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.

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.

INTRODUÇÃO: O ozônio (O3) é encontrado na natureza e também pode ser produzido no corpo humano através da ativação de anticorpos. Seus efeitos anti-bactericidas são descritos na literatura, mas esses dados são controversos quanto a um potencial efeito benéfico da ozonioterapia no tratamento de certos tipos de infecção. OBJETIVO: Avaliar os efeitos da aplicação intraperitoneal (i.p.) de uma mistura gasosa de ozônio em um modelo de ligadura e punção de ceco (LPC) em ratos, através da dosagem de interleucinas (IL)-6, IL-10 e da quimiocina CINC-1 (cytokine-induced neutrophil chemoattractant), da lesão pulmonar aguda (LPA) e da análise das taxas de sobrevida. MÉTODO: Quatro grupos de ratos Wistar foram utilizados para análise de cada objetivo (CTR, LPC, LPC+O2 e LPC+O3). Os animais do grupo CTR foram submetidos somente a laparotomia. O grupo LPC foi submetido aos procedimentos de LPC. Os outros grupos foram submetidos à LPC e receberam injeção (i.p.) da mistura gasosa correspondente, administrada a cada 12 horas durante o período de observação. Os níveis séricos de IL-6, CINC-1 e IL-10 foram determinados por imuno- ensaio (enzyme linked immunosorbent assay- ELISA). A LPA foi avaliada através da histologia pulmonar e quantificada através do método do extravasamento pulmonar do Azul de Evans. Os animais da análise de sobrevivência foram observados por cinco dias. Os valores obtidos foramexpressos como médias ± erro-padrão da média (EP) ou medianas mais percentis 25 e 75(P25; P75), de acordo com a distribuição dos dados. Considerou-se significante p<0,05. RESULTADOS: Os ratos do grupo CTR exibiram os menores níveis de CINC-1 (p<0,01). O grupo LPC+O3 teve níveis menores de CINC-1 comparado a LPC+O2 e LPC (p<0,05). Os níveis de IL-10 do grupo CTR foram menores do que nos outros 3 grupos(p=0,02) . Não houve diferenças entre os outros 3 grupos (p=0,85). IL-6 foi significativamente menor para o grupo CTR (30,8± 4,8) quando comparado a todos os outros grupos (p<0,001). LPC+O3 e LPC+O2 exibiram níveis menores quando comparados ao grupo LPC (p<0,01). Não houve diferença entre os grupos LPC+O3 e LPC+O2 (p=0,54). O escore de histologia pulmonar foi menor para CTR (p=0,02). Os outros grupos não apresentaram diferenças significantes intergrupos (p=0,3). Os valores dos coeficientes de extravasamento pulmonar do Azul de Evans foram menores para LPC+O3 quando comparado aos grupos LPC+O2 e LPC (p=0,02), porém não houve diferença na comparação com CTR O grupo CTR teve o maior tempo de sobrevida (110±10h) comparado com os outros grupos, ou seja, LPC (57,3± 10,4h), LPC+O2 (71 ± 12,9h) e LPC+O3 (52,1 ± 8), os quais não apresentaram diferenças entre si quanto à sobrevida (p=0,4). CONCLUSÃO: No presente estudo experimental em ratos, a ozonioterapia teve um benefício potencial na modulação da resposta inflamatória e na LPA, mas não influenciou as taxas de sobrevida dos animais. / INTRODUCTION: Ozone (O3) is found in nature and also can be produced in the human body through activation of antibodies. Its antibacterial effect has been described in the literature, but these data are controversial regardi ng a benefic role of O3 therapy in the treatment of certain types of infection. OBJECTIVE: To evaluate the effects of intraperitoneal (i.p.) application of an O3 gas mixture in a rat model of cecal ligation and puncture (CLP), by analyzing interleukin (IL)-6, IL-10 and cytokine-induced neutrophil chemoattractant (CINC)-1 levels, acute lung injury (ALI) and survival rates. METHOD: Four animal groups were used (SHAM, CLP, CLP+O2 and CLP+O3). SHAM animals were submitted solely to laparotomy. CLP group was submitted to cecal ligation and puncture. The other groups were submitted to CLP and received injections (i.p.) of the corresponding gas mixture every 12 hours during the observation period. The serum concentrations of IL-6, CINC-1 and IL-10 were determined by the enzyme-linked immunosorbent assay (ELISA). ALI was evaluated with pulmonary histology and quantitated by means of the Evans blue dye (EBD) lung leakage method. For survival analysis, animals were observed for 5 days. Values were expressed as means ± SEM or medians (P25; P75), according to the data distribution. A p<0,05 was considered significant.RESULTS: SHAM rats had the lowest levels of CINC-1 compared to all other groups (p<0,01). CLP+O3 group had lower levels of CINC-1 compared to CLP+O2 and CLP (p<0,05). SHAM IL-10 levels were the lowest compared to the 3 other groups (p=0,02). There were no differences between the other 3 groups (p=0,85). IL-6 was significantly lower for SHAM compared to all groups (p<0,001). CLP+O3 and CLP+O2 had lower levels when compared to CLP (p<0,01). Comparison between groups CLP+O3 and CLP+O2 showed no significant difference (p=0,54). Pulmonary histology score was lower for SHAM (p=0,02). The other groups presented no statistical difference when compared to each other (p=0,3). EBD lung leakage values were lower to CLP+O3 compared to CLP+O2 and CLP (p=0,02). SHAM group had the longest survival time (110±10h) compared to all other groups (p=0,002). CLP (57,3± 10,4h), CLP+O2 (71 ± 12,9h) and CLP+O3 (52,1 ± 8h), which did not show difference on survival compared to each other (p=0,4). CONCLUSION: In this rat model of sepsis, ozone therapy had a potential benefit in the modulation of inflammatory response and ALI, but no improvement on survival rates was observed.

Sepsis-induced organ failure is associated with high morbidity and mortality rates. The onset of an exaggerated host response to microbial invasion and/or trauma, is believed to be the primary cause of excessive inflammation and the subsequent tissue hypoperfusion observed in patients with severe sepsis. In our mouse model of sepsis induced by cecal ligation and perforation (CLP), symptoms indicative of the disease, including diarrhea, increased ventilation and persistent hypothermia, are present at six hours after the surgery (T6). In the untreated CLP mice, mortality occurs starting at T15. As induced hypothermia has shown to exert immunomodulatory effects, this study is aimed at assessing its potential in attenuating inflammation and improving survival in experimental sepsis. Our data has shown that deep hypothermia initiated at T6, by means of cold chamber-induced cooling, prolongs survival. Plasma cytokine quantification by enzyme-linked immunosorbent assays (ELISA) also reveals that induced deep hypothermia reduces tumour necrosis factor(TNF)-α and interleukin (IL)-6 production in untreated CLP mice. In contrast, induced moderate hypothermia does not have such effect. Antibiotic (cefotaxime) and saline resuscitation initiated immediately following CLP ensures survival. However, when these supportive treatments are initiated at T6, >50% mortality is observed in the CLP mice with or without induced hypothermia. In summary, this preliminary study provides proof for a downregulated inflammatory response mediated by external cooling. However, to achieve a survival benefit, treatment strategies in addition to cooling and antibiotics may be required.

Sepsis-induced organ failure is associated with high morbidity and mortality rates. The onset of an exaggerated host response to microbial invasion and/or trauma, is believed to be the primary cause of excessive inflammation and the subsequent tissue hypoperfusion observed in patients with severe sepsis. In our mouse model of sepsis induced by cecal ligation and perforation (CLP), symptoms indicative of the disease, including diarrhea, increased ventilation and persistent hypothermia, are present at six hours after the surgery (T6). In the untreated CLP mice, mortality occurs starting at T15. As induced hypothermia has shown to exert immunomodulatory effects, this study is aimed at assessing its potential in attenuating inflammation and improving survival in experimental sepsis. Our data has shown that deep hypothermia initiated at T6, by means of cold chamber-induced cooling, prolongs survival. Plasma cytokine quantification by enzyme-linked immunosorbent assays (ELISA) also reveals that induced deep hypothermia reduces tumour necrosis factor(TNF)-α and interleukin (IL)-6 production in untreated CLP mice. In contrast, induced moderate hypothermia does not have such effect. Antibiotic (cefotaxime) and saline resuscitation initiated immediately following CLP ensures survival. However, when these supportive treatments are initiated at T6, >50% mortality is observed in the CLP mice with or without induced hypothermia. In summary, this preliminary study provides proof for a downregulated inflammatory response mediated by external cooling. However, to achieve a survival benefit, treatment strategies in addition to cooling and antibiotics may be required.

Sepsis-induced organ failure is associated with high morbidity and mortality rates. The onset of an exaggerated host response to microbial invasion and/or trauma, is believed to be the primary cause of excessive inflammation and the subsequent tissue hypoperfusion observed in patients with severe sepsis. In our mouse model of sepsis induced by cecal ligation and perforation (CLP), symptoms indicative of the disease, including diarrhea, increased ventilation and persistent hypothermia, are present at six hours after the surgery (T6). In the untreated CLP mice, mortality occurs starting at T15. As induced hypothermia has shown to exert immunomodulatory effects, this study is aimed at assessing its potential in attenuating inflammation and improving survival in experimental sepsis. Our data has shown that deep hypothermia initiated at T6, by means of cold chamber-induced cooling, prolongs survival. Plasma cytokine quantification by enzyme-linked immunosorbent assays (ELISA) also reveals that induced deep hypothermia reduces tumour necrosis factor(TNF)-α and interleukin (IL)-6 production in untreated CLP mice. In contrast, induced moderate hypothermia does not have such effect. Antibiotic (cefotaxime) and saline resuscitation initiated immediately following CLP ensures survival. However, when these supportive treatments are initiated at T6, >50% mortality is observed in the CLP mice with or without induced hypothermia. In summary, this preliminary study provides proof for a downregulated inflammatory response mediated by external cooling. However, to achieve a survival benefit, treatment strategies in addition to cooling and antibiotics may be required.

INTRODUÇÃO: O ozônio (O3) é encontrado na natureza e também pode ser produzido no corpo humano através da ativação de anticorpos. Seus efeitos anti-bactericidas são descritos na literatura, mas esses dados são controversos quanto a um potencial efeito benéfico da ozonioterapia no tratamento de certos tipos de infecção. OBJETIVO: Avaliar os efeitos da aplicação intraperitoneal (i.p.) de uma mistura gasosa de ozônio em um modelo de ligadura e punção de ceco (LPC) em ratos, através da dosagem de interleucinas (IL)-6, IL-10 e da quimiocina CINC-1 (cytokine-induced neutrophil chemoattractant), da lesão pulmonar aguda (LPA) e da análise das taxas de sobrevida. MÉTODO: Quatro grupos de ratos Wistar foram utilizados para análise de cada objetivo (CTR, LPC, LPC+O2 e LPC+O3). Os animais do grupo CTR foram submetidos somente a laparotomia. O grupo LPC foi submetido aos procedimentos de LPC. Os outros grupos foram submetidos à LPC e receberam injeção (i.p.) da mistura gasosa correspondente, administrada a cada 12 horas durante o período de observação. Os níveis séricos de IL-6, CINC-1 e IL-10 foram determinados por imuno- ensaio (enzyme linked immunosorbent assay- ELISA). A LPA foi avaliada através da histologia pulmonar e quantificada através do método do extravasamento pulmonar do Azul de Evans. Os animais da análise de sobrevivência foram observados por cinco dias. Os valores obtidos foramexpressos como médias ± erro-padrão da média (EP) ou medianas mais percentis 25 e 75(P25; P75), de acordo com a distribuição dos dados. Considerou-se significante p<0,05. RESULTADOS: Os ratos do grupo CTR exibiram os menores níveis de CINC-1 (p<0,01). O grupo LPC+O3 teve níveis menores de CINC-1 comparado a LPC+O2 e LPC (p<0,05). Os níveis de IL-10 do grupo CTR foram menores do que nos outros 3 grupos(p=0,02) . Não houve diferenças entre os outros 3 grupos (p=0,85). IL-6 foi significativamente menor para o grupo CTR (30,8± 4,8) quando comparado a todos os outros grupos (p<0,001). LPC+O3 e LPC+O2 exibiram níveis menores quando comparados ao grupo LPC (p<0,01). Não houve diferença entre os grupos LPC+O3 e LPC+O2 (p=0,54). O escore de histologia pulmonar foi menor para CTR (p=0,02). Os outros grupos não apresentaram diferenças significantes intergrupos (p=0,3). Os valores dos coeficientes de extravasamento pulmonar do Azul de Evans foram menores para LPC+O3 quando comparado aos grupos LPC+O2 e LPC (p=0,02), porém não houve diferença na comparação com CTR O grupo CTR teve o maior tempo de sobrevida (110±10h) comparado com os outros grupos, ou seja, LPC (57,3± 10,4h), LPC+O2 (71 ± 12,9h) e LPC+O3 (52,1 ± 8), os quais não apresentaram diferenças entre si quanto à sobrevida (p=0,4). CONCLUSÃO: No presente estudo experimental em ratos, a ozonioterapia teve um benefício potencial na modulação da resposta inflamatória e na LPA, mas não influenciou as taxas de sobrevida dos animais. / INTRODUCTION: Ozone (O3) is found in nature and also can be produced in the human body through activation of antibodies. Its antibacterial effect has been described in the literature, but these data are controversial regardi ng a benefic role of O3 therapy in the treatment of certain types of infection. OBJECTIVE: To evaluate the effects of intraperitoneal (i.p.) application of an O3 gas mixture in a rat model of cecal ligation and puncture (CLP), by analyzing interleukin (IL)-6, IL-10 and cytokine-induced neutrophil chemoattractant (CINC)-1 levels, acute lung injury (ALI) and survival rates. METHOD: Four animal groups were used (SHAM, CLP, CLP+O2 and CLP+O3). SHAM animals were submitted solely to laparotomy. CLP group was submitted to cecal ligation and puncture. The other groups were submitted to CLP and received injections (i.p.) of the corresponding gas mixture every 12 hours during the observation period. The serum concentrations of IL-6, CINC-1 and IL-10 were determined by the enzyme-linked immunosorbent assay (ELISA). ALI was evaluated with pulmonary histology and quantitated by means of the Evans blue dye (EBD) lung leakage method. For survival analysis, animals were observed for 5 days. Values were expressed as means ± SEM or medians (P25; P75), according to the data distribution. A p<0,05 was considered significant.RESULTS: SHAM rats had the lowest levels of CINC-1 compared to all other groups (p<0,01). CLP+O3 group had lower levels of CINC-1 compared to CLP+O2 and CLP (p<0,05). SHAM IL-10 levels were the lowest compared to the 3 other groups (p=0,02). There were no differences between the other 3 groups (p=0,85). IL-6 was significantly lower for SHAM compared to all groups (p<0,001). CLP+O3 and CLP+O2 had lower levels when compared to CLP (p<0,01). Comparison between groups CLP+O3 and CLP+O2 showed no significant difference (p=0,54). Pulmonary histology score was lower for SHAM (p=0,02). The other groups presented no statistical difference when compared to each other (p=0,3). EBD lung leakage values were lower to CLP+O3 compared to CLP+O2 and CLP (p=0,02). SHAM group had the longest survival time (110±10h) compared to all other groups (p=0,002). CLP (57,3± 10,4h), CLP+O2 (71 ± 12,9h) and CLP+O3 (52,1 ± 8h), which did not show difference on survival compared to each other (p=0,4). CONCLUSION: In this rat model of sepsis, ozone therapy had a potential benefit in the modulation of inflammatory response and ALI, but no improvement on survival rates was observed.

Sepsis-induced organ failure is associated with high morbidity and mortality rates. The onset of an exaggerated host response to microbial invasion and/or trauma, is believed to be the primary cause of excessive inflammation and the subsequent tissue hypoperfusion observed in patients with severe sepsis. In our mouse model of sepsis induced by cecal ligation and perforation (CLP), symptoms indicative of the disease, including diarrhea, increased ventilation and persistent hypothermia, are present at six hours after the surgery (T6). In the untreated CLP mice, mortality occurs starting at T15. As induced hypothermia has shown to exert immunomodulatory effects, this study is aimed at assessing its potential in attenuating inflammation and improving survival in experimental sepsis. Our data has shown that deep hypothermia initiated at T6, by means of cold chamber-induced cooling, prolongs survival. Plasma cytokine quantification by enzyme-linked immunosorbent assays (ELISA) also reveals that induced deep hypothermia reduces tumour necrosis factor(TNF)-α and interleukin (IL)-6 production in untreated CLP mice. In contrast, induced moderate hypothermia does not have such effect. Antibiotic (cefotaxime) and saline resuscitation initiated immediately following CLP ensures survival. However, when these supportive treatments are initiated at T6, >50% mortality is observed in the CLP mice with or without induced hypothermia. In summary, this preliminary study provides proof for a downregulated inflammatory response mediated by external cooling. However, to achieve a survival benefit, treatment strategies in addition to cooling and antibiotics may be required.

Dans un contexte de limitation de l'utilisation des antibiotiques, de nouvelles stratégies doivent être mises en place pour préserver la santé des animaux, en particulier lors du sevrage. En cuniculture, les stratégies de limitation de l'ingestion permettent de réduire les troubles digestifs en post sevrage et améliorent l'efficacité alimentaire, mais induisent inévitablement un retard de croissance et une baisse du rendement à l'abattage. Ce travail de thèse avait pour objectif d'une part d'optimiser les performances de croissance et de rendement à l'abattage des animaux soumis à une restriction alimentaire via une modulation de la quantité et de la qualité énergétique des aliments et d'autre part d'expliciter les mécanismes physiologiques sous-jacents aux effets bénéfiques d'une ingestion restreinte sur la santé et l'efficacité alimentaire. Nous avons démontré que dans le cadre d'une stratégie de restriction alimentaire un aliment riche en énergie digestible permet d'optimiser la croissance (+2%), le rendement à l'abattage (+0,6 points) et l'efficacité alimentaire (+11%), via notamment une amélioration de l'efficacité digestive, sans pénaliser les paramètres sanitaires, mais ne permet de rattraper que partiellement le retard de croissance induit par la restriction alimentaire (-3% de poids à l'abattage par rapport aux animaux nourris à volonté). Un aliment riche en amidon était favorable à la croissance de ces animaux et en particulier au rendement à l'abattage et n'avait pas d'effet délétère sur les paramètres sanitaires. La restriction alimentaire et la concentration énergétique alimentaire dans une moindre mesure, modulaient la réponse immunitaire via une réduction de la réponse humorale digestive (IgA intestinaux) et systémique (IgA et IgG circulants) sans modification de la réponse inflammatoire. L'activité du microbiote caecal était quant à elle modulée par la masse d'aliment ingérée alors que le profil de la communauté bactérienne caecale, sa diversité et sa richesse ne semblaient pas modifiés par la restriction alimentaire. Toutefois l'abondance relative de certaines familles (Eubacteriaceae, Peptococcaceae et Christensenellaceae) et genres montraient des variations spécifiques à la restriction ou à la concentration énergétique de l'aliment. L'ensemble de ces résultats indiquent que les relations entre les mesures physiologiques et les effets bénéfiques de la restriction alimentaire sur la santé digestive restent complexes et suggèrent l'implication d'autres paramètres métaboliques non évalués. Des hypothèses complémentaires de travail doivent ainsi être envisagées Toutefois nos travaux établissent l'intérêt économique de la restriction alimentaire, quel que soit le contexte sanitaire, et celui de l'utilisation d'aliments à haute valeur énergétique sur les performances zootechniques. Notre étude ouvre dès lors des perspectives de recherches complémentaires d'optimisation des stratégies alimentaires via une modulation de la durée de restriction alimentaire et de la qualité énergétique des aliments. / With the reduction in antibiotic use new strategies are required in order to preserve animal health, particularly around weaning. In rabbit breading feed restriction strategies allow for the reduction of post-weaning digestive disorders and improve feed efficiency, but are inevitably responsible for a reduced growth and carcass yield at slaughter. This work aimed to optimize the growth performances and slaughter yield of restricted fed rabbits through a modulation of the dietary energy concentration and quality. The second objective of this work was to explain the physiological mechanisms underlying the beneficial effects of feed restriction upon health and feed efficiency. We demonstrated that, under a restriction strategy, a diet rich in digestible energy increased growth (+2%), slaughter yield (+0.6 points) and feed efficiency (+11%), through an improved digestive efficiency, without penalizing the sanitary parameters. The reduced growth induced by feed restriction was however only partially compensated for (-3% in final weight compared to the ad libitum fed animals). A diet rich in starch induced a higher growth and slaughter yield and did not penalize the sanitary parameters under a restriction strategy. Feed restriction and, to a lesser extent, the dietary energy concentration, modulated the immune response through a reduced humoral response at the digestive (fecal IgA) and systemic (plasmatic IgA and IgG) levels without modifying the inflammatory response. The cecal microbial activity was modulated by the amount of feed ingested while the cecal bacterial community profile, diversity and richness were not affected by the feed intake level. However, the relative abundance of certain families (Eubacteriaceae, Peptococcaceae and Christensenellaceae) and generas demonstrated specific variations according to the feed intake level and the dietary energy concentration. These results indicate that the relationship between the physiological measurements and the beneficial effects of feed restriction upon health remain complex and suggest the implication of other metabolic parameters that weren't measured. Complementary work hypothesis must therefore be considered. This work however enabled us to establish the economic advantage of feed restriction strategies regardless of the sanitary status and demonstrated the beneficial effects of a high energy diet on growth parameters. Our study opens the door to new complementary research projects regarding the optimization of the feeding strategies through for instance the modulation of the restriction length and the dietary energy quality.

<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/Fe³⁺ 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>