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narmq2@hotmail.com / Objetivo: La inflamación sistémica empeora los trastornos circulatorios en el paciente cirrótico y recientemente el síndrome de respuesta inflamatoria sistémica (SRIS) podría ser un indicador pronóstico en ellos. El objetivo del estudio fue determinar si la presencia de SRIS al ingreso en pacientes cirróticos hospitalizados está asociada a complicaciones o mortalidad. Materiales y métodos: Estudio de cohortes retrospectiva, realizado en el Hospital Nacional Daniel Alcides Carrión. Se admitieron pacientes cirróticos hospitalizados desde julio 2008 hasta diciembre 2010 sin comorbilidades importantes, neoplasia maligna, infección VIH, o estancia fue menor a 72 horas. Se evaluó presencia de SRIS al ingreso y la aparición de complicaciones o muerte después de 72 horas del ingreso. Resultados: Fueron 150 pacientes cirróticos admitidos, se excluyeron 6, tres por supervivencia menor a las 72 horas, uno por neoplasia, uno por insuficiencia cardiaca severa y dos por insuficiencia renal crónica. En total 144 pacientes ingresaron al estudio, 95 (66%) pacientes presentaron SRIS al ingreso. No hubo diferencia significativa en cuanto a edad, sexo, etiología, en ambos grupos. SRIS estuvo asociado a mayores puntajes de MELD y Child-Pugh Turcotte. De los pacientes con SRIS, 41 (43%) se complicaron y 16 (16,8%) fallecieron, mientras que del grupo sin SRIS 5 (10,2%) se complicaron y 2 (4%) fallecieron , ( p <0,0001y p =0,028 respectivamente). Las complicaciones más frecuentes fueron las infecciones y encefalopatía hepática. En el análisis multivariado SRIS estuvo asociado a complicaciones ( p <0,006) mas no a mortalidad ( p <0,276). Conclusiones: SRIS es frecuente en pacientes cirróticos hospitalizados y está asociado a complicaciones intrahospitalarias. / Objective: The systemic inflammation worsens circulatory disorders in cirrhotic patients and recently the systemic inflammatory response syndrome (SIRS) may be a prognostic indicator therein. The aim of the study was to determine whether the presence of SIRS at admission in hospitalized cirrhotic patients is associated with complications or mortality. Materials and methods: A retrospective cohorts study was conducted at the Daniel Alcides Carrion National Hospital. Hospitalized cirrhotic patients admitted from July 2008 to December 2010 without significant comorbidities, malignancy, HIV infection, or stay less than 72 hours were included. Presence of SIRS at admission and the occurrence of complications or death after 72 hours of admission were evaluated. Results: 150 cirrhotic patients were admitted, six were excluded; three for lower survival at 72 hours, one for neoplasia, one for severe heart failure and two for chronic renal failure. One hundred forty four patients were included, 95 (66%) patients had SIRS at admission. There was no significant difference in age, sex, etiology, in both groups. SIRS was associated with higher scores of MELD and Child-Turcotte Pugh. Of the group of patients with SIRS, 41 (43%) had complications and 16 (16.8%) died, while the group without SIRS 5 (10.2%) had complications and two (4%) died ( p <0.0001 and p =0.028 respectively). The most common complications were infections and hepatic encephalopathy. In multivariate analysis SIRS was associated with complications ( p <0.006) but not with mortality ( p <0.276). Conclusions: SIRS is common in hospitalized cirrhotic patients and is associated with in-hospital complications.

Objetivo: La inflamación sistémica empeora los trastornos circulatorios en el paciente cirrótico y recientemente el síndrome de respuesta inflamatoria sistémica (SRIS) podría ser un indicador pronóstico en ellos. El objetivo del estudio fue determinar si la presencia de SRIS al ingreso en pacientes cirróticos hospitalizados está asociada a complicaciones o mortalidad. Materiales y métodos: Estudio de cohortes retrospectiva, realizado en el Hospital Nacional Daniel Alcides Carrión. Se admitieron pacientes cirróticos hospitalizados desde julio 2008 hasta diciembre 2010 sin comorbilidades importantes, neoplasia maligna, infección VIH, o estancia fue menor a 72 horas. Se evaluó presencia de SRIS al ingreso y la aparición de complicaciones o muerte después de 72 horas del ingreso. Resultados: Fueron 150 pacientes cirróticos admitidos, se excluyeron 6, tres por supervivencia menor a las 72 horas, uno por neoplasia, uno por insuficiencia cardiaca severa y dos por insuficiencia renal crónica. En total 144 pacientes ingresaron al estudio, 95 (66%) pacientes presentaron SRIS al ingreso. No hubo diferencia significativa en cuanto a edad, sexo, etiología, en ambos grupos. SRIS estuvo asociado a mayores puntajes de MELD y Child-Pugh Turcotte. De los pacientes con SRIS, 41 (43%) se complicaron y 16 (16,8%) fallecieron, mientras que del grupo sin SRIS 5 (10,2%) se complicaron y 2 (4%) fallecieron, (p<0,0001y p=0,028 respectivamente). Las complicaciones más frecuentes fueron las infecciones y encefalopatía hepática. En el análisis multivariado SRIS estuvo asociado a complicaciones (p<0,006) mas no a mortalidad (p<0,276). Conclusiones: SRIS es frecuente en pacientes cirróticos hospitalizados y está asociado a complicaciones intrahospitalarias. / Objective: The systemic inflammation worsens circulatory disorders in cirrhotic patients and recently the systemic inflammatory response syndrome (SIRS) may be a prognostic indicator therein. The aim of the study was to determine whether the presence of SIRS at admission in hospitalized cirrhotic patients is associated with complications or mortality. Materials and methods: A retrospective cohorts study was conducted at the Daniel Alcides Carrion National Hospital. Hospitalized cirrhotic patients admitted from July 2008 to December 2010 without significant comorbidities, malignancy, HIV infection, or stay less than 72 hours were included. Presence of SIRS at admission and the occurrence of complications or death after 72 hours of admission were evaluated. Results: 150 cirrhotic patients were admitted, six were excluded; three for lower survival at 72 hours, one for neoplasia, one for severe heart failure and two for chronic renal failure. One hundred forty four patients were included, 95 (66%) patients had SIRS at admission. There was no significant difference in age, sex, etiology, in both groups. SIRS was associated with higher scores of MELD and Child-Turcotte Pugh. Of the group of patients with SIRS, 41 (43%) had complications and 16 (16.8%) died, while the group without SIRS 5 (10.2%) had complications and two (4%) died (p<0.0001 and p=0.028 respectively). The most common complications were infections and hepatic encephalopathy. In multivariate analysis SIRS was associated with complications (p<0.006) but not with mortality (p<0.276). Conclusions: SIRS is common in hospitalized cirrhotic patients and is associated with in-hospital complications. Key words: Liver cirrhosis; Systemic inflammatory response syndrome; Complications (source: MeSH NLM).

Early detection of infection, identification of microorganism, and correct choice of antibiotics are critical in the management of sepsis. Quantitative real-time polymerase chain reaction (RT-PCR) has the potential to improve the timeliness, sensitivity, and accuracy of detecting pathogens. In this study we utilize this method to detect the enteric bacterial counts in the blood from patients with systemic inflammatory response syndrome (SIRS) in the emergency department (ED). The universal primers utilized in RT-PCR are specific for 23S ribosomal DNA (rDNA) and wec F gene. The results show that in SIRS patients with positive culture results from specimen collected within 10 days after presenting to ED, and patients surviving for less than 28 days, the serum bacterial DNA load of enteric Gram negative bacilli is higher. In SIRS patients with shock, patients fulfilling both white blood cell counts and respiratory criteria of SIRS, and patients fulfilling both white blood cell counts and respiratory criteria of SIRS with Acute Physiology and Chronic Health Evaluation II score more than 20, the serum bacterial DNA load of enteric Gram negative bacilli and 28-day mortality are both higher. These results suggest that bacterial translocation may happen in patients with SIRS and may be related to higher mortality in patients with SIRS.

There were three objectives of this study. The first was to determine if commercially available fluorochromes could be used to determine the glucocorticoid receptor (GR) density and binding affinity (BA) in equine peripheral blood mononuclear cells. The second was to determine if there was a correlation between elevated plasma cortisol and GR density or binding affinity in healthy adult horses. The third objective was to evaluate the HPA axis in adult horses presenting with systemic inflammatory response syndrome (SIRS), and to determine where any alterations in HPA axis function occur in these patients compared to healthy adults. For the first part of the study, peripheral venous blood was collected from 3 healthy research horses on 3 days. Peripheral blood mononuclear cells were isolated using Ficoll gradient centrifugation. Phycoerythrin (PE)-CD44 was then used to extracellularly label leukocytes, and then an intracellular GR antibody was used to determine a baseline measurement of GR density and fluorescein isothiocyanate (FITC)-dexamethasone was used to determine binding affinity via flow cytometric analysis. Comparison of control samples to those for CD44, GR density, and GR binding affinity showed a statistically significant difference for all samples (P<0.0001, P<0.0001, and P<0.0001 respectively). This showed that the CD44, GR antibody, and FITC-dexamethasone could successfully be used to analyze equine peripheral blood mononuclear cells for GR activity. For the second part of the study, an ACTH stimulation test was performed on 8 healthy horses in order to induce an increase in endogenous cortisol production. Plasma cortisol levels, GR density, and GR binding affinity were measured at baseline, 4, 8, and 24 hours after treatment. Median basal cortisol concentration was 4.9, range 3.2-6.1 μg/dl. This initially increased following ACTH stimulation to 5.6, range 4.8-7.4 μg/dl, then showed a significant decrease by 8 hours post ACTH administration to 1.4, range 1.1-2.7 μg/dl (P=0.0221). No correlation was observed between plasma cortisol concentration in healthy horses and GR density or binding affinity (r=-0.145, P=0.428 and r=0.046, P=0.802, respectively). For the third phase of the study, horses (N=10) with systemic inflammatory response syndrome (SIRS) were compared to healthy, age and sex matched controls (N=10) presenting for lameness evaluation or ophthalmologic examination. Blood was collected from SIRS cases and controls on presentation to the Equine Medical Center. A CBC, serum biochemistry, and serum ACTH and cortisol measurements were performed. GR density and binding affinity were also determined. Nonsurvivors had a significantly decreased GR binding affinity (P=0.008) and demonstrated a trend towards an increase in the ACTH:cortisol ratio. ROC analysis was performed for serum ACTH and cortisol concentrations, the ACTH:cortisol ratio, GR density and GR binding affinity, and triglycerides to determine cut-off values associated with nonsurvival. These were then used to analyze this population using Fischer's exact test to determine the odds ratio (OR) associated with nonsurvival for each variable. This revealed that a serum triglyceride concentration greater than 28.5 mg/dl was associated with nonsurvival (OR=117, 95% CI, 1.94-7060). The other variables were not found to be significantly associated with nonsurvival, although a Delta BA% of less than 35.79% was found to be closely associated with nonsurvival (OR=30.33, 95% CI, 0.96-960.5). Additionally, a significant negative correlation was detected between the plasma ACTH concentration and Delta BA% (r=-0.685, P=0.029) and the ACTH:cortisol ratio and the Delta BA% (r=-0.697, P=0.025). This study showed that nonsurviving horses with SIRS had a significantly decreased GR binding affinity compared to survivors, and a tendency toward an increase in their ACTH:cortisol ratios. This confirms that HPA axis dysfunction occurs in adult horses with SIRS as tissue resistance to glucocorticoids, and potentially relative adrenal insufficiency as well. These results suggest that there are horses with SIRS that might benefit from "physiologic" doses of synthetic glucocorticoids to complement their relative adrenal insufficiency in addition to their poor tissue sensitivity. Further research should focus on methods to more rapidly determine which horses might benefit from treatment with glucocorticoids on presentation, as well as to more accurately determine prognosis for survival. / Master of Science

A Coagulação Intravascular Disseminada (CID) é uma síndrome caracterizada pela ativação sistêmica da coagulação sanguínea, levando à trombose microvascular difusa e podendo comprometer a função de múltiplos órgãos. O acelerado consumo de plaquetas e fatores de coagulação pode, no entanto, dar origem a um estado de hipocoagulabilidade, o que confere à CID uma característica paradoxal na qual o excesso de coagulação pode causar uma diátese hemorrágica. Doenças que levam à Síndrome de Resposta Inflamatória Sistêmica (SIRS) estão entre os principais gatilhos da CID. A pancreatite é uma dessas doenças. O maior desafio para o médico veterinário é diagnosticar a CID na fase precoce, silenciosa e de hipercoagulabilidade, visto que os testes laboratoriais de rotina, como contagem de plaquetas, tempo de protrombina (TP) e tempo de tromboplastina parcial ativada (TTPA), detectam apenas o estado de hipocoaguabilidade, que se estabelece na fase mais avançada da síndrome. Nesse contexto ganham importância os analisadores tromboelastográficos, equipamentos que avaliam a coagulação em sangue total e que, ao menos em tese, podem informar a velocidade de formação do coágulo, a força máxima que ele atinge e os padrões de sua dissolução. Este estudo é o primeiro realizado em cães com o aparelho ReoRox G2 (MediRox), uma da marcas disponíveis no mercado. Limites de referência para as variáveis do aparelho foram definidos a partir da análise do sangue de 49 animais clinicamente saudáveis para três tipos de reação: acelerada com fator tecidual (TF), acelerada com TF e um antagonista de agregação plaquetária (abciximab) e apenas com sangue recalcificado. Em seguida, foram comparados a esse intervalo de referência os valores obtidos pela análise tromboelastográfica do sangue de seis pacientes com pancreatite recém-diagnosticada. Nos três tipos de reação pelo menos 50% dos pacientes do Grupo Pancreatite apresentaram alterações sugestivas de hipercoagulabilidade. A variável MAXELAST (força máxima do coágulo) foi a que esteve alterada com mais frequência entre os animais doentes. Não houve alteração nos marcadores de velocidade de fibrinólise. Estudos prospectivos que associem outras variáveis de trombose, protocolos de tratamento e prognóstico de pacientes com doenças subjacentes que predisponham à CID são necessários para que se possa afirmar que o traçado obtido pela tromboelastografia realmente representa um estado de hipercoagulabilidade in vivo em pacientes com pancreatite. / Disseminated Intravascular Coagulation (DIC) is a syndrome characterized by systemic activation of blood clotting, leading to diffuse microvascular thrombosis and may compromise multiple organ function. The accelerated consumption of platelets and coagulation factors may, however, originate a state of hypocoagulability, which gives the DIC a paradoxical characteristic in which excess coagulation can lead to a hemorrhagic diathesis. Diseases which cause Systemic Inflammatory Response Syndrome (SIRS) are among the major triggers of DIC, including pancreatitis. The greatest challenge for veterinarians is to diagnose DIC in the early, silent and hypercoagulable phase, since routine laboratory tests, such as platelet count, prothrombin time (PT) and activated partial thromboplastin time (APTT), detect only the state of hypocoagulability, which occurs in the most advanced stage of the syndrome. In this context, thromboelastography analyzers stand out. They are equipment which evaluate coagulation in whole blood and, at least in theory, inform the speed of clot formation, its maximum force and how it dissolves. This is the first study performed in dogs with the ReoRox G2 (MediRox), one of the brands available in the market. Limits of reference were defined from blood analysis of 49 healthy animals for three reaction types: accelerated with tissue factor (TF), accelerated with TF and a platelet aggregation antagonist (abciximab) and with only recalcified blood. Next, values obtained by blood thromboelastographic analysis of six patients with newly diagnosed pancreatitis were compared to this reference range. In all three types of reactions, at least 50% of patients in the Pancreatitis Group presented alterations suggestive of hypercoagulability. The variable MAXELAST (maximum clot strength) was the one that was most frequently altered among ill animals. There was no change in fibrinolysis rate markers. Prospective studies associating other thrombosis variables, treatment protocols, and prognosis of patients with underlying diseases predisposing to DIC are necessary to confirm that the pathway obtained by thromboelastography actually represents a state of hypercoaguability in vivo in patients with pancreatitis.

A Coagulação Intravascular Disseminada (CID) é uma síndrome caracterizada pela ativação sistêmica da coagulação sanguínea, levando à trombose microvascular difusa e podendo comprometer a função de múltiplos órgãos. O acelerado consumo de plaquetas e fatores de coagulação pode, no entanto, dar origem a um estado de hipocoagulabilidade, o que confere à CID uma característica paradoxal na qual o excesso de coagulação pode causar uma diátese hemorrágica. Doenças que levam à Síndrome de Resposta Inflamatória Sistêmica (SIRS) estão entre os principais gatilhos da CID. A pancreatite é uma dessas doenças. O maior desafio para o médico veterinário é diagnosticar a CID na fase precoce, silenciosa e de hipercoagulabilidade, visto que os testes laboratoriais de rotina, como contagem de plaquetas, tempo de protrombina (TP) e tempo de tromboplastina parcial ativada (TTPA), detectam apenas o estado de hipocoaguabilidade, que se estabelece na fase mais avançada da síndrome. Nesse contexto ganham importância os analisadores tromboelastográficos, equipamentos que avaliam a coagulação em sangue total e que, ao menos em tese, podem informar a velocidade de formação do coágulo, a força máxima que ele atinge e os padrões de sua dissolução. Este estudo é o primeiro realizado em cães com o aparelho ReoRox G2 (MediRox), uma da marcas disponíveis no mercado. Limites de referência para as variáveis do aparelho foram definidos a partir da análise do sangue de 49 animais clinicamente saudáveis para três tipos de reação: acelerada com fator tecidual (TF), acelerada com TF e um antagonista de agregação plaquetária (abciximab) e apenas com sangue recalcificado. Em seguida, foram comparados a esse intervalo de referência os valores obtidos pela análise tromboelastográfica do sangue de seis pacientes com pancreatite recém-diagnosticada. Nos três tipos de reação pelo menos 50% dos pacientes do Grupo Pancreatite apresentaram alterações sugestivas de hipercoagulabilidade. A variável MAXELAST (força máxima do coágulo) foi a que esteve alterada com mais frequência entre os animais doentes. Não houve alteração nos marcadores de velocidade de fibrinólise. Estudos prospectivos que associem outras variáveis de trombose, protocolos de tratamento e prognóstico de pacientes com doenças subjacentes que predisponham à CID são necessários para que se possa afirmar que o traçado obtido pela tromboelastografia realmente representa um estado de hipercoagulabilidade in vivo em pacientes com pancreatite. / Disseminated Intravascular Coagulation (DIC) is a syndrome characterized by systemic activation of blood clotting, leading to diffuse microvascular thrombosis and may compromise multiple organ function. The accelerated consumption of platelets and coagulation factors may, however, originate a state of hypocoagulability, which gives the DIC a paradoxical characteristic in which excess coagulation can lead to a hemorrhagic diathesis. Diseases which cause Systemic Inflammatory Response Syndrome (SIRS) are among the major triggers of DIC, including pancreatitis. The greatest challenge for veterinarians is to diagnose DIC in the early, silent and hypercoagulable phase, since routine laboratory tests, such as platelet count, prothrombin time (PT) and activated partial thromboplastin time (APTT), detect only the state of hypocoagulability, which occurs in the most advanced stage of the syndrome. In this context, thromboelastography analyzers stand out. They are equipment which evaluate coagulation in whole blood and, at least in theory, inform the speed of clot formation, its maximum force and how it dissolves. This is the first study performed in dogs with the ReoRox G2 (MediRox), one of the brands available in the market. Limits of reference were defined from blood analysis of 49 healthy animals for three reaction types: accelerated with tissue factor (TF), accelerated with TF and a platelet aggregation antagonist (abciximab) and with only recalcified blood. Next, values obtained by blood thromboelastographic analysis of six patients with newly diagnosed pancreatitis were compared to this reference range. In all three types of reactions, at least 50% of patients in the Pancreatitis Group presented alterations suggestive of hypercoagulability. The variable MAXELAST (maximum clot strength) was the one that was most frequently altered among ill animals. There was no change in fibrinolysis rate markers. Prospective studies associating other thrombosis variables, treatment protocols, and prognosis of patients with underlying diseases predisposing to DIC are necessary to confirm that the pathway obtained by thromboelastography actually represents a state of hypercoaguability in vivo in patients with pancreatitis.

Pattern recognition receptors (e.g., S100A12 or S100A8/A9) hold promise as inflammatory biomarkers. We prospectively determined and compared serum S100A12 and S100A8/A9 concentrations in dogs with sepsis (n = 11) or systemic inflammatory response syndrome (SIRS; n = 8) over a 3-d period with each other, healthy controls (n = 50), and other clinical and clinicopathologic variables. Serum S100A12 and S100A8/A9 concentrations were significantly higher in dogs with sepsis or SIRS (all p < 0.05) at the time of hospital admission (day 1) compared to healthy controls, with no differences between patient groups. However, septic dogs had significantly lower serum S100A12 concentrations on day 2 and day 3 (both p < 0.05) compared to dogs with SIRS. Likewise, dogs with sepsis had significantly lower S100A8/A9 concentrations on day 2 (p < 0.05). Neither serum S100A12 nor S100A8/A9 concentrations were associated with survival to discharge. Our results suggest a differential expression of the S100/calgranulins between dogs with sepsis and those with SIRS. Serum S100A12 or S100A8/A9 concentration at the time of hospital admission did not differentiate dogs with sepsis from those with SIRS, but the trend of S100/calgranulin concentrations during the following 24–48 h may be a useful surrogate marker for differentiating sepsis from SIRS.

Introdução: O trauma é um fenômeno que cursa com lesão tecidual, sendo que o trauma cirúrgico (TC) apresenta a referida lesão como consequência de um ato cirúrgico. A isquemia seguida de reperfusão (IR) é um evento comum em várias condições patológicas, bem como em diversos procedimentos cirúrgicos, principalmente transplantes. É frequente o desenvolvimento de lesões teciduais locais e remotas após trauma e após a I/R, parte de um fenômeno conhecido como síndrome da resposta inflamatória sistêmica (SRIS), frequentemente seguida pela falência de múltiplos órgãos (FMO). Estudos provaram o envolvimento do neutrófilo em tais síndromes como resultado da ação de enzimas proteolíticas secretadas a partir de grânulos citoplasmáticos, radicais livres produzidos por explosão respiratória e citocinas liberadas após a infiltração nos tecidos. Nesse contexto, foi provado que o pré-condicionamento isquêmico (PCI), definido como curtos episódios de isquemia precedendo a IR, protege contra essas lesões, com menor ativação de neutrófilos. No entanto, o conhecimento a respeito dos mecanismos operantes nos neutrófilos após o trauma cirúrgico, a isquemia seguida de reperfusão ou o pré-condicionamento isquêmico, ainda são preliminares. Objetivo: Analisar com maior profundidade o impacto dessas condições (TC, IR e PCI) no proteoma e fosfoproteoma do neutrófilo. Métodos: Foi realizada a análise de parâmetros hematológicos juntamente com a análise proteômica e fosfoproteômica de neutrófilos de ratos submetidos a TC, IR e PCI, comparados ao grupo controle. A análise proteômica foi realizada em sistema de nLC-MS/MS orbitrap de alto desempenho, usando marcação com iTRAQ, enriquecimento de fosfopeptídios e pré-fracionamento por HILIC. A análise estatística baseada em clusters utilizando scripts em R mostrou proteínas com abundância relativa diferencial em todas as condições. Resultados: A avaliação dos parâmetros hematológicos antes e depois de TC, IR e IPC demonstrou alterações no número, forma e tamanho de linfócitos, hemácias, plaquetas e, principalmente, neutrófilos (granulócitos). Observou-se um claro aumento na contagem de neutrófilos após TC e IR, sendo que tal aumento foi prevenido pelo PCI. Um total de 393 proteínas foram determinadas como reguladas para abundância relativa entre o grupo controle e o grupo TC. A maioria das proteínas encontradas como reguladas em comum nos grupos TC e IR estão relacionadas à apoptose (caspase-3), motilidade celular (PAK2), transdução de sinal (IL-5, IL-6 e TNF) e degradação pelo sistema proteassoma no neutrófilo. Maior produção de espécies reativas de oxigênio e disfunção da migração direcional de neutrófilos (PKC-delta) com aumento do tempo de vida dos neutrófilos são eventos iniciais importantes que podem resultar em mais dano tecidual e em infecção. A análise proteômica de neutrófilos de ratos após PCI levou à identificação de 2437 grupos de proteínas atribuídos a 5 clusters diferentes, contendo proteínas de abundância relativa significativamente aumentada ou diminuída em IR e PCI. O estudo de vias desses clusters baseado no KEGG revelou aumento nas vias de fagocitose mediada por Fc-gama R, sinalização por quimiocinas, adesão focal e migração transendotelial, citoesqueleto de actina, metabolismo e diminuição nas vias ribossomais, de transporte de RNA, de processamento de proteínas. A regulação da fosforilação de proteínas após IR e PCI mostrou algumas vias como quimiocinas, Fc-gama, GPCR, migração celular e vias pró e antiapoptóticas, sendo que a via de splicing alternativo foi a que apresentou regulação mais evidente (p < 0.0001). A regulação da abundância, bem como da fosforilação, presença de motivos e de domínios levou à identificação de fosfatases, como Fgr, GRK2, PKC delta, ptpn6 e ptprc reguladas por IR, bem como stk38, pkn1, syk e inpp5d reguladas por PCI. A interação mais marcante entre proteínas foi demonstrada como sendo entre os receptores de Fgr e Ptp. Conclusão: Concluímos que as alterações causadas por TC, IR e PCI levaram a intenss alterações na abundância de algumas proteínas e em eventos de fosforilação em neutrófilos, levando ao efeito destrutivo observado após a IR e ao efeito protetor consequente ao PCI / Introduction: Trauma is a phenomenon that involves tissue injury, whereas the surgical trauma (ST) has such injury as a consequence of a surgery. Ischemia reperfusion is common event in many surgical procedures, especially in transplants, as well as in many pathological conditions. Local and remote tissue injuries usually develop after trauma and ischemic reperfusion, part of a phenomenon known as systemic inflammatory response syndrome, frequently followed by multiple organ failure (MOF). Studies have proven the involvement of the neutrophil in all these injuries as a result of proteolytic enzymes secreted from cytoplasmic granules, free radicals produced by respiratory burst, cytokines released after tissue infiltration. In that context, ischemic preconditioning (IPC), that are short episodes of ischemia before ischemia reperfusion, was proved to be protective against these injuries with less activation of neutrophils. However the knowledge about the underlying mechanism operating in the neutrophil after surgical trauma, ischemia reperfusion and preconditioning is preliminary. Objective: To deeply analyze the impact of these conditions (ST, IR and IPC) on the neutrophil proteome and phosphoproteome. Methodology: We did hematological analysis along proteomics and phospho proteomics through high throughput nLC-MS/MS analysis by orbitrap using iTRAQ labeling, phospho peptide enrichments, and HILIC pre-fractionation. Neutrophils from control, ST, IR and IPC conditions after extraction were processed for proteomic analysis. Statistical package using R based on cluster analysis led to the detection of differentially regulated proteins in all conditions. Results: The evaluation of the hematological parameters before and after ST, IR or IPC on blood cells stated alteration in size, number and shape of lymphocytes, RBCs, platelets and specially neutrophils (granulocytes). In the analysis, a clear increase in neutrophil count after ST and IR with such increase prevented by IPC. A total of 393 proteins were found differentially regulated between control and trauma groups. Most of the common proteins found regulated in trauma and IR seem to be related to apoptosis (caspase-3), cell motility (PAK2) and signal transduction in IL5, IL6 and TNF and proteasomal degradation in neutrophil. Higher oxygen species production and dysfunction of directional neutrophil migration (PKC delta) with increase in the life span of neutrophils are early important events that can finally result into more tissue damage and infection. The total proteomic analysis of rat neutrophils after IPC led to the identification of 2437 protein groups assigned to five different clusters with significantly up and downregulated proteins in IR and IPC. Cluster based KEGG pathways analysis revealed up-regulation of chemokine signaling, focal adhesion, leukocyte transendothelial migration, actin cytoskeleton, metabolism and Fc gamma R mediated phagocytosis, whereas downregulation in ribosome, spliceosome, RNA transport, protein processing in endoplasmic reticulum and proteasome, after intestinal ischemic preconditioning. The phosphoregulated proteins containing domains and motifs in the regulated peptides after IR and IPC led to the identification of some of important players such as chemokine, Fc gamma, GPCR, migration and pro/anti-apoptotic pathways. The phosphoproteins from alternative splicing was the pathway presenting the most remarkable regulation with a p-value of 0.0001. The regulation in expression as well as in phosphorylation, the presence of motifs and domains led to the identification of kinases and phosphatases including Fgr, GRK2, PKC delta, ptpn6 and ptprc in neutrophils after IR whereas stk38, pkn1, syk, and inpp5d in neutrophil due to IPC. The highest protein-protein interaction was shown by Fgr and Ptp receptors. Conclusion: We concluded that the changed stimulus produced after ST, IR and IPC led to the huge alteration in proteins expression and phosphorylation events in the neutrophil proteome as mentioned in our work, that leads to final destructive and protective phenotype of neutrophils respectively

O presente estudo tem por objetivo principal avaliar as alterações funcionais precoces de mitocôndrias hepáticas de ratos wistar submetidos ao estímulo de sepse através da técnica de ligadura cecal e punção (cecal ligation and puncture-CLP) e indução de tolerância ao lipopolissacarídeo (LPS) de Escherichia coli. As mitocôndrias exercem papel na alteração do metabolismo celular de pacientes sépticos. Os objetivos do presente trabalho foram: (1) padronizar a técnica de indução a tolerância para ratos wistar com LPS de E. coli (2) avaliar a função mitocondrial fosforilativa e oxidativa; (3) quantificar DNA mitocondrial em tecido hepático de animais submetidos à CPL e tolerância; (4) verificar a expressão dos genes responsáveis pela biogênese mitocondrial e replicação do DNA mitocondrial: nuclear respiratory factor (NRF-1), mitochondrial transcription factor A (TFAM) e peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1alfa); (5) avaliar a função dos complexos respiratórios I e IV. Os resultados encontrados no presente estudo revelaram que: (a) a taxa de mortalidade dos animais submetidos a tolerância foi de 10% quando submetidos à dose letal de LPS, enquanto a taxa de mortalidade dos animais controle foi de 100% quando submetidos à dose letal de LPS; (b) observou-se que o grupo do controle respiratório que recebeu doses controladas de LPS e foi submetido à CLP apresentou razão igual ao grupo Controle, sugerindo que a fosforilação oxidativa se manteve igual ao basal, enquanto o grupo que foi submetido ao procedimento de CLP sem indução a tolerância apresentou piora da razão do controle respiratório em relação ao grupo controle; (c) a quantificação de DNA mitocondrial mostrou-se maior nos animais submetidos a CLP sem prévia indução a tolerância, com igual aumento da expressão dos fatores de biogênese mitocondrial em relação aos demais grupos; (d) houve diferença significativa na avaliação da funcionalidade dos complexo I, porém o complexo IV se manteve igual em todos os grupos. Concluiu-se que a indução a tolerância altera positivamente a função mitocondrial em animais submetidos à CLP / The aim of this study was to evaluate the early functional alterations of hepatic mitochondria of wistar rats submitted to the stimulation of sepsis through the technique of cecal ligation and puncture (CLP) and induction of tolerance to lipopolysaccharide (LPS) of Escherichia coli. Mitochondria play a role in altering the cellular metabolism of septic patients. The objectives of the present study were: (1) to standardize the tolerance induction technique for wistar rats with E. coli LPS (2) to evaluate the mitochondrial phosphorylation and oxidative function; (3) quantify mitochondrial DNA in hepatic tissue of animals submitted to CPL and tolerance; (4) to verify the expression of genes responsible for mitochondria biogenesis and mitochondrial DNA replication nuclear mitochondrial biogenesis (NRF-1), mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1alpha); (5) to evaluate the function of respiratory complexes I and IV. The results found in the present study revealed that: (a) the mortality rate of the animals submitted to tolerance was 10% when submitted to the lethal dose of LPS, whereas the mortality rate of the control animals was 100% when submitted to the lethal dose of LPS; (B) it was observed that the group receiving controlled doses of LPS and submitted to CLP presented a ratio equal to the control group, suggesting that oxidative phosphorylation remained the same at baseline, whereas the group that underwent CLP procedure without induction of tolerance presented worsening of the respiratory control ratio in relation to the control group; (C) the mitochondrial DNA quantification was higher in the animals submitted to CLP without prior tolerance induction, with an equal increase in mitochondrial biogenesis factors expression in relation to the other groups; (D) there was significant difference in the evaluation of the functionality of complexes I, but no difference in complex IV in all groups. It was concluded that induction of tolerance positively alters mitochondrial function in animals submitted to CLP