No description available.

No description available.

No description available.

No description available.

<p>Amb a 1 is the major allergen found in ragweed. Our observations have suggested that Amb a 1 may bind lipopolysaccharide (LPS), which would likely contribute to the allergenicity of Amb a 1. In order to assess whether Amb a 1 can bind LPS, peptide sequences from Amb a 1 were assayed for their ability to bind to LPS using an ELISA based LPS binding assay. A 15 amino acid sequence in the β- chain of Amb a 1 demonstrated affinity for biotin labeled <em>E. coli </em>LPS. The sequence also bound to <em>P.</em> <em>aeruginosa</em> LPS, which is structurally disparate in the lipid A region, indicating that the sequence has flexibility in recognizing different lipid A moieties, or that the binding site may not include the lipid A portion of the LPS molecule. An IL-10 ELISA was also used to determine whether the LPS bound to the peptides induced an immunological response in leukocytes. Peptides containing the LPS-binding sequence were able to bind to LPS and induce IL-10 production, suggesting the interaction between Amb a 1 and LPS may have immunological consequences. We have identified a sequence within the major ragweed allergen Amb a 1 that has the potential to bind to LPS. This indicates that the allergen may provide its own adjuvant when encountered by the immune system, leading to an enhanced immunological response to an otherwise innocuous environmental protein.</p> / Master of Science (MSc)

Macrophages can be activated by a variety of extracellular signals to polarize to either the M1 (inflammatory and antimicrobial) or to the M2 (wound repair and inflammation resolution) phenotype. Expression of arginase 1 in macrophages is a key marker of the M2 phenotype. Arginase 1 expression is induced by interleukin 4 (IL-4), a cytokine secreted by Th2 helper cells. All-trans retinoic acid (ATRA) is a product of metabolism of dietary retinol (vitamin A). In a manner analogous to hormones, ATRA binds to nuclear receptors in cells and influences gene expression and cell physiology. ATRA is important in the resolution of inflammation systemically and on the cellular level, however it has not been linked to M2 activation or arginase 1 expression. Testing the hypothesis that ATRA can induce arginase 1 in macrophages either directly or indirectly, it was found that ATRA alone cannot cause murine bone marrow-derived macrophages (BMDM) to activate in the M2 phenotype (as indicated by arginase 1 expression), however it can dramatically potentiate induction of arginase 1 expression and activity by IL-4. This is the first observation positively linking ATRA to arginase 1. Lipopolysaccharide (LPS), is a conserved structural component of the outer membrane of Gram negative bacteria, and a potent pyrogen. In metabolic endotoxemia, LPS concentration in the blood is slightly elevated, and over the long term this contributes to diverse inflammatory diseases such as atherosclerosis, obesity, and diabetes. LPS promotes the M1 phenotype and suppresses the M2 phenotype, but its contribution at low doses such as those found in metabolic endotoxemia are not well studied. In order to investigate mechanisms of LPS suppression at low doses, mice deficient in IRAK1 and tollip, key mediators or proinflammatory LPS signaling, were used to study IL-4, ATRA, and LPS crosstalk. LPS suppression of arginase 1 was found to be dependent on IRAK1 and tollip, but only at low doses of LPS. / Ph. D.

Obesity over the last several has become a major health concern in our country as well as the world. Obesity is also one of the risk factors which lead to several inflammatory complications such as diabetes, artherosclerosis, etc. Two leading factors involved in the causes of inflammatory complications include leptin and low dose endotoxin lipopolysaccharide (LPS). However, the mechanism underlying the involvement of these two mediators is not clearly understood. The purpose of this study is to understand the mechanism underlying inflammatory complications caused by leptin and low dose endotoxin most recently coined metabolic endotoxemia. Interleukin-Receptor Associated Kinase 1 (IRAK-1) is an intracellular signaling component shown to activate NFκB which leads to the induction of proinflammatory mediators. Deletion of IRAK-1 in mice has beneficial effects in alleviating inflammatory complications and human variations in IRAK-1 gene are correlated with higher risks for inflammatory diseases. Therefore, we hypothesized that IRAK-1 is critically involved for the induction of proinflammatory mediators induced by leptin and low dose LPS. IL-6 mRNA levels were measured in THP-1 (human monocytic cells) and wild type and IRAK-deficient bone marrow derived macrophages (BMDM) challenged with different combinations of leptin and LPS. Data shows that leptin alone will not induce inflammatory mediators. However, increased induction of IL-6 was observed in a synergistic manner involving both LPS and leptin in an IRAK-1 dependent manner causing a robust inflammatory response. With regard to the effect of low dose LPS, we observed that human monocytic cells treated with low concentrations of LPS showed a mild yet sustained induction of proinflammatory cytokines, which is contrast to the robust and transient induction of cytokines by a high dose LPS. To further determine the molecular mechanisms, we measured several key signaling molecules that include IRAK-1, IKKepsilon, and C/EBPdelta. Our study revealed a novel mechanism that appears to be distinct from the traditional NFï «B pathway responsible for the effect of low dose LPS. / Ph. D.

The establishment of non-resolving inflammation underlies the pathogenesis of chronic inflammatory diseases in humans. Super low dose (SLD) endotoxin has been associated with exacerbating inflammation and the pathogenesis of chronic inflammatory diseases. However, the underlying molecular mechanisms are not well studied. In this study, I tested the hypothesis that SLD endotoxin may potentiate non-resolving innate immune cell inflammation through disrupting cellular endoplasmic reticulum (ER) homeostasis. We chose to study the dynamics of ER homeostasis in macrophages stimulated with SLD endotoxin. In naïve cells, ER stressor such as tunicamycin (TM) not only will induce cellular stress and inflammation through JNK and NFkβ activation, but also will cause subsequent compensatory homeostasis through inducing homeostatic molecules such as XBP1 and GRP78/BiP. We observed that cells challenged with SLD endotoxin have significantly reduced expression of homeostatic molecules XBP1 and BiP. Mechanistically, we observed that SLD-LPS increases phosphorylated HCK expression in TM treated cells. Phosphorylated HCK activation resulted in the phosphorylation of Golgi protein GRASP, leading to unstacking of Golgi cisterna and overall dysfunction of the Golgi apparatus. Dysfunctional Golgi apparatus and its effect on protein transport and secretion, may account for decreased levels of Site 2 Protease, reduced generation of ATF6 and its transcriptional target BiP. Taken together, our study reveal that super low dose endotoxin exacerbates low grade inflammation through increasing phosphorylation of HCK, inducing Golgi dysfunction, and decreasing BiP /homeostatic protein expression in innate immune cells. / Ph. D. / Non-resolving inflammation is a common factor shared in in many chronic inflammatory diseases such as atherosclerosis and diabetes mellitus type 2. Low levels of endotoxin have been shown to increase inflammation as well as further increase disease development. However, how such low levels of endotoxin is able to produce this effect is not well understood. This research focuses on how low levels of endotoxin can increase inflammation by decreasing the ability of the cell to restore homeostasis. It was found that a super low dose (SLD) endotoxin decreased activation of the unfolded protein response pathway (UPR). The UPR pathway is a prominent signaling pathway utilized by the cell to restore homeostasis and is activated following an accumulation of unfolded proteins in the endoplasmic reticulum of the cell. The disruption of this pathway by SLD endotoxin resulted in increased inflammatory signaling and decreased cellular homeostasis.

Lipopolissacarídeos (LPS), pode tanto proteger quanto exacerbar o desenvolvimento da asma. LPS inicia a ativação da resposta imune via ligação da molécula Toll-like receptor 4 (TLR4) que sinaliza por duas vias distintas, as moléculas adaptadoras MyD88 e TRIF. LPS é um adjuvante que induz resposta do tipo Th1, enquanto que o hidróxido de alumínio (Alum) desperta respostas Th2, porém, a mistura de ambos adjuvantes na indução da resposta alérgica pulmonar ainda não foi investigada. No presente estudo, nós determinamos o efeito de dois agonistas de TLR4, um natural (LPS) e outro sintético (ER-803022) adsorvidos ao Alum sobre o desenvolvimento de doença alérgica pulmonar. Os animais foram sensibilizados pela via subcutânea com os antígenos, Ovoalbumina (OVA) ou Toxóide Tetânico (TT) na presença ou ausência de agonistas de TLR4 co-adsorvidos ao Alum e desafiados com os respectivos antígenos pela via intranasal. Nossos resultados mostraram que a sensibilização com OVA ou TT e LPS coadsorvidos ao Alum, impede o estabelecimento da resposta alérgica mediada por linfócitos Th2, tais como, influxo de eosinófilos, produção de citocinas do tipo 2, hiperreatividade brônquica, secreção de muco, e produção de IgE ou IgG1 anafilática. Apesar dos níveis de IgG2a, isotipo associado com as respostas Th1 estarem aumentados, análise da histopatologia pulmonar não revelou um desvio para o padrão Th1 de inflamação. Verificamos que a presença das moléculas TLR4, MyD88, IL-12/IFN-g mas não TRIF foram necessários para LPS exercer seu efeito inibitório. O agonista sintético de TLR4, menos tóxico que LPS, também protegeu contra o desenvolvimento de inflamação alérgica pulmonar. Em conclusão, nosso trabalho esclarece o efeito da sinalização do TLR4 na sensibilização alérgica e indica que agonista sintético de TLR4 com baixa toxicidade, pode ser utilizado para modular a capacidade adjuvante do Alum e conseqüentemente diminuir a indução de alergias. / Epidemiological and experimental data suggest that bacterial lipopolysaccharides (LPS) can either protect from or exacerbate allergic asthma. LPS triggers immune responses through Toll-like receptor (TLR) 4 that in turn activates two major signaling pathways via either MyD88 or TRIF adaptor proteins. LPS is a pro-Th1 adjuvant while aluminum hydroxide (Alum) is a strong Th2 adjuvant, but the effect of mixing both adjuvants on development of lung allergy has not been investigated. We determined whether natural (LPS) or synthetic (ER-803022) TLR4 agonists adsorbed onto alum adjuvant affect allergen sensitization and development of airway allergic disease. To dissect LPS-induced molecular pathways we used TLR4, MyD88, TRIF, or IL-12/IFN-g deficient mice. Mice were sensitized subcutaneously to allergens such as ovalbumin (OVA) or tetanus toxoid (TT) with or without TLR4 agonists coadsorbed onto Alum and challenged twice via intranasal route with the same allergens. The development of type 2 immunity was evaluated 24 h after last allergen challenge. We found that sensitization with OVA or TT plus LPS co-adsorbed onto Alum impaired allergeninduced Th2-mediated responses such as airway eosinophilia, type 2 cytokines secretion, airway hyperreactivity, mucus hyper production and serum levels of IgE or IgG1 anaphylactic antibodies. Although the levels of IgG2a, a Th1 affiliated isotype increased, investigation into the lung-specific effects revealed that LPS did not induce a Th1 pattern of inflammation. LPS impaired the development of Th2 immunity, signaling via TLR4 and MyD88 molecules via the IL-12/IFN-g axis, but not through TRIF pathway. Moreover, the synthetic TLR4 agonists that proved to have a less systemic inflammatory response than LPS also protected against allergic asthma development. TLR4 agonists co-adsorbed with allergen onto Alum down modulate Th2 immunity and prevent the development of polarized T cell-mediated airway inflammation. Thus, our work clarifies the effect of TLR4 signaling in allergic sensitization and indicates that TLR4 agonists with low toxicity might be useful for down regulating the pro-Th2 adjuvant activity of alum and consequently decrease the induction of allergy.

Epidemiologische Daten belegen eine Assoziation von Infektionen mit der Exazerbation von Autoimmunerkrankungen, wobei man aber die Wege, die dahin führen noch nicht voll versteht. Die gängigste Hypothese sieht die Ursache für die Entstehung von Autoimmunerkrankungen in der Kreuzreaktivität zwischen mikrobiellen und Selbst-Antigenen, die von der selben T-Zelle erkannt werden. Dieser Antigen-spezifische Mechanismus konnte allerdings bisher nicht durch die Forschung belegt werden. In dieser Arbeit wird gezeigt, dass im Modell der experimentellen autoimmunen Enzephalitis durch die Aktivierung mit Lipopolysacchariden gramnegativer Bakterien (LPS) Schübe in normalen Mäusen ausgelöst werden können. Diese Art der Behandlung führt in-vitro zur Proliferation und zur Zytokinproduktion bei einem Teil der T-Helfer (Th)-Effektor/Gedächtniszellen. Dabei ist zwar der physische Kontakt zwischen Th-Zellen und CD4--LPS-responsiven Zellen essentiell, jedoch geschieht die Aktivierung nicht über den T-Zellrezeptor. Als entscheidend hat sich die Bindung von kostimulatorischen Rezeptoren auf Th-Zellen durch kostimulatorische Moleküle auf CD4--Zellen erwiesen. Diese Form der Bystander-Aktivierung bietet eine Antigen-unabhängige Erklärung für die Zusammenhänge von Infektion und Autoimmunität, die mit den klinischen und epidemiologischen Daten besser vereinbar ist als die Antigen-spezifischen Modelle. / Infections sometimes associate with exacerbations of autoimmune diseases through pathways that are poorly understood. Antigen-specific mechanisms such as cross-reactivity between a microbial antigen and a self-antigen have received no direct support. Here it is shown that activation by lipopolysaccharide (LPS) induces relapses of experimental autoimmune encephalitis (EAE) in normal mice. This form of treatment induces proliferation and cytokine production in a fraction of effector/memory T helper (Th) lyphocytes in vitro via physical contact of Th cells with CD4--LPS-responsive cells. TCR mediated signals are not necessary; rather what is required is ligation of costimulatory receptors on Th cells by costimulatory molecules on the CD4- cells. This form of bystander activation provides an antigen-independent link between infection and autoimmunity that might fit the clinical and epidemiological data on the connection between infection and autoimmunity better than the antigen-specific models.