Detta projekt genomfördes på företaget Sangair som för närvarande utvecklar medicinsk utrustning för att behandla bakteriemi med ozon. Bakteriemi uppstår när bakterier hamnar i blodomloppet, vilket kan trigga sepsis och septisk chock, med potentiellt dödligt utfall om obehandlat. För närvarande används antibiotika för att behandla bakteriemi, men det ökande hotet med antibiotikaresistens världen över innebär att forskare behöver hitta nya vägar för att behandla bakteriemi. Som en del av Sangairs utveckling gjordes en undersökning för att se hur bakterier påverkas av ozon. Projektet syftade till att 1) utveckla en metod för att detektera lysering av bakterier och 2) använda metoden för att se om ozon hade lyserande effekt på model-organismen Escherichia coli. För att testa metoden transformerades stam BL21 av E. coli med en pUC-19 plasmid för att producera beta-galaktosidas. Proteinets aktivitet mättes sedan i sonikerade bakterier, vilket visade att det var en effekitiv metod för att lysera bakterier och sedan mäta aktiviteten. För att undersöka om ozon hade en lyserande effekt, och samtidigt bestämma om man kunde mäta protein fann man att det optimala experimentella upplägget var att använde en ozonkoncentration på 6 g/m3, ett gasflöde på 5 ml/min och ett vätskeflöde på 25 ml/min. Resultatet från studien indikerade att ozon har en lyserande effekt, men fler studier behövs göras för att verifiera resultatet. Man undersökte också om detektionen av Beta-galaktosidas kunde förbättras genom att tillsätta Bovin serumalbumin (BSA) för att inhibera resterande ozon i prover som annars skulle kunna inaktivera Beta-galaktosidas. Resultaten indikerade dock inte någon effekt. Som ett sista experiment mättes även endotoxiner som frigjordes vid behandling, och det visade sig att när bakterier behandlas med ozon frigörs lipopolysackarider (LPS) och peptidoglykaner, vilket också kan tyda på lysis. / This project was conducted at Sangair. The company is currently developing a medical device aimed at treating bacteremia with ozone. Bacteremia is a condition that occurs when bacteria enters the bloodstream, and can trigger sepsis and septic shock, potentially leading to death if untreated. Antibiotics have traditionally been the way to treat bacteremia, but the looming threat of antibiotic resistance worldwide threatens this way of treatment, and research into novel approaches to eradicate the bacteria needs to be done. As part of Sangairs development, an investigation was done to see how ozone interacts with bacteria. The project aimed to 1) develop a method to detect bacterial lysis and 2) use the method to detect if ozone had a lysing effect on the bacterial model organism Escherechia coli. To test the method, the BL21 strain of  E. coli was transformed with the pUC-19 plasmid to produce the reporter enzyme Beta-galactosidase. The Beta-galactosidase activity was then measured in a supernatant of sonicated bacteria, which confirmed the suitability of the method to detect bacteria cell lysis. To be able to see if ozone had a lysing effect, while still being able to measure Beta-galactosidase, it was found that the optimal setup for this was using an ozone concentration of 6 g/m3, a gas flow of 5 ml/min and a liquid flow of 25 ml/min. The results acquired with this setup indicated that ozone had a lysing effect on E. coli but more studies are needed to verify this. It was further investigated if Beta-galactosidase detection could be improved by addition of bovine serum albumin (BSA) to quench residual ozone in the samples, but the results showed that it did not have any effect on the Beta-galactosidase enzymatic activity. As a final experiment, endotoxins that were released upon treatment were also measured, and it was found that when bacteria are treated with ozone, lipopolysaccharides (LPS) and peptidoglycans are released, further confirming lysis of the bacterial cells.

Myeloid-derived suppressor cells (MDSCs) prolong sepsis by promoting immunosuppression. We reported that sepsis MDSC development requires long non-coding RNA Hotairm1 interactions with S100A9. Using a mouse model that simulates the immunobiology of sepsis, we find that histone demethylase KDM6A promotes Hotairm1 transcription by demethylating transcription repression H3K27me3 histone mark. We show that chemical targeting of KDM6A by GSK-J4 represses Hotairm1 transcription, which coincides with decreases in transcription activation H3K4me3 histone mark and transcription factor PU.1 binding to the Hotairm1 promoter. We further show that immunosuppressive IL-10 cytokine promotes KDM6A binding at the Hotairm1 promoter. IL-10 knockdown repletes H3K27me3 and reduces Hotairm1 transcription. GSK-J4 treatment also relocalizes nuclear S100A9 protein to the cytosol. To support translation to human sepsis, we demonstrate that inhibiting H3K27me3 demethylation by KDM6A ex vivo in MDSCs from patients with protracted sepsis decreases Hotairm1 transcription. These findings suggest that epigenetic targeting of MDSCs in human sepsis might resolve post-sepsis immunosuppression and improve sepsis survival.

Abortion and severe pre-eclampsia/eclampsia remain the major causes of maternal mortality in Zimbabwe. Based on this problem, factors associated with maternal mortality due to abortion and severe pre-eclampsia/eclampsia were investigated to improve pregnancy outcomes. Cases and controls were selected from 4895 abortion and 318 severe preeclampsia/ eclampsia obstetric records to conduct a retrospective case-control study. Significant risk factors identified for reducing maternal mortality due to postabortion complications included the administration of oxytocic drugs and evacuations of the uterus whilst anaemia and sepsis apparently reduced these women's chances of survival. No significant factors could be identified which influenced maternal deaths among women suffering from severe pre-eclampsia/eclampsia. Magnesium sulphate was not routinely administered, as recommended internationally. In both groups, cases apparently received better reported quantitative care than controls. Recommendations based on this research report include improved midwifery education and in-service training, regular audits of patients' records and changed policies for managing these conditions more effectively in Zimbabwe. / Health Studies / D. Litt. et Phil. (Advanced Nursing Sciences)

Sepsis is the systemic inflammatory response to an infection. Severe sepsis with multi organ failure is one of the commonest causes of admission to intensive care units, and is associated with poor early and late outcomes. The pathophysiology of sepsis is complex, and poorly understood. This is reflected in the limited and contentious treatment options for sepsis. Genetic factors have been shown to be associated with the risk of and subsequent outcomes from infection. However, clear associations with bacterial sepsis are rare, and even when associations are present their functional effects are often unknown. Gene expression signatures in sepsis are investigated in this project using serial samples obtained from patients admitted to intensive care units with community-acquired pneumonia or faecal peritonitis. The evolving gene expression signatures that define the response to sepsis were identified with large changes seen in genes coding for ribosomal proteins RPS4Y1 and RPS26P54. The differences in the sepsis response between the two diagnostic classes were examined. The gene expression predictors of mortality in sepsis were determined and include genes from the class II MHC HLA-DRB4, HLA-DRB5 and the T cell differentiation protein MAL. The effects of important covariates on gene expression were investigated and their impact on survival related expression determined. The findings were confirmed in a validation cohort. A novel clustering of samples representing distinct inflammatory patterns in a clinically homogeneous population of sepsis patients was identified and related to differences in clinical behaviour. The biological relevance of the differentially expressed genes was ascertained by identifying enriched gene sets. The gene expression changes in sepsis were examined in the context of related clinically relevant immune phenomena: the sterile systemic inflammatory response in patients undergoing elective cardiac surgery and the phenomenon of endotoxin tolerance in PBMCs derived from healthy volunteers. The results highlight the complexities of clinical sepsis and identify hypotheses for future investigations.

Avant propos L'objectif de cette thèse est d'explorer l'inflammation et l'infection au niveau du transcriptome, à l'aide de la technologie des puces à ADN. Pour cela, nous avons dans un premier temps travaillé sur des données publiques. Nous avons construit une base de données de signatures transcriptionnelles annotées, et développé un logiciel modulaire d'analyse. Ce logiciel permet d'explorer aisément les données publiques en effectuant des recherches par nom de gène ou par mots-clés. Nous avons ensuite exploré la modulation temporelle de l'expression des gènes du parenchyme pulmonaire dans un modèle murin d'inflammation aiguë par injection d'acide oléique. Dans un second modèle murin d'infection par Coxiella burnetii, nous avons analysé le rôle du sexe dans la modulation de la réponse transcriptionnelle hépatique, et identifié des voies métaboliques impliquées dans le contrôle de l'infection. Dans un troisième modèle in-vitro d'infection par différentes souches du virus de la grippe, nous avons identifié une signature transcriptionnelle commune de réponse à l'infection. Par une approche bio-informatique originale, cette signature a conduit à l'identification de nouveaux anti-viraux à large spectre, dont l'efficacité a été démontrée in-vitro sur les souches utilisées pour l'analyse, et sur la souche H1N1, responsable de la dernière pandémie grippale. Enfin, nous avons analysé les modulations du transcriptome lors de pneumonies associées à la ventilation mécanique compliquant l'évolution de sujets traumatisés graves admis en réanimation.

<p>Uvod: Sepsa je jedan od vodećih uzroka smrtnosti u jedinicama intenzivnog lečenja i van njih uprkos implementaciji novih dijagnostičkih i terapijskih protokola &scaron;irom sveta. Multiorganska disfunkcija (MODS), koja predstavlja najtežu formu nepovoljnog toka sepse, je u osnovi svojih patofiziolo&scaron;kih de&scaron;avanja obeležena promenama, koje se de&scaron;avaju na nivou kapilara, pre svega u endotelu. Poremećaji koagulacije koji se javljaju kao posledica ovih promena u endotelu su prepoznati kao jedan od dijagnostičkih kriterijuma prema najnovijim smernicama za dijagnostiku i lečenje sepse, međutim njihov značaj u predviđanju toka i ishoda ovog oboljenja jo&scaron; uvek nije precizno definisan. Cilj istraživanja: Odrediti koncentraciju markera endotelne aktivacije, aktivacije koagulacije, aktivnost prirodnih inhibitora koagulacije i funkcionalnost fibrinolize kod obolelih od sepse u odnosu na njihove vrednosti u zdravoj populaciji. Ispitati mogućnost upotrebe markera endotelne disfunkcije i pokazatelja poremećaja mehanizma hemostaze za postavljanje dijagnoze sepse i predikciju pojave komplikacija. Ispitati mogućnost upotrebe markera endotelne disfunkcije i pokazatelja poremećaja mehanizma hemostaze za procenu ishoda kod obolelih od sepse. Materijal i metode: Istraživanje je sprovedeno analitičkom metodom u formi studije preseka, a obuhvatilo je pacijente lečene na Odeljenju anestezije i reanimacije Urgentnog centra Kliničkog centra Vojvodine i na Klinici za infektivne bolesti Kliničkog centra Vojvodine, u Novom Sadu. Istraživanje je sprovođeno tokom 2012. i 2013. godine u trajanju od dve godine. U studiju je bilo uključeno 180 ispitanika od kojih je 150 imalo postavljenu dijagnozu sepse,a 30 ispitanika su činili kontrolnu grupu su klinički i biohemijski zdravih ispitanika, dobrovoljni davaoci krvi. Ispitanici su kategorisani u četiri grupe u odnosu na kliničko stanje i laboratorijske nalaze unutar prvih 24 časa od prijema: bolesnici sa sepsom, te&scaron;kom sepsom, septičkim &scaron;okom i multiorganskom disfunkcijom na prijemu. Nakon kategorizacije ispitanika, izračunati su APACHE II i SOFA numerički pokazatelji procene težine bolesti ispitanika. U roku 24 časa od trenutka postavljanja dijagnoze sepse, iz uzoraka krvi ispitanika, izvr&scaron;ene su predviđene laboratorijske analize u cilju praćenja endotelne aktivacije, aktivacije koagulacije i inhibicije antikoagulantnih mehanizama. U toku 48 časova od prijema, bolesnici koji nisu imali MODS na prijemu su intenzivno praćeni u cilju evidentiranja razvoja multiorganske disfunkcije, dok su bolesnici koji su imali MODS praćeni radi evidentiranja perzistiranja ili eventualne rezolucije MODS-a. Zdravstveno stanje bolesnika je praćeno tokom 28 dana od trenutka uključivanja u studiju i nakon tog perioda je evidentiran ishod lečenja u smislu preživljavanja ili smrtnog ishoda. Statistička analiza je izvr&scaron;ena pomoću statističkog paketa IBM SPSS 20 Statistics. Podaci su predstavljeni tabelarno i grafički, a statistička značajnost određivana je na nivou p&lt; 0,05. Rezultati: Vrednosti biolo&scaron;kih markera endotelne aktivacije i aktivacije koagulacije su statistički značajno povi&scaron;ene kod obolelih od sepse u odnosu na njihove vrednosti u zdravoj populaciji, dok su vrednosti prirodnih inhibitora koagulacije statistički značajno snižene kod obolelih od sepse u odnosu na njihove vrednosti u zdravoj populaciji. Vrednosti APTT-a, PT-a, D-dimera, fibrinogena, prirodnih inhibitora koagulacije i markera endotelne aktivacije (endokan i vWF antigena i aktivnosti) imaju značajan i veoma visok dijagnostički potencijal. Vrednosti biomarkera endotelne disfunkcije i pokazatelja poremećaja hemostaznog mehanizma su značajni prediktori komplikacija kod bolesnika sa sepsom. APTT, PT, D-dimer, broj trombocita, vrednosti priorodnih inhibitora koagulacije, trombomodulina, endokana i ETP-a su jednako validni u inicijalnoj proceni toka kliničke slike sepse kao i prediktivni APACHE II i SOFA skorovi. Koncentracija trombomodulina, D-dimera, ETP-a i PC su dobri prediktori nastanka MODS-a u prvih 48 časova u toku sepse. Endokan, PT, APTT, koncentracija fibrinogena, prirodnih inhibitora koagulacije i vrednosti ETP-a su značajni u predikciji mortaliteta kod bolesnika sa sepsom. Zaključci: Ukoliko bi pokazatelji aktivacije endotela i mehanizma hemostaze bili inkorporirani u određeni sistem skorovanja u cilju procene težine bolesti u smislu ishoda kod bolesnika sa sepsom, to bi moglo doneti doprinos boljoj klasifikaciji bolesnika, te primeni pravovremene i adekvatne terapije u cilju postizanja pozitivnog ishoda kod bolesnika sa sepsom. Prilikom interpretacije pokazatelja inflamacije i koagulacije neophodno je steći uvid u celokupnu sliku pro-i antikoagulantnih de&scaron;avanja koja se odvijaju tokom sepse, odnosno adekvatno proceniti pravac toka disbalansa mehanizma hemostaze da bi se eventualnim terapijskim merama mogao postići pozitivan učinak.</p> / <p>Introduction: Sepsis is one of the main causes of death in intensive care units and other hospital wards in spite of implementation of new sepsis treatment guidelines in everyday hospital practice worldwide. Changes that occur in the microvasculature, affecting primarily endothelial cell, are the basis of the pathophysiology of multiorgan dysfunction (MODS) in sepsis. Coagulation abnormalities which occur as a consequence of endothelial changes are recognized as diagnostic criteria for sepsis, but significance of these changes in the outcome prognosis and prediction of the course of sepsis is still not accurately defined. Aims: Evaluation of hemostasis related parameters and endothelial activation biomarkers values in patients with sepsis and healthy volunteers. Determination whether the levels of hemostasis-related parameters and biomarkers of endothelial activation have diagnostic significance and are they associated with MODS development and persistence in the first 48 hours of hospitalization and 28-day mortality in patients with sepsis. Material and methods: This is cross-sectional study conducted in 2012 and 2013 in the Department of Anesthesia and Reanimation at the Emergency Center of the Clinical Center of Vojvodina and in the Clinic of Infectious Disease at the Clinical Center of Vojvodina. 150 patients who fulfilled criteria for diagnosis of sepsis were included in the study. Patients were divided into 4 groups: sepsis, severe sepsis, septic shock and MODS. 30 healthy volunteers, blood donors were the control group. After the categorization of patients, during the first 24 hours of hospitalization, predictive APACHE II and SOFA scores were calculated. Hemostasis related parameters and endothelial activation biomarkers concentrations were determined within the first 24 hours of the onset of the disease. To assess the development of complication of the disease, patients were monitored for 48 hours for MODS development and persistence or resolution and for 28 days from the onset of sepsis for outcome assessment. Data were analyzed using SPSS 20.0 software and are presented in tables and graphs, statistical significance was set at p&lt; 0,05. Results: Biomarkers of endothelial and coagulation activation are significantly higher in patients with sepsis in comparison to their values in healthy volunteers, while concentrations of natural anticoagulants are significantly lower in patients with sepsis than in healthy volunteers. APTT, PT, D-dimer, fibrinogen, natural anticoagulants and biomarkers od endothelial activation (endocan and vWF antigen and activity) have diagnostic significance in patients with sepsis. Hemostasis related parameters and endothelial activation biomarkers are good prognostic factors for complication development in patients with sepsis. APTT, PT, D-dimer, platelet count, natural anticoagulants, thrombomodulin, endocan and ETP are equally valuable in early prediction of sepsis development as APACHE II and SOFA scores. Thrombomodulin, D-dimer, ETP and PC are good predictors of MODS development during the first 48 hours from sepsis onset. Endocan, PT, APTT, fibrinogen concentration, values of natural anticoagulants and ETP values are significant in 28-day mortality prediction in patients with sepsis. Conclusion: A combination of markers of endothelial dysfunction with widely used ICU scores and organ failure assessment could contribute to an early recognition of complication development and consequent death in patients with sepsis. It is necessary to obtain the full insight in pro-and anticoagulant dynamic evaluation while interpreting coagulation and inflammation processes in sepsis development, in order to accurately lead early resuscitation therapy.</p>

Prenant appui sur l’occultation de la mémoire verbale par la théorie théâtrale actuelle, le présent mémoire questionne l’exercice mémoriel des acteurs contemporains à partir de la dialectique mémoire/oubli dans les théories et pratiques de l’énonciation de Larry Tremblay, de Daniel Danis et de Christian Lapointe. Le premier chapitre s’intéresse aux fondements de l’énonciation privilégiés par les praticiens dans leurs discours théoriques – Le crâne des théâtres (Tremblay), « La mémoire intime au théâtre » (Danis), « Petit guide de l’apparition à l’usage de ceux qu’on ne voit pas » (Lapointe) –, plus précisément à la valeur accordée à la mémorisation verbale. Le deuxième s’attache à montrer que les textes dramatiques des auteurs-metteurs en scène – The Dragonfly of Chicoutimi (Tremblay), Mille anonymes (Danis), Sepsis (Lapointe) – engagent aussi un rapport oblique à l’apprentissage par cœur. Enfin, le troisième cherche à définir la notion de mémoire oublieuse à la lumière de laquelle les théories et les pratiques de Tremblay, de Danis et de Lapointe peuvent être analysées ainsi qu’à en identifier certaines conséquences dans leurs mises en scène. / Based on the concealment of verbal memory by theatrical theory, this master’s dissertation questions the memorial exercise of contemporary actors from the memory/oblivion dialectic in the enunciation theories and practices of Larry Tremblay, Daniel Danis and Christian Lapointe. The first chapter focuses on the main foundations of enunciation defended in the theoretical discourses of the practioners – Le crâne des théâtres (Tremblay), « La mémoire intime au théâtre » (Danis), « Petit guide de l’apparition à l’usage de ceux qu’on ne voit pas » (Lapointe) –, more specifically in the value they place on verbal memory. The second chapter shows that their dramatic texts – The Dragonfly of Chicoutimi (Tremblay), Mille anonymes (Danis), Sepsis (Lapointe) – also undertake a slant to learning by heart. Finally, the third chapter seeks to define the concept of forgetful memory in the light of which the theories and practices of Tremblay, Danis and Lapointe can be analyzed and to identify some implications in their staging.

Introdução: Os receptores Fc são proteínas de importância crucial no processo saúde-doença. São responsáveis pela ativação de mecanismos efetores e modulam a resposta imune e inflamatória. Têm papel central na patogênese de doenças autoimunes, sepse e doenças neoplásicas. O lúpus, protótipo das doenças autoimunes e a sepse, infecção grave que causa disfunção orgânica, são doenças inflamatórias nas quais o papel dos receptores Fc vêm sendo desvendados. Essas patologias têm alta morbidade e mortalidade, impondo enormes custos para sociedade. A descoberta que a E. coli se liga ao receptor CD16 (FcGamaRIII) para evadir-se do sistema imune, através da ligação com a proteína wzxe presente em sua membrana, torna esse receptor um alvo terapêutico interessante. O CD16 é um FcGamaR com ITAM que classicamente tem função ativadora e gera respostas inflamatórias ao se ligar a imunocomplexos. Porém, na sepse, a ligação direta com a E. coli induz uma ativação ITAMi, que bloqueia a produção de ROS e inibe a fagocitose e a morte desta bactéria. A manipulação dessa ativação inibitória (ITAMi), aparentemente anti-inflamatória, pode ser uma estratégia efetiva para o tratamento de doenças inflamatórias como a sepse e o lúpus. Inicialmente visamos avaliar a importância fisiológica e o papel terapêutico do peptídeo ligante do CD16, em modelo de sepse e em modelo de lúpus induzido por pristane, respectivamente. De maneira similar ao CD16, o CD89 (FcAlfaRI) é capaz de mediar uma sinalização dual, ativatória ou inibitória, que depende da forma como se dá sua ligação às imunoglobulinas e imunocomplexos. Por isso, num segundo momento, averiguamos se o FcAlfaRI (CD89) poderia se ligar à bactérias de maneira direta, na ausência de ligantes cognatos e mediar respostas pro ou anti-inflamatórias, protegendo ou não o hospedeiro. Metodologia: Camundongos C57Bl/6, WT e CD16KO com lúpus induzido por pristane, foram tratados com o peptídeo CYWGGTEGAC(IRG Bioscience,USA). A expressão gênica e protéica de diversas citocinas, assim como genes associados a assinaturas de interferon foram avaliados nos pulmões desses animais. Utilizamos também um modelo de sepse através da injeção intra-peritoneal de E. coli WT e E. coli mutante wzxe -/-, no qual avaliamos mortalidade e produção de citocinas. Realizamos experimentos in vitro com BMM e BMDC murinos, fagócitos humanos e bactérias. A expressão de CD89 e de receptores cognatos foi avaliada através de citometria de fluxo. Empregamos a citometria de fluxo com imagem para análise da fagocitose. Foram realizados também, experimentos in vivo com camundongos WT e transgênicos: CD89tg, CD89R209Ltg CD89tgCD16KO, CD16KO e PCRKO. Comparamos mortalidade, produção de citocinas, quantidade de bactérias e lesão tecidual em modelos de CLP e de pneumonia por administração nasal de S. pneumoniae. Produção de ROS pelos BMM foi avaliada por microscopia confocal e, nos PMN, por quimioluminescência. Imunoprecipitação e immunoblotting foram utilizados para avaliar recrutamento de syk e SHP-1. Utilizamos ELISA para ensaios de ligação de bactérias com CD89 e para quantificar TNF-Alfa, IL-1 e IL-6. Resultados: Camundongos injetados com a bactéria mutante wzxe-/- sobreviveram mais e produziram quantidade menor de citocinas reforçando o papel chave da proteína wzxe, no mecanismo de evasão imune da E. coli. Camundongos WT e CD16KO com lupus induzido por pristane, tratados ou não com peptídeo CYWGGTEGAC, não apresentaram diferenças na expressão gênica nem protéica de citocinas nem em genes associados a assinaturas de interferon em seus pulmões. O CD89 interage diretamente com bactérias gram-positivas e gram-negativas. A interação bactéria-CD89 em macrófagos murinos induz ativação celular, fagocitose e morte bacteriana, que são dependentes da cadeia FcRGama. Essa mesma interação protege contra a mortalidade em dois modelos de sepse (CLP e pneumonia) e é dependente da cadeia FcRGama e indepedente de PCR e IgA anti-bactéria. Conclusões: O CD16 e o CD89 são FcRs com ITAM que apresentam uma dualidade na forma de ativação através do ITAM, que em algumas situações pode ser inibitória (ITAMi). Nessa tese reforçamos o papel chave da proteína wzxe, ligante do CD16, como responsável pela evasão bacteriana da E. coli através de sinalização ITAMi. Por outro lado, falhamos em demostrar diferenças após o tratamento de camundongos com lúpus induzido por pristane usando o peptídeo CYWGGTEGAC. Acreditamos que isso tenha ocorrido devido a dose inadequada do peptídeo ou proteólise por enzimas endógenas do camundongo, assim que o peptídeo é injetado. Novas doses ou a manipulação da estrutura do peptídeo são perspectivas futuras para este projeto. Quanto ao CD89, provamos seu papel extremamente importante na imunidade inata. Esse receptor, à semelhança do CD16, foi capaz de ligar-se diretamente a bactérias, na ausência de opsoninas e ligantes cognatos. O CD89 foi protetor tanto para infecção por gram-positivo quanto por gram-negativo enquanto o CD16 foi protetor apenas em modelo de pneumonia por gram-positivo / Introduction: Fc receptors are proteins of crucial importance in the health-disease process. They are responsible for the activation of effector mechanisms and modulate the immune and inflammatory responses. They play a central role in the pathogenesis of autoimmune diseases, sepsis and neoplastic diseases. Lupus, a prototype of autoimmune diseases and sepsis, a serious infection that causes organ dysfunction, are inflammatory diseases in which the role of Fc receptors has been unraveled. These pathologies have high morbidity and mortality, imposing enormous costs for society. E. coli has been found to bind directly to the FcGammaRIII(CD16) receptor to evade the immune system. This is due to the binding to the wzxe protein present in its membrane, making this receptor an interesting therapeutic target. CD16 is an FcGammaR with ITAM that classically has an activating function and generates inflammatory responses when binding to immunocomplexes. However, in sepsis, direct binding with E. coli induces an ITAMi activation, which blocks ROS production and inhibits phagocytosis and death of this bacterium. The manipulation of this apparently anti-inflammatory inhibitory signaling (ITAMi) may be an effective strategy for the treatment of inflammatory diseases such as sepsis and lupus. Initially we aimed to evaluate the physiological importance and therapeutic role of the CD16 binding peptide, in a sepsis model and pristane-induced lupus model, respectively. Similarly to CD16, CD89 (FcAlphaRI) is capable of mediating dual, activating or inhibitory signaling, which depends on how it binds to immunoglobulins and immunocomplexes. Therefore, we assessed whether FcAlphaRI (CD89) could bind to bacteria directly, in the absence of cognate ligands and mediate pro or anti-inflammatory responses, protecting or not the host. Methods: C57Bl/6 mice, WT and CD16KO with pristane-induced lupus were treated with the peptide CYWGGTEGAC (IRG Bioscience, USA). Gene and protein expression of cytokines, as well as genes associated to interferon signatures were evaluated in the lungs of these animals. We also used a sepsis model through the intra-peritoneal injection of E. coli WT and E. coli mutant wzxe-/-, in which we evaluated mortality and production of cytokines. We performed in vitro experiments with murine BMM and BMDC, human phagocytes and bacteria. Expression of CD89 and cognate receptors was assessed by flow cytometry. Flow cytometry with imaging was employed for phagocytosis analysis. In vivo experiments were also performed on WT and transgenic mice: CD89tg, CD89R209Ltg CD89tgCD16KO, CD16KO and CRPKO. We compared the mortality, cytokine production, amount of bacteria and tissue injury in CLP and pneumonia by nasal administration of S. pneumoniae. ROS production by BMM was evaluated with confocal microscopy and, in PMN, by chemiluminescence. Immunoprecipitation and immunoblotting were used to evaluate recruitment of syk and SHP-1. We used ELISA for binding assays with CD89 and bacteria and quantification of TNF-Aphla, IL-1 and IL-6. Results: Mice injected with wzxe-/- mutant E. coli survived more and produced smaller amounts of cytokines, reinforcing the key role of the wzxe protein in the mechanism of immune evasion of E. coli. WT and CD16KO pristane induced lupus mice, treated or not with the peptide CYWGGTEGAC didn\'t show differences in gene or protein expression of cytokines nor in interferon signature genes in their lungs. The bacterial-CD89 interaction in murine macrophages induces cellular activation, phagocytosis and bacterial death, which are dependent on the FcRGamma chain. This same interaction protects against mortality in two models of sepsis (CLP and pneumonia) and is dependent on the FcRGamma chain and independent of PCR and IgA anti-bacterium. Conclusions: CD16 and CD89 are ITAM-bearing FcRs that present a duality in the form of activation through ITAM, which in some situations may be inhibitory (ITAMi). In this thesis we reinforce the key role of wzxe protein, a CD16 ligand, as responsible for the bacterial evasion of E. coli through ITAMi signaling. On the other hand, we failed to demonstrate differences after treatment of pristane-induced lupus mice using the CYWGGTEGAC peptide. We believe that this was due to inadequate dose of the peptide or proteolysis by endogenous mouse enzymes, so the peptide is injected. New doses or manipulation of the peptide structure are future prospects for this project. As to CD89, we proved the extremely important role of CD89 in innate immunity. That receptor, similarly to CD16, was able to bind directly to bacteria, in the absence of opsonins and cognate ligands. CD89 was protective for both gram-positive and gram-negative infection while the CD16 was protective only in a model of gram-positive pneumonia

Sepse, a resposta do organismo a uma infecção, está associada a altas taxas de mortalidade. A razão pela qual um mecanismo protetor resulta num quadro clínico fatal permanece inexplicada. Em trabalho prévio nosso grupo demonstrou que exossomos de origem plaquetária são os mais freqüentes em plasma de pacientes com choque séptico e que estes podem induzir apoptose em células musculares lisas vasculares e células endoteliais em cultura. Demonstramos ainda que tais exossomos possuíam uma fonte enzimática de ROS, uma NADPH oxidase cuja atividade poderia estar associada à indução da apoptose (Janiszewski et al., 2004). No presente trabalho, nós buscamos criar um modelo de geração ex vivo de exossomos similares aos encontrados em pacientes sépticos e identificar possíveis vias responsáveis pela liberação destes e seus efeitos. Choque séptico é uma condição relacionada com exposição a lipopolissacarídeo (LPS) e geração de alta quantidade de trombina, TNF e espécies reativas de nitrogênio. Através de citometria de fluxo revelamos que plaquetas humanas expostas ao doador de NO dietilamina-NONOato e ao LPS geraram exossomos similares àqueles encontrados em pacientes com choque séptico, expondo alta quantidade de tetraspaninas CD9, CD63 e CD81 mas pouca fosfatidilserina. Por outro lado, plaquetas expostas à trombina ou TNF liberaram partículas com características claramente distintas, com alta exposição de fosfatidilserina e baixa de tetraspaninas. Assim como os exossomos sépticos, os exossomos obtidos pela exposição de NO e LPS geraram radical superóxido e NO, como demonstrado pela quimioluminescência da lucigenina (5M) e celenterazinina (5M) e pela fluorescência da 4,5-diaminofluoresceína (10mM) e 2,7-diclorofluoresceína (10mM). A análise por Western Blot nos permitiu identificar as subunidades Nox1, Nox2 e p22phox da NADPH oxidase e a isoforma induzível da enzima NO sintase (NOS) nesses exossomos. Como esperado, inibidores da NOS e da NADPH oxidase reduziram significamente os sinais fluorescentes e quimioluminescentes. Em adição, as células endoteliais em cultura expostas aos exossomos gerados por dietilamina-NONOato e LPS sofreram significativo aumento da taxa de apoptose quando comparadas àquelas expostas a exossomos controle. A inibição da NADPH oxidase assim como da NOS reduziu expressivamente tal efeito. Adição de urato (1mM), mostrou efeito aditivo sobre a inibição do sinal fluorescente, assim como redução adicional da taxa apoptótica, sugerindo papel importante do radical peroxinitrito. Nós propomos, assim, que exossomos derivados de plaquetas podem representar papel adicional no já complexo cenário da sinalização vascular redox. Nesse sentido, uma abordagem baseada em exossomos pode fornecer novas ferramentas para o entendimento e até tratamento da disfunção vascular na sepse / Sepsis, the bodys response to infection, is associated with high mortality rates. Why a protective mechanism turns into a deadly clinical picture is a matter of debate, and goes largely unexplained. In previous work we demonstrated that plateled derived exosomes are found in the plasma of septic patients with septic shock and can induce endothelial and vascular smooth muscle cell apoptosis in culture through an enzymatic superoxide source (Janiszewski et al., 2004). In this work we sought to create a model for ex vivo generation of exosomes, and to identify the pathways responsible for ROS release by exosomes and their effects. Septic shock is a condition related to exposure of lipopolysaccharide (LPS), generation of high amounts of thrombin, TNF and nitrogen reactive species. Through flow cytometry we demonstrated that human platelets exposed to the NO-donor diethylamine-NONOate, and to LPS, generated exosomes similar to those found in the blood of septic shock patients, with high exposure of the tetraspanin CD9, CD63, and CD81, but little phosphatidylserine. On the other hand, platelets exposed to thrombin or TNF released particles with clearly distinct characteristics, such as high phosphatidylserine and low tetraspanin. Like the septic exosomes, the exosomes obtained by NO and LPS exposure generated superoxide radical and NO, as disclosed by lucigenin and coelenterazine chemiluminescence and by 4,5-diaminofluorescein and 2,7-dichlorofluorescein fluorescence. Western Blot analysis revealed the presence of Nox1, Nox2 and p22phox NADPH oxidase subunits and the inducible isoform of NO synthase (NOS) in these exosomes. As expected, NOS inhibitors or NADPH oxidase inhibitors significantly reduced the fluorescence and chemiluminescente signals. In addition, endothelial cells exposed to NO or LPS generated exosomes underwent apoptotic death, while control exosomes had no effects on apoptosis. NADPH oxidase as well as NOS inhibition significantly reduced apoptosis rates. Concomitant generation of NO and superoxide suggests biological effects of the highly reactive radical peroxynitrite. In fact, the peroxynitrite scavenger urate (1 mM) showed an additive effect on fluorescent signal inhibition, as well as on endothelial apoptosis rate reduction. We thus propose that platelet-derived exosomes may be another class of actors in the complex play known as vascular redox signaling. In this sense, an exosome-based approach can provide novel tools for further understanding and even treating vascular dysfunction related to sepsis

A sepse após a injúria térmica é a maior causa de morbidade e mortalidade em pacientes queimados, uma vez que profundas alterações ocorrem na farmacocinética de agentes antimicrobianos. Investigaram-se trinta e um pacientes, portadores de sepse documentada e apresentando lesões ativas; utilizou-se o tratamento empírico conforme seguem os regimes de dose: 1 g, 12/12 h para a vancomicina, 1 g, 6/6 h para o imipenem e 2 g, 8/8 h para o cefepime. Sete coletas seriadas de sangue foram realizadas através de cateter venoso (2 mL/cada); o plasma foi obtido pela centrifugação e armazenado no congelador (-80o C) até o ensaio. A concentração plasmática dos antimicrobianos foi determinada simultaneamente pela aplicação do método bioanalítico desenvolvido no estudo. O método de cromatografia líquida de alta eficiência demonstrou boa linearidade, precisão e exatidão para a determinação simultânea da vancomicina, cefepime e imipenem plasmáticos; a plicação desse método bioanalítico permitiu o monitoramento plasmático terapêutico e o estudo farmacocinético. Com base nos resultados obtidos de concentração plasmática versus tempo, aplicou-se a modelagem para investigar a farmacocinética desses agentes antimicrobianos nos pacientes queimados. Os parâmetros cinéticos foram estimados com base no modelo aberto de um compartimento pela aplicação do programa PK Solutions v. 2.0; a estatística foi realizada pela utilização do programa GraphPad Prism v. 4.0. Com base na farmacocinética alterada, as concentrações obtidas para a vancomicina e imipenem se mostraram abaixo dos valores recomendados para atingir eficácia; por outro lado, as concentrações obtidas para o cefepime se mostraram dentro da faixa recomendada para atingir eficácia, uma vez que não se registrou alteração da farmacocinética deste antimicrobiano nos pacientes queimados. Desta forma, o monitoramento plasmático terapeutico se mostrou importante, permitindo o ajuste de dose para a vancomicina e para o imipenem, uma vez que a concentração minima efetiva (CME) não foi atingida para ambos pela utilização do regime de dose empírica nos pacientes queimados. Por outro lado, o monitoramento do cefepime plasmático também se mostrou de relevância, uma vez que os pacientes queimados com longa permanência na terapia intensiva podem apresentar disfunção renal em alguma fase da internação; consequentemente, a individualização de dose será recomendada para esses pacientes. Adicionalmente, investigou-se a disposição cinética da vancomicina em nove pacientes queimados após duas diferentes intervenções cirúrgicas. Comparou-se a farmacocinética da vancomicina pós-desbridamento versus pos-enxerto com base no monitoramento plasmático após o regime de dose empírica (1 g, 12/12 h). Após multiplas infusões, o vale da vancomicina plasmática foi obtido pela coleta de sangue imediatamente antes da infusão subsequente e está relacionado ao acúmulo no estado de equilíbrio. Em conseqüência da depuração aumentada e meia-vida biológica reduzida pós-desbridamento comparado ao pós-enxerto, registrou-se para a vancomicina vale abaixo da concentração efetiva mínima nos pacientes queimados. Finalmente, os resultados obtidos no presente estudo permitem concluir que a farmacocinética da vancomicina e do imipenem está alterada nos pacientes queimados com sepse, e recomenda-se o monitoramento das concentrações plasmáticas para garantir a eficácia de forma a previnir a emergência bacteriana. / Sepsis after thermal injury is the major cause of morbidity and mortality in burn patients, once deep changes on the pharmacokinetics of antimicrobials agents are expected. Thirty one burn patients were investigated, all of them had documented sepsis and presented active lesions; they were treated with empirical dose regimen as follows: 1 g, 12/12 h for vancomycin, 1 g, 6/6 h for imipenem and 2 g, 8/8 h for cefepime. A serial of seven blood samples were collected from the venous catheter (2 mL/each); plasma was obtained by centrifugation and storaged in an ultra-low freezer (-80o C) until assay. Drug plasma concentration was determined simultaneously by application of a bioanalytical method described previously. High performance liquid chromatographic method showed good linearity, precision and accuracy for vancomycin, cefepime and imipenem plasma measurements; its application permitted therapeutic drug monitoring and pharmacokinetic studies. Pharmacokinetic modeling was applied to data obtained based on drug plasma concentrations versus time, to investigate those antimicrobial agents in burn patients. Estimated kinetic parameters were based on the one compartment open model by application the software PK Solutions v. 2.0; statistics was performed by using the software GraphPad Prism v. 4.0. Based on altered pharmacokinetics, obtained plasma concentrations to reach drug efficacy were below the recommended values for vancomycin and imipenem; on the other hand, cefepime plasma concentrations to reach drug efficacy were in the recommended range, once its pharmacokinetics didnt change in burn patients. Then, therapeutic plasma monitoring was cost-effective permitting dose adjustment for vancomycin and imipenem, once the minimum effective concentration (MEC) wasnt reached for both antimicrobial agents by using the empirical dose regimen for burn patients. On the other hand, cefepime plasma monitoring was also cost-effective, since burn patients long term therapy can present renal dysfunction at the minimum one period in the intensive care unit; consequently, dose adjustment could be required for them. Additionally, vancomycin kinetic disposition was investigated in nine burn patients after two different surgical interventions. Vancomycin pharmacokinetics post-debridement versus post-skin grafting procedure was compared based on drug plasma monitoring by using the empirical dose regimen (1 g, 12/12 h). Trough vancomycin plasma level after multiple infusions, obtained by blood collection before de next dose, is related to drug accumulation at the steady state level; then, trough below the minimum effective concentration (MEC) were obtained after both surgical procedures performed in burn patients. Meanwhile, increased plasma clearance and reduced biological half-life were obtained after debridement compared skin grafting procedure. Finally, data obtained in the present study permit to conclude that the pharmacokinetics is altered for vancomycin and imipenem in burn patients with sepsis, and drug plasma monitoring is recommended to guarantee drug efficacy and to prevent the bacterial emergency.