Claritromicina e clofazimina são utilizadas no tratamento da hanseníase e em infecções causadas pelo complexo Mycobacterium avium, comuns em portadores do HIV. Devido à escassez de dados sobre a toxicidade de esquemas terapêuticos que associam estes fármacos, este estudo teve por objetivo avaliar os efeitos adversos desta terapia, em ratos machos Wistar, por meio da determinação de parâmetros hematológicos, hemostáticos e bioquímicos e correlação destes parâmetros com a dose e concentração plasmática dos medicamentos, em regime de doses únicas e múltiplas. Para tanto foram realizados: a) contagem global e específica de leucócitos (método manual) e ensaios de fagocitose e burst oxidativo de neutrófilos (citometria de fluxo); b) contagem de plaquetas (método manual), tempo de protrombina, tempo de tromboplastina parcial ativada, níveis plasmáticos dos fatores VII e X (método automatizado); c) níveis séricos de gama-glutamiltransferase (método cinéticocolorimétrico) e bilirrubinas total e direta (método colorimétrico); d) concentrações plasmáticas dos fármacos (Cromatografia Líquida de Alta Eficiência). Não houve diferenças entre as concentrações plasmáticas dos fármacos administrados em monoterapia ou politerapia. Entretanto, tanto clofazimina como claritromicina tiveram redução das concentrações plasmáticas em regime de doses múltiplas, quando comparadas à dose única. Houve aumento do número de leucócitos (dose múltipla) e de células polimorfonucleares (doses única e múltipla) nos grupos tratados com claritromicina em monoterapia ou associada à clofazimina, e redução das células mononucleares, em doses única e múltipla, nos mesmos grupos. Os fármacos parecem inverter a proporção entre células mono e polimorfonucleares. Observou-se aumento do burst oxidativo nos animais tratados com os fármacos tanto em monoterapia como em regime de politerapia. Entretanto, não houve diferença entre os tratamentos com os fármacos em relação ao controle DMSO, em dose única. Em doses múltiplas, os tratamentos com clofazimina e claritromicina em monoterapia ou politerapia estimularam o aumento do burst oxidativo (p < 0,0001) em relação ao controle DMSO. Não foram verificadas diferenças na fagocitose entre os grupos tratados e controle, tanto em dose única como em doses múltiplas. Tempo de protrombina e tempo de tromboplastina parcial ativada não foram alterados com o uso dos fármacos. Os fatores VII e X da coagulação tiveram aumento de suas atividades quando os ratos foram tratados em regime de dose múltipla com claritromicina, em regime de mono e politerapia. Houve perda de cerca de 8 % do peso de ratos tratados com clofazimina e 18 % daqueles tratados com claritromicina ou com a associação dos dois fármacos, no esquema de doses múltiplas, entretanto não houve diferença entre os grupos quando foram avaliados os níveis de gama-glutamiltransferase e bilirrubinas total e direta. Concluindo, clofazimina e claritromicina provocam alterações hematológicas, hemostáticas e bioquímicas e os resultados de concentração plasmática são valiosos para avaliação de efeitos adversos em estudos comparativos de monoterapia e politerapia entre os medicamentos. / Clarithromycin and clofazimine have been used to treat leprosy and infections caused by Mycobacterium avium complex in HIV patients. Because there are few data about the toxicity of treatment regimens involving these drugs, this study aimed to evaluate the adverse effects of this therapy in male Wistar rats through the determination of hematological, haemostatic and biochemical parameters and correlate them with the dose and plasma concentrations of drugs, under a single and multiple dose regimen. Evaluation was performed as follows: a) Global and specific count of leukocytes (manual method), phagocytosis and oxidative burst of neutrophils assays (flow cytometry), b) platelet count (manual method), prothrombin time, activated partial thromboplastin time, plasma levels of factors VII and X (automated method), c) Gamma-glutamyltransferase (kinetic-colorimetric method) and total and direct bilirubin serum levels (colorimetric method), d) plasma concentrations of drugs (High-Performance Liquid Chromatography). There were no differences between plasma concentrations of the drugs administered in monotherapy or polytherapy. However, the concentrations of both clofazimine and clarithromycin have decreased in plasma in multiple dose regimen compared to single dose. There was an increase in the number of leukocytes (multiple dose) and polymorphonuclear cells (single and multiple doses) in the groups treated with clarithromycin in monotherapy or in association with clofazimine, and a decrease in the number of mononuclear cells in single and multiple doses, in the same groups. Both drugs seemed to reverse the proportion between mononuclear and polymorphonuclear cells. The oxidative burst was observed in animals treated with drugs in polytherapy or in monotherapy, however there was no difference between the treatment with drugs and the control with DMSO in single dose. In multiple doses, treatment with clofazimine and clarithromycin in monotherapy or polytherapy stimulated the increase of oxidative burst (p <0.0001) compared to control. There were no differences in phagocytosis between the treated and control groups in single and multiple doses. Prothrombin time and activated partial thromboplastin time have not changed with the use drugs. In contrast, the activities of factors VII and X of coagulation have increased when rats were treated with multiple doses regimes with clarithromycin alone or in association with clofazimine. There was weight loss of 8% in rats treated with clofazimine and 18% in those treated with clarithromycin or with association of the drugs in the multiple doses regimen. However, there was no difference between the groups when gammaglutamyltransferase and total and direct bilirubin levels were analyzed. Therefore, clofazimine and clarithromycin induce hematological, hemostatic and biochemical changes and the results of plasma concentration is valuable for assessing adverse effects in comparative studies of monotherapy and polytherapy of these drugs.

Pre-B Cell Colony-Enhancing Factor (PBEF) is a cytokine-like molecule that functions as a nicotinamide phosphoribosyl transferase (Nampt) in a salvage pathway of NAD biosynthesis. PBEF has well-characterized activity as an extracellular inflammatory mediator and has been proposed to signal through the insulin receptor (IR). As neutrophils are key effectors of the innate immune response to infection and injury, we hypothesized that PBEF promotes pro-inflammatory function in neutrophils and that these pro-inflammatory effects may occur through interactions with the neutrophil IR or through PBEF���s enzymatic Nampt activity. Our studies focused on two important facets of neutrophil inflammatory function: their ability to generate reactive oxygen species (ROS) and undergo constitutive apoptosis. We found that, although PBEF does not activate oxidative burst on its own, it primes for ROS generation through the NADPH oxidase. PBEF promotes membrane translocation of cytosolic NADPH oxidase subunits p40phox and p47phox, but not p67phox, induces p40phox phosphorylation and activates Rac. Priming, translocation and phosphorylation are dependent on activation of p38 and ERK mitogen activated protein kinases. PBEF priming of neutrophils occurs independent of its Nampt capacity or of interactions with IR. We next investigated the effects of PBEF on neutrophil constitutive apoptosis. Our lab previously established that extracellular PBEF delays neutrophil apoptosis. Accordingly, we next investigated the mechanism through which this delay was occurring. PBEF-induced delayed apoptosis was enhanced in the presence of Nampt substrates, and NAD alone could delay apoptosis to an extent comparable to PBEF. Delayed apoptosis was blocked by a Nampt inhibitor and was lacking when a mutated PBEF deficient in Nampt activity was utilized. The cell-surface NAD glycohydrolase, CD38, can convert NAD to cyclic ADP-ribose (cADPR). Blocking CD38 activity with a blocking antibody partially reversed the delay of apoptosis induced by PBEF in conjunction with its substrates, and delayed apoptosis could be achieved by addition of the CD38 product cADPR. Finally, we found that delayed apoptosis induced by PBEF did not involve IR. These results indicate that PBEF can prime for enhanced oxidative burst and delay apoptosis in neutrophils, and that these phenomena occur through distinct mechanisms.

Pre-B Cell Colony-Enhancing Factor (PBEF) is a cytokine-like molecule that functions as a nicotinamide phosphoribosyl transferase (Nampt) in a salvage pathway of NAD biosynthesis. PBEF has well-characterized activity as an extracellular inflammatory mediator and has been proposed to signal through the insulin receptor (IR). As neutrophils are key effectors of the innate immune response to infection and injury, we hypothesized that PBEF promotes pro-inflammatory function in neutrophils and that these pro-inflammatory effects may occur through interactions with the neutrophil IR or through PBEF’s enzymatic Nampt activity. Our studies focused on two important facets of neutrophil inflammatory function: their ability to generate reactive oxygen species (ROS) and undergo constitutive apoptosis. We found that, although PBEF does not activate oxidative burst on its own, it primes for ROS generation through the NADPH oxidase. PBEF promotes membrane translocation of cytosolic NADPH oxidase subunits p40phox and p47phox, but not p67phox, induces p40phox phosphorylation and activates Rac. Priming, translocation and phosphorylation are dependent on activation of p38 and ERK mitogen activated protein kinases. PBEF priming of neutrophils occurs independent of its Nampt capacity or of interactions with IR. We next investigated the effects of PBEF on neutrophil constitutive apoptosis. Our lab previously established that extracellular PBEF delays neutrophil apoptosis. Accordingly, we next investigated the mechanism through which this delay was occurring. PBEF-induced delayed apoptosis was enhanced in the presence of Nampt substrates, and NAD alone could delay apoptosis to an extent comparable to PBEF. Delayed apoptosis was blocked by a Nampt inhibitor and was lacking when a mutated PBEF deficient in Nampt activity was utilized. The cell-surface NAD glycohydrolase, CD38, can convert NAD to cyclic ADP-ribose (cADPR). Blocking CD38 activity with a blocking antibody partially reversed the delay of apoptosis induced by PBEF in conjunction with its substrates, and delayed apoptosis could be achieved by addition of the CD38 product cADPR. Finally, we found that delayed apoptosis induced by PBEF did not involve IR. These results indicate that PBEF can prime for enhanced oxidative burst and delay apoptosis in neutrophils, and that these phenomena occur through distinct mechanisms.

This thesis is an investigation of the interactions of insulin resistance (IR), metabolic markers, neutrophil function, and reproductive health in peripartum dairy cows, including the evaluation of a hand-held glucometer for diagnosis of IR. The neutrophil functions of interest were oxidative burst and phagocytosis capacity, and reproductive diseases were endometritis and cervicitis. A total of 81 Holstein cows were enrolled 3 wk prior to expected calving date from November 2010 until October 2011, and were followed until 5 wk postpartum. Known markers of IR, neutrophil function, and disease were monitored through this period. The hand-held glucometer was identified as a useful alternative to laboratory measurements of glucose. Markers of IR influenced phagocytosis capacity and reproductive disease. High haptoglobin concentrations were associated with increased risk of reproductive disease and diminished oxidative burst function. Metabolically related inhibition of neutrophil function may be important in development of reproductive disease. / National Sciences and Engineering Research Council of Canada, Ontario Graduate Scholarship Program

Made available in DSpace on 2014-06-11T19:26:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-18Bitstream added on 2014-06-13T18:30:24Z : No. of bitstreams: 1 faria_cmqg_me_arafcf.pdf: 1839645 bytes, checksum: 622ea7e8a83cf33004224bc20f5561fb (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / A infecção pelo vírus da imunodeficiência humana (HIV) ocorre com aumento dos níveis de anticorpos séricos e com grande quantidade de imunocomplexos circulantes. A associação entre as disfunções da fagocitose mediadas por receptores do tipo FcR, na infecção pelo HIV e, principalmente, na co-infecção com o Mycobacterium tuberculosis, têm sido amplamente descrita na literatura científica. A tuberculose (TB) é a infecção oportunista mais comum entre pacientes infectados pelo HIV e a causa de morte mais frequente entre os pacientes com a síndrome da imunodeficiência adquirida (AIDS). Sabe-se que a infecção pelo HIV torna o paciente susceptível às infecções oportunistas, como a tuberculose, e, essas co-infecções aumentam o potencial de replicação viral. A ineficiência do mecanismo de clearance de imunocomplexo (IC) e de fagocitose pode contribuir para o aumento da suscetibilidade às infecções oportunistas em indivíduos infectados pelo HIV. Sendo assim, a determinação dos genótipos para as variantes polimórficas funcionais dos receptores para a porção Fc da imunoglobulina de classe G (FcR), bem como das combinações dos diferentes genótipos, pode constituir um marcador importante para a patogênese da infecção pelo HIV, na co-infecção pelo M. tuberculosis e em infecções pelo M. tuberculosis isoladamente, ou seja, independente da infecção pelo vírus da AIDS. Desta forma, o objetivo deste estudo foi determinar os genótipos para os polimorfismos dos receptores FcRIIa (CD32; H/R131) e FcRIIIb (CD16; NA1/NA2) em portadores da infecção pelo HIV-1, co-infectados pelo M. tuberculosis e infectados apenas pelo M. tuberculosis, assim como a avaliação da função fagocítica através do burst oxidativo (formação de espécies reativas de oxigênio - EROs) induzido por polissacarídeos... / Infection with human immunodeficiency virus (HIV) occurs along with increased levels of serum antibodies and a large amount of circulating immune complexes. The association between FcR-dependent phagocytosis dysfunction in HIV infection and, especially, when in co-infection with Mycobacterium tuberculosis, has been widely described in scientific literature. Tuberculosis (TB) is the most common opportunistic infection among HIV-infected patients and the most frequent cause of death among patients with acquired immunodeficiency syndrome (AIDS). It is known that HIV infection makes the patient susceptible to opportunistic infections such as tuberculosis, and these co-infections increase the potential for viral replication. The inefficiency of the immune complex (IC) clearance mechanism and phagocytosis may contribute to increased susceptibility to opportunistic infections in HIV-infected individuals. Thus, determination of the functional polymorphic variants‟ genotypes for class G immunoglobulin Fc receptors (FcR) as well as combinations of different genotypes may be an important marker for the pathogenesis of HIV infection, for co- infection with M. tuberculosis and for M. tuberculosis infection alone, i.e. independent of infection with AIDS virus. Thus, the aim of this study was to determine the genotypes for FcRIIa (CD32; H/R131) and FcRIIIb (CD16; NA1/NA2) polymorphisms in patients carrying HIV-1 infection, co-infected with M. tuberculosis and infected with M. tuberculosis alone, as well as evaluating the phagocytic function via oxidative burst (formation of reactive oxygen species - ROS) induced by cell wall polysaccharides from Saccharomyces cerevisiae, known as zymosan, in peripheral blood neutrophils from these patients. Our results demonstrated reduced capacity... (Complete abstract click electronic access below)

As influências etárias do sistema imune de bezerros são descritas na primeira fase de vida desses animais e a hipótese de também ocorrerem ariações nos principais mecanismos de resposta inata do pulmão pode identificar períodos de maior suscetibilidade às principais doenças respiratórias que acometem os bezerros nesse período. Com a finalidade de minimizar os prejuízos econômicos associados às doenças respiratórias em bezerros, este estudo objetivou avaliar a dinâmica imunológica inata do sistema respiratório de bezerros sadios nos três primeiros meses de vida, no qual nove bezerros sadios foram acompanhados por três meses e submetidos a oito avaliações imunológicas. O material recuperado do lavado broncoalveolar colhido por broncoscopia foi submetido à avaliação funcional dos macrófagos alveolares utilizando as provas de fagocitose (SaPI e E.coli), burst oxidativo, quantificação de imunoglobulinas e expressão de CD14. Os dados foram avaliados pelo teste ANOVA oneway (unstacked) (paramétricos) e pelo teste Mann-Whitney (não paramétricos). Verificaramse alterações funcionais de fagócitos CD14+, que apesar de se manterem constantes em seus valores relativos durante todo o período, apresentou intensidade de fagocitose elevada pontual na terceira semana de vida e um aumento da fagocitose por mononucleares CD14+ aos 45 dias de idade com diminuição da intensidade da fagocitose por essas mesmas células a partir dessa idade. Conclui-se que a partir de 45 dias de vida os animais começam a montar uma resposta imune própria, porém pontual e que até os 90 dias não atingem a estabilidade necessária para atestar a conclusão do processo de maturação da resposta inata local. / The influences of age in calves\' immune system are described in their first phase of life. The hypothesis that variations occur in the main mechanisms of lung innate response can help to identify periods of greater susceptibility to the respiratory diseases that affect calves in the first stage of their life. With the purpose of minimizing the economic losses associated with respiratory disease in calves, this study aimed to evaluate the innate immune dynamics of the respiratory system of healthy calves in the first three months of life. Nine healthy calves were monitored for three months and eight immunologic evaluations were performed. Bronchoalveolar lavage samples were recovered by bronchoscopy. Then, the alveolar macrophages in samples were identified by protein expression of CD14 and undergone functional evaluation of phagocytosis (SAPI and E.coli) and oxidative burst. Immunoglobulin were also quantified in samples. Data was assessed by one-way ANOVA (unstacked) (parametric) and the Mann-Whitney test nonparametric). Functional alterations in phagocytes CD14 + were observed, and although their relative values were kept throughout the period, higher intensity of phagocytosis in the third week and increased phagocytosis by macrophages CD14 + at 45 days of life was observed. Decreased intensity of phagocytosis was observed after this age. It is concluded that from 45 days of life on, calves began to maintain their immune response, but until 90 days of life they did not achieve the stability to conclude the maturation of local innate response.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2018-08-03T12:16:33Z No. of bitstreams: 1 gabriellafreitasferreira.pdf: 1111733 bytes, checksum: 4b8a7e3965e36b834dcb1890165ad008 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-08-03T12:21:08Z (GMT) No. of bitstreams: 1 gabriellafreitasferreira.pdf: 1111733 bytes, checksum: 4b8a7e3965e36b834dcb1890165ad008 (MD5) / Made available in DSpace on 2018-08-03T12:21:08Z (GMT). No. of bitstreams: 1 gabriellafreitasferreira.pdf: 1111733 bytes, checksum: 4b8a7e3965e36b834dcb1890165ad008 (MD5) Previous issue date: 2015-07-03 / PROQUALI (UFJF) / Embora os principais mecanismos de ação do fluconazol, itraconazol e anfotericina B estejam relacionados ao egosterol, é possível que essas drogas tenham outros efeitos nas células fúngicas. Além dos mais, a heterorresistência é considerada um mecanismo de adaptação frente a um estresse induzido por concentrações crescentes de antifúngicos no ambiente. Sendo o itraconazol um dos azólicos usados no tratamento da criptococose, o objetivo desse estudo foi avaliar a influência do estresse oxidativo e nitrosativo gerado pelos antifúngicos em células de C. gattii e sua influência no surgimento de clones heterorresistentes. Foram estudados distintos parâmetros para avaliar os estresses oxidativo e nitrosativo induzidos pelo fluconazol, itraconazol e anfotericina B em C. gattii. Já os efeitos da heterorresistência ao itraconazol foram estudados por meio de ensaios in vitro e em modelo murino. O itraconazol simultaneamente reduziu o conteúdo de ergosterol das células de C. gattii e induziu a produção de espécies reativas de oxigênio no início do tratamento, o que levou ao aumento da atividade das enzimas peroxidase e superoxidodismutase. O mesmo não aconteceu com o fluconazol. Já a anfotericina B promoveu grande estresse oxidativo e nitrosativo nas células de C. gattii, o que levou a uma elevada peroxidação lipídica e ineficiente ativação do sistema antioxidante celular. A heterorresistência ao itraconazol foi intrínseca para todas as linhagens testaddas, alterou parâmetros farmacodinâmicos, diminuiu o diâmetro celular e o tamanho da cápsula e ativação do sistema antioxidante celular. Observou-se uma correlação positiva entre a razão superfície/volume e o nível de heterorresistência ao itraconazol. Além do mais, a heterorresistência levou a maior internalização das células criptocócicas pelos macrófagos, mas também a uma maior proliferação dentro dessa célula fagocítica, o que culminou com o aumento da virulência dos clones heterorresistentes e alta carga fúngica nos pulmões e cérebro dos camundongos. Diante desses resultados, concluiu-se que o estresse oxidativo possuiu um importante papel no mecanismo de ação do itraconazol e pode ser um dos mecanismos que levam a heterorresistência e o aumento da virulência das células de C. gattii. / Although the most accepted mechanisms of action of amphotericin B and azoles are related to ergosterol, it is possible that these drugs have other effects on the fungal cell. Moreover, heteroresistance is an adaptive mechanism developed by the microorganism to counteract the stress of increasing drug concentration in the environment. Since itraconazole is used in the therapy of cryptococcosis, the aim of this study was to evaluate the role of endogenous reactive oxygen species (ROS) and peroxynitrite produced by azoles and amphotericin B in the fungus C. gattii and its influence on emergence of heteroresistante clones to itraconazole. We studied distinct parameters to evaluate the effect of oxidative and nitrosative stresses induced by fluconazole, itraconazole and amphotericin B in C. gattii cells. The effects of the heteroresistance to itraconazole were studied by performing tests in vitro and in a murine model. Itraconazole reduces the level of ergosterol and led to ROS production in C. gattii cells in the early stages of the treatment, enhancing the antioxidant activity. The same did not happen with fluconazole. Amphotericin B caused lipid peroxidation in C. gattii cells through a greatly enhanced production of oxidative and nitrosative radicals with increased lipid peroxidation and inefficient ativaction of antioxidant cellular system. Heteroresistance to itraconazole was intrinsic in all strains tested and changed pharmacodynamics parameters, diminished cell and capsule sizes, reduced ergosterol content and enhanced the antioxidant system of heteroresistant clones. Indeed, heteroresistance to itraconazole led to the increased internalization of cryptococcal cells by macrophages, but also to a prominent proliferation inside these phagocytic cells, culminating in the higher virulence of heteroresistant clones. Based on these results, we conclude that oxidative bursts play an important role in the antifungal activity of itraconazole and may be one of the mechanisms that lead to heteroresistance and the increased virulence of C. gattii.

Claritromicina e clofazimina são utilizadas no tratamento da hanseníase e em infecções causadas pelo complexo Mycobacterium avium, comuns em portadores do HIV. Devido à escassez de dados sobre a toxicidade de esquemas terapêuticos que associam estes fármacos, este estudo teve por objetivo avaliar os efeitos adversos desta terapia, em ratos machos Wistar, por meio da determinação de parâmetros hematológicos, hemostáticos e bioquímicos e correlação destes parâmetros com a dose e concentração plasmática dos medicamentos, em regime de doses únicas e múltiplas. Para tanto foram realizados: a) contagem global e específica de leucócitos (método manual) e ensaios de fagocitose e burst oxidativo de neutrófilos (citometria de fluxo); b) contagem de plaquetas (método manual), tempo de protrombina, tempo de tromboplastina parcial ativada, níveis plasmáticos dos fatores VII e X (método automatizado); c) níveis séricos de gama-glutamiltransferase (método cinéticocolorimétrico) e bilirrubinas total e direta (método colorimétrico); d) concentrações plasmáticas dos fármacos (Cromatografia Líquida de Alta Eficiência). Não houve diferenças entre as concentrações plasmáticas dos fármacos administrados em monoterapia ou politerapia. Entretanto, tanto clofazimina como claritromicina tiveram redução das concentrações plasmáticas em regime de doses múltiplas, quando comparadas à dose única. Houve aumento do número de leucócitos (dose múltipla) e de células polimorfonucleares (doses única e múltipla) nos grupos tratados com claritromicina em monoterapia ou associada à clofazimina, e redução das células mononucleares, em doses única e múltipla, nos mesmos grupos. Os fármacos parecem inverter a proporção entre células mono e polimorfonucleares. Observou-se aumento do burst oxidativo nos animais tratados com os fármacos tanto em monoterapia como em regime de politerapia. Entretanto, não houve diferença entre os tratamentos com os fármacos em relação ao controle DMSO, em dose única. Em doses múltiplas, os tratamentos com clofazimina e claritromicina em monoterapia ou politerapia estimularam o aumento do burst oxidativo (p < 0,0001) em relação ao controle DMSO. Não foram verificadas diferenças na fagocitose entre os grupos tratados e controle, tanto em dose única como em doses múltiplas. Tempo de protrombina e tempo de tromboplastina parcial ativada não foram alterados com o uso dos fármacos. Os fatores VII e X da coagulação tiveram aumento de suas atividades quando os ratos foram tratados em regime de dose múltipla com claritromicina, em regime de mono e politerapia. Houve perda de cerca de 8 % do peso de ratos tratados com clofazimina e 18 % daqueles tratados com claritromicina ou com a associação dos dois fármacos, no esquema de doses múltiplas, entretanto não houve diferença entre os grupos quando foram avaliados os níveis de gama-glutamiltransferase e bilirrubinas total e direta. Concluindo, clofazimina e claritromicina provocam alterações hematológicas, hemostáticas e bioquímicas e os resultados de concentração plasmática são valiosos para avaliação de efeitos adversos em estudos comparativos de monoterapia e politerapia entre os medicamentos. / Clarithromycin and clofazimine have been used to treat leprosy and infections caused by Mycobacterium avium complex in HIV patients. Because there are few data about the toxicity of treatment regimens involving these drugs, this study aimed to evaluate the adverse effects of this therapy in male Wistar rats through the determination of hematological, haemostatic and biochemical parameters and correlate them with the dose and plasma concentrations of drugs, under a single and multiple dose regimen. Evaluation was performed as follows: a) Global and specific count of leukocytes (manual method), phagocytosis and oxidative burst of neutrophils assays (flow cytometry), b) platelet count (manual method), prothrombin time, activated partial thromboplastin time, plasma levels of factors VII and X (automated method), c) Gamma-glutamyltransferase (kinetic-colorimetric method) and total and direct bilirubin serum levels (colorimetric method), d) plasma concentrations of drugs (High-Performance Liquid Chromatography). There were no differences between plasma concentrations of the drugs administered in monotherapy or polytherapy. However, the concentrations of both clofazimine and clarithromycin have decreased in plasma in multiple dose regimen compared to single dose. There was an increase in the number of leukocytes (multiple dose) and polymorphonuclear cells (single and multiple doses) in the groups treated with clarithromycin in monotherapy or in association with clofazimine, and a decrease in the number of mononuclear cells in single and multiple doses, in the same groups. Both drugs seemed to reverse the proportion between mononuclear and polymorphonuclear cells. The oxidative burst was observed in animals treated with drugs in polytherapy or in monotherapy, however there was no difference between the treatment with drugs and the control with DMSO in single dose. In multiple doses, treatment with clofazimine and clarithromycin in monotherapy or polytherapy stimulated the increase of oxidative burst (p <0.0001) compared to control. There were no differences in phagocytosis between the treated and control groups in single and multiple doses. Prothrombin time and activated partial thromboplastin time have not changed with the use drugs. In contrast, the activities of factors VII and X of coagulation have increased when rats were treated with multiple doses regimes with clarithromycin alone or in association with clofazimine. There was weight loss of 8% in rats treated with clofazimine and 18% in those treated with clarithromycin or with association of the drugs in the multiple doses regimen. However, there was no difference between the groups when gammaglutamyltransferase and total and direct bilirubin levels were analyzed. Therefore, clofazimine and clarithromycin induce hematological, hemostatic and biochemical changes and the results of plasma concentration is valuable for assessing adverse effects in comparative studies of monotherapy and polytherapy of these drugs.

Toll-like receptor 4 (TLR4) belongs among chief bacteria-sensing Pattern recognition receptors. Endotoxin (lipopolysaccharide, LPS) recognition by TLR4 triggers signalling leading to release of cytokines that direct leukocyte infiltration into the inflammatory site and cause swelling. Effector mechanisms that ensure pathogen elimination include phagocytosis and oxidative burst. It has been repeatedly reported that the polymorphism in TLR4 may affect host resistance to various diseases. TLR4 may be, therefore, an important molecule in host-parasite co-evolution. Herein, I focused on TLR4 amino acid substitution Q549R which is associated with ornamentation in great tits. In tits I describe immune responsiveness to LPS stimulation on morphological and molecular level and examine effects of the Q549R substitution on inflammation and general body condition. In LPS- treated individuals I found decrease in heterophil-lymphocyte ratio (H/L) that might be caused by attraction of the blood-borne cells into the inflamed tissue. This is in striking contrast with increase in H/L in PBS-treated animals resulting from the stress response. There was no effect of Q549R on general condition and haematological parameters but I revealed a significant effect of the interaction between host Q549R genotype and the type...

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No. of bitstreams: 1 wolf_vg_me_arafcf.pdf: 3123091 bytes, checksum: 0e5f498e0767893a065858a9ae558c1c (MD5) Previous issue date: 2017-06-22 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Helicobacter pylori é um dos principais causadores de gastrite crônica e úlcera péptica, e embora o mecanismo envolvido na inflamação gástrica por esta bactéria não esteja completamente elucidado, sabe-se do importante papel das espécies reativas de oxigênio (EROs) produzidas por polimorfonucleares neutrófilos, que são atraídos e ativados pelo agente da infecção, sem que, entretanto, consigam debelar a mesma, mas que contribuem fortemente para a lesão tecidual e o processo inflamatório crônico. Diante disso, tem aumentado a busca de novas estratégias de tratamento que possam levar à redução do estresse oxidativo gerado no sítio da infecção, com consequente redução do processo inflamatório. Neste sentido, ácido gálico, juntamente com seus ésteres (galato de metila, propila, hexila e octila), foram utilizados neste estudo, com o objetivo de avaliar suas ações como inibidores da liberação de EROs por neutrófilos ativados, bem como seus efeitos antimicrobianos sobre H. pylori. Para a avaliação da atividade antioxidante dessas substâncias foram realizados ensaios livres de células (efeito supressor sobre o radical DPPH e sobre radicais peroxila), e o efeito anti-EROs foi avaliado utilizando neutrófilos isolados de sangue humano estimulados por H. pylori, Zymosan ou PMA, através de ensaio quimiluminescente dependente de luminol ou lucigenina, ensaio com WST-1, ensaio de inibição da produção de HOCl, e o teste do NBT. Ensaio antimicrobiano foi realizado através da técnica de microdiluição em caldo. A presença da cadeia lateral carbônica levou a um significativo aumento na capacidade dos ésteres em inibir a produção de EROs por neutrófilos ativados, quando comparados ao ácido precursor, com destaque para os galatos de hexila e octila, que inibiram em quase 100% a produção de radical ânion superóxido, bem como de todas as EROs do burst oxidativo de forma geral. Galatos de octila e hexila também mostraram-se ser as moléculas com maior atividade antimicrobiana sobre H. pylori, com um valor de CIM de 125 e 250g/mL, respectivamente, ao passo que as demais substâncias apresentaram valor de CIM acima de 1000 g/mL. Os resultados obtidos mostram o grande potencial dos ésteres do ácido gálico quanto à suas atividades anti-H. pylori e anti-EROs, e além disso demonstram a importância da presença de uma cadeia carbônica lateral, conferindo maior hidrofobicidade à molécula, para obter-se a máxima atividade antimicrobiana in vitro e a máxima atividade antioxidante em modelo ex vivo. Assim, os ésteres do ácido gálico apresentam-se como moléculas promissoras no tratamento da infecção por Helicobacter pylori, apresentando ação antimicrobiana sobre o mesmo, bem como na redução do estresse oxidativo gerado no sítio da infecção. / Helicobacter pylori is one of major cause of chronic gastritis and peptic ulcer disease, and although the mechanism involved in gastric inflammation by this bacterium is not fully understood, it is know the important role of reactive oxygen species (ROS) produced by polymorphonuclear neutrophils (PMNs), which are attracted and activated by infection agent, without, however, to be able to overcome the same, but which contribute strongly to the tissue damage and chronic inflammation. Therefore, it has increased the search for new strategies of treatment that can lead to the reduction of the oxidative stress generated at the infection site, with consequent reduction of the inflammatory process. In this sense, gallic acid, together with its esters (methyl, propyl, hexyl and octyl gallate), were used in this study, with the aim of evaluating their actions as inhibitors of ROS release by activated neutrophils, as well as their antimicrobial effects on H. pylori. Cell-free assays (suppressor effect on the DPPH radical and peroxyl radicals) were performed to evaluate the antioxidant activity of these substances, and the anti-EROs effect was evaluated using neutrophils isolated from human blood, stimulated by H. pylori, Zymosan or PMA, through luminol-dependent or lucigenin-dependent chemiluminescent assay, WST-1 assay, inhibition of HOCl production assay, and the NBT assay. Antimicrobial assay was performed by broth microdilution technique. The presence of the carbonic side chain led to a significant increase in the ability of the esters to inhibit the ROS production by activated neutrophils when compared to the precursor acid, especially hexyl and octyl gallates, which inhibited practically 100% of the superoxide anion radical production, as well as all ROS of the oxidative burst in general. Octyl and hexyl gallates were also shown to be the molecules with the highest antimicrobial activity on H. pylori, with a MIC value of 125 and 250 μg/mL, respectively, while the other substances had a MIC value higher than 1000 g/ml. The results show the great potential of the esters of gallic acid for their anti-H. pylori and anti-EROs activities, and furthermore demonstrate the importance of the presence of a lateral carbonic chain, giving greater hydrophobicity to the molecule, to obtain the maximum antimicrobial activity in vitro and the maximum antioxidant activity in an ex vivo model. Thus, esters of gallic acid are promising molecules in the treatment of Helicobacter pylori infection, presenting antimicrobial action on the same, as well as reducing the oxidative stress generated at the site of infection. / CNPq: 130667/2015-3 / FAPESP: 2015/21693-0