München, Techn. Univ., Diss., 2010.

Memoria para optar al Título Profesional de Químico Farmacéutico / Los fibroblastos cardíacos (FC), han sido denominados células centinelas del corazón, pues responden de manera variada y compleja ante cualquier elemento externo o interno a fin de mantener la homeostasis. Esta respuesta se lleva a cabo mediante una maquinaria conformada por una amplia red de receptores, entre ellos los receptores tipotoll y, en particular, el TLR4. La activación de este receptor ha mostrado mediar respuestas inflamatorias y fibróticas tanto en FC, como en otras células; en donde la expresión de proteínas de adhesión y reclutamiento de células del sistema inmune componen un eje vital en estos procesos. Por tanto se buscó determinar si la activación de TLR4 en el FC de ratas adultas genera una mayor adhesión de monocitos a través de un aumento en los niveles de E-selectina. La activación de TLR4 por LPS en FC, indujo un aumento en la expresión de Eselectina, situación que es revertida con el pre tratamiento con TAK-242 (inhibidor de TLR4). Adicionalmente, la activación de TLR4 generó un aumento en la adhesión de monocitos sobre los FC, mientras que la inhibición de TLR4 y las vías transduccionales ERK1/2, PI3K/Akt y NF-κB previo al estímulo con LPS, revirtió el aumento de adhesión de monocitos. De igual forma, el bloqueo de E-selectina, mediante el uso de un anticuerpo bloqueante, también revirtió el aumento de la adhesión, dando luces de que esta proteína de adhesión es fundamental para el reclutamiento de monocitos y por tanto para mediar la respuesta inflamatoria orquestada por el FC / Cardiac fibroblasts (CF) have been considered as sentinel cells, since they respond in a complex and diverse manner to external and internal stimulus, in orden to maintein homeostasis of the heart. This is mediated through a wide number of receptor, among then Toll-Like receptors, an particulary TLR4, play a critical role in cardiac inflammation. TLR4 activation has been involved in the regulation of inflammatory and fibrotic response both in CF and other cells; adhesion molecules and recruiment of immune cells are key elements of these process. Therefore we sought to evaluate whether TLR4 activation in CF obteined from adults rats increased monocyte adhesion trough increased levels of E-selectin. TLR4 activation by LPS in CF, induce increased in E-selectin expression, which was abolished when CF were pre treatment with TAK-242 (TLR4 inhibitor). In adittion, TLR4 activation increased monocyte adhesion to CF, whereas the inhibition of this receptor and releated signaling pathway ERK1/2, PI3K/Akt and NF-κB, previous LPS exposure reverse monocyte adhesion. Similarly, E-selectin blockade, by the use of blocking antibody, also blocked monocyte adhesion, which a yeast the importance of this adhesion molecule in monocyte recruitment and inflammatory response orchestrated by CF

Toll-like receptor 4 (TLR4) is an important vertebrate innate immune receptor. TLR4 recognizes both endogenous and exogenous danger signals to trigger an NF-kB dependent inflammatory response. While exogenous danger signal recognition is an essential part of pathogen response by the innate immune system, endogenous danger signal recognition by TLR4 can lead to chronic and pathological inflammation. Understanding the differences in recognition of these two types of danger signals would allow for independent modulation of pathogen and host triggered inflammatory response through TLR4. Here, we examine the evolution of activation of TLR4 by two agonists, pathogen-derived lipopolysaccharide and host-produced S100A9. We show that these two types of signals evolved earlier than previously thought. We identified TLR4 cofactors MD-2 and CD14 in amphibians and fish, and validated that zebrafish TLR4 can recognize LPS. By contrast, we find that S100 activation evolved in the ancestor of amniotes. We identified an ortholog of S100A9 in birds and reptiles capable of activating TLR4. Using comparative immunology, we found that the requirements for LPS and S100A9 activation are different. In addition to our evolutionary studies, we used molecular approaches to probe if zinc binding to S100A9 is necessary for TLR4 activation. We found that activation of TLR4 by S100A9 occurs even in the absence of zinc. Finally, we describe how our evolutionary approach led to mechanistic hypotheses regarding TLR4 activation by both LPS and S100A9. This has led to ongoing projects in the Harms lab. This dissertation includes previously published and unpublished co-authored material. / 2021-04-30

Resumo: Não disponível / Abstract: Acute chorioamnionitis is a response to microbial infection of the amniotic fluid. The innate immune system constitutes the host's first line of defense against pathogens and, in this regard, Toll-like receptors (TLRs) are important regulators of such nonspecific response. However, the expression of these receptors in chorioamniotic membranes in pregnancies complicated by chorioamnionitis has not been well established. Objective:The purpose of this study was to examine the localization of TLR-1, TLR-2, TLR-4 and TLR-6 in fetal membranes and determine whether histologic chorioamnionitis is associated with changes in gene expression of these receptors. One hundred and fifteen chorioamniotic membranes were included in the study. They were collected at the Obstetrics Service of the Botucatu Medical School, São Paulo State University, UNESP, from pregnant women with preterm delivery or term delivery with or without labor. Both groups were stratified on the basis of the presence of histologic chorioamnionitis. Fragments of the chorioamniotic membranes were sent for histopathologic analysis in order to confirm histologic chorioamnionitis. Other membranes fragments measuring 1cm2 were placed into RNA later and submitted to total RNA extraction. After RNA extraction, the samples with concentration between 0.02 and 0.2 g/L of RNA were submitted to cDNA collection for later use in quantifying the expression of TLR-1, TLR-2, TLR-4 and TLR-6 by the real-time PCR technique using the TaqMan® Gene Expression Assays System. All membranes analyzed expressed TLR-1 and TLR-4, whereas 99.1% expressed TLR-2 and 77.4% expressed TLR-6. TLR-1 and TLR-2 expression were statistically higher in the membranes of preterm pregnancies in the presence of chorioamnionitis as compared with preterm membranes in the absence of the inflammatory infiltrate. Among the membranes of term pregnancies, there was... (Complete abstract click electronic access below) / Orientador: Márcia Guimarães da Silva / Coorientador: José Carlos Peraçoli / Banca: Angela Maria Victoriano de Campos Soares / Banca: Rosiane Mattar / Mestre

Erlangen, Nürnberg, Universiẗat, Diss., 2007. / Enth. 1 Sonderabdr. aus: American journal of reproductive immunology ; Vol. 53. 2005. - Beitr. teilw. dt., teilw. engl.

Burns often leads to infection, due to damage to the skin's protective barrier. Burn injury has been repeatedly shown to induce considerable inflammatory and immune dysfunction. The innate immune system is a universal and ancient form of host defense against infection. Activation of innate immunity constitutes the first line of host defense against infection. Neutrophils are white blood cells and part of the immune system. They are the most common PMN (polymorphonuclear neutrophils) and accounted for 70% of all leukocytes. Neutrophils provided the first line of defense of the innate immune system by phagocytosing, killing, and digesting bacteria and fungi. Priming means a process whereby the response of neutrophils to an activating stimulus is potentiated, sometimes greatly, by prior to exposure to priming agents such as tumor necrosis factor-alpha(TNF-alpha), platelet-activating factor (PAF). Neutrophil priming causes a dramatic increase in the response of these cells to an activating agent; this process has been shown to be critical for neutrophil-mediated tissue injury both in vitro and in vivo. However, the intracellular signaling pathways used by neutrophil in response to pro-inflammatory stimuli have not been elucidated. The discovery of TLRs has made us understanding of the mechanisms of innate immune recognition. The innate immune system detectes the invasion of microorganism through TLRs, which recognize microbial components and trigger inflammatory responses. Severe burn injury produces shock and induces acute gastrointestinal derangement that may disrupt gastrointestinal mucosa integrity and facilitate the bacterial translocation (BT) to Mesenteric lymph node (MLN), liver, and spleen. Hypertonic saline (HTS) has been advocated in thermal injury resuscitation because of the possibility of giving less total volume of resuscitation fluid, with a resulting decrease in edema and the need for escharotomy. In this study, I found that priming effect of BM neutrophils is TNF-alpha and p38 dependent and TLRs play a critical role to the innate immunity by recognizing bacteria and HTS enhance host response to bacterial challenge by increasing TLRs of inflammatory cells.

The most abundant microflora is present in the distal parts of gut and the majority of the bacteria are Gram-negative anaerobes. Toll-like receptor 4 (TLR4), one of the ¡§pathogen-recognition molecules¡¨, recognize the lipopolysaccharide (LPS), derived from Gram-negative bacteria. TLR4 recognized the intestinal microflora and triggers the inflammatory responses. Although the effect of intestinal microflora on enhancing host to response the challenge from bacteria has been established, the mechanism has not been well studied. In this study, the relationship between TLR4 expression and the inflammatory response under intestinal microflora depletion was investigated. Mice fed with antibiotics for 4 weeks to delete the intestinal commensals and supplemented with or without LPS to stimulate TLR4 at week 3 were under sham or burn treatment. Results showed that thermal injury intestinal permeability, bacterial translocation to mesenteric lymph nodes, and neutrophil deposition in lung. Also, the activation of NF-£MB, expression of HSP70 and TLR4 were induced in intestinal after thermal injury. Moreover, TLR4 expression was increased in lung and peritoneal cells, ICAM and TNF-£\ expression were increased in peritoneal cells after thermal injury. However, NF-£MB activation, expression of TLR4, ICAM, and HSP70 were decreased in intestinal mucosa of mice with microflora depletion after thermal injury. Microflora depletion also significantly decreased the MPO activity in lung, and the phagocytic activity, the TLR4, ICAM, and TNF-£\ expression of peritoneal cells after thermal injury. Interestingly, LPS supplement reversed the effect of microflora depletion, suggest that intestinal microflora can trigger the host defense through TLR4 signaling pathway in thermal injured mice.

Receptor glycosylation is critical in receptor-ligand interactions in immune cells, but the exact role of glycosylation in receptor activation upon ligand binding has not been elucidated. In neuronal cells, we have shown that when neurotrophic factors bind their respective Trk tyrosine kinase receptors, receptor activation and subsequent neurotrophin-mediated signaling is dependent upon the induction and activity of an endogenous sialidase enzyme. In this thesis, we report that toll-like receptor (TLR) activation upon ligand binding is similarly dependent on the induction of a cellular sialidase, which we have identified as Neu1 sialidase, which specifically targets and hydrolyses alpha-2,3-linked sialic acid residues on the receptor. Blocking Neu1 sialidase activity with specific inhibitor Tamiflu detrimentally impacts ligand-induced TLR4/MyD88 interaction, NFkappaB activation and TLR-mediated effector responses like nitric oxide and pro-inflammatory cytokine production. Diminished cytokine production is also seen in vivo in Neu1-deficient mice. We propose a mechanism for the induction of Neu1 sialidase, upon ligand binding to TLR, that involves the activation of heterotrimeric G-alpha protein-dependent G-protein coupled receptor (GPCR) signaling to activate a matrix metalloproteinase (MMP) enzyme, likely MMP-9. It is suggested that MMP-(9) targets the cell surface elastin receptor complex of Neu1/protective protein cathepsinA/elastin binding protein (EBP), which potentially catalytically activates Neu1. In addition, we report an association between Neu1 and TLR2, TLR3 and TLR4 on the plasma membrane that has not previously been described. The idea that the multiple functionality and diversity of TLRs and TLR-mediated signaling may be an immunologic paradigm capable of explaining all human disease is provocative but plausible. Certainly, the structural integrity of TLRs, their ligand interactions and activation are essential for immunological protection. Thus, understanding the molecular mechanism of Neu1 sialidase regulation of TLR activation will provide important opportunities for disease control through TLR manipulation. The future directions of this research will also open a new area of glycobiology research (the glycomics of innate immune responses) and will widen the scope for the development of novel therapeutic drugs to combat infections and inflammatory diseases. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2009-01-26 12:33:32.743

Univ., Diss., 2009--Marburg.

Hohenheim, Univ., Diss., 2006.