Introdução: A polineuromiopatia do doente crítico (PNMDC) é uma freqüente complicação neuromuscular adquirida na Unidade de Terapia Intensiva (UTI). O principal fator de risco está relacionado à sepse e à disfunção de múltiplos órgãos sendo que sua incidência pode variar entre 50 a 100%. A confirmação do diagnóstico é feita pela eletroneuromiografia, porém sugere-se que as características clínicas (fraqueza muscular, alterações sensoriais e de reflexos e dificuldade de desmame da ventilação mecânica) possam ser suficientes para o diagnóstico clínico. Objetivo: Identificar sinais clínicos que possam ser utilizados como marcadores para auxiliar no diagnóstico de PNMDC à beira do leito em pacientes sépticos. Resultados: Cinquenta pacientes foram avaliados e divididos em dois grupos conforme o resultado da eletroneuromiografia (PNMDC e não PNMDC). Os pacientes com PNMDC tinham idade maior e eram mais graves (APACHE IV e SAPS 3), permaneceram maior período hospitalizados e necessitaram de suporte ventilatório por mais tempo. Os pontos de corte: <40 para escore Medical Research Council (MRC); <7 kg para dinamometria em homens e <4 kg em mulheres; <34 cmH2O para pressão expiratória máxima e >-40 cmH2O para pressão inspiratória máxima conseguiram identificar com bons níveis de sensibilidade e de especificidade os pacientes com PNMDC. Conclusões: A força muscular esquelética pelo escore MRC, dinamometria e pressões respiratórias máximas é uma método simples que permite a identificação de PNMDC em uma avaliação clínica à beira do leito. / Introduction: Critical illness polyneuromyopathy (CIPNM) is a frequent acquired neuromuscular complication in the Intensive Care Units (ICU). The main risk factor is related to sepsis and multiple organs dysfunction and the incidence of this disorder can reach 50-100%. The diagnosis is made by electromyography, but it is suggested that the clinical features (muscle weakness, sensory and reflexes changes and difficulty in weaning patients from mechanical ventilation) may be sufficient for clinical diagnosis. Objective: To identify clinical signs that may be used as markers to help in the bedside diagnosis of CIPNM in septic patients. Results: Fifty patients were evaluated and divided into two groups according to the results of electromyography (CIPNM and non-CIPNM). The patients with CIPNM were older, showed more severe illness (Apache IV and Saps 3), remained hospitalized for longer period of time, and required longer period of ventilatory support than non-CIPNM. The cutoffs that could identify the patients with CIPNM with good levels of sensitivity and specificity were: Medical Research Council (MRC) score <40; dynamometry <7 kg for men and <4 kg for women, maximal expiratory pressure (MEP) <34 cmH2O and maximal inspiratory pressure (MIP) > -40 cmH2O . Conclusions: The assessment of skeletal muscle strength by MRC score, dynamometry and maximum respiratory pressure is a simple method that allows the diagnosis of CIPNM through a clinical examination at the bedside.

A sepse é causada por microorganismos tais como: bactérias Gram-negativas, Gram-positivas, fungos e vírus. A sepse grave e o choque séptico estão associados a taxas de mortalidade de 40 a 60%. O evento mais importante para a evolução do quadro séptico é a apoptose das células efetoras do sistema imune. A eliminação de um grande número de células do sistema imune compromete a defesa efetiva do hospedeiro. Para estudarmos o papel das células imunes na sepse utilizamos o modelo de tolerância ao LPS. Em nossos estudos induzimos tolerância ao LPS em camundongos Balb-C e analisamos os padrões da expressão gênica nos linfócitos do baço. Para o mapeamento dos genes, utilizamos o macroarray, identificamos o grupo funcional de genes que tem maior relevância na proteção induzida pela tolerância, e em seguida confirmamos os resultados encontrados através do RT-PCR, Western Blotting, atividade de caspase 1 e citometria de fluxo. Encontramos uma importante redução na expressão de genes, como heat shock proteins, óxido nítrico e apoptose. Após análise da membrana contendo os genes integradores da resposta produzida pela tolerância, optamos por enfatizar inicialmente os genes envolvidos no processo apoptótico devido à relevância deste processo no quadro séptico conforme mostraram os trabalhos encontrados na literatura. Demonstramos que animais tolerantes ao LPS apresentam diminuição dos eventos apoptóticos, com redução na expressão dos genes ligados às caspases 7, 8 e 11, assim como a redução dos genes ligados ao Bid e Apaf-1. Ao analisarmos o RNAm através do RT-PCR, encontramos redução na Caspase 3, Bax e Bcl2, resultado que se confirmou ao analisarmos a expressão protéica através do Western- Blotting. Realizamos citometria de fluxo para avaliarmos a ocerrência de apoptose nos linfócitos dos animais submetidos à ligadura cecal. Confirmamos uma redução da apoptose e necrose em linfócitos dos animais tolerantes em relação aos controles. Com estes resultados podemos propor que a tolerância seria benéfica na redução da apoptose e controle do quadro séptico, além disso, a diminuição na expressão do gene ligado à caspase 11 estaria contribuindo para a diminuição do quadro inflamatório, pois a caspase 11 é um mediador essencial na resposta ao choque séptico. / Sepses are caused by microorganisms such as: Gram-negative bacteria, Gram-positive bacteria, fungus and virus. Several sepses and the septic shock have been associated with the mortality rates from 40 to 60%. One of the most important events for the septic evolution is the apoptosis cells of the immune system. The cells immune system elimination compromises the host. We induced LPS tolerance in Balb-C mice and analyzed genes expressions in spleens; we found an important reduction in the genes expressions like: heat shock proteins, nitric oxide and apoptosis. After analysis of the membrane containing the sepses genes produced by tolerance, we emphasized initially the genes involved in the apoptosis. Demonstrated that animals LPS tolerants presented decrease in apoptotic events, with reduction in the genes expression related caspases 7, 8, 11, Bid and Apaf-1. In the mRNA by RT-PCR, we found a reduction in Caspase 3, Bax and Bcl2, and these results were confirmed by Western-blot. We realized flow cytometry and we showed that the results are maintained, presenting reduction in both the apoptotic and necrotic events in tolerants animals. These results showed that the tolerance would be favorable in the apoptosis reduction and control of the sepsis, moreover, the expression reduction to caspase 11 genes would be contributing to the inflammatory reduction because Caspase 11 is an essential mediator in the septic shock.

A sepse é a principal causa de morte em unidades de terapia intensiva (UTIs) no mundo. A reduzida disponibilidade do aminoácido mais abundante do organismo, a glutamina contribui para o complicado estado catabólico da sepse. No presente estudo investigamos os efeitos da suplementação oral com L-glutamina e L-alanina (GLN+ALA), ambos na norma livre e como dipeptídeo, L-alanil-L-glutamina (DIP), sobre o eixo glutamina-glutationa (GSH), sistema imune, inflamação, proteínas de choque térmico (HSPs) e expressão de genes envolvidos com vias de sinalização proteica em animais endotoxêmicos. Camundongos C57/B6 foram submetidos à endotoxemia (Escherichia coli LPS, 5 mg.kg-1, grupo LPS) e suplementados por 48 horas com L-glutamina (1 g.kg-1) e L-alanina (0,61 g.kg-1, grupo GLN+ALA-LPS) ou 1,49 g.kg-1 de DIP (grupo DIP-LPS). A endotoxemia promoveu depleção da concentração de glutamina no plasma (71%), músculo esquelético (50%) e fígado (49%), quando comparado ao grupo CTRL, sendo restauradas nos grupos DIP-LPS e GLN+ALA-LPS (P<0,05), fato que atenuou a redução da GSH e o estado redox (taxa GSSG/GSH) em eritrócitos circulantes, musculo e fígado (P<0,05). A suplementação em animais endotoxêmicos resultou em uma upregulation dos genes GSR, GPX1 e GCLC no músculo e fígado. A concentração das citocinas plasmáticasTNF-&#945;, IL-6, IL-1&#946; e IL-10 foi atenuada pelas suplementações, bem como a expressão de mRNAs envolvidos com a resposta inflamatória, ativadas pela via do NF-&#954;B(P<0,05). Concomitantemente, verificou-se aumento da capacidade proliferativa de linfócitos T e B circulantes nos grupos GLN+ALA-LPS e DIP-LPS. A expressão de mRNAs e a concentração de HSPs no tecido muscular foi restabelecida pelas suplementações, contudo, a expressão mRNAs relacionados às vias de síntese e degradação proteica foi somente estimulada no tecido hepático(P<0,05). Os resultados do presente estudo demonstram que a suplementação por via oral com GLN+ALA ou DIP podem ser utilizados clinicamente como métodos nutricionais em reverter o quadro de depressão da disponibilidade de glutamina corporal da sepse induzida por LPS, tendo impacto no eixo glutamina-glutationa, sistema imune e inflamatório. / Sepsis is the leading cause of death inintensive care units (ICUs) in the world.The availability ofthe most abundant amino acid in the body, glutamine, is reduced in this situation, fact that contribute to the complicated catabolic state of sepsis. In the present study, we investigated the effects of oral supplementation with L-glutamine and L-alanine (GLN+ALA), both in their free form and as a dipeptide, L-alanyl-L-glutamine (DIP) on glutamine-glutathione axis (GSH), immune and inflammatory system, heat shock proteins (HSPs) expression and gene expressions involved in protein signaling pathways during endotoxemia. C57/B6 mice were subjected to endotoxemia (Escherichia coli LPS, 5 mg.kg-1, LPS group) and supplemented for 48 hours with L-glutamine (1 g.kg-1) plus L-alanine(0.61 g.kg-1, GLN+ALA-LPS group) or 1.49 g.kg-1of DIP (DIP-LPS group). Endotoxemia promoted depletion glutamine concentration in plasma (71%), skeletal muscle (50%) and liver (49%), when compared to the CTRL group, and was restored in the DIP-LPS e GLN+ALA-LPS (P<0.05), fact that attenuate the reduction of GSH and the redox state (GSSG/GSH rate) in circulating erythrocytes, liver and muscle (P<0.05). Supplementations in endotoxemic mice resulted in upregulation of GSR, GCLC and GPX1 genes in muscle and liver. Plasma concentration of TNF-&#945;, IL-6, IL-1&#946; and IL-10 were attenuated by supplementation as well as the expression of mRNAs involved in the inflammatory response, activated by NF&#954;-B pathway (P <0.05). At the same time, high proliferative capacity of circulating T and B lymphocytes GLN+ALA-LPS e DIP-LPS were observed. HSPs (protein and mRNAs) and in muscle were restored by the supplements, however, the mRNAs expression related to the synthesis and degradation of protein pathways was only stimulated in the liver (P <0.05). Our results demonstrate that oral supplementation with GLN+ALA or DIP can be used as clinically nutritional methods to reverse the depression of body glutamine availability during sepsis induced by LPS, impacting on the glutamine-glutathione axis, immune and inflammatory system.

Sympathetic nerves are important for renal physiology and sepsis pathophysiology. A recent study showed sprouting of sympathetic nerves in spleen of septic mice. This study was done to test if renal sprouting of sympathetic nerves also happens and to investigate renal morphology in septic mice. Cecal ligation and puncture (CLP) was used to induce sepsis and kidneys were removed for evaluation. Bowman’s space was diminished with cortical bubble cells present suggestive of acute renal pathology, however, renal function was unchanged. Acute sepsis did not affect either renal sympathetic innervation or non-neuronal cholinergic cells. Mouse kidneys had more epinephrine (EPI) than norepinephrine (NE) in both groups. This is most likely due to uptake of epinephrine by renal sympathetic nerves and may have no correlation with sepsis. In conclusion, septic mice showed minor renal pathology and no evidence of acute sympathetic nerve sprouting. Further studies are needed to understand the mechanism and consequences of elevated EPI in mice kidney.

Sepsis is a severe life-threatening critical illness that damages multiple physiological systems. After hospital discharge, more than 70% of severe sepsis survivors report profound weakness which significantly impacts quality of life. Such weakness gives rise to new limitations of daily living, which ultimately leads to loss of independence in many patients. Despite wide recognition of this serious issue by clinicians and researchers alike, the mechanisms contributing to chronic skeletal muscle dysfunction after sepsis are not well understood. Lack of progress in this field is largely due to the absence of an appropriate animal model; current models are either too mild to induce muscle weakness or too severe and cause death within a few days. As such, this dissertation work first focused on establishing a clinically-relevant animal model of sepsis which yields surviving mice with chronic skeletal muscle weakness (Aim 1). This aim involved refining the cecal slurry injection model of polymicrobial sepsis in young adult animals, as well as optimizing the timing, duration, and dose of multiple therapeutic agents. The resulting resuscitation protocol was adapted for use in late-middle-aged animals, and muscle strength was evaluated using an ex vivo system which confirmed significant muscle weakness in sepsis survivors, long after sepsis was resolved. Next, using this novel model, we sought to characterize sepsis-induced long-term muscle dysfunction at the molecular level (Aim 2). The first set of experiments under this aim was designed to identify the primary global mechanism(s) (i.e. atrophy, polyneuropathy, and/or myopathy) responsible for muscle weakness in sepsis survivors. Analysis of the force-frequency curves and specific force measurements led to the conclusion that myopathy is the primary cause. Electron micrograph observation, functional assays, and protein analysis then showed that sepsis survivors’ skeletal muscles are characterized by profound mitochondrial abnormalities and oxidative damage. Collectively, these studies demonstrate that long-term muscle weakness is apparent in sepsis-surviving animals, and the functional decline is associated with unresolved mitochondrial damage and dysfunction. This work suggests that medical treatments beyond targeting muscle wasting alone could allow sepsis survivors to regain function and return to productive lives.

The metabolic pathways required for adenosine triphosphate (ATP) production within the cell are well understood, however recent publications suggest that metabolic pathways are closely linked to immune cell activation and inflammatory diseases. There has been little examination of the metabolic pathways that modulate mast cell activation and the feedback regulator lactic acid. Here we examine metabolic pathways and regulation within mast cells in the context of lipopolysaccharide (LPS) and interleukin (IL-33) activation, for which there has been little to no reported studies. First, we examine the effects of lactic acid, previously considered only a by-product of glycolysis and now understood to act as a negative feedback regulator of inflammation in the context of LPS activation and sepsis. Lactic acid is elevated in septic patients and associated with mortality, potentially due to suppressive effects on LPS signaling and contribution to late phase immunosuppression. By attenuating glycolysis and reducing ATP availability for signaling and cytokine transcription, lactic acid impairs the function of immune cells to fight the initial or subsequent infections. We support this with in vitro and in vivo data. Additionally, our lab has published that lactic acid can suppress IL-33 activation, potentially by metabolic modulation as with LPS activation; however there has been no study of the metabolic requirements for IL-33 activation. We report here that glycolysis is required for ATP and reactive oxygen species (ROS) production to augment signaling and cytokine production downstream of the IL-33 receptor. Together, these studies examine the contribution of metabolism to mast cell activation and may provide potential targets for treatments of diseases that involve LPS- or IL-33-dependent mast cell activation.

Beobachtung nosokomialer Infektionen bei intensivhospitalisierten Patienten im Rahmen einer einjährigen prospektiven, multizentrischen Kohortenstudie. Besondere Beachtung von Komplikationen wie Sepsis und Peritonitis sowie des Auftretens multiresistenter Keime bei der Infektentstehung. / Surveillance of nosocomial ICU-acquired infections in intensive care unit patients in a 1-year prospective multicenter cohort study. Monitoring of complications like sepsis and peritonitis and acquisition of multi-resistant infectious agents.

Uma das alterações comuns na sepse são os distúrbios microcirculatórios podem ocorrer mesmo com parâmetros macro hemodinâmicos normais. Vários protocolos são indicados para a anestesia de paciente em sepse. Um fármaco geralmente utilizado em pacientes críticos em humanos é a dexmedetomidina, um potente e seletivo alfa 2 agonista com propriedade sedativas, analgésicas e relaxante muscular que promove aumento de pressão arterial e vasoconstrição periférica. O objetivo do presente estudo é avaliar comparativamente a infusão contínua da dexmedetomidina versus fentanil em cadelas sépticas no que diz respeito a três parâmetros relacionados a microcirculação, parâmetros hemodinâmicos e metabólicos. Foram avaliadas 33 cadelas com piometra submetidas à cirurgia terapêutica de OSH, e triadas pelo score quick SOFA. Os animais foram randomizados em duplo estudo de infusão contínua de 3 &micro;g/kg/hora de dexmedetomidina ou 5 &micro;g/kg/hora de fentanil, durante anestesia com isofluorano e em ventilaçao mecânica. Parâmetros hemodinâmicos, microcirculatórios, ventilatórios e metabólicos foram utilizados para comparação entre grupos. Esses foram coletados antes da indução anestésica, durante e pós a anestesia. Os dados foram submetidos a teste de normalidade e variâncias iguais, para então avaliar comparativamente os grupos pelo test t student ou Wilcoxon não pareado quando necessário. Todas as variáveis referentes à microcirculação não apresentaram diferença significativa entre os grupos. A infusão contínua de dexmedetomidina apresentou melhores resultados de pressão arterial e clearance de lactato, sugerindo não comprometer o transporte de oxigênio da periferia e perfusão de órgãos, quando comparado com o uso de fentanil, e ainda com semelhantes desfechos clínicos como mortalidade, tempo de extubação e ocorrências de hipotensão ou bradiarritmia. Os valores semelhantes de microcirculação e superiores de pressão arterial demonstram que a dexmedetomidina não compromete a microcirculação em relação ao fentanil, mas mantém a macrohemodinâmica com valores superiores. / One of the common changes in sepsis is microcirculatory disorders can occur even with normal hemodynamic macro parameters. Several protocols are indicated for sepsis anesthesia. A drug commonly used in critically ill patients in humans is dexmedetomidine, a potent and selective alpha 2 agonist with proprietary sedatives, analgesics and muscle relaxant that promotes increased blood pressure and peripheral vasoconstriction. The objective of the present study is to compare the continuous infusion of dexmedetomidine versus fentanyl in septic dogs using three parameters related to microcirculation, hemodynamic parameters and metabolic parameters. Thirty - three bitches with pyometra submitted to OSH therapeutic surgery, and triaged by the quick SOFA score, were evaluated. The animals were randomized into a double study using continuous rate infusion of 3 &micro;g/kg/h dexmedetomidine or 5 &micro;g/kg/h of fentanyl, during isoflurane anesthesia and under mechanical ventilation. Hemodynamic, microcirculatory, ventilatory and metabolic parameters were used for comparison between groups. These were collected prior to anesthetic induction, during and after anesthesia. The data were submitted to normality test and the same variances, and then comparatively evaluate the groups by the unpaired Wilcoxon test t student or when necessary. All variables related to microcirculation did not present significant difference between the groups. The continuous infusion of dexmedetomidine presented better blood pressure and lactate clearance results, suggesting that it did not compromise peripheral oxygen transport and organ perfusion when compared to fentanyl, and with similar clinical outcomes such as mortality, extubation time and occurrences of hypotension or bradyarrhythmia. Similar values of microcirculation and higher blood pressure demonstrate that dexmedetomidine does not compromise microcirculation over fentanyl, but maintains macrohemodynamics with higher values.

Les plaquettes jouent un rôle majeur dans l'hémostase primaire ainsi que dans l'inflammation. Elles contiennent et sécrètent une grande variété de facteurs solubles et parmi les nombreux récepteurs qu'elles expriment à leur surface, les plaquettes expriment les " Toll-Like Receptor " (TLR), récepteurs clés de l'interaction entre l'immunité innée et adaptative. En réponse à un stimulus infectieux, comme le lipopolysaccharide (LPS) des bactéries Gram-négative, ligand naturel du TLR4, ou des peptides issus d'une partie de la protéine d'enveloppe du VIH (gp41), les plaquettes vont s'activer de manière différentielle. L'activation plaquettaire est variable en fonction de leur activation par à un stimulus hémostatique (exemple : la thrombine) vs. infectieux (exemple : le LPS) ; le panel de cytokines libérées dans le surnageant plaquettaire semble en fait finement régulé. De plus, nous avons démontré la présence intra-plaquettaire de la majorité des protéines composant les voies de signalisation du TLR4 eucaryote. Nous avons ensuite montré que ces voies pouvaient être modulées. L'engagement du TLR4 plaquettaire par deux types biochimiques de LPS entraîne un relargage différentiel des facteurs solubles immunomodulateurs dans le surnageant de culture et que ce surnageant dernier génère une activation différentielle des cellules cibles, comme les cellules mononucléées du sang circulant. Ces travaux montrent que la réponse inflammatoire plaquettaire est régulée en fonction du stimulus. Ainsi, mes travaux s'inscrivent dans la ré-exploration de la fonction inflammatoire des plaquettes sanguines et l'étude du rôle des plaquettes comme cellules de l'immunité innée et inflammatoire

<p>Septic shock is frequently seen in intensive care units and is associated with significant mortality. Endotoxin – a major mediator of the pathophysiologic responses – is released during lysis of Gram-negative bacteria. These responses can be mimicked in the endotoxaemic pig.</p><p>This thesis focuses on the following topics: the inflammatory and pathophysiological responses to various endotoxin doses and infusion patterns; covariations between endotoxin induced inflammatory and pathophysiological responses; whether the biological effects of endotoxin can be modulated by clopidogrel and whether tobramycin or ceftazidime reduce plasma cytokine levels.</p><p>Endotoxin induced linear log-log cytokine and F2-isoprostane responses. Leukocyte and platelet responses, pulmonary compliance, circulatory variables as well as indicators of plasma leakage and hypoperfusion exhibited log-linear responses to the endotoxin dose. Biological responses to endotoxaemia such as inflammation, hypotension, hypoperfusion and organ dysfunction were more expressed when the organism was exposed to endotoxin at a higher rate. These results may facilitate the possibility to choose relevant endotoxin administration, when experiments are set up in order to evaluate certain responses to endotoxaemia.</p><p>Correlation studies between cytokines, leukocytes, platelets and the endotoxin dose were in agreement with the well-known ability of endotoxin to induce cytokine expression and to activate both primary haemostasis and leukocytes. Free radical mediated lipid peroxidation and COX-mediated inflammation correlated to cytokine expression and organ dysfunction in endotoxaemic shock. </p><p>Endotoxaemic pigs pretreated with clopidogrel, exhibited a trend towards less expressed deterioration of renal function, although blocking of ADP-induced primary haemostasis is not a key mediator of endotoxin induced deterioration of renal function.</p><p>Tobramycin did not neutralise the biological effects of endotoxin or the plasma levels of endotoxin, suggesting that these antibiotics do not bind to endotoxin.</p><p>Reduction in IL-6 was greater in pigs treated with ceftazidime and tobramycin as compared with those given saline, indicating a possible anti-inflammatory effect of both antibiotics.</p>