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Avaliação da atividade de enzimas que degradam nucleotídeos de adenina e do perfil oxidativo em ratos com sepse induzida / Evaluation activity enzymes that degrade adenine nucleotides and oxidative profile in rats with induced sepsisBertoncheli, Claudia de Mello 30 July 2014 (has links)
Conselho Nacional de Desenvolvimento Científico e Tecnológico / Sepsis is recognized as a systemic inflammatory response (SIRS) to infection with the
presence of progressive tissue damage, where multiple organ failure is the most severe
expression. The purinergic signaling plays an important role in inflammatory response
modulation as well as in immune responses through its extracellular biomolecules, such
as adenine nucleotides and its derivative nucleoside adenosine. These signalling
molecules are released by cells in response to damage or cellular stimuli induced by
pathogens. Adenine nucleotides and adenosine effects are promoted by activation of
specific purinergic receptors controlled by an enzymatic cascade on cell surface. In
addition to immunologic changes, oxidative stress has an important part in sepsis
pathophysiology, contributing to its deleterious systemic effects such as tissue hypoxia
and organ failure. This study aims to evaluate the activity of the E-NTPDase, which
degrade adenine nucleotides in lymphocytes, as well as analyse the oxidative profile in
brain, heart, liver and kidney in rats submitted to experimental sepsis. The sepsis was
induced by cecal ligation and puncture (CLP). The evaluation of the effects of sepsis on
E-NTPDase activity in lymphocytes, as well as on the oxidative stress parameters in
different tissues, was divided into two stages. On the first stage, the animals were split
into two groups: (1) negative controls and (2) septic, which evaluated the activity of the
E-NTPDase and histological analysis of the kidneys, liver and lung. On the second
stage, the animals were divided into three groups: (1) negative controls, (2) sham e (3)
septic. An increase in ATP hydrolysis was observed in sepsis-induced rats when
compared to the control group. Nevertheless, the E-NTPDase activity remained
unchanged when ADP was applied as substrate. Histological analyses of kidneys, liver and lung have shown vascular congestion, necrosis and inflammatory mononuclear cell
infiltration when compared to control group. Regarding the antioxidant activity, no
difference was observed in the NPSH content and SOD activity in the organs analysed.
Concerning the oxidative stress parameters, the carbonyl protein content showed no
significant difference in brain and liver, whilst in heart and kidney a decrease was
observed in the septic group. No significant difference in TBARS levels was observed in
brain, however an increase was observed in kidney while a decrease was observed in
heart and liver. E. coli was identified as the etiological agent. On the septic group,
significant hematological changes such as leucocytosis and thrombocytopenia were
observed. This reduction in TBARS levels in heart and liver does not agree with data on
the literature; this may be due to the employment of different induction techniques
among studies, added to altered neutrophil function and and also the characteristics
inherent to the pathogenic microorganism. Our findings suggest that the increase in ATP
hydrolysis in induced sepsis may be a dynamic response in order to eliminate the
increased ATP levels resulting from cell death. Regarding the oxidative stress
parameters, the reduction in heart and liver may be due differences in the sepsis
induction of CLP model by between different research groups added to altered
neutrophil function and and also the characteristics inherent to the pathogenic
microorganism. / A sepse pode ser definida como síndrome da resposta inflamatória sistêmica
(SIRS) decorrente da reação do sistema imunológico à infecção, denotando um
processo progressivo de dano tecidual, onde a disfunção múltipla dos órgãos é a
expressão mais grave. O sistema de sinalização purinérgica desempenha um papel
importante na modulação das respostas inflamatórias e imunes através de biomoléculas
extracelulares, como os nucleotídeos de adenina e seu derivado nucleosídeo
adenosina. Essas moléculas sinalizadoras são liberadas do meio intracelular em
resposta ao dano ou ao estímulo celular por ação de patógenos. Os efeitos dos
nucleotídeos de adenina e da adenosina são promovidos através da ativação de
receptores purinérgicos específicos e controlados por uma cascata enzimática
localizada na superfície das células. Além das alterações imunes, o estresse oxidativo
desempenha um importante papel na patofisiologia desta doença, contribuindo para os
principais efeitos sistêmicos deletérios da sepse como a hipóxia tecidual e a falência de
órgãos. Sendo assim, visando à melhor compreensão desta patologia, este trabalho
teve por objetivo avaliar a atividade da enzima E-NTPDase em linfócitos bem como
analisar o perfil oxidativo em cérebro, coração, fígado e rins em ratos submetidos à
sepse experimental utilizando a técnica de ligação e perfuração do ceco (CLP). Os
procedimentos para avaliação dos efeitos da sepse sob a atividade das enzimas ENTPDase
em linfócitos e sob os parâmetros de estresse oxidativo nos tecidos foram
divididos em duas etapas. Na primeira etapa, os animais foram divididos em dois
grupos: (1) controle negativo e (2) séptico, onde se avaliou a atividade da E-NTPDase e a análise histológica dos rins, fígado e pulmão. Para a segunda etapa, os animais foram
divididos em 3 grupos: (1) controle negativo, (2) Sham e (3) séptico, na qual se
determinou os parâmetros de estresse oxidativo, bem como o perfil bacteriano e
hematológico. Observou-se um aumento na hidrólise de ATP em ratos com sepse
induzida quando comparado ao grupo controle. Contudo, a atividade da E-NTPDase
não foi alterada, quando utilizado ADP como substrato. Pelas análises histológicas de
rins, fígado e pulmão verificou-se no grupo séptico a presença de congestão vascular,
necrose e infiltrado inflamatório mononuclear quando comparados com o grupo
controle. Em relação à atividade antioxidante não se observou diferença significativa no
conteúdo de NPSH e na atividade da SOD em nenhum órgão analisado. Quanto aos
parâmetros do estresse oxidativo, o teor de proteína carbonil não apresentou diferença
significativa no cérebro e no fígado enquanto nos tecidos cardíaco e renal observou-se
um decréscimo no grupo séptico em relação aos demais grupos. Não foi observada
nenhuma diferença significativa nos níveis de TBARS no cérebro, no entanto, estes
níveis estavam reduzidos no coração e no fígado e aumentados no tecido renal. Foi
identificado como agente etiológico da sepse E. coli. Observou-se significativas
alterações hematológicas no grupo séptico como: leucocitose e trombocitopenia. Com o
presente trabalho conclui-se que, na sepse induzida, o aumento da hidrólise do ATP
seja provavelmente consequência de uma resposta dinâmica para reduzir os níveis
elevados de ATP resultantes da morte celular. Já a redução nos parâmetros de
estresse oxidativo no coração e no fígado pode ter ocorrido devido a diferenças na
indução da sepse pelo modelo CLP entre os diferentes grupos de pesquisa, adicionado
à função dos neutrófilos alterados e também às características inerentes do tipo de
micro-organismo patogênico.
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Effects of Vasoflux on DNA-Histone Complexes in Vitro and on Organ Function and Survival Outcome in a Murine Model of SepsisSharma, Neha January 2018 (has links)
Sepsis is life-threatening organ dysfunction produced by a dysregulated host response to infection in which neutrophils release neutrophil extracellular traps (NETs). NETs consist of DNA, histones, and antimicrobial peptides which kill pathogens. However, DNA and histones also exert damage by activating the intrinsic pathway of coagulation and inducing endothelial cell death, respectively. AADH, a 15kDa non-anticoagulant unfractionated heparin (UFH), prevents histone-mediated cytotoxicity in vitro and improves survival in septic mice. We explored the effectiveness of Vasoflux, a 5.5kDa low-molecular-weight-heparin as an anti-sepsis treatment as compared to enoxaparin and UFH. Vasoflux has reduced anticoagulant functions and hence reduces the risk of bleeding as compared to enoxaparin or UFH. We showed that UFH, enoxaparin, or Vasoflux at concentrations of up to 13.3uM, 40uM, or 40uM, neutralize histone-mediated cytotoxicity. These results suggest that these glycosaminoglycans (GAGs) are able to neutralize histone-mediated cytotoxicity independent of the AT-binding pentasaccharide. To quantitate the binding affinity between GAGs and histones, surface plasmon resonance was conducted. UFH is a more potent inhibitor of histone-mediated cytotoxicity compared to Vasoflux as UFH has a 10-fold greater binding affinity to histones compared to Vasoflux. To translate our in vitro findings to in vivo, Vasoflux, enoxaparin, and UFH were administered in a murine model of sepsis. Vasoflux at 8mg/kg - 50mg/kg reduced survival and exhibited damage in the lung, liver, and kidney in septic mice compared to 10 mg/kg of UFH or 8mg/kg of enoxaparin. This may be due to Vasoflux and UFH disrupting the DNA-histone complex, thereby releasing free procoagulant DNA. This is evident by our gel electrophoresis experiments, where addition of 1uM Vasoflux or 3.3uM UFH to DNA-histone complexes lead to histone dissociation from DNA. UFH bound to histones may be able to inhibit DNA-mediated thrombin generation, as it retains its anticoagulant properties, demonstrated by UFH-histone complexes attenuating DNA and TF-mediated thrombin generation. In contrast, Vasoflux may not neutralize the procoagulant DNA leading to a hypercoagulable state in the mice. Our study may have important clinical implications as there is an ongoing trial, HALO, which will administer intravenous UFH to patients suspected to have septic shock to reduce mortality. Based on our results, future clinical trials should consider the antithrombin-dependent anticoagulant activity of UFH being used as a sepsis treatment. / Thesis / Master of Science (MSc) / Sepsis is a life threatening condition caused by the body’s extreme response to microbial infection of the blood, whereby neutrophils release traps composed of cell-free DNA (cfDNA), histones, and antimicrobial proteins. In addition to fighting off infections, these traps also exert harmful effects like triggering clotting and killing host cells. Currently, no specific anti-septic drugs exist. Studies have shown that DNase1 (a recombinant protein that digests double stranded cfDNA) or a modified form of heparin (neutralizes histones) improves survival in septic mice. Our goal was to explore the protective effects of Vasoflux, (a non-anticoagulant heparin) and DNase1 in a mouse model of sepsis. We hypothesize that the combined therapy of DNase1 and Vasoflux will improve survival. We found that Vasoflux has minimal blood thinning activity and can prevent histones from killing cells. However, Vasoflux administered into septic mice worsened organ damage and decreased survival. We hypothesize that this damage may be due to Vasoflux’s ability to displace histones from histone-DNA complexes, thereby releasing free DNA, which promotes excessive blood clotting in sepsis.
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DEVELOPMENT OF A LIPOPOLYSACCHARIDE ANTAGONIST FOR THE TREATMENT OF SEPSISSimseok Yuk (9173015) 10 September 2022 (has links)
<p>Sepsis and septic shock are life-threating conditions, which
resulted from a continuum of the body’s response to overwhelming infection.
Elimination of bacteria through antibiotics is not sufficient, because the host
is still left with a large amount of lipopolysaccharide (LPS) that prevents the
host immune system from returning to normal homeostasis. Synthetic LPS antagonists that
can bind to LPS via electrostatic and/or hydrophobic interactions cause systemic toxicities. Moreover, LPS elimination alone may not address already
established complications of sepsis. To address these
challenges, we propose to develop nanoparticle formulations of LPS antagonists (D-TZP)
that can be delivered systemically. Specifically, cholecalciferol (vitamin D) was encapsulated in a self-assembly of
tannic acid/Fe<sup>3+</sup> coordination complex (pTA) capsule, forming a core
that could be surface-modified with LPS
adsorbents, such as low molecular weight succinylated chitosan (LMZWC) and polymyxin B (PMB). D-TZP suppressed pro-inflammatory effects of LPS on the
engineered human monocytes with significantly less cytotoxicity than free PMB
at the equivalent dose. D-TZP increased the maximum tolerated dose of PMB by
both intraperitoneal and intravenous administration. In the LPS-induced mouse model
of sepsis, systemic administration of D-TZP immediately after LPS challenge
neutralized the lethal effect of LPS. D-TZP also reduced the mortality of mice when given 2 h after the LPS challenge.
D-TZP inhibited the mortality in the cecal
ligation and puncture (CLP)-induced bacteremia mouse model when given IV 2 h after the insult. In
the CLP model, the D-TZP-treated animals also showed lower levels of both TNF-α
and IL-10 cytokines as well as D-dimer levels, reflecting the attenuation of disseminated
intravascular coagulation, compared to the vehicle-treated control group.
Collectively, these results support that the D-TZP is a safe and effective
systemic intervention of sepsis.<br></p>
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Papel do receptor toll-like 9 na falência de migração dos neutrófilos na sepse / The role of toll-like receptor 9 on failure of neutrophil migration during sepsis.Trevelin, Silvia Cellone 20 December 2010 (has links)
O recrutamento de neutrófilos para o sítio da infecção é um evento crucial para o combate aos microrganismos e sobrevivência na sepse. A migração destes polimorfonucleares é dirigida através de um gradiente quimiotático por meio do reconhecimento de quimiocinas por receptores acoplados a proteína G (GPCRs), os quais são regulados por quinases específicas (GRKs). Estudos prévios demonstraram que na sepse ocorre uma falência na migração de neutrófilos para o foco infeccioso em função da dessensibilização de receptores quimiotáticos via GRKs induzida pela ativação de receptores toll-like (TLRs), TLR2 e TLR4. Apesar de a ausência de TLR9 em células dendriticas ter sido relacionada a maior sobrevivência de camundongos sépticos, o papel do TLR9 atuando diretamente em neutrófilos não foi avaliado. Objetivando preencher esta lacuna, propôs-se avaliar o papel direto de TLR9 na falência de migração de neutrófilos na sepse. Os camundongos TLR9-/- apresentaram maior sobrevivência a sepse polimicrobiana avaliada por meio do modelo de ligadura e perfuração do ceco (CLP). A deficiência de TLR9 também acarretou em aumento na migração de neutrófilos para o foco da infecção, menor seqüestro de neutrófilos no pulmão, bem como, menor número de bactérias no lavado peritoneal e sangue. A ativação de TLR9 por oligodeoxinucleotídeo contendo o dinucleotídeo CpG não metilado (ODN CpG) nos neutrófilos reduziu a quimiotaxia destes em direção a quimiocina CXCL2 e expressão do receptor quimiotático CXCR2. Além disso, neutrófilos estimulados com ODN CpG apresentaram aumento na expressão da quinase tipo 2 relacionada a receptores acoplados a proteína G (GRK2). Dessa forma, a ativação de TLR9 em neutrófilos circulantes no sangue é prejudicial na sepse por reduzir a quimiotaxia destes para o foco da infecção ao induzir a dessensibilização de CXCR2 via GRK2. / The recruitment of neutrophils to the site of infection is a crucial event for combating the microorganisms and survival on sepsis. The neutrophil migration is directed by a chemotactic gradient through the recognition of chemokines by G protein-coupled receptors (GPCRs), which are regulated by specific kinases (GRKs). Previous studies have shown a failure of neutrophil migration into infectious focus on sepsis due to chemotactic receptor desensitization via GRKs induced by activation of toll- like receptors (TLRs), TLR2 and TLR4. Despite the absence of activation of TLR9 in dendritic cells have been related to increase survival of septic mice, the role of TLR9 acting directly on neutrophils was not evaluated. We proposed to verify the direct role of TLR9 in the failure of neutrophil migration on sepsis. The TLR9 knockout mice (TLR9-/-) showed high survival to polymicrobial sepsis using cecal ligation and puncture model (CLP). TLR9-/- mice had high neutrophil migration to the focus of infection, low neutrophil sequestration in the lung, as well as, few bacteria in the peritoneal exudates and blood. The activation of TLR9 by oligodeoxinucleotide containing unmethylated dinucleotide CpG (CpG ODN) in neutrophils also reduced chemotaxis toward CXCL2 and the expression of chemokine receptor CXCR2. In addition, neutrophils stimulated with CpG ODN showed increased expression of kinase-related G protein-coupled receptor type 2 (GRK2). Thus, the activation of TLR9 in blood circulating neutrophils is harmful on sepsis by reducing their chemotaxis into the site of the infection by inducing CXCR2 desensitization via GRK2.
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Papel do receptor toll-like 9 na falência de migração dos neutrófilos na sepse / The role of toll-like receptor 9 on failure of neutrophil migration during sepsis.Silvia Cellone Trevelin 20 December 2010 (has links)
O recrutamento de neutrófilos para o sítio da infecção é um evento crucial para o combate aos microrganismos e sobrevivência na sepse. A migração destes polimorfonucleares é dirigida através de um gradiente quimiotático por meio do reconhecimento de quimiocinas por receptores acoplados a proteína G (GPCRs), os quais são regulados por quinases específicas (GRKs). Estudos prévios demonstraram que na sepse ocorre uma falência na migração de neutrófilos para o foco infeccioso em função da dessensibilização de receptores quimiotáticos via GRKs induzida pela ativação de receptores toll-like (TLRs), TLR2 e TLR4. Apesar de a ausência de TLR9 em células dendriticas ter sido relacionada a maior sobrevivência de camundongos sépticos, o papel do TLR9 atuando diretamente em neutrófilos não foi avaliado. Objetivando preencher esta lacuna, propôs-se avaliar o papel direto de TLR9 na falência de migração de neutrófilos na sepse. Os camundongos TLR9-/- apresentaram maior sobrevivência a sepse polimicrobiana avaliada por meio do modelo de ligadura e perfuração do ceco (CLP). A deficiência de TLR9 também acarretou em aumento na migração de neutrófilos para o foco da infecção, menor seqüestro de neutrófilos no pulmão, bem como, menor número de bactérias no lavado peritoneal e sangue. A ativação de TLR9 por oligodeoxinucleotídeo contendo o dinucleotídeo CpG não metilado (ODN CpG) nos neutrófilos reduziu a quimiotaxia destes em direção a quimiocina CXCL2 e expressão do receptor quimiotático CXCR2. Além disso, neutrófilos estimulados com ODN CpG apresentaram aumento na expressão da quinase tipo 2 relacionada a receptores acoplados a proteína G (GRK2). Dessa forma, a ativação de TLR9 em neutrófilos circulantes no sangue é prejudicial na sepse por reduzir a quimiotaxia destes para o foco da infecção ao induzir a dessensibilização de CXCR2 via GRK2. / The recruitment of neutrophils to the site of infection is a crucial event for combating the microorganisms and survival on sepsis. The neutrophil migration is directed by a chemotactic gradient through the recognition of chemokines by G protein-coupled receptors (GPCRs), which are regulated by specific kinases (GRKs). Previous studies have shown a failure of neutrophil migration into infectious focus on sepsis due to chemotactic receptor desensitization via GRKs induced by activation of toll- like receptors (TLRs), TLR2 and TLR4. Despite the absence of activation of TLR9 in dendritic cells have been related to increase survival of septic mice, the role of TLR9 acting directly on neutrophils was not evaluated. We proposed to verify the direct role of TLR9 in the failure of neutrophil migration on sepsis. The TLR9 knockout mice (TLR9-/-) showed high survival to polymicrobial sepsis using cecal ligation and puncture model (CLP). TLR9-/- mice had high neutrophil migration to the focus of infection, low neutrophil sequestration in the lung, as well as, few bacteria in the peritoneal exudates and blood. The activation of TLR9 by oligodeoxinucleotide containing unmethylated dinucleotide CpG (CpG ODN) in neutrophils also reduced chemotaxis toward CXCL2 and the expression of chemokine receptor CXCR2. In addition, neutrophils stimulated with CpG ODN showed increased expression of kinase-related G protein-coupled receptor type 2 (GRK2). Thus, the activation of TLR9 in blood circulating neutrophils is harmful on sepsis by reducing their chemotaxis into the site of the infection by inducing CXCR2 desensitization via GRK2.
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