Endothelial cell dysfunction contributes to sepsis induced initiate immune response and the infiltration of immune cells into organs, resulting in organ injury. Heat shock protein A12B (HSPA12B) is predominantly expressed in endothelial cells. The present study investigated whether endothelial HSPA12B could regulate macrophage pro-inflammatory response during sepsis. Wild type (WT) and endothelial cell-specific HSPA12B deficient (HSPA12B–/–) mice were subjected to CLP sepsis. Mortality and cardiac function were monitored. Higher mortality, worsened cardiac dysfunction, and greater infiltrated macrophages in the myocardium and spleen were observed in HSPA12B–/– septic mice compared with the WT septic mice. The serum levels of TNF-α and IL-1β were higher and the levels of IL-10 were lower in HSPA12B–/– septic mice than in WT septic mice. Importantly, endothelial exosomes contain HSPA12B which can be uptaken by macrophages. Interestingly, endothelial exosomal HSPA12B significantly increases IL-10 levels and decreases TNF-α and IL-1β production in LPS-stimulated macrophages. Mechanistic studies show that endothelial exosomal HSPA12B downregulates NF-κB activation and nuclear translocation in LPS stimulated macrophages. These data suggest that endothelial HSPA12B plays a novel role in the regulation of macrophage pro-inflammatory response via exosomes during sepsis and that sepsis induced cardiomyopathy and mortality are associated with endothelial cell deficiency of HSPA12B.

Background. Cardiac dysfunction is present in >40% of sepsis patients and is associated with mortality rates of up to 70%. Recent evidence suggests that glycolytic metabolism plays a critical role in host defense and inflammation. Activation of Toll-like receptors on immune cells can enhance glycolytic metabolism. This study investigated whether modulation of glycolysis by inhibition of hexokinase will be beneficial to septic cardiomyopathy. Methods. Male C57B6/J mice were treated with a hexokinase inhibitor (2-deoxy-d-glucose [2-DG], 0.25-2 g/kg, n = 6-8) before cecal ligation and puncture (CLP) induced sepsis. Untreated septic mice served as control. Sham surgically operated mice treated with or without the 2-DG inhibitor served as sham controls. Cardiac function was assessed 6 hours after CLP sepsis by echocardiography. Serum was harvested for measurement of inflammatory cytokines and lactate. Results. Sepsis-induced cardiac dysfunction was significantly attenuated by administration of 2-DG. Ejection fraction and fractional shortening in 2-DG-treated septic mice were significantly (P < .05) greater than in untreated CLP mice. 2-DG administration also significantly improved survival outcome, reduced kidney and liver injury, attenuated sepsis-increased serum levels of tumor necrosis factor α and interleukin 1β as well as lactate, and enhanced the expression of Sirt1 and Sirt3 in the myocardium, which play an important role in mitochondrial function and metabolism. In addition, 2-DG administration suppresses sepsis-increased expression of apoptotic inducers Bak and Bax as well as JNK phosphorylation in the myocardium. Conclusions. Glycolytic metabolism plays an important role in mediating sepsis-induced septic cardiomyopathy. The mechanisms may involve regulation of inflammatory response and apoptotic signaling.

Regulation of the inflammatory response is essential for the successful resolution of inflammation, and restoration of normal tissue homeostasis. Eosinophils are granulocytic cells of the innate immune system historically considered to be primarily involved in the defence against parasitic infection. Eosinophils are also key effector cells in the allergic inflammatory response, initiation of which is associated with the recruitment and activation of eosinophils culminating in the release of their intracellular granule contents. Eosinophil granules contain a range of cytotoxic proteins (major basic protein, eosinophil cationic protein and eosinophil peroxidase) that act to destroy infectious and parasitic organisms. However, these cytotoxic proteins can also cause damage to surrounding host tissue cells. The resolution of the inflammatory response acts to limit the extent of eosinophil-mediated tissue damage. Programmed cell death (apoptosis) of eosinophils represents an important component of this resolution process, limiting release of granule contents and triggering efferocytosis (the removal of apoptotic cells by phagocytes). Apoptosis is initiated by the activation of intracellular caspases, a family of cysteine proteases. Caspase activation primarily occurs as a result of changes in the balance of intracellular pro- and anti-apoptotic Bcl-2 family proteins. Mcl-1, an anti-apoptotic Bcl-2 protein has been shown to play a pivotal role in the regulation of neutrophil apoptosis. Pharmacological down-regulation of Mcl-1 initiates apoptosis and promotes the resolution of neutrophil-dominant inflammation. The importance of Mcl-1 in the regulation of apoptosis was shown using cyclin-dependent kinase inhibitors (CDKis), where induction of neutrophil apoptosis by CDKis was due to down-regulation of intracellular Mcl-1. Apoptotic cells display distinct surface molecules known as ‘eat-me’ signals that identify them for phagocytosis by macrophages and other phagocytes. One key receptor involved in the removal of apoptotic cells from tissue is the receptor tyrosine kinase Mer, a member of the Tyro3/Axl/Mer (TAM) family, which recognises the ‘eat me’ signal phosphatidylserine expressed on apoptotic cells. In the absence of Mer expression, clearance of apoptotic cells is compromised delaying the resolution of neutrophil-dominant inflammation. However, the roles of Mcl-1 and Mer in eosinophil apoptosis and clearance, respectively, and the resolution of allergic inflammation are not known. Asthma is a chronic inflammatory lung disease characterised by shortness of breath, airway obstruction, wheeze, non-specific bronchial hyper-responsiveness, excessive airway mucus production and an eosinophil dominant inflammatory infiltrate. The persistent presence of eosinophils in the lung, in chronic asthma, is likely due to a combination of excessive eosinophil recruitment and activation together with impaired eosinophil apoptosis. Investigation into the underlying mechanisms of these processes in allergic airway disease is of critical importance, as blocking eosinophil recruitment and/or promoting eosinophil apoptosis could provide a therapeutic approach to reduce associated eosinophil-mediated tissue damage. Understanding the regulation of eosinophil apoptosis and phagocytic clearance may identify novel pharmacological targets to enhance the resolution of allergic inflammation. We hypothesise that Mcl-1 and Mer play vital roles in the successful resolution of allergic airway inflammation. To investigate this hypothesis, we have used pharmacological and genetic manipulation of intracellular eosinophil Mcl-1 levels, and phagocyte Mer expression, to determine the role they play in the regulation of eosinophil apoptosis and phagocytic clearance of apoptotic eosinophils, respectively. Human and mouse eosinophils were cultured, and rates of constitutive and CDKi-induced apoptosis were determined, to investigate eosinophil apoptosis in vitro. Mice expressing human Mcl-1 (hMcl-1) were used to determine the effect of over-expression of Mcl-1 on eosinophil viability in vitro. The effect of hMcl-1 on eosinophil viability and disease severity in vivo was determined using an ovalbumin-induced model of allergic airway inflammation, which mimicked the symptoms of human asthma. Apoptotic eosinophils were co-incubated with macrophages in vitro to investigate the capacity for phagocytosis by different macrophage populations. Apoptotic cell clearance was further investigated using a Mer-kinase-dead mouse, which lacked Mer expression, to determine the role of Mer-dependent phagocytosis on the process of resolution of inflammation in vivo. Over-expression of Mcl-1 in eosinophils significantly delayed both constitutive and CDKi-induced apoptosis in vitro. In vivo in the ovalbumin-induced model of allergic airway inflammation, over-expression of Mcl-1 resulted in a significantly increased number of eosinophils in the lung and delayed rate of resolution of allergic airway inflammation. Alveolar and bone marrow-derived macrophages exhibited Mer-dependent phagocytosis of eosinophils, which was significantly reduced by an inhibitor of Mer kinase activity (BMS777607) or lack of macrophage Mer expression. The absence of Mer expression resulted in a significant increase in the number of apoptotic eosinophils in the lung together with a delayed rate of resolution of allergic airway inflammation in vivo. Together this work has shown that delayed rates of eosinophil apoptosis and impaired phagocytic clearance both delayed the resolution of allergic airway inflammation. These data suggest that both Mcl-1 and Mer are pivotal for the successful regulation of eosinophil apoptosis and phagocytic clearance of apoptotic eosinophils in asthma and may provide attractive novel therapeutic targets.

No / This review focuses on treatment targets for the most common inflammatory skin diseases, eczema and psoriasis with an emphasis on cytokines expressed in the uppermost layer of the skin which is easily accessible for diagnostic and therapeutic approaches. Recently, a significant body of research has highlighted the influence of the skin barrier and the patients’ microbiome on skin inflammatory responses and we will comment on their impact on mediator regulation. Itch is a prominent dermatology symptom which is influenced by cytokines and can via itch–scratch cycle impact on the skin barrier and mediator expression associated with damage. Taking the contribution of pruritus and superficial skin damage into account, we address cytokines as targets for stratified treatment approaches in subgroups of eczema and psoriasis.

Contaminated poultry and egg products are major sources of Salmonella enterica serovar Enteritidis (S. enteritidis, SE) infections in humans. Colonization of SE in chicken reproductive tract results in the production of contaminated commercial shell-eggs and fertilized hatchery eggs. The complex pathogen-host interactions during SE colonization of chicken reproductive tract are largely unknown. This study was aimed at determining the pathogenic roles of the type three secretion systems (TTSS-1 and TTSS-2) in SE infection of chicken oviduct epithelial cells (COEC). A series of SE strains carrying mutations in the genes encoding structure or effector proteins of TTSS-1 and TTSS-2 were constructed. The invasiveness and intracellular survival rate of each SE strain as well as the host innate immune responses induced by the infections were evaluated. The results demonstrate that both TTSS-1 and TTSS-2 are required by SE to invade COEC which involve genes encoding effector proteins SipA, SopB, SopE2, and PipB. In addition to their involvement in host cell invasion, sipA and sipB are also necessary for the survival or replication of SE inside COEC. Inactivation of TTSS-2 genes (ssaV and pipB) resulted in an enhanced bacterial proliferation inside COEC. The data from this study also show that SE infection triggers pro-inflammatory responses in COEC and TTSS-1 is involved in the expression of iNOS and IL-8, a CXC chemokine. TTSS-1 and TTSS-2 are not necessary for induction of K203, MIP-1β, and IL-10 or suppression of TGF-β3 in COEC.

Orientador: Denise Vaz de Macedo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Educação Fisica / Made available in DSpace on 2018-08-15T19:33:35Z (GMT). No. of bitstreams: 1 Ide_BernardoNeme_M.pdf: 1916014 bytes, checksum: abba7d8be6180d34542170cfe621bf21 (MD5) Previous issue date: 2010 / Resumo: O objetivo do presente estudo foi analisar as adaptações ao treinamento com ênfase nas ações musculares excêntricas (EXC). Os dados foram coletados durante uma disciplina eletiva oferecida aos alunos do curso de Educação Física da UNICAMP. Ao longo do semestre, além dos treinamentos, ministramos aulas expositivas, discussões em grupo sobre a forma de tratamentos dos dados para a pesquisa, e também fundamentações acerca da metodologia do treinamento de força. Realizamos também uma revisão sobre as adaptações musculares ao treinamento de força com ênfase nas ações excêntricas, contida na Introdução do trabalho, e que foi utilizada como material didático na disciplina. No primeiro experimento observamos as adaptações promovidas por 13 sessões de treino na magnitude de lesão à célula, respostas inflamatórias, composição corporal, força muscular (FM), resistência de força (RF), consumo máximo de oxigênio (VO2max), limiar ventilatório, e ponto de compensação respiratório. No segundo experimento verificamos se protocolos com ações musculares concêntricas (ACON), concêntricas e excêntricas (ACON/EXC) e somente excêntricas (AEXC) durante o exercício prévio, geraria respostas distintas no tocante a ocorrência da potencialização pós-ativação (PPA). No primeiro estudo testes de FM, VO2max, e de RF foram realizados após as 13 sessões de treinamento, 7 dias após e 14 dias após o término do programa. As avaliações de composição corporal (CC) e coletas de sangue (CS) foram realizadas em cinco momentos ao longo do treinamento (após 2, 7, 9, 11, 13 sessões). As séries vermelha e branca não se alteraram ao longo do programa de treinamento. A atividade sérica da CK aumentou significativamente somente após 2 sessões de treino. Já as concentrações séricas de PCR aumentaram significativamente em diferentes momentos e em diferentes sujeitos, mesmo após 96h de descanso da última sessão de treino. Esses dados sugerem que a carga utilizada, prescrita com 80% de 1RMexc e ajustada no início de cada sessão promoveu respostas inflamatórias, e incrementos significativos na FM e RF dos indivíduos, sem quedas significativas até 2 semanas após o término do treinamento. Já no estudo 2 observamos que o exercício prévio de meio agachamento, com ênfase nas ações EXC, afetou de forma negativa a performance muscular nos estímulos subsequentes. Contrariamente, o exercício prévio de meio agachamento com predomínio CON induziu a PPA / Abstract: The objective of this study was to analyze the adaptations to training with emphasis on eccentric muscle actions (EXC). Data were collected during an elective discipline offered to Physical Education students of State University of Campinas - UNICAMP. During the semester, in addition to training, expositive classes, group discussions about the processing of the data for research, and also grounding about the strength training methodology. We also reviewed the muscular adaptations to strength training with emphasis on eccentric actions contained in the Introduction of the thesis, to be used as teaching material in the course. In the first experiment we observed the changes promoted by 13 training sessions in the magnitude of muscle damage, inflammatory responses, body composition, muscular strength (FM), strength endurance (RF), maximal oxygen uptake (VO2max), ventilatory threshold, and point respiratory compensation. In a second experiment we verified if protocols with concentric muscle actions (ACON), concentric and eccentric (ACON / EXC), and eccentric only (AEXC) during the previous exercise, would generate different responses regarding the occurrence of post-activation potentiation (PAP). In the first study the FM, VO2max, and RF were performed after 13 training sessions (P13), 7 days after the end of the program (7D), and 14 days after the end of the program (14D). Body composition assessments (CC) and blood samples (CS) were carried out during the training program (P2, P5, P7, P11 and P13). The red series did not change during the training. The serum activity of CK increased only after the first two training sessions. Since PCR concentrations increased substantially and at other times, but at the end of 13 sessions all subjects showed normal values. The 13 training sessions induced significant increases in thigh circumference and muscle mass of the individuals. The FM and RF increased by 20 and 30%, respectively, without decreases until 14 days after the end of the experiment. In the study 2 we showed that the prior exercise predominantly EXC in the squat affected negatively the performance in the subsequent stimulus. In contrast, predominantly CON exercise induced PPA / Mestrado / Biodinamica do Movimento Humano / Mestre em Educação Física

Les azasulfurylpeptides sont des mimes peptidiques auxquels le carbone en position alpha et le carbonyle d’un acide aminé sont respectivement remplacés par un atome d’azote et un groupement sulfonyle (SO2). Le but premier de ce projet a été de développer une nouvelle méthode de synthèse de ces motifs, également appelés N-aminosulfamides. À cette fin, l’utilisation de sulfamidates de 4-nitrophénol s’est avérée importante dans la synthèse des azasulfuryltripeptides, permettant le couplage d’hydrazides avec l’aide d’irradiation aux micro-ondes (Chapitre 2). Par la suite, en quantité stoechiométrique d’une base et d’un halogénure d’alkyle, les azasulfurylglycines (AsG) formés peuvent être chimiosélectivement alkylés afin d’y insérer diverses chaînes latérales. Les propriétés conformationnelles des N-aminosulfamides à l’état solide ont été élucidées grâce à des études cristallographiques par rayons X : elles possèdent une structure tétraédrique autour de l’atome de soufre, des traits caractéristiques des azapeptides et des sulfonamides, ainsi que du potentiel à favoriser la formation de tours gamma (Chapitre 3). Après le développement d’une méthode de synthèse des N-aminosulfamides en solution, une approche combinatoire sur support solide a également été élaborée sur la résine amide de Rink afin de faciliter la génération d’une librairie d’azasulfurylpeptides. Cette étude a été réalisée en employant le growth hormone releasing peptide 6 (GHRP-6, His-D-Trp-Ala-Trp-D-Phe-Lys-NH2). Ce dernier est un hexapeptide possédant une affinité pour deux récepteurs, le growth hormone secretagogue receptor 1a (GHS-R1a) et le récepteur cluster of differenciation 36 (CD36). Une affinité sélective envers le récepteur CD36 confère des propriétés thérapeutiques dans le traitement de la dégénérescence maculaire liée à l’âge (DMLA). Six analogues d’azasulfurylpeptides de GHRP-6 utilisés comme ligands du CD36 ont été synthétisés sur support solide, mettant en évidence le remplacement du tryptophane à la position 4 de GHRP-6 (Chapitre 4). Les analogues de GHRP-6 ont été ensuite analysés pour leur capacité à moduler les effets de la fonction et de la cascade de signalisation des ligands spécifiques au Toll-like receptor 2 (TLR2), en collaboration avec le Professeur Huy Ong du département de Pharmacologie à la Faculté de Pharmacie de l’Université de Montréal. Le complexe TLR2-TLR6 est reconnu pour être co-exprimé et modulé par CD36. En se liant au CD36, certains ligands de GHRP-6 ont eu un effet sur la signalisation du TLR2. Par exemple, les azasulfurylpeptides [AsF(4-F)4]- et [AsF(4-MeO)4]-GHRP-6 ont démontré une capacité à empêcher la surproduction du monoxyde d’azote (NO), un sous-produit réactif formé suite à l’induction d’un signal dans les macrophages par des ligands spécifiques liés au TLR2, tel le fibroblast-stimulating lipopeptide 1 (R-FSL-1) et l’acide lipotéichoïque (LTA). En addition, la sécrétion du tumor necrosis factor alpha (TNFa) et du monocyte chemoattractant protein 1 (MCP-1), ainsi que l’activation du nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), ont été réduites. Ces résultats démontrent le potentiel de ces azasulfurylpeptides à pouvoir réguler le rôle du TLR2 qui déclenche des réponses inflammatoires et immunitaires innées (Perspectives). Finalement, le potentiel des azasulfurylpeptides d’inhiber des métallo-bêta-lactamases, tels le New-Delhi Metallo-bêta-lactamase 1 (NDM-1), IMP-1 et le Verona Integron-encoded Metallo-bêta-lactamase 2 (VIM-2), a été étudié en collaboration avec le Professeur James Spencer de l’Université de Bristol (Royaumes-Unis). Certains analogues ont été des inhibiteurs micromolaires du IMP-1 (Perspectives). Ces nouvelles voies de synthèse des azasulfurylpeptides en solution et sur support solide devraient donc permettre leur utilisation dans des études de relations structure-activité avec différents peptides biologiquement actifs. En plus d'expandre l'application des azasulfurylpeptides comme inhibiteurs d'enzymes, cette thèse a révélé le potentiel de ces N-aminosulfamides à mimer les structures secondaires peptidiques, tels que les tours gamma. À cet égard, l’application des azasulfurylpeptides a été démontrée par la synthèse de ligands du CD36 présentant des effets modulateurs sur le TLR2. Compte tenu de leur synthèse efficace et de leur potentiel en tant qu’inhibiteurs, les azasulfurylpeptides devraient trouver une large utilisation dans les sciences de peptides pour des applications dans la médecine et de la chimie biologique. / The azasulfurylpeptides are peptide mimics in which the alpha carbon and the carbonyl of an amino acid residue are respectively replaced by a nitrogen atom and a sulfonyl group (SO2). The primary goal of this doctorate project was to develop a new effective method for the synthesis of these motifs, also called N-aminosulfamides. Towards this aim, the use of 4-nitrophenyl sulfamidates turned out to be important in the synthesis of azasulfuryltripeptides, allowing hydrazide couplings under micro-wave irradiation (Chapter 2). Side-chain diversity was then added using a stoichiometric amount of base and different alkyl halides to alkylate chemoselectively the azasulfurylglycine (AsG) residue. The conformational properties of the N-aminosulfamides in the solid state were studied using X-Ray crystallography, which showed a tetrahedral geometry about the sulfur atom, features of azapeptides and sulfonamides, as well as potential to favor the formation of gamma turns (Chapter 3). Following the development of the synthesis of these N-aminosulfamides in solution, a combinatorial approach on solid support was elaborated on Rink amide resin to generate a library of azasulfurylpeptides. The study was performed using the Growth Hormone Releasing Peptide 6 (GHRP-6, His-D-Trp-Ala-Trp-D-Phe-Lys-NH2). The latter is a hexapeptide that has affinity for two receptors, the Growth Hormone Secretagogue Receptor 1a (GHS-R1a) and the Cluster of Differenciation 36 (CD36) receptor. Selective binding to the CD36 receptor has therapeutic potential in the treatment of age-related macular degeneration (AMD). Six azasulfurylpeptide analogs were synthesized on solid support by replacing tryptophan at the 4th position of GHRP-6 with different N-aminosulfamide residues (Chapter 4). The GHRP-6 analogs were tested for their ability to mediate the effects of receptor-specific ligands on the function and downstream signaling of the Toll-Like Receptor 2 (TLR2), in collaboration with Professor Huy Ong at the department of Pharmacology in the Faculty of Pharmacy at the Université de Montréal. The TLR2-TLR6 complex is known to be co-expressed and modulated by CD36. On binding to CD36, certain GHRP-6 ligands exhibited effects on the signaling of TLR2. For example, the azasulfurylpeptides [4-F-AsF4]- and [4-MeO-AsF4]-GHRP-6 prevented the overproduction of nitric oxide (NO), a reactive oxygen species formed following the induction of signal in macrophages on binding of TLR2-specific ligands, such as the Fibroblast-Stimulating Lipopeptide 1 (R-FSL-1) and lipoteichoic acid (LTA). Furthermore, the secretion of the Tumor Necrosis Factor Alpha (TNFa) and Monocyte Chemoattractant Protein 1 (MCP-1), as well as the activation of the Nuclear Factor Kappa-light-chain-enhancer of activated B cells (NF-kB), all were reduced. These results offer promise for regulating Toll-like receptor roles in triggering innate immunity and inflammatory responses (Perspectives). Finally, the potential of the azasulfurylpeptides to inhibit metallo-bêta-lactamases, such as the New-Delhi Metallo-β-lactamase 1 (NDM-1), IMP-1 and the Verona Integron-encoded Metallo-bêta-lactamase 2 (VIM-2), has been studied in collaboration with Professor James Spencer at the University of Bristol (United-Kingdom). Some analogs were micromolar inhibitors of IMP-1 (Perspectives). These new approaches for the synthesis of azasulfurylpeptides in solution and on solid support should enable their use in studies of structure-activity relationships with different biologically active peptides. In addition to expanding the application of azasulfurylpeptides as enzyme inhibitors, this thesis has revealed the potential of these N-aminosulfamides to mimic the peptide secondary structures, such as gamma turns. Application of azasulfurylpeptides in this respect has been demonstrated by the synthesis of CD36 ligands exhibiting modulatory effects on the TLR2. Considering their effective synthesis and potential as inhibitors, azasulfurylpeptides should find broad use in peptide science for applications in medicine and chemical biology.

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