Orientador: Marcia Carneiro Valera Garakis / Coorientador: Flávia Goulart da Rosa Cardoso / Banca: Claudio Talge Carvalho / Banca: Flaviana Bombarda de Andrade / Resumo: Os objetivos deste trabalho são: 1) Quantificar por checkerboard a carga microbiana e pelo método de LAL endotoxinas (EU/mL) nas infecções endodônticas primárias; 2) Realizar o monitoramento dos níveis de endotoxinas (EU/mL) e de carga microbiana antes do tratamento, após o preparo biomecânico com sistemas de instrumentação rotatória, reciprocante e híbrida e após o uso da medicação intracanal; 3) Relacionar sinais e sintomas clínicos com níveis de endotoxinas, micro-orgnismos e com complexos bacterianos; 4) Relacionar volumetria dos canais radiculares por meio de TCFC com níveis de endotoxina, micro-organismos e complexos bacterianos. Trinta dentes com infecção endodôntica primária e presença de lesão periapical foram submetidos a TCFC antes do tratamento e avaliados quanto a presença de sinais e sintomas clínicos. Após abertura coronária, foi realizada a coleta inicial nos canais radiculares, e em seguida, procedeu-se com o tratamento endodôntico, sendo os dentes divididos em diferentes grupos experimentais de acordo com o sistema de instrumentação utilizado (n=10): rotatório Mtwo (MTWO), reciprocante Reciproc (REC), e híbrido Genius (GEN). Durante o preparo biomecânico, os canais foram irrigados com 24 mL de NaOCl 2,5%. Foram realizadas coletas do conteúdo dos canais radiculares: logo após a abertura coronária (1 col), após a instrumentação (2 col), e após a MIC por 14 dias, realizada com pasta de hidróxido de cálcio associada a solução salina fisiológica (3 col). A detecç... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aims of this study are: 1) Quantify by checkerboard test the microbial load and endotoxins through LAL method (EU/ml) in primary endodontic infections; 2) To monitore levels of endotoxin (EU/ml) and microbial load before treatment, after biomechanical preparation with rotatory, reciprocating and hybrid instrumentation systems, and after use of intracanal medication; 3) To associate clinical signs and symptoms with endotoxin levels, microorganism and bacterial complexes; 4) To relate volumes of root canals through cone beam computed tomography (CBCT), with endotoxin levels, microorganisms and bacterial complexes. Thirty teeth with primary endodontic infection and periapical lesion were submitted to endodontic treatment after CBCT and evaluated the presence of clinical signs and symptoms. After coronary opening, the initial samples were collected to verify the presence of infection in root canals. Then, teeth were divided into different experimental groups according to the instrumentation system used (n=10): rotatory Mtwo (MTWO), reciprocating Reciproc (REC), and hybrid Genius (GEN). During biomechanical preparation, the canals were irrigated with 24 mL of 2.5% NaOCl. Samples were collected: after coronary opening (S1), after the instrumentation (S2) and after intracanal medication for 14 days with calcium hydroxide paste and physiological saline solution (S3). The detection of microorganisms was performed by checkerboard DNA-DNA hybridization technique. The endotoxin quantification was performed by chromogenic kinetic test of the Limulus amoebocyte lysate. The root canal volumetries were performed by Nemotec® software. All data were analyzed statistically. The results showed the detection of microorganisms and endotoxins in 100% of the S1, with the most prevalent bacteria being C. ochracea and F. nucleatum (53%). After biomechanical preparation, the most found microorganisms were F. nucleatum.. (Resumo completo, clicar acesso eletrônico abaixo / Mestre

The objective of the study was to investigate and characterize the effects of a continuous rate infusion of dextrose or dextrose and insulin on glucose and insulin dynamics in both healthy and endotoxin-exposed horses. Administration of a low dose of endotoxin has been used in horses to mimic the clinicopathologic changes seen in endotoxemia, including the development of an inflammatory response. Our hypothesis was that a continuous rate infusion of insulin at a rate of 0.07 IU/kg/hr would prevent the development of hyperglycemia induced by administration of dextrose in both healthy and endotoxin-exposed horses. Nine healthy adult horses were used in the study. In Phase 1 of the experiment, horses received a saline infusion or a dextrose infusion in a balanced crossover design. In Phase 2 of the experiment, horses received a dextrose and insulin infusion, both prior to and after receiving a low dose of endotoxin (no LPS group and LPS group respectively) in a balanced crossover design. Blood samples were collected at regular intervals throughout both phases for measurement of plasma glucose and insulin concentrations. Infusion of dextrose alone resulted in hyperglycemia for nearly the entire study period. Insulin concentration was also increased in comparison to the saline infusion. When comparing the dextrose treatment group to the combined dextrose and insulin treatment group (no LPS group), the insulin levels were significantly greater over time in the latter group and resulted in maintenance of euglycemia. When comparing the no LPS group to the LPS group, both the glucose and insulin concentrations were higher in the LPS group but euglycemia was still achieved. These results serve to validate the dose of insulin used in this study (0.07 IU/kg/hr) in regards to effective prevention of hyperglycemia when administered concurrently with a dextrose infusion. Hyperglycemia was prevented in both healthy and endotoxin-exposed horses. In addition, the dose of insulin used was demonstrated to be safe, as hypoglycemia did not occur in any of the horses. / Master of Science

Obesity-related metabolic derangements have been linked to toll-like receptor 4 (TLR4), an innate immune system receptor, due to its role in proinflammatory pathways. Lipopolysaccharide (LPS), a gram-negative bacteria cell wall component, is the ligand for TLR4, and has been shown to be elevated in states of metabolic disease. Heightened levels of circulating endotoxin is termed metabolic endotoxemia and has been linked to systemic inflammation which is associated with obesity, type 2 diabetes mellitus (T2DM), and cardiovascular disease (CVD). Immune cells exhibit a protective ability to develop endotoxin tolerance. The objective of this study was to determine if endotoxin tolerance exists in skeletal muscle cells, and if a condition that mimics a state of over nutrition, such as elevated levels of fatty acids, affect this tolerance. To this end, L6 skeletal muscle cells were treated with low (50 pg/mL)- and high (500 ng/mL)-doses of LPS, with and without the presence of free fatty acids (FFAs). Tolerance was assessed by measuring: 1) changes in mRNA expression of interleukin-6 (IL-6) and monocyte chemoattractant-1 (MCP-1) as markers of a pro-inflammatory response; and 2) mRNA levels of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1-°) and mitochondrial oxidative capacity via an XF24 Flux Analyzer (Seahorse Bioscience) as measures of the metabolic response. Tolerance to LPS was observed in response to low- and high-doses with MCP-1 mRNA transcription but not IL-6. Changes in PGC1-° and mitochondrial OCR exhibited a tolerant effect in response to the high dose of LPS but not the low dose. The addition of free fatty acids to LPS treatments did not prevent the tolerant effects under any conditions. In conclusion, LPS tolerance exists in skeletal muscle cells but appears to differ depending on pro-inflammatory target and LPS concentration. Additionally, fatty acids, in the current model, have no effect on LPS tolerance. / Master of Science

Eine intakte Endothelbarriere ist eine unabdingbare Voraussetzung für die uneingeschränkte Funktion sämtlicher Organe. Wird die Barrierefunktion durch entzündliche Prozesse gestört, so kommt es zum Austritt von Gefäßflüssigkeit ins Interstitium. Dies resultiert in Organversagen und ist Mitverursacher der hohen Sterblichkeit bei systemischen Entzündungsreaktionen und Sepsis. Vorangehende Untersuchungen haben bereits wichtige Mechanismen aufgedeckt, die zum Barriereverlust führen (Schlegel et al., 2009). In dieser Studie wurde untersucht, ob die Aktivierung des Transkriptionsfaktors NF ĸB für den LPS-induzierten Zusammenbruch der Endothelbarriere von Bedeutung ist. In der vorliegenden Arbeit wurde gezeigt, dass die Einwirkung von LPS in zwei verschiedenen Endothelzelllinien, den makrovaskulären PSEC sowie den mikrovaskulären HDMEC, zu einer signifikanten Aktivierung von NF ĸB führte. Dies wurde sowohl mittels Kernextraktionsversuchen als auch durch Immunfluoreszenzfärbungen nachgewiesen. Messungen des TER zeigten eine Abnahme des endothelialen Widerstands und folglich der Barrierefunktion nach Applikation von LPS, gefolgt von einer spontanen Regeneration der Barriere nach einer Inkubationszeit von 24 h. Eine Erhöhung des intrazellulären cAMP Spiegels durch Applikation von Forskolin/Rolipram verhinderte zwar die LPS induzierte Bildung interzellulärer Lücken, nicht jedoch die Aktivierung des Transkriptionsfaktors NF ĸB. Vielmehr verstärkte eine cAMP Erhöhung sogar eine NF ĸB Aktivierung in HDMEC nach 4 h, ohne die Morphologie der Endothelzelljunktionen zu schädigen. Der selektive NF ĸB Inhibitor NBD Peptid vermochte im Gegensatz dazu die LPS induzierte NF ĸB Aktivierung deutlich zu hemmen, verhinderte allerdings weder die interzelluläre Lückenbildung noch den Abfall des TER durch LPS. Im Gegenteil – da unter Einwirkung von NBD Peptid die spontane Regeneration des TER nach LPS Applikation ausblieb, schienen barrierekompromittierende Effekte von LPS durch Hemmung der NF ĸB Aktivierung mittels NBD-Peptid sogar verstärkt zu werden. In Übereinstimmung mit diesen Ergebnissen hemmte auch die Repression von NF ĸB p65 durch eine spezifische p65 siRNA den LPS induzierten Zusammenbruch der Endothelbarriere nicht. Weitere NF ĸB abhängige Proteine wie VASP und Caveolin 1, deren Beteiligung am Pathomechanismus von anderen Arbeitsgruppen vorgeschlagen wurde, blieben in unseren Experimenten unter LPS Exposition unverändert. Zusammenfassend scheint die NF ĸB Aktivierung initial nicht entscheidend am LPS induzierten Zusammenbruch der Endothelbarriere beteiligt zu sein. Unsere Ergebnisse legen vielmehr nahe, dass die Aktivierung des Transkriptionsfaktors möglicherweise Teil eines „Rescue“ Mechanismus sein könnte. / An intact endothelial barrier is indispensable for the functioning of all organs. Inflammatory processes like sepsis lead to increased endothelial permability, causing edema which often result in organ failure. Previous studies uncovered important mechanisms leading to endothelial barrier breakdown (Schlegel et al., 2009). This study analyses if activation of NF ĸB is involved in LPS-induced damage to intercellular junctions. It is demonstrated that LPS leads to activation of NF ĸB in two different endothelial cell lines, PSEC and HDMEC, shown by nuclear extraction and immunofluorescence experiments. Measurements of TER visualise reduction of endothelial resistance and thus integrity of the endothelial barrier after application of LPS, followed by spontaneous regeneration after 24 hours. Increase of intracellular cAMP levels by application of Forskolin/Rolipram prevented LPS-induced intercellular gap formation, while activation of NF ĸB was not affected. Rather, increased cAMP levels intensified activation of NF ĸB in HDMEC after 4 h, without damaging morphology of endothelial cell junctions. The selective NF ĸB-inhibitor NBD-peptide prevented LPS-induced NF ĸB activation but not intercellular gap formation and reduction of TER. Because NBD-peptide blocked spontaneous regeneration of TER after LPS application, barrier compromising effects of LPS appeared to be intensified by inhibition of NF ĸB activation. In accordance with these results repression of NF ĸB p65 by siRNA did not prevent LPS-induced barrier breakdown. Other NF ĸB-dependent proteins like VASP and Caveolin-1, both suggested to be involved in the pathomechanism, remained unaffected. In summary, NF ĸB activation appears not to be initially involved in LPS-induced endothelial barrier breakdown. The results of this study suggest activation of NF ĸB might be part of a “rescue” mechanism.

This study tested the role of the calcium dependent cytosolic protein calpain in neutrophilic hepatitis. We hypothesized that inhibition of calpain would protect against LPS-induced neutrophilic liver damage. To test our hypothesis, a reliable LPS-mediated hepatitis model to investigate the mechanisms of hepatic neutrophil infiltration following LPS administration was developed by repeat intravenous injection of LPS at a dose of 10 mg/kg to rats. Blood was collected for hematologic and biochemical analysis and multiple organs including liver were collected for evaluation of histopathologic changes. Flow cytometry was employed to investigate L-selectin (CD 62L) and MAC-1 (CD11b/18) expression on neutrophils both in vivo and in vitro. Significant hematologic changes included neutrophilia, elevation in neutrophil to lymphocyte ratio with toxic changes and left shift. Biochemical changes were observed in several liver (AST, GGT) and kidney (BUN) parameters generally at the earliest time points. Histopathology revealed a time-dependent neutrophil and mononuclear infiltration around the periportal areas in the single dose study and mid-zonal inflammation with multifocal coagulative necrosis in the repeated dose study. CD 11b was up-regulated both in vitro and in vivo. After development of a suitable model, the first goal was to investigate the role of the intracellular enzyme calpain in the development of neutrophilic hepatitis and midzonal necrosis. A second goal was to compare the observed protective effects of calpain inhibition with a relatively selective inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine and an inhibitor of coagulation, heparin. When compared to rats administered LPS alone, administration of calpain 1 inhibitor prior to LPS significantly reduced hepatic iNOS expression, hepatic neutrophil infiltration and attenuated midzonal hepatic necrosis. Administration of heparin and aminoguanidine prior to LPS also decreased liver iNOS expression, hepatic neutrophil infiltration and liver pathology comparable to calpain inhibition. Blood neutrophil activation, as measured by the neutrophil adhesion molecule CD11b integrin, was upregulated in all the LPS treated groups regardless of inhibitor administration. We conclude that amelioration of liver pathology via calpain inhibition is likely dependent on the down-regulation of iNOS expression in the rat model of LPS mediated hepatitis.

This study tested the role of the calcium dependent cytosolic protein calpain in neutrophilic hepatitis. We hypothesized that inhibition of calpain would protect against LPS-induced neutrophilic liver damage. To test our hypothesis, a reliable LPS-mediated hepatitis model to investigate the mechanisms of hepatic neutrophil infiltration following LPS administration was developed by repeat intravenous injection of LPS at a dose of 10 mg/kg to rats. Blood was collected for hematologic and biochemical analysis and multiple organs including liver were collected for evaluation of histopathologic changes. Flow cytometry was employed to investigate L-selectin (CD 62L) and MAC-1 (CD11b/18) expression on neutrophils both in vivo and in vitro. Significant hematologic changes included neutrophilia, elevation in neutrophil to lymphocyte ratio with toxic changes and left shift. Biochemical changes were observed in several liver (AST, GGT) and kidney (BUN) parameters generally at the earliest time points. Histopathology revealed a time-dependent neutrophil and mononuclear infiltration around the periportal areas in the single dose study and mid-zonal inflammation with multifocal coagulative necrosis in the repeated dose study. CD 11b was up-regulated both in vitro and in vivo. After development of a suitable model, the first goal was to investigate the role of the intracellular enzyme calpain in the development of neutrophilic hepatitis and midzonal necrosis. A second goal was to compare the observed protective effects of calpain inhibition with a relatively selective inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine and an inhibitor of coagulation, heparin. When compared to rats administered LPS alone, administration of calpain 1 inhibitor prior to LPS significantly reduced hepatic iNOS expression, hepatic neutrophil infiltration and attenuated midzonal hepatic necrosis. Administration of heparin and aminoguanidine prior to LPS also decreased liver iNOS expression, hepatic neutrophil infiltration and liver pathology comparable to calpain inhibition. Blood neutrophil activation, as measured by the neutrophil adhesion molecule CD11b integrin, was upregulated in all the LPS treated groups regardless of inhibitor administration. We conclude that amelioration of liver pathology via calpain inhibition is likely dependent on the down-regulation of iNOS expression in the rat model of LPS mediated hepatitis.

Zugl.: Berlin, Freie Univ., Diss., 2008

Universiẗat, Diss., 2005--Giessen.

Made available in DSpace on 2014-06-11T19:31:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-03Bitstream added on 2014-06-13T20:22:00Z : No. of bitstreams: 1 borges_jhs_dr_jabo.pdf: 283985 bytes, checksum: b9c4395bd3d21e16f86c5a71e29c87e1 (MD5) / A endotoxemia é um distúrbio grave na clínica veterinária, sendo uma das principais causas de mortalidade em equinos.Trabalhos recentes relatam a eficácia do azul de metileno na prevenção dos danos impostos pelo óxido nítrico. Este estudo foi concebido com o fito de avaliar os efeitos do azul de metileno sobre as respostas clínica, celular e bioquímica, na endotoxemia experimental em equinos. Os animais foram divididos em três grupos. LPS+AM recebeu LPS e foi tratado com 3mg/kg de azul de metileno 60 minutos após a indução da endotoxemia; LPS+NaCl também recebeu LPS e foi tratado com 3mg/kg de azul de metileno 255 minutos após a indução da endotoxemia, e NaCl+AM que recebeu NaCl e foi tratado com azul de metileno 60 minutos após a infusão do NaCl. Foram realizados exames clínicos e laboratoriais durante 12 horas em quatro momentos. Observou-se aumento de temperatura, leucopenia e aumento no fibrinogênio nos animais pré-tratados com LPS, a partir do momento 90minutos. Não foi possível afirmar se houve efeito benéfico do azul de metileno sobre a resposta dos equinos frente à endotoxemia experimental. / Endotoxemy is a severe disturb in Veterinary Clinics and one of the most important causes of deaths in equine. Recent papers report the efficiency of methylene blue in preventing the damage caused by nitric oxide. The aim of this work was to study the effects of methylene blue in clinic, cellular and biochemistry responses in experimental endotoxemy of horses. The animals were divided in three groups. The group LPS+AM received LPS and was treated with 3mg/Kg of methylene blue 60 minutes after the induction of endotoxemy; LPS+NaCl also received LPS and was treated with 3mg/Kg of methylene blue 255 minutes after the induction of endotoxemy and NaCl+AM that received NaCl and was treated with methylene blue 60 minutes after NaCl infusion. Clinical and laboratorial exams were done in four moments during 12 hours. There was an increase in temperature levels, leucopenia and increase in fibrinogen in the animals treated with LPS, beginning within 90 minutes. It was not possible to affirm if there was any benefit in the use of methylene blue in inflammatory response of horses with induced endotoxemy.

Lipopolysaccharide (LPS) is the major surface molecule of Gram-negative bacteria and consists of three distinct structural domains: O-antigen, core, and lipid A. The lipid A (endotoxin) domain of LPS is a unique, glucosamine-based phospholipid that serves as the hydrophobic anchor of LPS and is the bioactive component of the molecule that is associated with Gram-negative septic shock. The structural genes encoding the enzymes required for the biosynthesis of Escherchia coli lipid A have been identified and characterized. Lipid A is often viewed as a constitutively synthesized structural molecule. However, determination of the exact chemical structures of lipid A from diverse Gram-negative bacteria shows that the molecule can be further modified in response to environmental stimuli. These modifications have been implicated in virulence of pathogenic Gram-negative bacteria and represent one of the molecular mechanisms of microbial surface remodeling used by bacteria to help evade the innate immune response. The intent of this review is to discuss the enzymatic machinery involved in the biosynthesis of lipid A, transport of the molecule, and finally, those enzymes involved in the modification of its structure in response to environmental stimuli.