Lipopolysaccharide (LPS) is the toxic chemical component of the cell wall in all gram-negative bacteria which can activate NF-£eB, also stimulate immune cells to release cytokines. These pro-inflammatory mediators induce systemic acute inflammation, multiple organs dysfunction syndrome¡]MODS¡^and sepsis. LPS could increase the permeability of capillary, and cause the acute formation of numerous endothelial gaps among venular endothelial cells that result in extensive plasma leakage in the inflammatory tissues. Plasma leakage from microvasculature is a hallmark of inflammation. Mammalian intestines have many goblet cells that synthesize mucus and discharge it into the intestinal lumen. The mucus film that covers the surface epithelium facing the lumen of digestive system, is an immune defense that can prevent gastrointestinal epithelium from chemical and physical damage and act as a lubricant. Goblet cells can discharge mucins in response to a wide variety of stimuli, including irritant gases, nerve activation, reactive oxygen species, inflammatory mediators. This study was aimed to investigate : (1) The degree of plasma leakage and goblet cell secretion in the small intestine of rats after an intravenous injection of a high dose of LPS (15 mg/kg), (2) The effect of £\2-adrenergic receptors antagonist, urethan, on endotoxin-induced plasma leakage and goblet cell secretion. For the study of plasma extravasation in small intestine during endotoxemia, India ink was used as the tracer to mark the inflamed leaky microvessels. The sections of the small intestine 3£gm in thickness were stained with Alcian blue and periodic acid-Schiff reagent to detect glycoproteins of goblet cells. Our results showed that LPS not only caused an increase in plasma leakage but also triggered degranulation of many goblet cells in the small intestine. LPS augment the expression of plasma leakage and mucus secretion for three times. A large amount of extracellular mucus was accumulated between intestinal villi after LPS stimulation. Pretreatment with urethan, the £\2-adrenergic receptor antagonist, significantly inhibited plasma leakage by 40-50% and goblet cell secretion by 25-30% induced by endotoxin. It is concluded that the plasma leakage and goblet cell hypersecretion induced by endotoxin shock was outstanding and associated with activation of £\2-adrenergic receptors.

IL-17 promotes inflammation through the recruitment of monocytes and induction of various chemokines and inflammatory cytokines. Monocytes respond to IL-17 through the heteromeric IL-17 receptor (IL-17R) composed of subunits IL-17RA and IL-17RC. Together, monocytes and IL-17 amplify inflammation. Controlling the cellular response to IL-17 is crucial to prevent hyperactivation of inflammatory responses, which could lead to chronic inflammatory diseases. The cellular response to increased IL-17 levels may be limited by controlling the receptor levels. Before we understand how monocytes respond to IL-17 during infection, we must first characterize the expression of IL-17R in these cells in response to LPS, a well-characterized pro-inflammatory signal. The aim of this study is to understand the mechanisms which regulate IL-17R levels in human monocytes. IL-17R mRNA and protein levels were measured in response to LPS by RT-PCR and Western blot analysis in primary human monocytes, peripheral blood mononuclear cells (PBMC), and the human monocytic cell line, THP-1. LPS enhanced IL-17RA and RC transcript levels in monocytes and PBMC. In contrast, IL-17RA protein levels decreased with LPS treatment in these cells. Investigation into mechanisms regulating IL-17RA protein levels lead to the observation that IL-17RA undergoes receptor degradation in response to LPS. This work identifies for the first time that 1) LPS enhances transcript levels of IL-17R and 2) after LPS treatment, IL-17RA protein levels are reduced via an endosome-dependent degradation pathway. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2011-06-21 11:53:28.706

TOMAZ, V. S. Papel do óxido nítrico no comportamento tipo-depressão induzido por LPS. 2013. 75 f. Dissertação (Mestrado em Microbiologia Médica) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2013. / Submitted by Carolinda Oliveira (ppgmm@ufc.br) on 2017-07-10T13:52:28Z No. of bitstreams: 1 2013_dis_vstomaz.pdf: 500496 bytes, checksum: a7fe3ea5438d8393b0dd8af83696208a (MD5) / Approved for entry into archive by denise santos (denise.santos@ufc.br) on 2017-07-10T14:14:26Z (GMT) No. of bitstreams: 1 2013_dis_vstomaz.pdf: 500496 bytes, checksum: a7fe3ea5438d8393b0dd8af83696208a (MD5) / Made available in DSpace on 2017-07-10T14:14:26Z (GMT). No. of bitstreams: 1 2013_dis_vstomaz.pdf: 500496 bytes, checksum: a7fe3ea5438d8393b0dd8af83696208a (MD5) Previous issue date: 2013-08-31 / Psychiatric disorders, including depression, are among the leading causes of disability in the world. Thus, research on new pathways involved in the pathophysiology of this mental disorder that allows the discovery of new targets for the treatment of this disorder has been extensively studied. Nitric oxide (NO) and its enzymatic nitric oxide synthase (NOS) synthetase have been associated with depression and other affective disorders. In this context, the effect of pre-treatment of drugs that modulate the NO pathway in animals submitted to immunological challenge was determined by the systemic administration of LPS (0.5 mg / kg, ip). For both behaviors related to depression, pre-pulse inhibition (PPI) and locomotor activity, 24 h after endotoxin administration, respectively, were the key points for the development of depressive and neurochemical behaviors through evaluation of TBARS, nitrite and GSH in the cerebral areas (prefrontal cortex - CPF, hippocampus - HC and striatum - CE) were evaluated. LPS-treated animals, as well as those pretreated with L-ariginine prior to LPS, significantly increased the time of immobility in forced swimming compared to controls. A significant reduction in the immobility time of animals pretreated with aminoguanidine was observed when compared to the animals in the control and LPS groups. Remarkably, animals pretreated with sildenafil and L-NAME showed a significant decrease in immobility time when compared to the group receiving only LPS. The results showed that 24 hours after LPS administration, the locomotor activity assessed through the number of crosses in the open field, remained unchanged, with only a significant increase in animals treated with imipramine. A significant decrease in PPI levels was observed 24 h after administration of LPS at the pre-pulse intensities of 70, 75 and 80 dB relative to the control animals. Administration of all other drugs (imipramine, L-arginine, sildenafil, L-NAME and aminoguanidine) was able to prevent the reduction of PPI levels caused after systemic administration of LPS. GSH levels decreased in all brain areas studied, ie, prefrontal cortex, hippocampus and striatum, of LPS treated animals compared to controls. This reduction was maintained by pretreatment with L-arginine in the prefrontal cortex compared to LPS and controlled and prevented by pretreatment with imipramine, L-arginine, sildenafil, L-NAME, and aminoguanidine drugs. In the evaluation of lipid peroxidation after LPS administration, a significant increase in this parameter was evidenced. Administration of imipramine maintained increased levels of TBARS in the prefrontal cortex and striatum compared to control animals, but reduced this parameter when compared to LPS-treated animals. Administration of L-arginine, sildenafil, L-NAME and aminoguanidine reduced levels of lipid peroxidation when compared to LPS-treated animals in all brain areas studied. The pre-administration of imipramine, 1-arginine and aminoguanidine was able to prevent the increase of BDNF levels caused by the systemic administration of LPS. On the other hand, analyzes of BDNF levels 24 h after administration of LPS from animals pretreated with sildenafil and L-NAME did not demonstrate significant changes. An increase in IL-1β content was observed 24 h after administration of LPS in the prefrontal cortex, hippocampus and striatum, all drugs were able to prevent such changes in the hippocampus and striatum. Nitric oxide (NO) plays an important neuromodulatory role in the central nervous system. Any pharmacological manipulation of the NO pathway can be considered as a novel therapeutic approach for the treatment of CNS disorders, more so for mental depression (Heiberg et al., 2002) (1) (1) (1) (1). / Os transtornos psiquiátricos, dentre eles a depressão, estão entre as principais causas de incapacidade no mundo. Desta forma, pesquisas a respeito de novas vias envolvidas na fisiopatologia deste transtorno mental que possibilitem a descoberta de novos alvos para o tratamento deste transtorno têm sido amplamente estudados. O óxido nítrico (NO) e a sua enzima de síntese óxido nítrico sintase (NOS) vem sendo relacionados com a depressão e outros transtornos afetivos. Neste contexto buscou-se determinar o efeito do pré-tratamento de drogas que modulam a via do NO em animais submetidos ao desafio imune pela administração sistêmica de LPS (0,5 mg / kg, ip). Para tanto os comportamentos relacionados à depressão, inibição pré-pulso (PPI) e atividade locomotora, 24 h após a administração da endotoxina, respectivamente, ponto de tempo chave para o desenvolvimento de comportamentos tipo depressivo, e neuroquímicas através da avaliação dos níveis de TBARS, Nitrito e GSH nas áreas cerebrais (córtex pré-frontal - CPF, hipocampo - HC e corpo estriado - CE) foram avaliados. Os animais tratados com LPS, bem como os pré-tratados com L-ariginina antes do LPS aumentaram significativamente o tempo de imobilidade no nado forçado em comparação com os controles. Observou-se uma redução significativa no tempo de imobilidade dos animais pré-tratados com aminoguanidina quando em comparação com os animais do grupo controle e LPS. Notavelmente os animais pré-tratados com sildenafil e L-NAME apresentaram uma diminuição significativa do tempo de imobilidade quando comparado ao grupo que recebeu apenas LPS. O resultados mostraram que 24 horas pós a administração de LPS, a atividade locomotora avaliada através do número de cruzamentos no campo aberto, manteve-se inalterada, apenas havendo aumento significativo nos animais tratados com imipramina. Observou-se uma diminuição significativa nos níveis de PPI 24 h após a administração de LPS nas intensidades de pré-pulso de 70, 75 e 80 dB em relação aos animais controle. A administração de todas as outras drogas (imipramina, L-arginina, sildenafil, L-NAME e aminoguanidina), foi capaz de prevenir a redução dos níveis de PPI causada após a administração sistêmica do LPS. Os níveis de GSH diminuiram em todas as áreas cerebrais estudadas, ou seja, córtex pré-frontal, hipocampo e corpo estriado, de animais tratados com LPS quando comparados aos controles. Esta redução foi mantida pelo pré-tratamento com L-arginina no córtex pré-frontal em comparação ao LPS e controle e prevenida pelo pré-tratamento com as drogas imipramina, L-arginina, sildenafil, L-NAME e aminoguanidina. Na avaliação da peroxidação lipídica após a administração do LPS foi evidenciado aumento significativo neste parâmetro. A administração da imipramina manteve o aumento nos níveis de TBARS no córtex pré-frontal e corpo estriado em relação aos animais controle, mas reduziu este parâmetro quando comparado aos animais tratados com LPS. A administração da L-arginina, sildenafil, L-NAME e aminoguanidina reduziu os nível de peroxidação lipídica quando comparado aos animais tratados com LPS em todas as áreas cerebrais estudadas. A pré-administração da imipramina, l-arginina e aminoguanidina foram capazes de prevenir o aumento dos níveis de BDNF causado pela administração sistêmica do LPS. Por outro lado, as análises dos níveis de BDNF 24 h após a administração de LPS dos animais pré-tratados com sildenafil e L-NAME, não demonstraram alterações sisgnificativas. Observou-se um aumento no conteúdo de IL-1β 24 h após a administração de LPS no córtex pré-frontal, hipocampo e corpo estriado, todas as drogas foram capazes de prevenir essas alterações no hipocampo e corpo estriado. O óxido nítrico (NO) desempenha um papel significativo neuromodulador no sistema nervoso central. Qualquer manipulação farmacológica da via de NO pode ser considerado como uma nova abordagem terapêutica para o tratamento de distúrbios do SNC, mais ainda para a depressão mental(Heiberg et al., 2002)(1)(1)(1)(1).

Submitted by Amanda Silva (amanda.osilva2@ufpe.br) on 2015-04-14T15:08:27Z No. of bitstreams: 2 DISSERTAÇÃO Rafael Pinto Ferreira de Queiroz.pdf: 636068 bytes, checksum: fc69901a9ee1e408621b1e492f72c21f (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-04-14T15:08:27Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO Rafael Pinto Ferreira de Queiroz.pdf: 636068 bytes, checksum: fc69901a9ee1e408621b1e492f72c21f (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2014 / FACEPE / Esta pesquisa analisa a permanência da cultura do vinil através das festas na cidade de Recife, que têm os DJs e os seus LPs como protagonistas. Buscamos conhecer esses profissionais, bem como os principais motivos que os levam a ainda tocar e consumir esse artefato cultural analógico, em plena era digital. Mesmo em face das facilidades que a música digitalizada oferece, com equipamentos mais leves e mais práticos, assim como uma oferta gratuita e quase irrestrita de fonogramas, esses DJs ainda preferem usar seus discos e dão muitas outras explicações além da qualidade sonora e da técnica de djing. Interessa-nos, também, como a circulação dos LPs através de rotas comerciais e de afeto, ao se encontrarem com os DJs em suas festas, podem formar uma interface ser humano – coisa e se constituir em processos de circularidade e cena. A partir do uso desses conceitos a hipótese é que espaços antes vazios podem ser transformados em lugares de significação, que abarcam sensibilidades e sociabilidade, assim como disputas e tensões. A tríade LP-DJs-Festas tem o poder de transformar ou influir na vida cultural de uma cidade, trazendo novos significados e experiências na maneira de vivenciar e olhar a cidade, e é o ponto nevrálgico dessa pesquisa. Procurou-se dar um entendimento a esse universo, articulando noções relacionadas a materialidade da mídia e práticas de consumo e coleção.

Sepsis, a life-threatening organ dysfunction caused by dysregulated host immune response to infection, is one of the leading causes of death within intensive care units (ICUs) in the United States. Developing specific therapies to treat sepsis is a current challenge for translational research, where over 100 drugs developed to target pro- and anti-inflammatory pathways in sepsis have failed to pass clinical trials. The recent challenge in translating effective sepsis pharmaceuticals from animals to humans has led some researchers to question the overall viability of using specific pathogen free (SPF) mice as an animal model for sepsis in human beings. SFP mice are raised in a barrier facility designed to prevent exposure to specific pathogens, while humans are exposed to a wide range of pathogens on a daily basis. Acknowledging the influence that prior environmental pathogen exposure has on the immune system’s response to future infections, researchers have developed the cohoused (CoH) mouse model as a potential alternative to SPF mice for use in research on immunological diseases such as sepsis. CoH mice are SPF mice cohoused with “dirty” pet store mice for 60 days, which increases their pathogen exposure and results in mice with immune experience more comparable to humans. Although the CoH model shows promise, a recent study conducted by Huggins et al shows increased sepsis-induced morbidity and mortality of CoH mice compared to SPF mice when using the lipopolysaccharide (LPS) and cecal ligation and puncture (CLP) models of sepsis. The experiments described in this thesis aim to further compare the inflammatory response of SPF, CoH, and pet store mice after intra-peritoneal injections of either LPS or cecal slurry (CS) to identify potential differences among these mouse groups and better understand why CoH mice experience increased mortality during sepsis. A baseline experiment was performed on each of these mice groups for comparison. Our baseline experiments demonstrate significant elevations in peritoneal immune cells within CoH mice compared to SPF mice. CS experiments demonstrate a significantly higher infiltration of immune cells in CoH mice following cecal slurry injection compared to SPF mice, suggesting that environmental pathogen exposure influences host inflammatory response within the peritoneum. However, LPS experiments were largely inconclusive. No significant differences were observed between SPF and CoH mice in regard to immune cell infiltration within the peritoneum, while blood analysis showed significant elevations in Tumor Necrosis Factor alpha (TNFa) and Interleukin 6 (IL-6) with increasing environmental pathogen exposure. Since the inflammatory response within the peritoneum to LPS was not significantly different between SPF and CoH mice, future studies could expand upon these results by investigating other tissue compartments in SPF and CoH mice following LPS injection into the peritoneum to provide a more complete comparison between these mice during LPS induced sepsis.

C1q/TNF-related protein 3 (CTRP3) is a secreted hormone that modulates hepatic glucose and lipid metabolism. Its circulating levels are reduced in human and rodent models of obesity, a metabolic state accompanied by chronic low-grade inflammation. Recent studies have demonstrated an anti-inflammatory role for recombinant CTRP3 in attenuating LPS-induced systemic inflammation, and its deficiency markedly exacerbates inflammation in a mouse model of rheumatoid arthritis. We used genetic mouse models to explore the immunomodulatory function of CTRP3 in response to acute (LPS challenge) and chronic (high-fat diet) inflammatory stimuli. In a sublethal dose of LPS challenge, neither CTRP3 deficiency nor its overexpression in transgenic mice had an impact on IL-1β, IL-6, TNF-α, or MIP-2 induction at the serum protein or mRNA levels, contrary to previous findings based on recombinant CTRP3 administration. In a metabolic context, we measured 71 serum cytokine levels in wild-type and CTRP3 transgenic mice fed a high-fat diet or a matched control low-fat diet. On a low-fat diet, CTRP3 transgenic mice had elevated circulating levels of multiple chemokines (CCL11, CXCL9, CXCL10, CCL17, CX3CL1, CCL22 and sCD30). However, when obesity was induced with a high-fat diet, CTRP3 transgenic mice had lower circulating levels of IL-5, TNF-α, sVEGF2, and sVEGFR3, and a higher level of soluble gp130. Contingent upon the metabolic state, CTRP3 overexpression altered chemokine levels in lean mice, and attenuated systemic inflammation in the setting of obesity and insulin resistance. These results highlight a context-dependent immunomodulatory role for CTRP3.

Recent evidence suggests that the macrophage scavenger receptor class A (SR-A, aka, CD204) plays a role in the induction of innate immune and inflammatory responses. We investigated whether SR-A will cooperate with Toll-like receptors (TLRs) in response to TLR ligand stimulation. Macrophages (J774/a) were treated with Pam2CSK4, (TLR2 ligand), Polyinosinic:polycytidylic acid (Poly I:C) (TLR3 ligand), and Lipopolysaccharides (LPS) (TLR4 ligand) for 15min in the presence or absence of fucoidan (the SR-A ligand). The levels of phosphorylated IκBα (p-IκBα) were examined by Western blot. We observed that Poly I:C and LPS alone, but not Pam2CSK4 or fucoidan increased the levels of p-IκBα. However, LPS-induced increases in p-IκBα levels were further enhanced when presence of the fucoidan. Immunoprecipitation and double fluorescent staining showed that LPS stimulation promotes SR-A association with TLR4 in the presence of fucoidan. To further confirm our observation, we isolated peritoneal macrophages from SR-A deficient (SR-A-/-), TLR4-/- and wild type (WT) mice, respectively. The peritoneal macrophages were treated with LPS for 15min in the presence and absence of fucoidan. We observed that LPS-stimulated TNFα and IL-1β production was further enhanced in the WT macrophages, but did not in either TLR4-/- or SR-A-/- macrophages, when fucoidan was present. Similarly, in the presence of fucoidan, LPS-induced IκBα phosphorylation, NF-κB binding activity, and association between TLR4 and SR-A were significantly enhanced in WT macrophages compared with LPS stimulation alone. The data suggests that SR-A is needed for LPS-induced inflammatory responses in macrophages.

Modification of the lipid A domain of lipopolysaccharide (LPS) has been reported to contribute to the virulence and pathogenesis of various Gram-negative bacteria. The Kdo (3-deoxy-D-manno-octulosonic acid)-lipid A domain of Helicobacter pylori LPS shows several differences to that of Escherichia coli. It has fewer acyl chains, a reduced number of phosphate groups, much lower immunobiological activity, and only a single Kdo sugar is attached to the disaccharide backbone. However, H. pylori synthesizes a minor lipid A species resembling that of E. coli, which is both bis-phosphorylated and hexa-acylated suggesting that the major species results from the action of specific modifying enzymes. This work describes two enzymes, a lipid A phosphatase and a phosphoethanolamine transferase, involved in the periplasmic modification of the 1-position of H. pylori lipid A. Furthermore, we report a novel Kdo trimming enzyme that requires prior removal of the 1-phosphate group for enzymatic activity. Discovery of the enzymatic machinery involved in the remodeling of H. pylori LPS will help unravel the importance of these modifications in H. pylori pathogenesis.d.

Hemocytes are an important part of a crayfish’s immune system in helping tackling both virus and bacterial infections. Dscam is a protein that can be found in hemocytes, as well as many other tissues like the brain. In the brain, Dscam is thought to be important in the establishment of neuronal connections. Previous studies have found that the neurons in the crayfish brain do not replenish themselves, but instead are replenished by hemocytes that enter through a vascular cavity that pass through the neurogenic niche. There might be a specific type of hemocyte that is drawn to the niche and because of the link between Dscam and establishment of neuronal connections, Dscam have been chosen as a potential factor for this attraction. Dscam could be upregulated at many places along the way from the HPT to the brain. In this study, antibodies have been used to view BrdU and Dscam presence in hemocytes from crayfish P. leniusculus to find out where Dscam is upregulated and in what cells they are located. It was found that Dscam is not present on newly synthesized cells but rather on more differentiated cells, suggesting that Dscam is upregulated in older HPT cells or in circulation. It was found that LPS injections are an efficient way to upregulate Dscam in hemocytes and that expression of extracellular Dscam is peaking 24 hours post LPS injection.

Periodontitis potentially contributes to many systemic diseases. Specific gram-negative bacteria such as Porphyromonas gingivalis (P.g) and Treponema denticola (T.d) contribute to the initiation and progression of periodontal disease via factors such as NF-κB, TNF-α, IL-1β, and CXCL1. Down-regulation of these factors by natural compounds or short hairpin RNAs (shRNAs) reduces periodontal bacteria-induced inflammation. Our preliminary data indicated that P.gingivalis / lipopolysaccharide (LPS) stimulates chemokine CXCL1 production in macrophages. CXCL1 stimulates LPS-induced TNF-α expression, resulting in inflammation. We hypothesize that inhibiting the expression of CXCL1 will reduce LPS-induced TNF-α production. We recently demonstrated that a CXCL1 shRNA inhibits LPS-stimulated CXCL1 production in macrophages and leads to reduced expression of LPS-stimulated pro-inflammatory cytokines TNF-α and IL-1β. This indicates that CXCL1 shRNAs have potential as inhibitors of LPS-induced inflammation. Further studies are needed to confirm these as well as to identify the signaling pathway.