A tolerância ou pré-condicionamento é uma estratégia de defesa do hospedeiro que reduz o impacto negativo da infecção na saúde. Diminui a susceptibilidade a danos teciduais, causados pelos patógenos ou pela resposta imune. Um desbalanço no fluido e pressão osmótica intra e extracelular causa o estresse osmótico e liberação de mediadores inflamatórios. A solução salina hipertônica (SSH) tem sido utilizada na ressuscitação volêmica, mostrando-se benéfica em pacientes com danos cerebrais e pulmonares (3), melhora a contratilidade e perfusão microvascular. Utilizamos esta solução como possível agente indutor de pre-condicionamento ou tolerância. A SSH em doses crescentes de 4, 6 e 8 ml/kg levou a diminuição da mortalidade em camundongos endotoxêmicos o mesmo ocorrendo com outras soluções hiperosmóticas. Analisamos o padrão do perfil de produção de citocinas pró e anti-inflamatórias em períodos de 2 e 4 horas pós SSH e pós LPS. No estudo sistêmico do pré-condicionamento os resultados revelaram uma diminuição da resposta inflamatória causada pelo LPS nas dosagens plasmáticas, o mesmo ocorrendo nos tecidos estudados. Concluímos que o pré-condicionamento com SSH é promissor como um prétratamento na endotoxemia / The tolerance or preconditioning is a host defense strategy able to reduce the infection negative effect in health. Decrease the tissue damage susceptibility caused by the imune response. A fluid imbalance and osmotic pressure intra and extracellular cause the osmotic stress and inflammatory mediators release. The hypertonic saline solution (SSH) has been used in fluid resuscitation, with good results in patients with brain and lung damage, it improves microvascular perfusion and contractility. We used this solution as a possible induct agent of preconditioning or tolerance. The Hypertonic saline solution in increasing doses of 4,6 and 8ml/kg was able to stablish a decrease in endotoxemic mice mortality, the same happened with other hyperosmotic solutions. We analyzed the pattern profile of inflammatory and anti-inflammatory cytokines in a period of two and four hours after SSH. In the systemic study of the precondicioning, the results showed a decrease in the inflammatory response caused by LPS in the plasmatic dosage, the same was observed in the studied tissues. We concluded that the preconditioning with SSH is a promising pre-treatment in endotoxemia

Made available in DSpace on 2014-06-11T19:25:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-28Bitstream added on 2014-06-13T18:53:48Z : No. of bitstreams: 1 carrenho_lca_me_araca.pdf: 1197310 bytes, checksum: 3d5470911a8b6fe2c7996a8ec61a673a (MD5) / A endotoxemia é um importante distúrbio sistêmico que se origina da resposta do hospedeiro a um componente das bactérias Gram-negativas, o lipopolissacarídeo (LPS) ou endotoxina, que é liberado após bacteriólise ou rápida multiplicação. A ativação do sistema imune inato pelo LPS é um fator chave para o disparo da resposta inflamatória pelo hospedeiro e que acarreta a produção de mediadores inflamatórios, responsáveis pelos eventos patológicos da endotoxemia. A interação dos receptores Toll-like (TLRs) com antígenos específicos deflagram a resposta inflamatória, sendo que o receptor Toll-like-4 (TLR-4) é ativado pela ação das endotoxinas, enquanto o receptor Toll-like-2 (TLR-2) interage com uma variedade de componentes microbianos. Uma exposição prévia a baixas concentrações de LPS pode tornar os cavalos “tolerantes” a um desafio letal subsequente, acarretando uma diminuição na produção de citocinas inflamatórias por um período transitório. Pouco se sabe a respeito do mecanismo celular deste fenômeno em equinos, supondo-se o envolvimento dos receptores Toll-like semelhante ao encontrado em outras espécies. Com este estudo investigaram-se os mecanismos celulares da tolerância à endotoxina em um modelo ex vivo com sangue total. Foi demonstrado redução na síntese de citocinas pró-inflamatórias (TNF-α, IL-1 e IL-6), aumento da expressão gênica da citocina anti-inflamatória IL-10, e ausência de expressão do TGF-β, após o desafio secundário com LPS. A maior expressão dos receptores TLR-2 e -4 após o segundo estímulo de LPS demonstrou que a tolerância à endotoxina não acarreta diminuição da expressão de ambos os receptores em equinos. / Endotoxemia is an important systemic disease originated from host response to a component of Gram-negative bacteria, lipopolysaccharide (LPS) or endotoxin, which is released after bacteria death or quick replication. The innate immune recognition of LPS has a key role triggering host inflammatory answer and is due to inflammatory mediator’s synthesis, which are responsible for pathologic events of endotoxemia. Signs initiated by interaction of Toll-like receptors (TLRs) with specific products induce the inflammatory response. Toll-like receptor-4 (TLR- 4) is activated by endotoxin action while Toll-like receptor-2 (TLR-2) interacts with a range of microbial compounds. Some studies demonstrate that both can act like LPS receptors, although by independent pathways. It was demonstrated that a previous exposition to low concentrations of LPS can render horses “tolerant” to a lethal subsequent challenge with endotoxin, leading to a diminished release of inflammatory cytokines during a transient period. However, little is known about the cellular mechanisms involved in this phenomenon in horses, suspecting that there is involvement of cell surface receptors, similarly to other species. This study investigated cellular mechanisms of endotoxin tolerance in a whole blood ex vivo model, demonstrating a reduction on pro-inflammatory cytokines synthesis (TNF- α, IL-1 and IL-6), increased gene expression of anti-inflammatory cytokine IL-10 and no alteration in TGF-β expression, after a secondary stimulus with LPS. The Toll-like receptors-2 and -4 increased expression after a second stimulus with LPS showed that endotoxin tolerance does not lead to a decreased expression of both receptors in horses.

Pacientes sépticos podem evoluir para choque séptico, destes 40% sobrevivem. Caracterizamos o modelo experimental, avaliamos fatores envolvidos na inflamação e avaliamos a modulação causada por purinas (ATP/ADP) na quantificação de superóxido (O2-) e na reatividade vascular da aorta isolada. Os resultados sugerem que na aorta isolada de animais endotoxêmicos, ATP e ADP aumentam a síntese de óxido nítrico (NO), porém somente o ATP reduz a biodisponibilidade de O2-, provavelmente pelo reacoplamento da NO sintase endotelial / Septic patients can evolve for septic shock and 40% of these survive. We characterize the experimental model we evaluate involved factors in the inflammation and evaluate the modulation caused by purines (ATP/ADP) in the superoxide quantification (O2-) and in the vascular reactivity of isolated aorta. The results suggest that in isolated aorta of endotoxemics rats, ATP and ADP increase the endothelial nitric oxide synthase (NOS) however just ATP reduces the bio availability of O2-, probably for the re-couples of the endothelial NOS synthase

Orientador: Marcio Alberto Torsoni / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T22:25:41Z (GMT). No. of bitstreams: 1 Santos_GustavoAparecidodos_M.pdf: 1529206 bytes, checksum: e9aae5229598f14274aea766dccadceb (MD5) Previous issue date: 2013 / Resumo: A hipoglicemia endotóxica tem papel importante na sobrevivência de ratos e pacientes com sepse. O hipotálamo e uma importante área no sistema nervoso central que regula a homeostase energética. Estudos recentes têm demonstrado à importante papel da proteína quinase ativada por AMP (AMPK) no controle da homeostase glicêmica, sendo um importante sensor energético ativado quando a uma diminuição de disponibilidade energética como é observado na hipoglicemia, e por controlar importantes estímulos que visão restabelecer a glicemia como a modulação da produção de glicose hepática. Até o momento não existem estudos que investiguem o papel da AMPK hipotalâmica na endotoxemia e na hipoglicemia associada a essa enfermidade. Por isso o objetivo do nosso estudo foi o de avaliar a participação das citocinas inflamatórias e da AMPK hipotalâmica na redução da resposta contrarregulatória hepática durante a endotoxemia promovida por LPS. Para isso foram utilizados camundongos (Swiss) e camundongos com mutação no receptor TLR4 (C3H/HeJ) e seus controles selvagens (Wild type (C3H/HeN)) ambos os animais receberam LPS (1mg/Kg) ou salina intraperitonealmente ou/e AICAR por via intracerebroventricular e após os tempos pré-estabelecidos obtivemos os resultados que mostram que a administração periférica de LPS induz o aumento nas citocinas TNF? e IL?, ocorreu a desfosforilação da AMPK hipotalâmica e menor fosforilação da sua proteína alvo a ACC se comparado ao grupo controle; desfosforilação da AMPK hepática e ACC hepática, aumento da fosforilação da STAT3 hipotalâmica e hepática, aumento da fosforilação a TAK1 hipotalâmica e quantidade da MyD88 hipotalâmica proteínas da via do TLR4 se comparado ao grupo controle, também foi observado aumento na fosforilação da JNK hepática, redução da ingestão alimentar, redução da glicemia basal e um possível aumento na captação de glicose observada pelo teste de tolerância à glicose nos animais que receberam LPS, também foi observado à diminuição da quantidade e expressão da proteína PEPCK e diminuição da G6Pase resultado que corroborou o resultado da diminuição da produção de glicose hepática observada no teste de tolerância ao piruvato (PTT). A redução na glicemia nos animais nos gerou uma hipótese de que esse efeito pode ter a participação da insulina onde observamos um significativo aumento nos níveis séricos deste hormônio, a ativação farmacológica da AMPK hipotalâmica reduziu o efeito hipoglicemiante do LPS, onde também observamos que a ativação farmacológica da AMPK inibiu a diminuição das proteínas PEPCK e G6Pase deflagrado pelo LPS sugerindo que a um controle hipotalâmico através da AMPK sobre a secreção de glicose. Nos animais mutantes do receptor TLR4 (C3H/HeJ) não houve diminuição da glicemia e desfosforilação da AMPK hipotalâmica e diminuição da PEPCK hepática como observamos nos animais Wild type (C3H/HeN). Mostrando que o receptor TLR4 possui uma atividade intrínseca na indução à hipoglicemia e desfosforilação da AMPK. Os dados do nosso trabalho mostram que a endotoxemia ocasionada pela administração de LPS exerce uma importante modulação da ação da AMPK hipotalâmica, modulação negativa da ingestão e glicemia plasmática, alem de inibir a produção de glicose e expressão e quantidades de proteínas neoglicogênicas e ativação de vias inflamatórias mostrando que a sepse e/ou endotoxemia por LPS leva a severos danos na homeostase glicêmica, contudo também mostramos que a ativação da AMPK hipotalâmica consegue minimizar os efeitos oriundos da endotoxemia, mostrando que mecanismos que atuem sobre a ativação da AMPK hipotalâmica podem contribuir para o tratamento da Sepse/endotoxemia / Abstract: Endotoxic hypoglycaemia has an important role in the survival rates of septic patients. Currently, the hypothalamus is the main area of the brain that regulates glycemic homeostasis. Previous studies have demonstrated that hypothalamic AMP-activated protein kinase (AMPK) activity is sufficient for nutrient-sensing mechanisms to modulate glucose production. However, the role of hypothalamic AMPK in hypoglycaemia associated with endotoxemia is unknown. The aims of this study were to examine hypothalamic AMPK dephosphorylation in lipopolysaccharide (LPS) treated mice and to determine whether pharmacological AMPK activation could reduce the effects of endotoxemia on the liver metabolism of glucose. Fasted Swiss mice and C3H/HeJ (TLR4-receptor mutant) and C3H/HeN (wild type) mices received intraperitoneal injections of LPS (1mg/kg). LPS-treated mice showed reduced food intake and diminished basal glycemia, increased serum TNF? and IL1? levels and hypothalamic p-TAK and TLR4/MyD88 association. These effects were accompanied by hypothalamic AMPK/ACC dephosphorylation and reduction of glucose production in the liver. Interestingly, the LPS treated mice liver also showed diminished expression of PEPCK/G6Pase and reduction in p-FOXO1, p-AMPK, p- STAT3 and p-JNK level. In contrast, the pharmacological hypothalamic AMPK activation blocked the effects of LPS on the hypothalamic AMPK phosphorylation, liver PEPCK expression and glucose production. Furthermore, the effects of LPS were TLR4-dependent because no effect on hypothalamic AMPK phosphorylation, liver PEPCK expression and basal glycemia was detected in C3H/HeJ (TLR4- receptor mutant) mice. These results suggest that hypothalamic AMPK activity may be an important pharmacological target to control glucose homeostasis during endotoxemia / Mestrado / Clinica Medica / Mestre em Ciências

It is well established that continual consumption of a diet high in fat leads to the development of chronic conditions such as obesity, cardio metabolic syndrome, and atherosclerosis that are associated with high incidence of cardiovascular disease. Recent studies have identified endotoxin-derived inflammation as a major diving force for the development of these conditions. Our laboratory has recently demonstrated that consumption of a single high-fat meal results in acute postprandial endotoxemia and alters monocyte cell surface adhesion molecule expression and scavenger receptor CD36 expression. These collective projects describe our efforts to understand the physiological significance of these postprandial changes and if supplementation with spore-based probiotics are able to provide any form of protection against these responses that are associated with the onset of atherogenesis.

Dysfunction of Mitochondrial Respiratory Chain in Rostral Ventrolateral Medulla During Experimental Endotoxemia Sepsis is a complex pathophysiologic state resulting from an exaggerated whole-body inflammatory response to infection or injury. Metabolic disturbances, abnormal regulation of blood flow and diminished utilization of oxygen at the cellular level may account for tissue damage and lead to multiple organ failure and death. As the primary site of cellular energy generation is the mitochondrion, it presents itself as an important target for the septic cascade. In this regard, the notion that bioenergetic failure due to mitochondrial dysfunction contributes to organ failure during sepsis has received attention. We established the low frequency fluctuations in the systemic arterial pressure signals are related to the sympathetic neurogenic vasomotor tone, and reflect the functional integrity of the brain stem. Their origin is subsequently traced to the premotor sympathetic neurons at the rostral ventrolateral medulla (RVLM), whose neuronal activity is intimately related to the ¡§life-and-death¡¨ process. Based on a rat model of experimental endotoxemia that provides continuous information on changes in neuronal activity in the RVLM, the present study was undertaken to evaluate whether changes in mitochondrial respiratory functions are associated with death arising from sepsis. We also evaluated the efficacy of a new water-soluble coenzyme Q10 (CoQ10, ubiquinone) formula in the protection against fatality during endotoxemia by microinjection into bilateral RVLM. Dysfunction of Mitochondrial Respiratory Chain in Rostral Ventrolateral Medulla During Experimental Endotoxemia in the Rat We investigated the functional changes in mitochondrial respiratory chain at the RVLM in an experimental model of endotoxemia that mimics systemic inflammatory response syndrome. Experiments were carried out in adult male Sprague-Dawley rats that were maintained under propofol anesthesia. Intravenous administration of E. coli lipopolysaccharide (LPS; 30 mg/kg) induced progressive hypotension, with death ensued within 4 hours. The sequence of cardiovascular events during this LPS-induced endotoxemia can be divided into a reduction (Phase I), followed by an augmentation (Phase II; ¡§pro-life¡¨ phase) and a secondary decrease (Phase III; ¡§pro-death¡¨ phase) in the power density of the vasomotor components (0-0.8 Hz) of systemic arterial pressure (SAP) signals. Enzyme assay revealed significant decrease of the activity of NADH cytochrome c reductase (Complex I+III) and cytochrome c oxidase (Complex IV) in the RVLM during all 3 phases of endotoxemia. On the other hand, the activity of succinate cytochrome c reductase (Complex II+III) remained unaltered. Neuroprotective Effects of Coenzyme Q10 at Rostral ventrolateral Medulla Against Fatality During Experimental Endotoxemia in the Rat CoQ10 is a highly mobile electron carrier in the mitochondrial respiratory chain that also acts as an antioxidant. We evaluated the neuroprotective efficacy of CoQ10 against fatality in an experimental model of endotoxemia, using a novel water-soluble formulation of this quinone derivative. In Sprague-Dawley rats maintained under propofol anesthesia, intravenous administration of E. coli LPS (30 mg/kg) induced experimental endotoxemia. Pretreatment by microinjection bilaterally of CoQ10 (1 or 2 mg) into RVLM significantly diminished mortality, prolonged survival time, and reduced the slope or magnitude of the LPS-induced hypotension. CoQ10 pretreatment also significantly prolonged the duration of Phase II endotoxemia and augmented the total power density of the vasomotor components of SAP signals in Phase II endotoxemia. The increase in superoxide anion production induced by LPS at the RVLM during Phases II and III endotoxemia was also significantly blunted. Conclusion The present study revealed that selective dysfunction of respiratory enzyme Complexes I and IV in the mitochondrial respiratory chain at the RVLM is closely associated with fatal endotoxemia. CoQ10 provides neuroprotection against fatality during endotoxemia by acting on the RVLM. We further found that a reduction in superoxide anion produced during endotoxemia at the RVLM may be one of the mechanisms that underlie the elicited neuroprotection of CoQ10. These findings therefore open a new direction for future development of therapeutic strategy in this critical, complicated and highly fatal condition known as sepsis.

Laminitis is a crippling disease of horses that can result in chronic lameness and debilitation, and sometimes warrants euthanasia. It is a complication of inflammatory conditions such as gastrointestinal disease, and also occurs in obese, insulin-resistant horses with Equine Metabolic Syndrome (EMS). Inflammation and insulin resistance are risk factors for laminitis, and these mechanisms might converge to induce laminitis in susceptible animals. Systemic inflammation is often attributed to endotoxemia, although circulating endotoxin concentrations are not commonly measured in the clinical setting. Although a theoretic basis exists for endotoxemia in the pathogenesis of laminitis, administration of endotoxin alone does not induce the condition. This could be related to differences between experimental models and naturally occurring disease. Studies presented in this dissertation address the overall hypothesis that systemic inflammation causes laminitis and new experimental models can be developed to better represent clinical disease. Associations between systemic inflammation and laminitis were first established by measuring blood inflammatory cytokine expression during a laminitis induction model. A clinically relevant endotoxin model that induced laminitis was then sought, but endotoxin administration alone was insufficient to cause laminitis and endotoxin tolerance developed. Endotoxemia was therefore evaluated in conjunction with predisposing factors such as obesity. In horses with EMS, endotoxin infusion caused exaggerated inflammatory responses, and derangements in glucose homeostasis were more pronounced. Laminitis, however, did not develop. Repeated inflammatory events are implicated in the pathogenesis of sepsis-associated organ failure, so a final study was performed to test whether preexisting endotoxemia increased the risk of laminitis during subsequent carbohydrate overload-induced systemic inflammation. This did not occur, however systemic inflammation was more pronounced in horses that developed laminitis compared to non-responders, and tissues rather than circulating leukocytes appeared to be the major source of inflammatory mediators. Our results do not support a role for endotoxin as the causal agent of laminitis, even when combined with predisposing factors. Tissues appear to be an important source of inflammatory mediators, therefore their role in laminitis should be further characterized. Additionally, future investigations should determine whether exaggerated inflammatory responses and loss of glycemic control increase the risk of laminitis in horses with EMS.

Diss. (sammanfattning) Linköping : Univ., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.