique de la condition initiale qui assure le processus de coagulation. Ensuite, nous étudions le modèle de la formation de caillot dans l'écoulement sanguin et déterminons la taille critique de la zone endommagée conduisante à l'occlusion complète du vaisseau par le caillot. Enfin, nous développons et analysons le modèle de l'activation du système de contact par les agrégats des LPS. Dans le quatrième chapitre, nous modélisons la structure supramoléculaire des LPS, qui a un impact crucial sur leur activité biologique. Nous développons des modèles de la dynamique moléculaire des LPS, de leurs agrégats et des membranes des compositions variées, et analysons le comportement conformationnel des LPS en fonction de leur environnement / The outer membrane of the Gram-negative bacteria cell wall is composed of lipopolysaccharide (LPS) molecules. Being released to the blood flow during sepsis, LPS induce strong immune response accompanied by pathological blood clotting. Blood coagulation is activated both due to the vessel wall damage, and the activation of the contact pathway. The details of the mechanisms involved remain obscure despite the extensive experimental studies. In the present work we develop theoretical models of the different time and space scales to elucidate the details of the LPS-induced blood coagulation during the Gram-negative sepsis. In the first two chapters we provide the state of the art of the problem and describe the methods we use. The third chapter is devoted to the analysis of the mathematical models of blood coagulation. We determine the conditions of the existence of the traveling wave solutions in the model of the self-sustained clot growth, estimate the speed of their propagation and demonstrate existence of the pulse solution determining the critical value of the initial condition. Then, we consider the model of blood coagulation under flow conditions and determine the critical size of the damaged zone leading to the complete vessel occlusion by the clot. Finally, we develop and analyze the model of the contact system activation by the LPS aggregates. In the fourth chapter we model the LPS supramolecular structure, which has crucial impact on the LPS biological activity. We develop molecular dynamics models of the LPS molecules, their aggregates and LPS-containing membranes of different composition and analyze LPS conformational behavior in different environment

Véritable enjeu de santé publique, l’obésité et ses complications seraient la conséquence d’un état inflammatoire chronique de bas-grade qui pourrait résulter de la présence dans le sang de composés bactériens, les lipopolysaccharides (LPS), état appelé endotoxémie métabolique. Le premier objectif de cette thèse était de comprendre pourquoi les LPS, initialement contenus dans le microbiote, sont capables de traverser l’intestin et d’entrer dans le système sanguin. Mon second objectif était d’étudier l’impact de la composition du microbiote dans le contrôle de la satiété par le nerf vague, lien de communication entre l’intestin et le cerveau. Pour cela, un modèle de rats soumis à un régime obésogène a été utilisée.Mes travaux ont montré que la consommation d’un régime obésogène induisait une perte de la fonction de barrière intestinale au niveau de l’iléon caractérisée par une baisse des défenses mucosales et une augmentation de la perméabilité au LPS. L’obésité est également caractérisée par une altération du comportement alimentaire, avec notamment une réduction de la sensibilité aux signaux de satiété. Nous avons montré que ni l’obésité ni le pourcentage de lipides du régime n’étaient responsables de cette perte de sensibilité aux signaux de satiété mais que l’altération du microbiote en serait le contributeur principal. Ainsi, l’endotoxémie métabolique serait le résultat d’une augmentation du passage transepithelial de LPS, qui, une fois dans le sang, pourraient atteindre, entre autres, le nerf vague où ils perturberaient les signaux intestinaux de satiété. / A real public health issue, obesity and its associated metabolic and behavioral disorders are the consequences of a state of low grade chronic inflammation that might originate from the presence in host plasma of gut-derived bacteria components, lipopolysaccharides (LPS). This present state is called metabolic endotoxemia. The first aim of my thesis was to understand why, in diet-induced obesity (DIO), LPS initially contained in the gut lumen, are able to cross the intestine and enter into the circulatory system. My second aim was to investigate the effect of gut microbiota composition and LPS on the satiety regulation by the vagus nerve, the main communication pathway between the gut and the brain. To answer these questions, we have mainly used a DIO rat model.We showed that consumption of WD induced a loss of ileal barrier function characterized by a reduction in mucosal defenses associated to elevated LPS permeability. Obesity is also characterized by an alteration in feeding behavior including a decreased sensitivity to intestinal satiety signals. We showed that neither obesity nor the lipid percentage of the diet triggers loss of sensitivity to satiety signals but that gut microbiota alterations could rather be the main driver. Hence, metabolic endotoxemia could result from an increased transepithelial passage of LPS, which once spread in the blood could reach, among other things, the vagus nerve where they could disrupt intestinal signals of satiety.